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                    Mauss − Berg − Rockstroh − Sarrazin − Wedemeyer
Hepatology 2012
Third Edition

This textbook was supported by a grant from
Roche Pharma, Germany

Hepatology 2012
A Clinical Textbook

Editors

Stefan Mauss
Thomas Berg
Juergen Rockstroh
Christoph Sarrazin
Heiner Wedemeyer

Flying Publisher

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English language and style:

Rob Camp
camporama@gmail.com

Disclaimer
Hepatology is an ever-changing field. The editors and authors of Hepatology − A Clinical
Textbook have made every effort to provide information that is accurate and complete as of the
date of publication. However, in view of the rapid changes occurring in medical science, as well
as the possibility of human error, this site may contain technical inaccuracies, typographical or
other errors. Readers are advised to check the product information currently provided by the
manufacturer of each drug to be administered to verify the recommended dose, the method and
duration of administration, and contraindications. It is the responsibility of the treating physician
who relies on experience and knowledge about the patient to determine dosages and the best
treatment for the patient. The information contained herein is provided "as is" and without
warranty of any kind. The editors and Flying Publisher & Kamps disclaim responsibility for any
errors or omissions or for results obtained from the use of information contained herein.
© 2012 by Mauss et al.
Design: Attilio Baghino, www.baghino.com
ISBN: 978-3-924774-73-8
Printed in Germany by Druckhaus Süd, www.druckhaus-sued.de

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Foreword
3rd Edition – 2012
We are pleased to present you with the 3rd Edition of Hepatology − A Clinical
Textbook. We especially want to offer our thanks to all the authors who have
worked so hard to keep their chapters fresh and up-to-date. We have included very
exciting updates, especially regarding the new oral treatments for HCV. We also are
expanding our project to try to reach a broader range of readers and look forward to
collaborating with you to connect with newer specialists as well as those not
necessarily in large urban centers or those who have less access to information on
the latest diagnostics and treatments.
We would be especially interested in hearing from you regarding your experience
with the book, and how it could be made better for you. Please let us know at
www.hepatolgytextbook.com where you can also download this book by chapter, by
section or the full book, 100% free. We hope you can give us a few minutes of your
time to help us make the next edition better for you and that this project can
continue to be a lasting success.
The Editors
Stefan Mauss, Thomas Berg, Jürgen Rockstroh, Christoph Sarrazin, Heiner Wedemeyer

Foreword
2nd Edition – 2010
Because hepatology is such a dynamic and exciting area of medicine, regular
updates are mandatory in keeping a clinical textbook useful. We are delighted to
present this second edition of Hepatology – A Clinical Textbook. The first edition
was a major success, with more than 80,000 downloads worldwide. In addition, a
Romanian translation was carried out by Camelia Sultana and Simona Ruta shortly
after the appearance of the first edition. We invite qualified people everywhere to do
the same, into any appropriate language! This web-based free-of-charge concept
made possible by unrestricted grants from Roche and Gilead has allowed the
material to reach countries usually not easily covered by print media, a special
quality of this project. We hope this second edition of Hepatology – A Clinical
Textbook will continue to be a vluable source of information for our readers.
The Editors
Stefan Mauss, Thomas Berg, Jürgen Rockstroh, Christoph Sarrazin, Heiner Wedemeyer

6 Hepatology 2012

Preface
Hepatology is a rapidly evolving field that will continue to grow and maintain
excitement over the next few decades. Viral hepatitis is not unlike HIV 10 or 15
years ago. Today, hepatitis B viral replication can be suppressed by potent antiviral
drugs, although there are risks regarding the emergence of resistance. Strategies to
enhance the eradication rates of HBV infection still need to be developed. On the
other hand, hepatitis C virus infection can be eradicated by treatment with pegylated
interferon plus ribavirin, although the sustained virologic response rates are still
suboptimal, particularly in those infected by genotype 1. Many new antiviral drugs,
especially protease and polymerase inhibitors, are currently in preclinical and
clinical development, and the first data from larger clinical trials provide some
optimism that the cure rates for patients with chronic hepatitis C will be enhanced
with these new agents. In other areas of hepatology, e.g., hereditary and metabolic
liver diseases, our knowledge is rapidly increasing and new therapeutic options are
on the horizon.
In rapidly evolving areas such as hepatology, is the book format the right medium
to gather and summarise the current knowledge? Are these books not likely to be
outdated the very day they are published? This is indeed a challenge that can be
convincingly overcome only by rapid internet-based publishing with regular
updates. Another unmatched advantage of a web-based book is the free and
unrestricted global access. Viral hepatitis and other liver diseases are a global
burden and timely information is important for physicians, scientists, patients and
health care officials all around the world.
The editors of this web-based book – Thomas Berg, Stefan Mauss, Jürgen
Rockstroh, Christoph Sarrazin and Heiner Wedemeyer – are young, bright, and
internationally renowned hepatologists who have created an excellent state-of-theart textbook on clinical hepatology. The book is well-written and provides in-depth
information without being lengthy or redundant. I am convinced that all five experts
will remain very active in the field and will continue to update this book regularly as
the science progresses. This e-book should rapidly become an international
standard.
Stefan Zeuzem – Frankfurt, January 2009

Preface
Therapeutic options and diagnostic procedures in hepatology have quickly advanced
during the last decade. In particular, the management of viral hepatitis has
completely changed since the early nineties. Before nucleoside and nucleotide
analogs were licensed to treat hepatitis B and before interferon α + ribavirin
combination therapy were approved for the treatment of chronic hepatitis C, very
few patients infected with HBV or HCV were treated successfully. The only option
for most patients with end-stage liver disease or hepatocellular carcinoma was liver
transplantation. And even if the patients were lucky enough to be successfully
transplanted, reinfection of the transplanted organs remained major challenges. In
the late eighties and early nineties discussions were held about rejecting patients
with chronic hepatitis from the waiting list as post-transplant outcome was poor.

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Today, just 15 years later, hepatitis B represents one of the best indications for liver
transplantations, as basically all reinfection can be prevented. In addition, the
proportion of patients who need to be transplanted is declining − almost all HBVinfected patients can nowadays be treated successfully with complete suppression of
HBV replication and some well-selected patients may even be able to clear HBsAg,
the ultimate endpoint of any hepatitis B treatment.
Hepatitis C has also become a curable disease with a sustained response of 5080% using pegylated interferons in combination with ribavirin. HCV treatment
using direct HCV enzyme inhibitors has started to bear fruit (we draw your attention
to the HCV Chapters).
Major achievements for the patients do sometimes lead to significant challenges
for the treating physician. Is the diagnostic work-up complete? Did I any recent
development to evaluate the stage and grade of liver disease? What sensitivity is
really necessary for assays to detect hepatitis viruses? When do I need to determine
HBV polymerase variants, before and during treatment of hepatitis B? When can I
safely stop treatment without risking a relapse? How to treat acute hepatitis B and
C? When does a health care worker need a booster vaccination for hepatitis A and
B? These are just some of many questions we have to ask ourselves frequently
during our daily routine practice. With the increasing number of publications,
guidelines and expert opinions it is getting more and more difficult to stay up-todate and to make the best choices for the patients. That is why HEPATOLOGY 2012
– A Clinical Textbook is a very useful new tool that gives a state-of-the art update
on many aspects of HAV, HBV, HCV, HDV and HEV infections. The editors are
internationally-known experts in the field of viral hepatitis; all have made
significant contributions to understanding the pathogenesis of virus-induced liver
disease, diagnosis and treatment of hepatitis virus infections.
HEPATOLOGY 2012 – A Clinical Textbook gives a comprehensive overview on
the epidemiology, virology, and natural history of all hepatitis viruses including
hepatitis A, D and E. Subsequent chapters cover all major aspects of the
management of hepatitis B and C including coinfections with HIV and liver
transplantation. Importantly, complications of chronic liver disease such as
hepatocellular carcinoma and recent developments in assessing the stage of liver
disease are also covered. Finally, interesting chapters on autoimmune and metabolic
non-viral liver diseases complete the book.
We are convinced that this new up-to-date book covering all clinically relevant
aspects of viral hepatitis will be of use for every reader. The editors and authors
must be congratulated for their efforts.
Michael P. Manns – Hannover, January 2009

8 Hepatology 2012

9

Contributing Authors
Fernando Agüero
Preventive Medicine and Public
Health
Parc de Salut Mar
Pompeu Fabra University
Public Health Agency of Barcelona
Barcelona, Spain
Hikmet Akkiz
Depatment of Gastroenterology and
Hepatology
Çukurova University, School of
Medicine
Adana, Turkey
Akif Altnibas
Yıldırım Beyazıt Education and
Research Hospital
Gastroenterology Clinic
Ankara, Turkey
Matthias J. Bahr
Dept. of Medicine I
Sana Kliniken Lübeck
Kronsforder Allee 71-73
23560 Lübeck, Germany
Lars P. Bechmann
Department of Gastroenterology and
Hepatology,
University Hospital Essen
Hufelandstr. 55
45122 Essen
Germany
Susanne Beckebaum
Department of Transplant Medicine
University Hospital Münster
Domagkstr. 3A
48149 Münster

Thomas Berg
Sektion Hepatologie
Klinik und Poliklinik für
Gastroenterologie & Rheumatologie
Universitätsklinikum Leipzig
Liebigstr. 20
04103 Leipzig, Germany
thomas.berg@medizin.uni-leipzig.de
Leber- und Studienzentrum am
Checkpoint
Charlottenstrasse 81
10969 Berlin
berg@leberzentrum-checkpoint.de
Albrecht Böhlig
Sektion Hepatologie
Klinik und Poliklinik für
Gastroenterologie & Rheumatologie
Universitätsklinikum Leipzig
Liebigstr. 20
04103 Leipzig, Germany
Florian van Bömmel
Sektion Hepatologie
Klinik und Poliklinik für
Gastroenterologie & Rheumatologie
Universitätsklinikum Leipzig
Liebigstr. 20
04103 Leipzig, Germany
Christoph Boesecke
Department of Medicine I
University Hospital Bonn
Sigmund-Freud-Strasse 25
53105 Bonn, Germany
Ali Canbay
Department of Gastroenterology and
Hepatology,
University Hospital Essen
Hufelandstr. 55
45122 Essen
Germany

10 Hepatology 2012
Carlos Cervera
Infectious Diseases Service
Hospital Clínic − IDIBAPS
University of Barcelona
Villarroel, 170
08036 Barcelona, Spain
Vito R. Cicinnati
Department of Transplant Medicine
University Hospital Münster
Domagkstr. 3A
48149 Münster
Markus Cornberg
Dept. of Gastroenterology,
Hepatology and Endocrinology
Medical School of Hannover
Carl-Neuberg-Str. 1
30625 Hannover, Germany
Maura Dandri
University Hospital HamburgEppendorf
Zentrum für Innere Medizin
I. Medizinische Klinik
Labor Hepatologie und Virus
Hepatitis
Martinistr. 52
20246 Hamburg, Germany
Neus Freixa
Psychiatry Department
Hospital Clínic − IDIBAPS
University of Barcelona
Villarroel, 170
08036 Barcelona, Spain
Juan-Carlos García-Valdecasas
Liver Transplant Unit, Department of
Surgery
Hospital Clínic − IDIBAPS
University of Barcelona
Villarroel, 170
08036 Barcelona, Spain

Guido Gerken
Department of Gastroenterology,
University Hospital Essen
Hufelandstr. 55
45122 Essen, Germany
Frank Grünhage
Medical Department II
Saarland University Hospital
Kirrbergerstr. 1
66421 Homburg, Germany
Svenja Hardtke
Dept. of Gastroenterology,
Hepatology and Endocrinology
Medical School of Hannover
Carl-Neuberg-Str. 1
30625 Hannover, Germany
Bernd Kupfer
Department of Medicine I
University Hospital Bonn
Sigmund-Freud-Strasse 25
53105 Bonn, Germany
Montserrat Laguno
Infectious Diseases Service
Hospital Clínic − IDIBAPS
University of Barcelona
Villarroel, 170
08036 Barcelona, Spain
Frank Lammert
Medical Department II
Saarland University Hospital
Kirrbergerstr. 1
66421 Homburg, Germany
Christian Lange
J. W. Goethe-University Hospital
Medizinische Klinik 1
Theodor-Stern-Kai 7
60590 Frankfurt am Main, Germany

Contributing Authors 11
Michael P. Manns
Dept. of Gastroenterology,
Hepatology and Endocrinology
Medical School of Hannover
Carl-Neuberg-Str. 1
30625 Hannover, Germany
Christian Manzardo
Infectious Diseases Service
Hospital Clínic − IDIBAPS
University of Barcelona
Villarroel, 170
08036 Barcelona, Spain
Stefan Mauss
Center for HIV and
Hepatogastroenterology
Grafenberger Allee 128a
40237 Duesseldorf, Germany
stefan.mauss@center-duesseldorf.de
José M. Miró
Infectious Diseases Service
Hospital Clínic − IDIBAPS
University of Barcelona
Villarroel, 170
08036 Barcelona, Spain
Asuncion Moreno
Infectious Diseases Service
Hospital Clínic − IDIBAPS
University of Barcelona
Villarroel, 170
08036 Barcelona, Spain
Claus Niederau
Katholische Kliniken Oberhausen
gGmbH, St. Josef Hospital
Department of Internal Medicine
Academic Teaching Hospital of the
University
Duisburg-Essen
Mülheimer Str. 83
46045 Oberhausen, Germany

Jörg Petersen
Liver Unit IFI Institute for
Interdisciplinary Medicine
Asklepios Klinik St George Hamburg
Lohmühlenstr. 5
University of Hamburg
20099 Hamburg, Germany
Sven Pischke
Dept. of Gastroenterology,
Hepatology and Endocrinology
Medical School of Hannover
Carl-Neuberg-Str. 1
30625 Hannover, Germany
Kerstin Port
Dept. of Gastroenterology,
Hepatology and Endocrinology
Medical School of Hannover
Carl-Neuberg-Str. 1
30625 Hannover, Germany
Karl-Philipp Puchner
Charité, Campus Virchow-Klinikum,
Universitätsmedizin
Medizinische Klinik m. S.
Hepatologie und Gastroenterologie
Augustenburger Platz 1
13353 Berlin, Germany
Antonio Rimola
Liver Transplant Unit - CIBEREHD
Hospital Clínic − IDIBAPS
University of Barcelona
Villarroel, 170
08036 Barcelona, Spain
J. K. Rockstroh
Department of Medicine I
University Hospital Bonn
Sigmund-Freud-Strasse 25
53105 Bonn, Germany
rockstroh@uni-bonn.de

12 Hepatology 2012
Christoph Sarrazin
J. W. Goethe-University Hospital
Medizinische Klinik 1
Theodor-Stern-Kai 7
60590 Frankfurt am Main, Germany
sarrazin@em.uni-frankfurt.de
Martin Schäfer
Department of Psychiatry
and Psychotherapy
Kliniken Essen-Mitte
Ev. Huyssenstift
Henricistraße 92
45136 Essen, Germany
Carolynne Schwarze-Zander
Department of Medicine I
University Hospital Bonn
Sigmund-Freud-Strasse 25
53105 Bonn, Germany
Ulrich Spengler
Department of Internal Medicine 1
University Hospitals of Bonn
University
Sigmund-Freud-Strasse 25
53105 Bonn, Germany
Christian P. Strassburg
Dept. of Gastroenterology,
Hepatology and Endocrinology
Medical School of Hannover
Carl-Neuberg-Str. 1
30625 Hannover, Germany
Montserrat Tuset
Pharmacy Department
Hospital Clínic − IDIBAPS
University of Barcelona
Villarroel, 170
08036 Barcelona, Spain
Jan-Christian Wasmuth
Department of Medicine I
University Hospital Bonn
Sigmund-Freud-Strasse 25
53105 Bonn, Germany

Heiner Wedemeyer
Dept. of Gastroenterology,
Hepatology and Endocrinology
Medical School of Hannover
Carl-Neuberg-Str. 1
30625 Hannover, Germany
wedemeyer.heiner@mh-hannover.de
Johannes Wiegand
Sektion Hepatologie
Klinik und Poliklinik für
Gastroenterologie & Rheumatologie
Universitätsklinikum Leipzig
Liebigstr. 20
04103 Leipzig, Germany
Stefan Zeuzem
J. W. Goethe-University Hospital
Medizinische Klinik 1
Theodor-Stern-Kai 7
60590 Frankfurt am Main, Germany

13

Table of Contents
1. Hepatitis A
Sven Pischke and Heiner Wedemeyer
The virus
Epidemiology
Transmission
Clinical course
Extrahepatic manifestations
Diagnosis
Treatment and prognosis
References

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29
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30
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2. Hepatitis B
Christoph Boesecke and Jan-Christian Wasmuth
Introduction
Transmission
Sexual transmission
Percutaneous inoculation
Perinatal transmission
Horizontal transmission
Blood transfusion
Nosocomial infection
Organ transplantation
Postexposure prophylaxis
Natural history and clinical manifestations
Acute hepatitis
Chronic hepatitis
Prognosis and survival
Extrahepatic manifestations
References

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3. Hepatitis C
Christoph Boesecke and Jan-Christian Wasmuth
Epidemiology
Transmission
Injection drug use
Blood transfusion
Organ transplantation
Sexual or household contact
Perinatal transmission
Hemodialysis
Other rare transmission routes
Needlestick injury
Clinical manifestations
Acute hepatitis
Chronic hepatitis C
Extrahepatic manifestations
Natural history

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Cirrhosis and hepatic decompensation
Disease progression
References

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4. Hepatitis E: an underestimated problem?
Sven Pischke and Heiner Wedemeyer
Introduction
HEV: genetic characteristics of the virus
Diagnosis of hepatitis E
Worldwide distribution of HEV infections
Transmission of HEV
Acute hepatitis E in immunocompetent individuals
Acute and chronic HEV infections in organ
transplant recipients
Hepatitis E in patients with HIV infection
Extrahepatic manifestations of hepatitis E
Treatment of chronic hepatitis E
Vaccination
Conclusions/Recommendations
References

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5. HBV Virology
Maura Dandri, Jörg Petersen
Introduction
Taxonomic classification and genotypes
HBV structure and genomic organization
HBV structural and non-structural proteins
The HBV replication cycle
Animal models of HBV infection
References

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6. HCV Virology
Bernd Kupfer
History
Taxonomy and genotypes
Viral structure
Genome organization
Genes and proteins
Viral lifecycle
Adsorption and viral entry
Translation and posttranslational processes
HCV RNA replication
Assembly and release
Model systems for HCV research
References

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7. Prophylaxis and Vaccination
Heiner Wedemeyer
Introduction
Prophylaxis of hepatitis viruses
Hepatitis A and E
Hepatitis B and D
Hepatitis C
Vaccination against hepatitis A
Vaccination against hepatitis B
Efficacy of vaccination against hepatitis B
Post-exposure prophylaxis
Safety of HBV vaccines
Long-term immunogenicity of hepatitis B vaccination
Prevention of vertical HBV transmission
Vaccination against hepatitis C
Vaccination against hepatitis E
References

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111
112
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8. Hepatitis B: Diagnostic Tests
Jörg Petersen
Introduction
Serological tests for HBV
Collection and transport
Hepatitis B surface antigen and antibody
Hepatitis B core antigen and antibody
Hepatitis B e antigen and antibody
Serum HBV DNA assays
HBV genotypes
Antiviral resistance testing
Assessment of liver disease
Acute HBV infection
Past HBV infection
Chronic HBV infection
Serum transaminases
Occult HBV infection
Assessment of HBV immunity
Liver biopsy and noninvasive liver transient elastography
References

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9. Hepatitis B Treatment
Florian van Bömmel, Johannes Wiegand, Thomas Berg
Introduction
Indication for antiviral therapy
Acute hepatitis B
Chronic hepatitis B
Summary of treatment indications in the German Guidelines of 2011
Endpoints of antiviral treatment
How to treat

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16 Hepatology 2012
Treatment options
Interferons
Nucleoside and nucleotide analogs
Choosing the right treatment option
Prognostic factors for treatment response
Monitoring before and during antiviral therapy
Treatment duration and stopping rules
References

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141
146
147
151
152
154

10. Management of Resistance in HBV Therapy
Stefan Mauss and Heiner Wedemeyer
Introduction
Antiviral HBV therapy – how to avoid resistance
Treatment endpoints
Resistance patterns of HBV polymerase inhibitors
Combination therapy of chronic hepatitis B to delay
development of resistance
Management of drug resistance
Special considerations in HIV/HBV coinfection
Immune escape and polymerase inhibitor
resistance
Conclusion
References

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161
162
163

11. Hepatitis D – Diagnosis and Treatment
Heiner Wedemeyer
Introduction
Virology of hepatitis delta
Epidemiology of hepatitis delta
Pathogenesis of HDV infection
Clinical course of delta hepatitis
Acute HBV/HDV coinfection
Chronic delta hepatitis
Diagnosis of delta hepatitis
Treatment of delta hepatitis
Nucleoside and nucleotide analogs
Recombinant interferon α
Pegylated interferon α
Liver transplantation for hepatitis delta
References

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174
175
176
178
179
179
179
180
181
181
182
182
183
184

12. Hepatitis C: Diagnostic Tests
Christian Lange and Christoph Sarrazin
Serologic assays
HCV core antigen assays
Nucleic acid testing for HCV
Qualitative assays for HCV RNA detection
Qualitative RT-PCR
Transcription-mediated amplification (TMA) of HCV RNA

189
189
190
191
191
192
192
192

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168
169
169

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Quantitative HCV RNA detection
Competitive PCR: Cobas® Amplicor HCV 2.0 monitor
Branched DNA hybridisation assay (Versant® HCV RNA 3.0
quantitative assay)
Real-time PCR-based HCV RNA detection assays
Cobas® TaqMan® HCV test
RealTime HCV test
HCV genotyping
Reverse hybridising assay (Versant® HCV Genotype 2.0 System (LiPA))
Direct sequence analysis (Trugene® HCV 5’NC genotyping kit)
Real-time PCR technology (RealTime™ HCV Genotype II assay)
Implications for diagnosing and managing acute and chronic hepatitis C
Diagnosing acute hepatitis C
Diagnosing chronic hepatitis C
Diagnostic tests in the management of hepatitis C therapy
References
13. Standard Therapy of Chronic Hepatitis C Virus Infection
Markus Cornberg, Svenja Hardtke, Kerstin Port,
Michael P. Manns, Heiner Wedemeyer
Goal of antiviral therapy
Basic therapeutic concepts and medication
Predictors of treatment response
Antiviral resistance
Treatment of HCV genotype 1
Treatment of naïve patients
Treatment of patients with prior antiviral treatment failure
PEG-IFN maintenance therapy
Treatment of HCV genotypes 2 and 3
Naïve patients
Treatment of HCV G2/3 patients with prior antiviral treatment failure
Treatment of HCV genotypes 4, 5, and 6
Optimisation of HCV treatment
Adherence to therapy
Management of side effects and complications
Drug interactions
Treatment of hepatitis C in special populations
Patients with acute hepatitis C
Patients with normal aminotransferase levels
Patients with compensated liver cirrhosis
Patients after liver transplantation
Hemodialysis patients
Drug abuse and patients on stable maintenance substitution
Patients with coinfections
Patients with hemophilia
Patients with extrahepatic manifestations
References

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18 Hepatology 2012
14. Hepatitis C: New Drugs
Christian Lange, Christoph Sarrazin
Introduction
HCV life cycle and treatment targets
NS3-4A protease inhibitors
Molecular biology
Ciluprevir (BILN 2061)
Telaprevir (Incivek/Incivo®) and boceprevir (Victrelis®)
Other NS3 protease inhibitors
Resistance to NS3-4A inhibitors
NS5B polymerase inhibitors
Molecular biology
Nucleoside analogs
Non-nucleoside analogs
NS5A inhibitors
Compounds targeting viral attachment and entry
Host factors as targets for treatment
Cyclophilin B inhibitors
Nitazoxanide
Silibinin
Miravirsen
Newer combination therapies
Quadruple therapy
All-oral therapy without ribavirin
All-oral therapy with ribavirin
Novel interferons
Conclusions
References

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15. Management of Adverse Drug Reactions
Martin Schaefer and Stefan Mauss
Introduction
Flu-like symptoms, fever, arthralgia and myalgia
Gastrointestinal disorders
Weight loss
Asthenia and fatigue
Cough and dyspnea
Disorders of the thyroid gland
Psychiatric adverse events
Incidence and profile of psychiatric adverse events
Preemptive therapy with antidepressants
Sleep disturbances
Hematological and immunologic effects
Skin disorders and hair loss
Adverse events with telaprevir and boceprevir
Adherence
Conclusion
References

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263
264
264
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267
268
269
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269

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16. Extrahepatic Manifestations of Chronic HCV
Karl-Philipp Puchner, Albrecht Böhlig and Thomas Berg
Introduction
Mixed cryoglobulinemia
Diagnosis
Clinical presentation
Malignant lymphoproliferative disorders/NHL
Etiology and pathogenesis of LPDs in patients with HCV infection
Treatment of lymphoproliferative disorders
Mixed cryoglobulinemia
Systemic vasculitis
Peripheral neuropathy
Further hematological manifestations
HCV-associated thrombocytopenia
HCV-related autoimmune hemolytic anemia
HCV-related glomerulonephritis
Endocrine manifestations
Dermatologic and miscellaneous manifestations
References

272
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272
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274
275
276
276
277
277
278
278
281
281
281
282
283
284
285

17. Management of HBV/HIV coinfection
Stefan Mauss and Jürgen Kurt Rockstroh
Introduction
HBV therapy in HBV/HIV-coinfected patients without HIV therapy
Treatment of chronic hepatitis B in HBV/HIV-coinfected patients
Management of resistance to HBV polymerase inhibitors
Conclusion
References

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296
298
298
298

18. Management of HCV/HIV Coinfection
Christoph Boesecke, Stefan Mauss, Jürgen Kurt Rockstroh
Epidemiology of HIV and HCV coinfection
Diagnosis of HCV in HIV coinfection
Natural course of hepatitis C in HIV coinfection
Effect of hepatitis C on HIV infection
Effect of HAART on hepatitis C
Treatment of hepatitis C in HIV coinfection
The choice of antiretrovirals while on HCV therapy
Treatment of HCV for relapsers or non-responders
Treatment of acute HCV in HIV
Liver transplantation in HIV/HCV-coinfected patients
Conclusion
References

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303
304
305
305
306
310
310
311
312
313
313

19. HBV/HCV Coinfection
Carolynne Schwarze-Zander and Jürgen Kurt Rockstroh
Epidemiology of HBV/HCV coinfection
Screening for HBV/HCV coinfection
Viral interactions between HBV and HCV

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20 Hepatology 2012
Clinical scenarios of HBV and HCV infection
Acute hepatitis by simultaneous infection of HBV and HCV
HCV superinfection
HBV superinfection
Occult HBV infection in patients with HCV infection
Chronic hepatitis in HBV/HCV coinfection
Cirrhosis
Hepatocellular carcinoma
Treatment of HBV and HCV coinfection
Conclusion
References

319
319
319
320
320
320
322
322
322
323
323

20. Assessment of Hepatic Fibrosis in Chronic Viral Hepatitis
Frank Grünhage and Frank Lammert
Introduction
Mechanisms of liver fibrosis in chronic viral hepatitis
Liver biopsy – the gold standard for staging of liver fibrosis
Surrogate markers of liver fibrosis in chronic viral hepatitis
Transient elastography
Other imaging techniques for the
assessment of liver fibrosis
Clinical decision algorithms
Summary
References

326
326
326
327
327
329
330
333
333
333
334
334

21. Diagnosis, Prognosis & Therapy of Hepatocellular Carcinoma
Ulrich Spengler
Classification of HCC
Epidemiology
Surveillance of patients at high risk and early HCC diagnosis
Diagnosis
Stage-adapted therapy for liver cancer
Potentially curative therapy in BCLC stages 0-A
Palliative therapy in BCLC stages B and C
Prophylaxis of liver cancer
References

338
338
338
339
339
340
341
341
343
345
346

22. Update in Transplant Hepatology
349
S. Beckebaum, G. Gerken, V. R. Cicinnati
349
Introduction
349
Timing and indications for liver transplantation
349
Patient evaluation
351
Pretransplant management issues
351
Waiting list monitoring of hepatitis B liver transplant candidates
352
Waiting list monitoring and treatment of hepatitis C liver transplant candidates
353
Adjunctive treatment and staging of HCC transplant candidates
353
Living donor liver transplantation: indications, donor evaluation,
and outcome
354
Perioperative complications
355

21
Long-term complications after liver transplantation
Opportunistic infections
Chronic ductopenic rejection
CNI-induced nephrotoxicity and alternative immunosuppressive
protocols
Other side effects of CNI
Corticosteroid minimization/avoidance protocols
De novo malignancies
Biliary complications
Metabolic bone disease
Recurrent diseases after liver transplantation
Recurrence of hepatitis B in the allograft
Recurrence of hepatitis C in the allograft
Recurrence of cholestatic liver diseases and autoimmune hepatitis
Outcome in patients transplanted for hepatic malignancies
Recurrent alcohol abuse after liver transplantation for
alcoholic liver disease
Experiences with liver transplantation in inherited
metabolic liver diseases in adult patients
Outcome after liver transplantation for acute hepatic failure
Conclusion
References
23. End-stage Liver Disease, HIV Infection and Liver Transplantation
José M. Miró, Fernando Agüero, Montserrat Laguno,
Christian Manzardo, Montserrat Tuset, Carlos Cervera,
Neus Freixa, Asuncion Moreno, Juan-Carlos García-Valdecasas,
Antonio Rimola, and the Hospital Clinic OLT in HIV Working Group
Introduction
Epidemiology
Clinical features of coinfected patients with ESLD
Prognosis after decompensation
Management of cirrhosis complications
Substance abuse
HCV/HBV management
Combination antiretroviral therapy (HAART)
Orthotopic liver transplant (OLT)
Liver disease criteria
HIV infection criteria
Clinical criteria
Immunological criteria
Virologic criteria
Other criteria

356
356
357
358
359
359
360
361
362
363
363
365
368
370
371
372
373
373
375
386

386
386
386
387
388
389
390
390
391
392
392
392
392
393
393
394

22 Hepatology 2012
Outcome of OLT in HIV-positive patients
HIV/HCV coinfection
HIV/HVB coinfection
Hepatocellular carcinoma
Liver retransplantation
Conclusions
References

394
395
397
398
398
399
399

24. Metabolic Liver Diseases: Hemochromatosis
Claus Niederau
Definition and classification of iron overload diseases
Type 1 HFE hemochromatosis
History
Epidemiology
Etiology and pathogenesis
Diagnosis
Early diagnosis and screening
Complications of iron overload
Therapy
Prognosis
Juvenile hereditary hemochromatosis
Transferrin receptor 2 (TFR2)-related type 3 hemochromatosis
Type 4 hemochromatosis – Ferroportin Disease
Secondary hemochromatosis
Pathophysiology
References

405
405
405
406
406
407
407
409
411
415
418
418
419
419
420
421
421
422

25. NAFLD and NASH
Claus Niederau
Introduction
Prevalence
Demographics and risk factors
Pathogenesis
Natural history
Diagnosis
Diet and lifestyle recommendations
Pharmacological treatment
Surgery for obesity
Liver transplantation (LTX) for NASH
References

427
427
427
427
428
428
429
430
431
432
432
433
433

26. Wilson’s Disease
Claus Niederau
Introduction
Clinical presentation

437
437
437
437

23
Diagnosis
Serum ceruloplasmin
Serum copper
Urinary copper excretion
Hepatic copper concentration
Radiolabelled copper
Liver biopsy findings
Neurology and MRI of the CNS
Genetic Studies
Treatment
Monitoring of treatment
References
27. Autoimmune Liver Diseases: AIH, PBC and PSC
Christian P. Strassburg
Autoimmune hepatitis (AIH)
Definition and diagnosis of autoimmune hepatitis
Epidemiology and clinical presentation
Natural history and prognosis
Who requires treatment?
Who does not require treatment?
Standard treatment strategy
Treatment of elderly patients
Alternative Treatments
Budesonide
Deflazacort
Cyclosporine A
Tacrolimus
Mycophenolic acid
Cyclophosphamide
Anti-TNF α antibodies
Ursodeoxycholic acid
Liver transplantation
Recurrence and de novo AIH after liver transplantation
Primary biliary cirrhosis
Introduction
Definition and prevalence of PBC
Diagnostic principles of PBC
Therapeutic principles in PBC
Primary sclerosing cholangitis
Diagnosis of primary sclerosing cholangitis (PSC)
Differential diagnosis: sclerosing cholangitis
Association of PSC with inflammatory bowel disease
PSC as a risk factor for cancer
Medical therapy of PSC
Therapy of IBD in PSC
References

440
440
441
442
442
442
442
443
443
443
447
449
453
453
453
453
455
457
457
458
458
461
462
463
463
464
464
464
464
465
465
466
466
467
467
468
469
470
473
473
473
475
476
477
478
479

24 Hepatology 2012
28. Alcoholic Hepatitis
Claus Niederau
Health and social problems due to alcohol
overconsumption
Classification and natural history of alcoholic liver disease
Clinical features and diagnosis of alcoholic
hepatitis
Course and severity
Mechanisms of alcohol-related liver injury
Treatment
Abstinence from alcohol
Supportive therapy
Corticosteroids
Pentoxifylline
N-acetyl cysteine
Anti-TNF-α therapy
Nutritional support
Other pharmacologic treatments
Liver transplantation
Summary
References

488
488
488
488
488
490
490
491
492
495
495
495
495
496
497
497
497
498
498
498
499

29. Vascular Liver Disease
Matthias J. Bahr
Disorders of the hepatic sinusoid
Sinusoidal obstruction syndrome
Peliosis hepatis
Disorders of the hepatic artery
Hereditary hemorrhagic teleangiectasia (Osler-Weber-Rendu syndrome)
Disorders of the portal vein
Portal vein thrombosis
Nodular regenerative hyperplasia
Hepatoportal sclerosis
Disorders of the hepatic veins
Budd-Chiari syndrome
References

509
509
509
509
513
514
516
518
518
521
521
522
522
524

30. Acute Liver Failure
Akif Altinbas, Lars P. Bechmann, Hikmet Akkiz, Guido Gerken, Ali Canbay
Introduction and definition
Epidemiology and etiologies
Intoxication
Amanita intoxication
Viral hepatitis
Immunologic etiologies
Wilson’s Disease
Vascular disorders
Pregnancy-induced liver injury
Undetermined

526
526
526
526
527
528
529
529
529
529
530
530

25
Molecular mechanisms and clinical presentation
Prognosis
Treatment
General management
Hepatic encephalopathy
Coagulopathy
Liver Transplantation
Extracorporal liver support systems
Specific treatment options
References

530
532
533
533
533
534
534
534
535
536

26 Hepatology 2012

Hepatitis A 27

1. Hepatitis A
Sven Pischke and Heiner Wedemeyer

The virus
Hepatitis A is an inflammatory liver disease caused by infection with the hepatitis A
virus (HAV). HAV is a single-stranded 27 nm non-enveloped, icosahedral RNA
virus, which was first identified by immune electron microscopy in 1973 (Feinstone
1973). The virus belongs to the hepadnavirus genus of the Picornaviridiae.
Seven different HAV genotypes have been described, of which four are able to
infect humans (Lemon 1992).
The positive-sense single-stranded HAV RNA has a length of 7.5 kb and consists
of a a 5’ non-coding region of 740 nucleotides, a coding region of 2225 nucleotides
and a 3’ non-coding region of approximately 60 nucleotides.

Epidemiology
HAV infections occur worldwide, either sporadically or in epidemic outbreaks. An
estimated 1.4 million cases of HAV infections occur each year. HAV is usually
transmitted and spread via the fecal-oral route (Lemon 1985). Thus, infection with
HAV occurs predominantly in areas with lower socio-economic status and reduced
hygienic standards, especially in developing, tropical countries. In industrialised
countries like the US or Germany the number of reported cases has decreased
markedly in the past decades, according to official data published by the Centers for
Disease Control and Prevention (CDC, Atlanta, USA) and the Robert Koch Institute
(RKI, Berlin, Germany) (Figure 1). This decrease is mainly based on improved
sanitary conditions and anti-HAV vaccination. Vaccination programs have also
resulted in fewer HAV infections in various endemic countries including Argentina,
Brazil, Italy, China, Russia, Ukraine, Spain, Belarus, Israel and Turkey (Hendrickx
2008).

Transmission
HAV is usually transmitted fecally-orally either by person-to-person contact or
ingestion of contaminated food or water. Five days before clinical symptoms
appear, the virus can be isolated from feces of patients (Dienstag 1975). The
hepatits A virus usually stays detectable in the feces up to two weeks after the onset

28 Hepatology 2012
of jaundice. Fecal excretion of HAV up to five months after infection can occur in
children and immunocompromised persons.
Risk groups for acquiring an HAV infection in Western countries are health care
providers, military personnel, psychiatric patients and men who have sex with men.
Parenteral transmission by blood transfusion has been described but is a rare event.
Mother-to-fetus transmission has not been reported (Tong 1981).

Figure 1. Number of reported cases of HAV infections in the USA and Germany
within the last decade. (Sources: CDC and Robert Koch Institut, data for 2010 for the
US are not yet available.)

Clinical course
The clinical course of HAV infection varies greatly, ranging from asymptomatic,
subclinical infections to cholestatic hepatitis or fulminant liver failure. Most
infections in children are either asymptomatic or unrecognized while 70% of adults
develop clinical symptoms of hepatitis with jaundice and hepatomegaly.
The incubation time ranges between 15 to 49 days with a mean of approximately
30 days (Koff 1992). Initial symptoms are usually non-specific and include
weakness, nausea, vomiting, anorexia, fever, abdominal discomfort, and right upper
quadrant pain (Lednar 1985). As the disease progresses, some patients develop
jaundice, darkened urine, uncoloured stool and pruritus. The prodromal symptoms
usually diminish when jaundice appears.
Approximately 10% of infections take a biphasic or relapsing course. In these
cases the initial episode lasts about 3-5 weeks, followed by a period of biochemical
remission with normal liver enzymes for 4-5 weeks. Relapse may mimic the initial
episode of the acute hepatitis and complete normalization of ALT and AST values
may take several months. (Tong 1995).

Hepatitis A 29
Cases of severe fulminant HAV infection leading to hepatic failure occur more
often in patients with underlying liver disease. Conflicting data on the course of
acute hepatitis A have been reported for patients with chronic hepatitis C. While
some studies showed a higher incidence of fulminant hepatitis (Vento 1998), other
studies do not confirm these findings and even suggest that HAV superinfection
may lead to clearance of HCV infection (Deterding 2006). Other risk factors for
more severe courses of acute hepatitis A are age, malnutrition and
immunosuppression.
In contrast to hepatitis E, there are no precise data on the outcome of HAV
infection during pregnancy. Some data suggest an increased risk of gestational
complications and premature birth (Elinav 2006).
HAV infection has a lethal course in 0.1% of children, in 0.4% of persons aged
15-39 years, and in 1.1% in persons older than 40 years (Lemon 1985). In contrast
to the other fecally-orally transmitted hepatitis, hepatitis E, no chronic courses of
HAV infection have been reported so far.

Extrahepatic manifestations
Extrahepatic manifestations are uncommon in HAV (Pischke 2007). If they occur,
extrahepatic symptoms usually show an acute onset and disappear upon resolution
of HAV infection in most cases. Possible extrahepatic manifestations of acute HAV
infection are arthralgia, diarrhea, renal failure, red cell aplasia, generalised
lymphadenopathy, and pancreatitis. Arthralgia can be found in 11% of patients with
hepatitis A.
Very uncommon are severe extrahepatic manifestations like pericarditis and/or
renal failure. An association of hepatitis A with cryoglobulinemia has been reported
but is a rare event (Schiff 1992). Furthermore, cutaneous vasculitis can occur. In
some cases, skin biopsies reveal anti-HAV-specific IgM antibodies and
complements in the vessel walls (Schiff 1992). In contrast to hepatitis B or C, renal
involvement is rare, and there are very few case reports showing acute renal failure
associated with HAV infection (Pischke 2007). Recently it has been shown that
approximately 8% of hepatitis A cases are associated with acute kidney injury (Choi
2011).

Diagnosis
Diagnosis of acute HAV infection is based on the detection of anti-HAV IgM
antibodies or HAV RNA. The presence of HAV IgG antibodies can indicate acute
or previous HAV infection. HAV IgM and IgG antibodies also become positive
early after vaccination, with IgG antibodies persisting for at least two to three
decades after vaccination. Available serological tests show a very high sensitivity
and specificity.
Delayed seroconversion may occur in immunocompromised individuals, and
testing for HAV RNA should be considered in immunosuppressed individuals with
unclear hepatitis. HAV RNA testing of blood and stool can determine if the patient
is still infectious. However, it has to be kept in mind that various in-house HAV
RNA assays may not be specific for all HAV genotypes and thus false-negative
results can occur.

30 Hepatology 2012
Elevated results for serum aminotransferases and serum bilirubin can be found in
symptomatic patients (Tong 1995). ALT levels are usually higher than serum
aspartate aminotransferase (AST) in non-fulminant cases. Increased serum levels of
alkaline phosphatase and gamma-glutamyl transferase indicate a cholestatic form of
HAV infection. The increase and the peak of serum aminotransferases usually
precede the increase of serum bilirubin. Laboratory markers of inflammation, like
an elevated erythrocyte sedimentation rate and increased immunoglobulin levels,
can also frequently be detected.

Treatment and prognosis
There is no specific antiviral therapy for treatment of hepatitis A. The disease
usually takes a mild to moderate course, which requires no hospitalisation, and only
in fulminant cases is initiation of symptomatic therapy necessary. Prolonged or
biphasic courses should be monitored closely. HAV may persist for some time in
the liver even when HAV RNA becomes negative in blood and stool (Lanford
2011), which needs to be kept in mind for immunocompromised individuals. Acute
hepatitis may rarely proceed to acute liver failure; liver transplantation is required in
few cases. In the US, 4% of all liver transplantations performed for acute liver
failure were due to hepatitis A (Ostapowicz 2002). In a cohort of acute liver failures
at one transplant center in Germany approximately 1% of patients suffered from
HAV infection (Hadem 2008). The outcome of patients after liver transplantation
for fulminant hepatitis A is excellent. Timely referral to liver transplant centers is
therefore recommended for patients with severe or fulminant hepatitis A.

References
Choi HK, Song YG, Han SH, et al. Clinical features and outcomes of acute kidney injury among
patients with acute hepatitis A. J Clin Virol 2011;52:192-7. (Abstract)
Deterding K, Tegtmeyer B, Cornberg M, et al. Hepatitis A virus infection suppresses hepatitis C
virus replication and may lead to clearance of HCV. J Hepatol 2006;45:770-8.
(Abstract)
Dienstag JL, Feinstone SM, Kapikian AZ, Purcell RH. Faecal shedding of hepatitis-A antigen.
Lancet 1975;1:765-7.
Elinav E, Ben-Dov IZ, Shapira Y, et al. Acute hepatitis A infection in pregnancy is associated
with high rates of gestational complications and preterm labor. Gastroenterology
2006;130:1129-34. (Abstract)
Feinstone SM, Kapikian AZ, Purceli RH. Hepatitis A: detection by immune electron microscopy
of a viruslike antigen associated with acute illness. Science 1973;182:1026-8.
(Abstract)
Hadem J, Stiefel P, Bahr MJ, et al. Prognostic implications of lactate, bilirubin, and etiology in
German patients with acute liver failure. Clin Gastroenterol Hepatol 2008;6:339-45.
(Abstract)
Hendrickx G, Van Herck K, Vorsters A, et al. Has the time come to control hepatitis A globally?
Matching prevention to the changing epidemiology. J Viral Hepat 2008;15 Suppl 2:115. (Abstract)
Koff RS. Clinical manifestations and diagnosis of hepatitis A virus infection. Vaccine 1992;10
Suppl 1:S15-7. (Abstract)
Lanford RE, Feng Z, Chavez D, et al. Acute hepatitis A virus infection is associated with a
limited type I interferon response and persistence of intrahepatic viral RNA. Proc Natl
Acad Sci U S A 2011;108:11223-8. (Abstract)
Lednar WM, Lemon SM, Kirkpatrick JW, Redfield RR, Fields ML, Kelley PW. Frequency of
illness associated with epidemic hepatitis A virus infections in adults. Am J Epidemiol
1985;122:226-33. (Abstract)

Hepatitis A 31
Lemon SM, Jansen RW, Brown EA. Genetic, antigenic and biological differences between
strains of hepatitis A virus. Vaccine 1992;10 Suppl 1:S40-4. (Abstract)
Lemon SM. Type A viral hepatitis. New developments in an old disease. N Engl J Med
1985;313:1059-67. (Abstract)
Ostapowicz G, Fontana RJ, Schiodt FV, et al. Results of a prospective study of acute liver
failure at 17 tertiary care centers in the United States. Ann Intern Med 2002;137:94754. (Abstract)
Pischke S, Vogel, A. Jaeckel, E., Manns, M.P. Immunopathogenesis of Extrahepatic
Manifestations in HAV, HBV, and HCV Infections. In: Liver Immunology. Totowa,
New Jersey: Humana Press, 2007:209-17.
Schiff ER. Atypical clinical manifestations of hepatitis A. Vaccine 1992;10 Suppl 1:S18-20.
(Abstract)
Tong MJ, el-Farra NS, Grew MI. Clinical manifestations of hepatitis A: recent experience in a
community teaching hospital. J Infect Dis 1995;171 Suppl 1:S15-8. (Abstract)
Tong MJ, Thursby M, Rakela J, McPeak C, Edwards VM, Mosley JW. Studies on the maternalinfant transmission of the viruses which cause acute hepatitis. Gastroenterology
1981;80:999-1004. (Abstract)
Vento S, Garofano T, Renzini C, et al. Fulminant hepatitis associated with hepatitis A virus
superinfection in patients with chronic hepatitis C. N Engl J Med 1998;338:286-90.
(Abstract)

32 Hepatology 2012

2. Hepatitis B
Christoph Boesecke and Jan-Christian Wasmuth

Introduction
It is estimated that approximately 30% of the world's population has had contact
with or are carriers of the hepatitis B virus (HBV). An estimated 350 million of
them are HBV carriers (Goldstein 2005). Thus, HBV infection is one of the most
important infectious diseases worldwide. Around one million persons die of HBVrelated causes annually. There is a wide range of HBV prevalence rates in different
parts of the world. HBV prevalence varies from 0.1% up to 20%. Low prevalence
areas (0.1-2%) are Western Europe (with wide variation within Europe), United
States and Canada, Australia and New Zealand; intermediate prevalence (3-5%) are
the Mediterranean countries, Japan, Central Asia, the Middle East, and Latin and
South America; and high prevalence areas (10-20%) include southeast Asia, China,
and sub-Saharan Africa. This diversity is probably related to differences in age at
infection, which correlates with the risk of chronicity. The progression rate from
acute to chronic HBV infection decreases with age. It is approximately 90% for an
infection acquired perinatally, and is as low as 5% (or even lower) for adults
(Stevens 1975, Wasley 2008).
The incidence of new infections has decreased in most developed countries, most
likely due to the implementation of vaccination strategies (Rantala 2008). However,
exact data is difficult to generate as many cases remain undetected due to the
asymptomatic nature of many infections (RKI 2007, CDC 2010). Nevertheless, in
Germany 2524 cases of acute hepatitis B were documented in the year 2006,
corresponding to an incidence rate of 1.4 per 100,000 inhabitants. In 1997 there
were 6135 documented cases of acute hepatitis B. Likewise, the incidence of acute
hepatitis B in the United States has decreased by 78% from 1990 to 2005 (Wasley
2008). It is expected that this number will further decrease in countries with
implementation of vaccination programs. In Germany 87% of all children starting
school were fully vaccinated in 2006 with a trend toward increasing coverage
(Poethko-Muller 2007). Interestingly, recent data from a Swiss clinic indicate that
uptake in HBV vaccinations is significantly higher when vaccination is endorsed by
nurses rather than the patients’ physician (Blanco 2011).

Hepatitis B 33
Although the incidence of acute HBV infection has decreased in most countries
due to the implementation of vaccination programs, HBV-related complications
such as cancers and deaths have been on the increase (Gomaa 2008, Hatzakis 2011).
Reasons might be the delay of vaccination effects, improved diagnosis, and better
documentation of HBV cases. Although a drop in prevalence has been observed in
many countries, estimates are difficult due to a continuously growing migration
from high or medium prevalence areas to low prevalence areas (Belongia 2008).

Transmission
The routes of HBV transmission:
− Sexual
− Percutaneous (Intravenous Drug Use)
− Perinatal
− Horizontal
− Transfusion
− Nosocomial infection (including needle-stick injury)
− Organ transplantation
There is considerable variation in the predominance of transmission modes in
different geographic areas. For example, in low prevalence areas such as Western
Europe, the routes are mainly unprotected sexual intercourse and intravenous drug
use. In high prevalence areas like sub-Saharan Africa perinatal infection is the
predominant mode of transmission. Horizontal transmission, particularly in early
childhood, is regarded as the major route of transmission in intermediate prevalence
areas.

Sexual transmission
In low prevalence areas sexual transmission is the major route of transmission.
Approximately 40% of new HBV infections in the United States is considered to be
transmitted via heterosexual intercourse, and 25% occurs in men who have sex with
men (MSM) (Wasley 2008). Measures to prevent HBV transmission are vaccination
and safer sex, i.e., use of condoms. However, there is ongoing debate regarding
what to advise low-viremic patients.

Percutaneous inoculation
Percutaneous transmission seems to be an effective mode of HBV transmission. The
most important route is sharing syringes and needles by intravenous drug users. In
low prevalence areas such as Europe and the United States about 15% of newly
diagnosed HBV infections is in the IVDU population (Wasley 2008). The risk of
HBV transmission increases with the number of years of drug use, frequency of
injection, and sharing of drug preparation equipment.
Other situations with possible percutaneous inoculation of HBV are sharing
shaving razors or toothbrushes, although the exact number remains unknown. In
addition, certain practices like acupuncture, tattooing, and body piercing have been
associated with transmission of hepatitis B. Public health education and the use of
disposable needles or equipment are important methods of prevention.

34 Hepatology 2012

Perinatal transmission
Transmission from an HBeAg-positive mother to her infant may occur in utero, at
the time of birth, or after birth. The rate of infection can be as high as 90%.
However, neonatal vaccination is highly efficacious (95%). Its efficacy indicates
that most infections occur at or shortly before birth. On the other hand, caesarean
section seems not be as protective as it is in other vertically transmitted diseases like
HIV.
The risk of transmission from mother to infant is related to the HBV replicative
rate in the mother. There seems to be a direct correlation between maternal HBV
DNA levels and the likelihood of transmission. In mothers with highly replicating
HBV the risk of transmission may be up to 85 to 90%, and continuously lowers with
lower HBV DNA levels (Burk 1994). In some studies there has been almost no
perinatal transmission if the mother has no significant ongoing replication (<105 log
copies/ml) (Li 2004).
It is possible to reduce the risk of perinatal transmission in several ways. The first
step is identification of persons at risk. Testing for HBsAg should be performed in
all women at the first prenatal visit and repeated later in pregnancy if appropriate
(CDC 2011). Newborns born to HBV-positive mothers can be effectively protected
by passive-active immunisation (>90% protection rate) (del Canho 1997, Dienstag
2008). Hepatitis B immunoglobulin for passive immunization should be given as
early as possible (within 12 hours), but can be given up to seven days after birth if
seropositivity of the mother is detected later. Active immunisation follows a
standard regimen and is given at three time points (10 µg at day 0, month 1, and
month 6). Anti-HBV treatment of the mother with nucleoside analogs may be
discussed, especially in mothers with high HBV DNA levels, i.e., HBV DNA >106
copies/ml or 2x105 IU/ml. In one randomised, prospective, placebo-controlled
study, treatment of the mother with telbivudine resulted in prevention of almost all
cases of vertical transmission compared to a vertical transmission rate of about 10%
in the arm receiving only active and passive immunisation (Han 2011). Telbivudine
or tenofovir seem to be the treatment of choice. Adefovir and entecavir are not
recommended in pregnancy (Cornberg 2011).
As mentioned earlier, caesarean section should not be performed routinely, except
in cases of high viral load. If the child is vaccinated, (s)he may be breastfed (Hill
2002).

Horizontal transmission
Children may acquire HBV infection through horizontal transmission via minor
breaks in the skin or mucous membranes or close bodily contact with other children.
In addition, HBV can survive outside the human body for a prolonged period; as a
result, transmission via contaminated household articles such as toothbrushes, razors
and even toys may be possible. Although HBV DNA has been detected in various
bodily secretions of hepatitis B carriers, there is no firm evidence of HBV
transmission via body fluids other than blood.

Blood transfusion
Blood donors are routinely screened for hepatitis B surface antigen (HBsAg).
Therefore incidence of transfusion-related hepatitis B has significantly decreased.
The risk of acquiring post-transfusion hepatitis B depends on factors like prevalence

Hepatitis B 35
and donor testing strategies. In low prevalence areas it is estimated to be one to four
per million blood components transfused (Dodd 2000, Polizzotto 2008). In high
prevalence areas it is considerably higher (around 1 in 20,000) (Shang 2007,
Vermeulen 2011).
There are different strategies for donor screening. Most countries use HBsAg
screening of donors. Others, like the United States, use both HBsAg and anti-HBc.
Routine screening of anti-HBc remains controversial, as the specificity is low and
patients with cleared hepatitis have to be excluded. Screening of pooled blood
samples or even individual samples may be further improved by nucleic acid
amplification techniques. However, this is an issue of continuous debate due to
relatively low risk reduction and associated costs.

Nosocomial infection
Nosocomial infection can occur from patient to patient, from patient to health care
worker and vice versa. HBV is considered the most commonly transmitted bloodborne virus in the healthcare setting.
In general, nosocomial infection of hepatitis B can and should be prevented.
Despite prevention strategies, documented cases of nosocomial infections do occur
(Williams 2004). However, the exact risk of nosocomial infection is unknown. The
number of infected patients reported in the literature is likely to be an underestimate
of true figures as many infected patients may be asymptomatic and only a fraction
of exposed patients are recalled for testing.
Strategies to prevent nosocomial transmission of hepatitis B:
− use of disposable needles and equipment,
− sterilization of surgical instruments,
− infection control measures, and
− vaccination of healthcare workers.
Due to the implementation of routine vaccination of health care workers the
incidence of HBV infection among them is lower than in the general population
(Duseja 2002, Mahoney 1997). Therefore, transmission from healthcare workers to
patients is a rare event, while the risk of transmission from an HBV-positive patient
to a health care worker seems to be higher.
Healthcare workers positive for hepatitis B are not generally prohibited from
working. However, the individual situation has to be evaluated in order to decide on
the necessary measures. Traditionally, HBeAg-negative healthcare workers are
considered not to be infective, whereas HBeAg-positive healthcare workers should
wear double gloves and not perform certain activities, to be defined on an individual
basis. However, there have been cases of transmission of hepatitis B from HBsAgpositive, HBeAg-negative surgeons to patients (Teams 1997). Hepatitis B virus has
been identified with a precore stop codon mutation resulting in non-expression of
HBeAg despite active HBV replication. Therefore, HBV DNA testing has been
implemented in some settings, although this may not always be reliable due to
fluctuating levels of HBV DNA. In most developed countries guidelines for
hepatitis B positive healthcare workers have been established and should be
consulted.

36 Hepatology 2012

Organ transplantation
Transmission of HBV infection has been reported after transplantation of
extrahepatic organs from HBsAg positive donors (e.g., kidney, cornea) (Dickson
1997). Therefore, organ donors are routinely screened for HBsAg. The role of antiHBc is controversial, as it is in screening of blood donors. Reasons are the
possibility of false positive results, the potential loss of up to 5% of donors even in
low endemic areas, and the uncertainty about the infectivity of organs, especially
extrahepatic organs, from donors who have isolated anti-HBc (Dickson 1997).
There is an increased risk of HBV infection for the recipient if organs of such
donors are transplanted as compared to anti-HBc negative donors.

Postexposure prophylaxis
In case of exposure to HBV in any of the circumstances mentioned above,
postexposure prophylaxis is recommended for all non-vaccinated persons. A
passive-active immunization is recommended. The first dose of active immunization
should be given as early as possible. 12 hours after the exposure is usually
considered the latest time point for effective postexposure prophylaxis. One dose of
hepatitis B-immunoglobulin (HBIG) should be administered at the same time, if the
source is known to be HBsAg-positive. The other two doses of vaccine should be
administered according to the usual schedule.
Vaccinated individuals with a documented response do not need postexposure
prophylaxis. Individuals who have had no post-vaccination testing should be tested
for anti-HBs titer as soon as possible. If this is not possible, or the anti-HBs titer is
insufficient (<100 IU/l), they will require a second course of vaccination.
Individuals who are documented non-responders will require two doses of HBIG
given one month apart.

Natural history and clinical manifestations
The spectrum of clinical manifestations of HBV infection varies in both acute and
chronic disease. During the acute phase, manifestations range from subclinical or
anicteric hepatitis to icteric hepatitis and, in some cases, fulminant hepatitis. During
the chronic phase, manifestations range from an asymptomatic carrier state to
chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Extrahepatic
manifestations can occur in both acute and chronic infection.

Acute hepatitis
After HBV transmission, the incubation period lasts from one to four months. A
prodromal phase may appear before acute hepatitis develops. During this period a
serum sickness-like syndrome may develop. This syndrome manifests with fever,
skin rash, arthralgia and arthritis. It will usually cease with the onset of hepatitis. At
least 70% of patients will then have subclinical or anicteric hepatitis, while less than
30% will develop icteric hepatitis. The most prominent clinical symptoms of
hepatitis are right upper quadrant discomfort, nausea, jaundice and other unspecific
constitutional symptoms. In case of coinfection with other hepatitis viruses or other
underlying liver disease the clinical course may be more severe. The symptoms
including jaundice generally disappear after one to three months, but some patients

Hepatitis B 37
have prolonged fatigue even after normalisation of serum aminotransferase
concentrations.
Concentrations of alanine and aspartate aminotransferase levels (ALT and AST)
may rise to 1000-2000 IU/L in the acute phase. ALT is typically higher than AST.
Bilirubin concentration may be normal in a substantial portion of patients. In
patients who recover, normalisation of serum aminotransferases usually occurs
within one to four months. Persistent elevation of serum ALT for more than six
months indicates progression to chronic hepatitis.
The rate of progression from acute to chronic hepatitis B is primarily determined
by the age at infection (Ganem 2004, McMahon 1985). In adult-acquired infection
the chronicity rate is 5% or less, whereas it is higher if acquired at younger ages. It
is estimated to be approximately 90% for perinatally-acquired infection, and 2050% for infections between the ages of one and five years.
Until recently it was assumed that patients who recover from acute hepatitis B
actually clear the virus from the body. However, there is a lot of evidence now
suggesting that even in patients positive for anti-HBs and anti-HBc HBV DNA may
persist lifelong in the form of covalently closed circular DNA (cccDNA) and this
latent infection maintains the T cell response that keeps the virus under control
(Yotsuyanagi 1998, Guner 2011). Complete eradication rarely occurs. This is an
important finding, as immunosuppression can lead to reactivation of the virus, e.g.,
after organ transplant or during chemotherapy.
Fulminant hepatic failure is unusual, occurring in approximately 0.1-0.5% of
patients. Reasons and risk factors for fulminant hepatitis B are not well understood
(Garfein 2004). There may be correlation with substance abuse or coinfections with
other viruses. Fulminant hepatitis B is believed to be due to massive immunemediated lysis of infected hepatocytes. This is why many patients with fulminant
hepatitis B have no evidence of HBV replication at presentation.
Antiviral treatment of patients with acute hepatitis B usually is not recommended
(Cornberg 2011). In adults, the likelihood of fulminant hepatitis B is less than 1%,
and the likelihood of progression to chronic hepatitis B is less than 5%. Therefore,
treatment of acute hepatitis B is mainly supportive in the majority of patients.
Treatment can be considered in certain subsets of patients, e.g., patients with a
severe or prolonged course of hepatitis B, patients coinfected with other hepatitis
viruses or underlying liver diseases, patients with immunosuppression, or patients
with fulminant liver failure undergoing liver-transplantation (Kondili 2004,
Tillmann 2006). However, early intervention may interfere with the immune
response and decrease the likelihood of immune control of HBV infection, thus
facilitating chronicity (Tillmann 2006).
In addition, contacts of the patient should be checked for exposure to hepatitis B.

Chronic hepatitis
The HBV chronicity rate is around 5% or less in adult-acquired infection, as
mentioned earlier. In perinatally-acquired infection it is estimated to be
approximately 90%, and 20-50% for infections between the age of one and five
years (Ganem 2004, McMahon 1985). Most patients will not have a history of acute
hepatitis.
Most patients with chronic hepatitis B are clinically asymptomatic. Some may
have nonspecific symptoms such as fatigue. In most instances, significant clinical

38 Hepatology 2012
symptoms will develop only if liver disease progresses to decompensated cirrhosis.
In addition, extrahepatic manifestations may cause symptoms.
Accordingly, physical examination will be normal in most instances. In advanced
liver disease there may be stigmata of chronic liver disease such as splenomegaly,
spider angiomata, caput medusae, palmar erythema, testicular atrophy,
gynecomastia, etc. In patients with decompensated cirrhosis, jaundice, ascites,
peripheral edema, and encephalopathy may be present.
Laboratory testing shows mild to moderate elevation in serum AST and ALT in
most patients, whereas normal transaminases occur rarely. During exacerbation,
serum ALT concentration may be as high as 50 times the upper limit of normal.
Alfa-fetoprotein concentrations correlate with disease activity. In exacerbations of
hepatitis B, concentrations as high as 1000 ng/mL may be seen.
The natural course of chronic HBV infection is determined by the interplay of
viral replication and the host immune response. Other factors that may play a role in
the progression of HBV-related liver disease include gender, alcohol consumption,
and concomitant infection with other hepatitis virus(es). The outcome of chronic
HBV infection depends upon the severity of liver disease at the time HBV
replication is arrested. Liver fibrosis is potentially reversible once HBV replication
is controlled.
There are two different states that are distinguished in chronic HBV infection:
first, a high-replicative state with active liver disease and elevated serum ALT.
HBV DNA and HBeAg are present. Second, a low or non-replicative phase, where
serum ALT may normalize, HBeAg disappears, and anti-HBe antibodies appear. In
some patients, viral replication stops completely, as demonstrated by sensitive HBV
DNA assays, although they remain HBsAg-positive. These patients have
undetectable HBV DNA in serum and normal ALT concentrations. No sign of
ongoing liver damage or inflammation is found on liver biopsy. This state is called
inactive carrier state.
A small percentage of patients continue to have moderate levels of HBV
replication and active liver disease (elevated serum ALT and chronic inflammation
on liver biopsies) but remain HBeAg negative. These patients with HBeAg-negative
chronic hepatitis may have residual wild type virus or HBV variants that cannot
produce HBeAg due to precore or core promoter variants.
The first high-replicative phase may switch into the non-replicative phase either
spontaneously or upon antiviral treatment. Conversely, the non-replicative phase
may reactivate to the high-replicative phase either spontaneously or with
immunosuppression (e.g., in HIV infection or with chemotherapy).
In perinatally-acquired chronic HBV infection there are three different states: An
immune tolerance phase, an immune clearance phase, and a late non-replicative
phase.
The immune tolerance phase, which usually lasts 10-30 years, is characterized by
high levels of HBV replication, as manifested by the presence of HBeAg and high
levels of HBV DNA in serum. However, there is no evidence of active liver disease
as seen by normal serum ALT concentrations and minimal changes in liver biopsy.
It is thought that this lack of liver disease despite high levels of HBV replication is
due to immune tolerance to HBV (Dienstag 2008), although the exact mechanisms
are unknown. This phenomenon of immune tolerance is believed to be the most
important reason for the poor response to interferon therapy in HBeAg-positive

Hepatitis B 39
patients with normal ALT levels. During this phase there is a very low rate of
spontaneous HBeAg clearance. It is estimated that the rate of spontaneous HBeAg
clearance is only 15% after 20 years of infection.
During the second to third decade, the immune-tolerance phase may convert to
one of immune clearance. The spontaneous HBeAg clearance rate increases. It is
estimated to be 10 to 20% annually. If HBeAg seroconversion occurs, exacerbations
of hepatitis with abrupt increases in serum ALT are very often observed. These
exacerbations follow an increase in HBV DNA and might be due to a sudden
increase in immune-mediated lysis of infected hepatocytes. Most often there are no
clinical symptoms during exacerbation, and rise of ALT is only detected by routine
examinations. Some patients may develop symptoms mimicking acute hepatitis.
Titers of anti-HBc IgM may rise as well as alfa-fetoprotein. If such patients are not
known to be HBV-infected, misdiagnosis of acute hepatitis B can be made. HBeAg
seroconversion and clearance of HBV DNA from the serum is not always achieved
after exacerbation. In these patients recurrent exacerbation with intermittent
disappearance of serum HBV DNA with or without HBeAg loss may occur. The
non-replicative phase is usually characterized by the absence of HBV DNA and
normalisation of serum ALT, like in adult chronic HBV.
Very few patients with chronic HBV infection become HBsAg-negative in the
natural course of infection. The annual rate of HBsAg clearance has been estimated
to be less than 2% in Western patients and even lower (0.1-0.8%) in patients of
Asian origin (Liaw 1991) following an accelerated decrease in HBsAg levels during
the 3 years before HBsAg seroclearance (Chen 2011). If loss of HBsAg occurs,
prognosis is considered favourable. However, clearance of HBsAg does not exclude
development of cirrhosis or hepatocellular carcinoma in some patients, although the
exact rate of these complications is not known. This phenomenon is thought to be
linked to the fact that HBV DNA may still be present in hepatocytes despite HBsAg
loss.

Prognosis and survival
As clinical course varies among patients, there is a wide variation in clinical
outcome and prognosis of chronic HBV infection. The lifetime risk of a liver-related
death has been estimated to be 40-50% for men and 15% for women. The risk of
progression appears to be higher if immune activation occurs. The estimated fiveyear rates of progression (Fattovich 2008):
− Chronic hepatitis to cirrhosis – 10-20%
− Compensated cirrhosis to hepatic decompensation – 20-30%
− Compensated cirrhosis to hepatocellular carcinoma – 5-15%
Accordingly, the survival rates are:
− Compensated cirrhosis - 85% at five years
− Decompensated cirrhosis - 55-70% at one year and 15-35% at five years
Viral replication
In patients with signs of viral replication (i.e., HBeAg-positive) survival is
consistently worse than in patients who are HBeAg-negative. However, in recent
decades, infections with HBeAg-negative precore mutants prevail by far in newlyacquired infections, resulting in a different pattern of HBeAg-negative and HBV
DNA-positive hepatitis with fibrosis progression and HCC in a substantial

40 Hepatology 2012
proportion of patients. In recent years, the amount of HBV DNA has also been
linked to disease progression and has replaced HBeAg-positivity as a marker for
disease activity (Chen 2006). This is true both for progression to cirrhosis as well as
for the risk of HCC. Therefore, most treatment guidelines today are based on the
level of HBV viremia. A reasonable cut-off to distinguish patients with a low risk of
progression from patients with a high risk and indication for antiviral treatment is
104 copies/ml (corresponding to approximately 2 x 103 IU/ml) (Cornberg 2011),
although other cut-offs may be used.
The duration of viral replication is obviously linked with the risk of development
of cirrhosis and HCC. As necroinflammation may persist longer in patients with a
prolonged replicative phase, the risk of disease progression is elevated. Conversely,
even in patients with decompensated cirrhosis, suppression of HBV replication and
delayed HBsAg clearance can result in improvement in liver disease (Fung 2008).
Alcohol use
HBV infection in heavy alcohol users is associated with faster progression to liver
injury and an elevated risk of developing cirrhosis and HCC (Bedogni 2008,
Marcellin 2008). Survival is reduced compared to HBV-negative heavy alcohol
users. However, there is no clear evidence that heavy alcohol use is associated with
an enhanced risk of chronic HBV infection, although prevalence of HBV is
estimated to be fourfold higher than in controls (Laskus 1992) with variation among
regions and cohorts (Rosman 1996).
Hepatitis C coinfection
If coinfection of HCV and HBV occurs, HCV usually predominates. This may lead
to lower levels of transaminases and HBV DNA (Jardi 2001). The rate of HBsAg
seroconversion even appears to be increased, although this finding may be due to
the fact that around one third of patients coinfected with HBV and HCV lack
markers of HBV infection (i.e., HBsAg) although HBV DNA is detectable. Despite
lower aminotransferases and HBV DNA levels, liver damage is worse in most
instances. The risks of severe hepatitis and fulminant hepatic failure seem to be
elevated if both infections occur simultaneously regardless of whether it is an acute
coinfection of HBV and HCV or acute hepatitis C in chronic hepatitis B (Liaw
2004).
Hepatitis D coinfection
Acute HBV and HDV coinfection tends to be more severe than acute HBV infection
alone. It is more likely to result in fulminant hepatitis. If HDV superinfection in
patients with chronic HBV infection occurs, HDV usually predominates, and HBV
replication is suppressed (Jardi 2001). Severity of liver disease is worse and
progression to cirrhosis is accelerated (Fattovich 2000, Grabowski 2010).
It is very difficult to predict the individual course of hepatitis B due to the many
factors influencing disease progression. Several predictive models of disease
progression that include clinical parameters (e.g., hepatic decompensation) and
laboratory parameters (e.g., bilirubin, INR) have been evaluated, but none of these
is used routinely in the clinic at present. In patients with cirrhosis, the MELD-score
(Model for End-Stage Liver Disease) and the CHILD-Pugh score are used (see
Chapter 3).

Hepatitis B 41

Extrahepatic manifestations
The two major extrahepatic complications of chronic HBV are polyarteritis nodosa
and glomerular disease. They occur in 10-20% of patients with chronic hepatitis B
and are thought to be mediated by circulating immune complexes (Han 2004).
Polyarteritis nodosa
The clinical manifestations are similar to those in patients with polyarteritis who are
HBV-negative. There may be some clinical benefit to antiviral therapy.
Nephropathy/Glomerulonephritis
HBV can induce both membranous nephropathy and, less often,
membranoproliferative glomerulonephritis. Most cases occur in children. The
clinical hallmark is proteinuria. In contrast to polyarteritis nodosa, there is no
significant benefit of antiviral treatment.
For further details, please refer to extrahepatic manifestations in Chapter 16.

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44 Hepatology 2012

3. Hepatitis C
Christoph Boesecke and Jan-Christian Wasmuth

Epidemiology
Hepatitis C is a disease with a significant global impact. According to the World
Health Organization there are 130-170 million people infected with the hepatitis C
virus (HCV), corresponding to 2-2.5% of the world’s total population. There are
considerable regional differences. In some countries, e.g., Egypt, the prevalence is
as high as 22% (WHO 2011). In Africa and the western Pacific the prevalence is
significantly higher than in North America and Europe (RKI 2004).
It is estimated that there are 2-5 million HCV-positive persons in Europe. The
prevalence of HCV antibodies in otherwise healthy blood donors is approximately
1.6% in the United States, 1.15% in Italy, 0.4% in Germany, and 0.23% in
Scandinavia (RKI 2004, Hatzakis 2011). The number of patients HCV RNApositive is estimated to be around 80 to 90% of all HCV antibody-positive persons.
Certain groups are preferentially affected: The highest risk factor in most cases is
injection drug use. But patients undergoing hemodialysis and persons who received
blood transfusions before 1991 are at risk also. In Europe and the United States
chronic hepatitis C is the most common chronic liver disease and the majority of
liver transplants performed are for chronic HCV.
It is difficult to determine the number of new HCV infections, as most acute cases
will not be noticed clinically. Fewer than 25% of acute cases of hepatitis C are
clinically apparent (Vogel 2009). In addition, the age of infection upon diagnosis is
not possible to determine in most cases. Nevertheless, it has to be assumed that the
number of new infections has considerably decreased over the past decades. For the
United States it is estimated that the number of new cases of acute HCV infection
has fallen from approximately 230,000 per year in the 1980s to about 20,000 cases
per year currently (Wasley 2008). This decrease is primarily associated with
reduced infections in injection drug users, a probable consequence of changes in
injection practices motivated by education about human immunodeficiency virus
(HIV) transmission. Transfusion-associated hepatitis C has had little impact on this
decline, as the number of cases has been reduced almost to zero. The only different
trend is an increase in acute hepatitis C infections in HIV-positive men who have
sex with men (MSM) globally over the last decade (Boesecke 2011).

Hepatitis C 45

Transmission
Parenteral exposure to the hepatitis C virus is the most efficient means of
transmission. The majority of patients infected with HCV in Europe and the United
States acquired the disease through intravenous drug use or blood transfusion. The
latter has become rare since routine testing of the blood supply for HCV began in
the early 1990s. Other types of parenteral exposure are important in specific regions
in the world.
The following possible routes of infection have been identified in anti-HCVpositive blood donors (in descending order of transmission risk):
− Injection drug use
− Blood transfusion
− Sex with an intravenous drug user
− Having been in jail more than three days
− Religious scarification
− Having been struck or cut with a bloody object
− Pierced ears or body parts
− Immunoglobulin injection
Very often in patients with newly diagnosed HCV infection no clear risk factor
can be identified.

Injection drug use
Injection drug use has been the most commonly identified source of acute HCV
infection. It is estimated that most newly acquired infections occur in individuals
who have injected illegal drugs. The seroprevalence of anti-HCV antibodies in
groups of intravenous drug users may be up to 70% with considerable variation
depending on factors such as region, risk behaviour, socioeconomic status, etc,
underscoring the efficiency of transmission via direct blood contact (Sutton 2008).
HCV infection also has been associated with a history of intranasal cocaine use,
presumably due to blood on shared straws or other sniffing paraphernalia. This may
explain partly the recent increase in cases of acute HCV infections in HIV-positive
MSM (Schmidt 2011).

Blood transfusion
In the past, blood transfusion or use of other blood products was a major risk factor
for transmission of HCV. In some historic cohorts 10% or more of patients who
received blood transfusions were infected with hepatitis C (Alter 1989). However,
blood donor screening for HCV since the early 1990s has nearly eliminated this
transmission route. Blood donors are screened for anti-HCV antibodies and HCV
RNA – at least in developed countries. The risk is now estimated to be between
1:500,000 and 1:1,000,000 units (Pomper 2003).
In cohorts of multiply transfused patients such as hemophiliacs, over 90% of
patients were infected with hepatitis C in the past (Francois 1993). Since the use of
routine inactivated virus (e.g., heat inactivation or pasteurization) or recombinant
clotting factors, new cases of hepatitis C infection have become uncommon in these
patients.

46 Hepatology 2012

Organ transplantation
Transplant recipients who receive organs from HCV-positive donors have a high
risk of acquiring HCV infection. Transmission rates in different cohorts vary from
30 to 80% (Pereira 1991, Roth 1994). Therefore, most transplant organisations have
developed strategies for screening and selective utilization of organs from anti-HCV
positive donors.

Sexual or household contact
Usual household contacts do not pose a risk of HCV transmission.
The efficiency of HCV transmission by sexual contact is very low. However,
there is no doubt that sexual transmission of hepatitis C is possible.
The exact risk of HCV transmission in monogamous heterosexual relationships
has been difficult to determine. It appears that the risk in long-term partnerships is
very low. In prospective cohorts of monogamous, heterosexual couples, there was a
long-term transmission risk of 0.01% or lower (Vandelli 2004). Factors that may
increase the risk of HCV infection include greater numbers of sex partners, history
of sexually transmitted diseases, and not using a condom. Whether underlying HIV
infection increases the risk of heterosexual HCV transmission to an uninfected
partner is unclear. Very often it is difficult to rule out the possibility that
transmission results from risk factors other than sexual exposure.
Outbreaks of cases of acute hepatitis C in several cities in Europe and the United
States among men who have sex with men (MSM) have focused attention on sexual
transmission of HCV (Boesecke 2011). There is clear evidence unprotected sex can
account for the transmission of HCV. Unprotected anal sex, fisting, having many
sex partners in a short time period, a concomitant sexually transmitted disease
including HIV and use of recreational drugs were identified as risk factors (Danta
2007, Schmidt 2011). It appears that mucosal damage is a prerequisite for HCV
transmission. According to these observations, the seroprevalence of HCV in MSM
ranges from about 4 to 8%, which is higher than the HCV prevalence reported for
general European populations.
Patients with acute or chronic HCV infection should be advised that transmission
to sexual contacts is a possibility, although the risk is extremely low in heterosexual
relationships. It is likely that the use of condoms will lower the risk of sexual
transmission further. However, in most countries there are no firm
recommendations to use barrier precautions in stable monogamous sexual
partnerships. The transmission risk in MSM is considerably higher so that – in
conjunction with the risk of other sexually transmitted diseases – safer sex practices
are advised for this group.

Perinatal transmission
The risk of perinatal transmission of HCV in HCV RNA-positive mothers is
estimated to be 5% or less (Ohto 1994). In mothers coinfected with HIV this risk
correlates with immunosuppression and has been described in up to 20%. Today,
there are no specific recommendations for prevention of perinatal transmission
(Pembrey 2005). Cesarean section has not been shown to reduce the transmission
risk. There is no evidence that breastfeeding is a risk for infection among infants
born to HCV-infected women. Early diagnosis of infection in newborns requires

Hepatitis C 47
HCV RNA testing since anti-HCV antibodies are passively transferred from the
mother.

Hemodialysis
Patients who participate in chronic hemodialysis programs are at increased risk for
hepatitis C. The prevalence of HCV antibodies in such patients reaches 15%,
although it has declined in recent years (Fissell 2004). A number of risk factors have
been identified for HCV infection among dialysis patients. These include blood
transfusions, duration of hemodialysis, prevalence of HCV infection in the dialysis
unit, and type of dialysis. The risk is higher with in-hospital hemodialysis as
opposed to peritoneal dialysis. The best strategy to prevent hemodialysis-associated
HCV transmission is subject to debate.

Other rare transmission routes
Rare sources of percutaneous transmission of HCV are contaminated equipment
used during medical procedures, procedures involved in traditional medicine (e.g.,
scarification, cupping), tattooing, and body piercing (Haley 2001). All these routes
bear the potential of transmitting HCV. However, in most instances it is not clear if
the risk is due to the procedure itself, or whether there are possible contacts with
persons involved who are HCV-positive. In addition, transmission via these routes
is so rare that persons with exposure are not at increased risk for acquiring hepatitis
C.

Needlestick injury
There is some risk of HCV transmission for health care workers after unintentional
needlestick injury or exposure to other sharp objects. The incidence of
seroconversion after exposure to an HCV-positive source is generally estimated to
be less than 2% (MMWR 2001). However, data are divergent and figures ranging
from 0 to 10% can be found (Mitsui 1992). Exposure of HCV to intact skin has not
been associated with HCV transmission.

Clinical manifestations
The spectrum of clinical manifestations of HCV infection varies in acute versus
chronic disease. Acute infection with HCV is most often asymptomatic (Vogel
2009) and leads to chronic infection in about 80% of cases. The manifestations of
chronic HCV range from an asymptomatic state to cirrhosis and hepatocellular
carcinoma. HCV infection usually is slowly progressive. Thus, it may not result in
clinically apparent liver disease in many patients if the infection is acquired later in
life. Approximately 20-30% of chronically infected individuals develop cirrhosis
over a 20-30 year period of time.

Acute hepatitis
After inoculation of HCV, there is a variable incubation period. HCV RNA in blood
(or liver) can be detected by PCR within several days to eight weeks.
Aminotransferases become elevated approximately 6-12 weeks after exposure
(range 1-26 weeks). The elevation of aminotransferases varies considerably among
individuals, but tends to be more than 10-30 times the upper limit of normal

48 Hepatology 2012
(typically around 800 U/l). HCV antibodies can be found for the first time around 8
weeks after exposure although in some patients it may take several months before
HCV antibodies can be detected by ELISA testing.
However, the majority of newly-infected patients will be asymptomatic and have
a clinically non-apparent or mild course. Jaundice as a clinical feature of acute
hepatitis C will be present in less than 25% of infected patients. Therefore, acute
hepatitis C will not be noticed in most patients (Vogel 2009). Periodic screening for
infection may be warranted in certain groups of patients who are at high risk for
infection, e.g., homosexually-active patients with HIV infection.
Other symptoms that may occur are similar to those in other forms of acute viral
hepatitis, including malaise, nausea, and right upper quadrant pain. In patients who
experience such symptoms of acute hepatitis, the illness typically lasts for 2-12
weeks. Along with clinical resolution of symptoms, aminotransferases levels will
normalize in about 40% of patients. Loss of HCV RNA, which indicates cure from
hepatitis C, occurs in fewer than 20% of patients regardless of normalisation of
aminotransferases.
Fulminant hepatic failure due to acute HCV infection is very rare. It may be more
common in patients with underlying chronic hepatitis B virus infection (Chu 1999).

Chronic hepatitis C
The risk of chronic HCV infection is high. 80-100% of patients remain HCV RNA
positive after acute hepatitis C (Alter 1999, Vogel 2009). Most of these will have
persistently elevated liver enzymes in further follow-up. By definition, hepatitis C is
regarded to be chronic after persistence of more than six months. Once chronic
infection is established, there is a very low rate of spontaneous clearance.
It is unclear why infection with HCV results in chronic infection in most cases.
Genetic diversity of the virus and its tendency toward rapid mutation may allow
HCV to constantly escape immune recognition. Host factors may also be involved
in the ability to spontaneously clear the virus. Factors that have been associated with
successful HCV clearance are HCV-specific CD4 T cell responses, high titers of
neutralising antibodies against HCV structural proteins, IL28B gene polymorphisms
and specific HLA-DRB1 and -DQB1 alleles (Lauer 2001, Thomas 2009, Rauch
2010). Infection with HCV during childhood appears to be associated with a lower
risk of chronic infection, approximately 50-60% (Vogt 1999). Finally, there seem to
be ethnic differences, with lower risk of chronicity in certain populations.
Most patients with chronic infection are asymptomatic or have only mild
nonspecific symptoms as long as cirrhosis is not present (Merican 1993, Lauer
2001). The most frequent complaint is fatigue. Less common manifestations are
nausea, weakness, myalgia, arthralgia, and weight loss. HCV infection has also been
associated with cognitive impairment. All these symptoms are non-specific and do
not reflect disease activity or severity (Merican 1993). Very often symptoms may be
caused by underlying diseases (e.g., depression), and it can be difficult to
distinguish between different diseases. Fatigue as the most common symptom may
be present in many other situations (including healthy control groups within clinical
studies). Hepatitis C is rarely incapacitating.
Aminotransferase levels can vary considerably over the natural history of chronic
hepatitis C. Most patients have only slight elevations of transaminases. Up to one
third of patients have a normal serum ALT (Martinot-Peignoux 2001, Puoti 2002).

Hepatitis C 49
About 25% of patients have a serum ALT concentration of more than twice normal,
but usually less than 5 times above the upper limit of normal. Elevations of 10 times
the upper limit of normal are very seldomly seen.
There is a poor correlation between concentrations of aminotransferases and liver
histology. Even patients with normal serum ALT show histologic evidence of
chronic inflammation in the majority of cases (Mathurin 1998). The degree of injury
is typically minimal or mild in these patients. Accordingly, normalisation of
aminotransferases after interferon therapy does not necessarily reflect histologic
improvement.

Extrahepatic manifestations
Around 30 to 40% of patients with chronic hepatitis C have an extrahepatic
manifestation of HCV (Zignego 2008). There is a wide variety of extrahepatic
manifestations described as being associated with HCV:
− Hematologic manifestations (essential mixed cryoglobulinemia, lymphoma)
− Autoimmune disorders (thyroiditis, presence of various autoantibodies)
− Renal disease (membranoproliferative glomerulonephritis)
− Dermatologic disease (porphyria cutanea tarda, lichen planus)
− Diabetes mellitus
For further details refer to Chapter 16.

Natural history
The risk of developing cirrhosis within 20 years is estimated to be around 10 to
20%, with some studies showing estimates up to 50% (Poynard 1997, Wiese 2000,
Sangiovanni 2006, de Ledinghen 2007). Due to the long course of hepatitis C the
exact risk is very difficult to determine, and figures are divergent for different
studies and populations. In fact, chronic hepatitis C is not necessarily progressive in
all affected patients. In several cohorts it has been shown that a substantial number
of patients will not develop cirrhosis over a given time. It is estimated that about
30% of patients will not develop cirrhosis for at least 50 years (Poynard 1997).
Therefore, studies with short observation periods sometimes fail to show an
increase in mortality. In addition, survival is generally not impaired until cirrhosis
has developed. On the other hand, there is no doubt that patients with chronic
hepatitis C have a high risk of cirrhosis, decompensation, and hepatocellular
carcinoma in long-term follow-up. For example, in a cohort of patients with posttransfusion hepatitis C evaluated more than 20 years after transfusion 23% had
chronic active hepatitis, 51% cirrhosis, and 5% hepatocellular carcinoma (Tong
1995). It is not completely understood why there are such differences in disease
progression. An influence of host and viral factors has to be assumed.

Cirrhosis and hepatic decompensation
Complications of hepatitis C occur almost exclusively in patients who have
developed cirrhosis. Interestingly, non-liver related mortality is higher in cirrhotic
patients as well. However, cirrhosis may be very difficult to diagnose clinically, as
most cirrhotic patients will be asymptomatic as long as hepatic decompensation
does not occur. Findings that can be associated with cirrhosis are hepatomegaly

50 Hepatology 2012
and/or splenomegaly on physical examination, elevated serum bilirubin
concentration, hyperalbuminemia, or low platelets. Other clinical findings
associated with chronic liver disease may be found such as spider angiomata, caput
medusae, palmar erythema, testicular atrophy, or gynaecomastia. Most of these
findings are found in less than half of cirrhotic patients, and therefore none is
sufficient to establish a diagnosis of cirrhosis.
Hepatic decompensation can occur in several forms. Most common is ascites,
followed by variceal bleeding, encephalopathy and jaundice. As mentioned earlier,
hepatic decompensation will develop only in cirrhotic patients. However, not all
patients with cirrhosis actually show signs of decompensation over time. The risk
for decompensation is estimated to be close to 5% per year in cirrhotics (Poynard
1997). Once decompensation has developed the 5-year survival rate is roughly 50%
(Planas 2004). For this group of patients liver transplantation is the only effective
therapy.
Similar to decompensation, hepatocellular carcinoma (HCC) develops solely in
patients with cirrhosis (in contrast to chronic hepatitis B). The risk for HCC has
been estimated to be less than 3% per year once cirrhosis has developed (Di
Bisceglie 1997, Fattovich 1997). However, HCV-associated HCC has significant
impact on survival (see Chapter 21).
Elevated concentrations of α-fetoprotein (AFP) do not necessarily indicate HCC.
AFP may be mildly elevated in chronic HCV infection (i.e., 10 to 100 ng/mL).
Levels above 400 ng/mL as well as a continuous rise in AFP over time are
suggestive of HCC.

Disease progression
Chronic hepatitis C has different courses among individuals. It is not completely
understood why there are differences in disease progression. Several factors have
been identified that may be associated with such differences. However, other factors
not yet identified may also be important.
Age and gender: Acquisition of HCV infection after the age of 40 to 55 may be
associated with a more rapid progression of liver injury, as well as male gender
(Svirtlih 2007). On the contrary, children appear to have a relatively low risk of
disease progression (Child 1964). In one cohort, for example, only 1 of 37 patients
with HCV RNA in serum had elevated levels of serum aminotransferases, and only
3 of 17 (18%) who had liver biopsies approximately 20 years after exposure had
histologic signs of progressive liver disease.
Ethnic background: Disease progression appears to be slower and changes in
liver histology less severe in African-Americans (Sterling 2004).
HCV-specific cellular immune response: The severity of liver injury is
influenced by the cellular immune response to HCV-specific targets. Inflammatory
responses are regulated by complex mechanisms and probably depend on genetic
determinants such as HLA expression (Hraber 2007). Whether this determines
progression of liver disease is not clear.
Alcohol intake: Alcohol increases HCV replication, enhances the progression of
chronic HCV, and accelerates liver injury (Gitto 2009). Even moderate amounts of
alcohol appear to increase the risk of fibrosis. Accordingly, in alcoholic patients
with cirrhosis and liver failure a high prevalence of anti-HCV antibodies has been

Hepatitis C 51
described. Alcohol intake should be avoided in all patients with chronic hepatitis C.
There is no clear amount of safe alcohol intake.
Daily use of marijuana: Daily use of marijuana has been associated with more
rapid fibrosis progression, possibly through stimulation of endogenous hepatic
cannabinoid receptors.
Other host factors: Genetic polymorphisms of certain genes might influence the
fibrosis progression rate (Jonsson 2008). For example, transforming growth factor
B1 (TGF B1) phenotype or PNPLA3 (adiponutrin) are correlated with fibrosis stage
(Zimmer 2011). Patients with moderate to severe steatosis are at higher risk for
developing hepatic fibrosis.
Viral coinfection: Progression of hepatitis C clearly is accelerated in HIVinfected patients (see section on coinfection). Acute hepatitis B in a patient with
chronic hepatitis C may be more severe. Chronic hepatitis B may be associated with
decreased HCV replication as opposed to HCV monoinfected patients, although
HCV usually predominates. Nevertheless, liver damage is usually worse and
progression faster in patients with dual HBV/HCV infections. Around one third of
patients coinfected with HBV and HCV lack markers of HBV infection (i.e.,
HBsAg) although HBV DNA is detectable.
Geography and environmental factors: There are some obvious geographic
differences (Lim 2008). For example, hepatocellular carcinoma is observed more
often in Japan than in the United States. The reason for this is not clear.
Use of steroids: It is well known that use of steroids increases the HCV viral
load, while the effect on aminotransferases is variable. They tend to decrease in
most patients, although increases in transaminases and bilirubin have also been
described. Reducing dosage of corticosteroids returns HCV viral load to baseline.
However, the clinical consequences of corticosteroid use are largely unknown. It
seems to be reasonable to assume that short-term use of corticosteroids is not
associated with significant changes in long-term prognosis.
Viral factors: The influence of viral factors on disease progression is unclear.
Overall, there seems to be no significant role of different genotypes and
quasispecies on fibrosis progression or outcome. However, coinfection with several
genotypes may have a worse outcome as compared to monoinfection.
It is very difficult to predict the individual course of hepatitis C due to the many
factors influencing disease progression. Today, assessment of liver fibrosis by noninvasive techniques such as transient elastography, AFRI or by liver biopsy is the
best predictor of disease progression (Gebo 2002). The grade of inflammation and
stage of fibrosis are useful in predicting further clinical course. In patients with
severe inflammation or bridging fibrosis virtually all patients will develop cirrhosis
within ten years. In contrast, patients with mild inflammation and no fibrosis have
an annual progression risk to cirrhosis of around 1%.
Several predictive models of disease progression that include clinical parameters
(e.g., hepatic decompensation) and laboratory parameters (e.g., bilirubin, INR) have
been evaluated, but none of these models is routinely used in the clinic at present. In
patients with cirrhosis, the MELD score (Model for End-Stage Liver Disease) and
the Child score (Table 1) are used to stage disease and to describe the prognosis (see
Chapters 22 & 23). The MELD Score is used especially to estimate relative disease
severity and likely survival of patients awaiting liver transplant. It is calculated as:
MELD Score = 10 x ((0.957 x ln(Creatinine)) + (0.378 x ln(Bilirubin)) + (1.12 x

52 Hepatology 2012
ln(INR))) + 6.43. An online calculator and further information can be found at the
website of The United Network for Organ Sharing (UNOS) (http://www.unos.org).
Table 1. Child-Pugh classification of severity of liver disease (Child 1964).*
Points assigned
Ascites
Bilirubin, mg/dL
Albumin, g/dL
Prothrombin time
Seconds over control
INR
Encephalopathy

1

2

3

Absent
<2
>3.5

Slight
2-3
2.8-3.5

Moderate
>3
<2.8

<4
<1.7
None

4-6
1.7-2.3
Grade 1-2

>6
>2.3
Grade 3-4

* A total score of 5-6 is considered stage A (well-compensated disease); 7-9 is stage B
(significant functional compromise); and 10-15 is stage C (decompensated disease). These
grades c orrelate with one- and two-year patient survival (stage A: 100 and 85 percent; stage B:
80 and 60 percent; stage C: 45 and 35 percent).

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Hepatitis E: an underestimated problem? 55

4. Hepatitis E: an underestimated problem?
Sven Pischke and Heiner Wedemeyer

Introduction
Hepatitis E is an inflammatory liver disease caused by the hepatitis E virus (HEV),
which is endemic in many tropical countries. Hepatitis E has been considered to be
a travel-associated, acute, self-limiting liver disease that only causes fulminant
hepatic failure in specific, high-risk groups. It has recently been estimated that HEV
infection causes approximately 70,000 deaths each year worldwide (Rein 2011). In
recent years sporadic cases of HEV infections have emerged also in industrialized
countries, mostly caused by HEV genotype 3, for which zoonotic transmission has
been described (Pischke 2010b).
In immunocompetent individuals infection with HEV usually leads to a clinically
silent seroconversion or to an acute self-limited inflammation of the liver. In
pregnant women and patients with pre-existing chronic liver diseases cases of
fulminant liver failure by HEV infection are reported (Pischke 2010b).
Moreover, cases of chronic HEV infection associated with progressive liver
disease have been described in several cohorts of immunocompromised individuals.
In this context, diagnosis of HEV infection should rely on detection of HEV RNA,
as testing for HEV-specific antibodies may lack sensitivity (Pischke 2010c).
Therapeutic options for chronic hepatitis E include reduction of
immunosuppressive medication (Kamar 2011a), treatment with α-interferon
(Haagsma 2010, Kamar 2010a) or therapy with ribavirin (Kamar 2010b, Mallet
2010).
Recently, results of a large Phase III study were presented investigating a novel
recombinant HEV vaccine in China. The vaccine had an efficacy to prevent acute
symptomatic hepatitis E of >90% (Zhu 2010). It is unknown yet if and when this
vaccine might become available for other countries.

HEV: genetic characteristics of the virus
The hepatitis E virus is a non-enveloped, single-stranded RNA virus classified into
the family of Hepeviridae and its own genus Hepevirus (Pischke and Wedemeyer
2010). There are 5 known genotypes. The HEV genome includes two short noncoding regions surrounding three open reading frames (ORF1 to 3). These ORFs
contain the genetic information for various proteins that are necessary for capsid

56 Hepatology 2012
formation, virus replication and infectivity of HEV. Various HEV isolates have
been differentiated by phylogenetic analysis based on a hypervariable region within
ORF1 (Meng 1999). Four of five HEV genotypes are able to infect humans, while
genotype 5, called “avian HEV”, has only been detected in birds.
HEV genotype 1 is responsible for endemic and epidemic infections by HEV in
Asia, while genotype 2 is endemic in Africa and Mexico (Figure 1). These
genotypes are usually transmitted orally-faecally by contaminated drinking water
under conditions of poor sanitation. There is no known animal reservoir for these
genotypes (Pischke 2010b).
HEV genotype 3 can be found in humans and animals in Europe, the US and Asia
(Pischke 2010b). For this genotype zoonotic transmission, foodborne or by contact
with infected animals has been described. HEV genotype 3 has been identified in
pigs, wild boars, shellfish, deer, oysters, cats, rats and various rodents (Pischke
2010b). Genotype 4 has also been detected in both humans and pigs in Asia (Geng
2009) and Europe (Hakze-van der Honing 2011).
Foodborne transmission can be avoided by cooking meat above 60°C, which
inactivates the virus (Emerson 2005).

Figure 1. Worldwide distribution of HEV genotypes.

Diagnosis of hepatitis E
In immmunocompetent patients the diagnosis of hepatitis E is based on the
detection of HEV-specific antibodies. While IgG antibodies indicate acute and past
HEV infections, IgM antibodies can only be found in patients with acute infections
(Pischke and Wedemeyer 2010). There are different commercial assays available for
detection of HEV-specific antibodies. Comparison of six of these assays revealed a
wide variation of diagnostic sensitivities and specificities as well as interassay
disagreements (Drobeniuc 2010). Thus, some of the remarkable discrepancies in
HEV seroprevalence rates reported in different studies may be explained by varying
sensitivities of the respective assays.
HEV-specific IgG antibodies can be detected in patients with previous contact
with HEV. They do not differentiate between ongoing HEV infection and past
contact with the virus. To prove current infection the detection of HEV RNA by
PCR has been established. Numerous assays using different primers have been

Hepatitis E: an underestimated problem? 57
developed (Meng 1999, Zhao 2007). Furthermore, few quantitative PCR assays
have been described (Ahn 2006, Enouf 2006).
In immunocompromised individuals, diagnosis of HEV infection may only be
based on the detection of HEV RNA as seroassays lack sensitivity especially in the
early phase of infection (Pischke 2010c). HEV RNA can not only be detected in
serum samples but also in stool (Pischke 2010b) and thus infectivity of HEV
infected persons can be determined by investigating stool for HEV RNA.

Worldwide distribution of HEV infections
In the last few years an increasing frequency of diagnosed cases of HEV infections
has been reported from various industrialised countries (Pischke 2010b). The
presence of HEV RNA in urban sewage samples from Spain, the US and France has
been shown, suggesting that HEV may be more prevalent in industrialised countries
than previously assumed (Clemente-Casares 2003). In each of these three countries
it was possible to discover HEV contamination in sewage samples in a notably high
frequency. These findings may partially explain the huge gap between
seroprevalence rates and the rather low numbers of diagnosed and reported cases of
acute hepatitis E in Western countries. For example, Germany has a seroprevalence
rate of 2% in a population of 80 million individuals (representing 1.6 million
persons with possible previous HEV infection) but only about 200 cases of hepatitis
E are diagnosed and reported each year (Pischke 2011a, Pischke 2010b). The
mismatch between high seroprevalence rates and the low number of symptomatic
cases has also been investigated in a recent study from Egypt. 919 anti-HEV
seronegative individuals from rural Egypt were followed and, interestingly, 3.7%
(n=34) of these individuals seroconverted to anti-HEV within 11 months of follow
up (Stoszek 2006). However, none of these 34 individuals suffered from
symptomatic hepatitis E. This finding corresponds with data from a recently
published large vaccine study performed in China where very few of the patients in
the placebo group who seroconverted during a follow-up period developed
symptomatic acute hepatitis E (Zhu 2010). Overall, these data suggest that far less
than 5% of all contacts with HEV lead to symptomatic hepatitis E (Wedemeyer and
Pischke 2011).
Even so, a rapid increase in reported HEV infections has been recognized in
several industrialized countries over the last 10 years. To investigate the potential
underlying reasons for this phenomenon, we analyzed the time trend of the antiHEV seroprevalence in healthy German individuals versus the number of reported
cases of acute hepatitis E. Even though the number of reported cases increased more
than 5-fold over the last ten years (Figure 1), the anti-HEV IgG seroprevalence rate
remained rather stable over the last 15 years (Pischke 2011a). In contrast, the
number of scientific articles on HEV infections published in PubMed increased
sharply during the same period (Figure 1). These findings could indicate that the
increase of reported HEV cases in Germany and other industrialized countries is
based on an increased awareness associated with more frequent diagnosis of
hepatitis E but not a true increase in incidence rates (Pischke 2011a).

58 Hepatology 2012

Figure 2. Number of reported HEV infections in Germany over the last decade (Figure 2a)
and number of publications on HEV over the same time period (Figure 2b).

Transmission of HEV
The vast majority of HEV infections worldwide happens via the faecal-oral route
(Figure 2). Patient-to-patient transmission is very rare but has been described from a
large outbreak in Northern Uganda (Teshale 2011) and from hematology wards in
Europe (Pischke 2010b). Bloodborne transmission of HEV has been suggested in
the late nineties (Fainboim 1999). Subsequent studies from Hong Kong, Japan,
Great Britain and France confirmed blood transfusions as a possible source of HEV
transmission (Pischke 2010b). A single case of HEV transmission by
transplantation of a liver graft from a patient with occult hepatitis E has been
reported (Schlosser 2011).
Zoonotic transmission of HEV has recently been assumed to be the main source
of HEV infections in industrialized countries (Figure 3). Both direct contact with
HEV-infected domestic animals and foodborne transmission are possible (Pischke
2010b). Commercial food products such as pig meat may be contaminated with
HEV as shown in studies from the Netherlands, France and Germany (Colson 2010,
Melenhorst 2007, Wenzel 2011). Meat should be heated to over 70°C to prevent
foodborne HEV infections (Emerson 2005).

Hepatitis E: an underestimated problem? 59

Figure 3. Possible sources of HEV infection.

Acute hepatitis E in immunocompetent individuals
In the vast majority of cases, contact with HEV takes an asymptomatic course
(Stoszek 2006, Wedemeyer and Pischke 2011), especially if the contact happens
during childhood (Buti 2008). Immunocompetent individuals should be able to clear
the virus spontaneously. In symptomatic cases the incubation period of HEV
infections ranges from three to eight weeks with a mean of 40 days (Pischke 2010b).
The peak of HEV viremia can be detected in the early phase of infection while the
peak of ALT elevations usually occurs around 6 weeks after infection (Pischke
2010b).
Initial symptoms in acute hepatitis E are typically unspecific and can include flulike myalgia, arthralgia, weakness and vomiting. In some patients jaundice, itching,
uncoloured stool and darkened urine occur accompanied by elevation of liver
transaminases, bilirubin, alkaline phosphatase and gamma-glutamyltransferase.
HEV infection can lead to more severe acute liver disease in pregnant women or
patients with underlying chronic liver diseases progressing to fulminant hepatic
failure in individual cases (Pischke 2010b). Possible explanations for the severe
courses in pregnant women are hormonal and immunological changes during
pregnancy (Navaneethan 2008). Recently an association between reduced
expression of the progesterone receptor and fatal outcome of hepatitis E in pregnant
women has been reported (Bose 2011).
Single cases of prolonged courses of HEV infection in immunocompetent
individuals with up to two years of viremia have been described in the US (Mallet
2010), Spain (Gonzalez Tallon 2011) and China (Liu and Liu 2011). However, no
case of HEV-associated liver cirrhosis or development of hepatocellular carcinoma
has been reported in immunocompetent individuals.

60 Hepatology 2012

Acute and chronic HEV infections in organ
transplant recipients
Chronic courses of HEV infections have been described in European liver or kidney
transplant recipients since 2008 (Gerolami 2008, Haagsma 2009, Kamar 2008,
Pischke 2010c). 14 cases of acute hepatitis E were initially reported in kidney- and
liver-transplanted patients from southwest France (Kamar 2008). Eight of them
developed a chronic course leading to persistently elevated ALT levels, significant
histological activity and fibrosis after a follow-up of more than 12 months (range 10
to 18). Subsequently, additional cases of chronic HEV infections have been reported
in transplant patients by several groups (Pischke and Wedemeyer 2010), clearly
demonstrating that chronic hepatitis E can be associated with progressive liver
disease in patients after organ transplantation (Kamar 2011c).
A study from Germany examined 226 liver-transplanted patients and 129 patients
with chronic liver disease to evaluate the frequency of chronic HEV infections in
liver transplant recipients in a low endemic country (Pischke 2010c). All patients
were tested for HEV RNA and anti-HEV IgG. Two cases of chronic HEV infections
in liver transplanted patients were identified showing different courses. One of them
developed significant liver fibrosis (ISHAK F3) within less than 2 years. Both
patients were infected with HEV genotype 3. The possibility of reverse zoonotic
transmission was experimentally confirmed by infecting pigs with the patient’s
blood. HEV RNA was detectable in various organs of the pigs including muscle.
Thus, these findings further support the recommendations that eating uncooked
meat should be avoided by organ transplant recipients as this may represent a source
for acquiring HEV infection.
A recent study summarized retrospective data on hepatitis E in transplant
recipients in 17 centres. Overall, 85 cases of HEV infections were described and 56
(66%) patients developed chronic hepatitis E. Of note, chronicity was associated
with the use of tacrolimus and with low platelet count (Kamar 2011c). However it
has to be considered that the vast majority of patients had been recruited by one
center (Toulouse) and experiences from other regions and transplant centres need to
be reported.
Chronic courses of HEV infection have also been reported in heart transplant
recipients (de Man 2011, Pischke 2011b). Overall, all recipients of solid organ
transplant with elevated liver enzymes should be tested for HEV RNA unless other
obvious reasons already explain the hepatitis. In immunosuppressed patients testing
for HEV RNA should be applied as antibody testing may lack sensitivity.

Hepatitis E in patients with HIV infection
Chronic hepatitis E was described for the first time in a patient with underlying HIV
infection in 2009 (Dalton 2009). This patient had a CD4 T cell count of less than
200 cells and high HIV RNA levels (>100,000 copies/ml). However, subsequent
studies from Spain (n=93) (Madejon 2009), Germany (n=123) (Pischke 2010a) and
England (n=138) (Keane 2012) could not identify cases of chronic hepatitis in HIVinfected individuals. HEV RNA was detected for more than 10 months in only one
out of 184 HIV-positive individuals in France (Kaba 2010). This patient had
particularly low CD4 counts (<50 cells/mm) while two additional patients with

Hepatitis E: an underestimated problem? 61
higher CD4 levels were able to clear HEV spontaneously. Thus, persistent HEV
infection is rarely observed in HIV-infected patients and only subjects with strongly
impaired immune system seem to be at risk for chronic hepatitis E.

Extrahepatic manifestations of hepatitis E
There is some evidence that HEV infections maybe associated with extrahepatic
manifestations. One case report described muscular weakness and a pyramidal
syndrome in a kidney transplant recipient with persistent HEV infection (Kamar
2011b). Moreover, neurological disorders including polyradiculopathy, GuillainBarre syndrome, bilateral brachial neuritis, encephalitis or proximal myopathy, have
been reported in patients with acute and chronic HEV infections (Kamar 2011b).
The underlying mechanisms and the clinical relevance of this association require
further investigation.

Treatment of chronic hepatitis E
Treatment options for chronic hepatitis include reduction of immunosuppression,
administration of pegylated-interferon α with ribavirin. The first step in the
treatment of chronic HEV infection should be to evaluate if it is possible to reduce
the immunosuppressive medication (Pischke and Wedemeyer 2010). Reduction of
immunosupression in 16 solid organ transplant recipients with chronic hepatitis E
led to clearance of HEV in 4 cases (25%) (Kamar 2011a). A second possible
treatment option is the use of pegylated-interferon α (Haagsma 2010, Kamar
2010a). Treatment durations varied between 3 and 12 months. Overall, 4 out 5
patients were successfully treated with sustained clearance of HEV RNA. However,
the use of interferon can be associated with significant side effects and may cause
rejection in organ transplant recipients. Interferon α is therefore not recommended
in heart or kidney transplant recipients. The antiviral efficacy of ribavirin
monotherapy has been evaluated by two French groups (Kamar 2010b, Mallet
2010). A sustained virological response was observed in 2/2 and 4/6 treated
patients, respectively. Ribavirin has also been used in a not-transplanted patient with
severe acute hepatitis E who showed rapid improvement of symptoms and liver
function tests during treatment (Gerolami 2011).

Vaccination
No commercial HEV vaccine is currently available. A vaccine developed by GSK
and the Walter Reed Army Institute that was successfully tested in a Phase II study
(Shrestha 2007). However, this vaccine has not been further developed. A group
from China reported data recently from a very large successful Phase III vaccine
trial (Zhu 2010). This trial included almost 110,000 individuals who received either
a recombinant HEV vaccine (“HEV 239”) or placebo. The vaccine efficacy after
three doses was 100%. It is currently not known if and when this vaccine will
become available in China and other countries. Moreover, the efficacy of this
vaccine needs to be evaluated in special risks groups such as patients with end-stage
liver disease or immunosuppressed individuals. It is also unknown if HEV-239 also
protects from HEV genotype 3 infection (Wedemeyer and Pischke 2011).

62 Hepatology 2012

Conclusions/Recommendations
− The prevalence of chronic HEV infections in liver transplant recipients
depends on the general prevalence in the population and is low in most
industrialized countries. However, chronic hepatitis E occurs and needs to be
considered in the differential diagnosis of graft hepatitis as persistent HEV
infection can be associated with progressive graft hepatitis and the
development of liver cirrhosis. Currently all reported cases of chronic HEV
infections in transplant recipients are caused by HEV genotype 3. It is not
known if chronic hepatitis E can also be caused by the other genotypes.
− The diagnosis of HEV infection should not be based on serological assays
alone in organ transplant recipients as these assays may lack sensitivity.
Detection of HEV RNA by PCR in serum or stool represents the gold standard
to determine the diagnosis of HEV infection.
− Organ transplant recipients and other immunocompromised individuals should
avoid eating uncooked meats to avoid infection with HEV.
− Additional studies investigating the use of ribavirin for treatment of chronic
hepatitis E are necessary.
− The relevance of extrahepatic manifestations associated with acute or chronic
HEV infections needs further examination.

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HBV Virology 65

5. HBV Virology
Maura Dandri, Jörg Petersen

Introduction
The human hepatitis B virus (HBV) is a small enveloped DNA virus causing acute
and chronic hepatitis. Although a safe and effective vaccine has been available for
the last two decades, HBV infection still represents a major global health burden,
with about 350 million people chronically infected worldwide and more than 1
million deaths per year due to HBV-associated liver pathologies (Block 2007).
Many epidemiological and molecular studies have shown that chronic HBV
infection represents the main risk factor for hepatocellular carcinoma development
(Shepard 2006, Lok 2004, Pollicino 2011). The rate for chronicity is approximately
5% in adult infections, but it reaches 90% in neonatal infections. HBV transmission
occurs vertically and horizontally via exchange of body fluids. In serum, up to 1012
HBV genome equivalents per ml serum can be found. Although HBV does not
induce direct cytopathic effects under normal infection conditions (Wieland 2004,
Thimme 2003), liver damage (fibrosis, cirrhosis, and eventually hepatocellular
carcinoma) is believed to be induced by the ongoing immune reaction and a
consistent inflammation of the liver (McMahon 2009, Chisari 2007).
HBV is the prototype member of the Hepadnaviridae family, which are the
smallest DNA-containing, enveloped animal viruses known. Characteristic of HBV
is its high tissue- and species-specificity, as well as a unique genomic organization
with asymmetric mechanism of replication (Nassal 2008). Since all hepadnaviruses
use a reverse transcriptase to replicate their genome, they are considered distantly
related to retroviruses. Despite decades of research and significant progresses in
understanding of the molecular virology of HBV, important steps of the infection,
such as the mechanism and cellular receptor(s) mediating viral entry, have not yet
been clarified (Glebe 2007). Only recently, innovative infection models and
molecular techniques have opened new possibilities to investigate specific steps of
the lifecycle, as well as the organization and the activity of the covalently closed
circular DNA (cccDNA), the viral minichromosome serving as the template of HBV
transcription in the nucleus of the infected hepatocytes, which enables maintenance
of chronic HBV infection (Levrero 2009).

66 Hepatology 2012

Taxonomic classification and genotypes
The Hepadnaviridae form their own taxonomic group, since their biological
characteristics are not observed in any other viral family. Based on host and
phylogenetic differences, the family of Hepadnaviridae contains two genera: the
orthohepadnaviruses infecting mammals, and the avihepadnaviruses that infect
birds. To date, orthohepadnaviruses have been found in human (HBV), woodchuck
(WHV) (Korba 1989), ground squirrel (GSHV), arctic squirrel (ASHV) and woolly
monkey (WMHBV) (Lanford 1998). Avihepadnaviruses include duck HBV
(DHBV) (Mason 1980), heron HBV (HHBV) (Sprengel 1988), Ross’s goose HBV,
snow goose HBV (SGHBV), stork HBV (STHBV) (Pult 2001) and crane HBV
(CHBV) (Roggendorf 2007, Funk 2007, Dandri 2005b, Schaefer 2007).
Due to the lack of proofreading activity of the viral polymerase, misincorporation
of nucleotide mutations occurs during viral replication. This has led to the
emergence of eight HBV genotypes, A-H, which differ in more than 8% of the
genome, as well as different subgenotypes, which differ by at least 4% (Fung and
Lok 2004, Guirgis 2010). The HBV genotypes have different geographic
distribution (Liaw 2010), with predominance of genotype A in northwestern
Europe, North and South America, genotype B and C in Asia and genotype D in
eastern Europe and in the Mediterranean basin. The less diffuse remaining
genotypes are mostly found in West and South Africa (genotype E), in Central and
South America (genotypes F and H), while genotype G has been detected in France
and in the US (Pujol 2009). The phylogenetic tree of HBV genomes is reviewed
elsewhere (Schaefer 2007)

HBV structure and genomic organization
Three types of viral particles can be visualized in the infectious serum by electron
microscopy: the infectious virions and the subviral particles (SVPs). The infectious
virus particles are the so-called Dane particles (Dane 1970), have a spherical,
double-shelled structure of 42-44 nm containing a single copy of the viral DNA
genome, covalently linked to the terminal protein of the virus. A hallmark of HBV
infection is the presence of two additional types of particles, the spheres and the
filaments, which are exclusively composed of hepatitis B surface proteins and hostderived lipids (Glebe and Urban 2007). Since they do not contain viral nucleic
acids, the subviral particles are non-infectious. The spherical structures measure
around 22 nm in diameter, while the filaments are similarly width, but display
variable lengths (Figure 1).
The viral membrane contains three viral surface proteins and is acquired by the
virus during budding into the endoplasmic reticulum (ER), whereas the viral
particles are transported via the secretory pathways through the ER and Golgi. The
surface proteins are named, according to their size, the preS1 (or large), the preS2
(or middle) and the S (or small), which corresponds to the HBsAg. As with nearly
all enveloped viruses, the HBV particle also contains proteins of host origin (Glebe
2007, Urban 2010).
The HBV genome consists of a partially double-stranded relaxed circular DNA of
approximately 3200 nucleotides in length, varying slightly from genotype to
genotype, that in concert with the core protein (HBcAg) forms the nucleocapsids

HBV Virology 67
(Nassal 2008). Within the Dane particle the negative strand of the viral DNA is
present in full-length, thus carrying the complete genetic information. In contrast,
the positive strand spans only ~ 2/3 of the genome in length, whilst its 3’ end is
variable in size (Summers 1988, Lutwick 1977). The viral polymerase is covalently
bound to the negative strand by a phosphotyrosine bond. At the 5’ end of the
positive strand a short RNA oligomer originating from the pre-genomic (pg) RNA
residually remains bound covalently after the viral DNA synthesis. The negative
strand also contains a small redundancy of 8-9 nucleotides in length on both the 5’
end and the 3’ end, named the R region. These redundant structures are essential for
viral replication (Seeger 1986, Seeger 2000, Nassal 2008, Lee 2004).

HBV virion
“Dane
particle”

Subviral particles

Figure 1. Schematic representation of the HBV virion and non-infectious empty
subviral particles (filaments and spheres). Within the nucleocapsid (HBcAg, shown in
black) is depicted the partial double-stranded viral genome (rcDNA) covalently linked to
the terminal protein of reverse transcriptase. The presence and distribution of the three
surface proteins L (preS1 or large), M (preS2 or middle) and S (small) are shown both on
HBV and subviral particles (adapted from Glebe 2007).

The HBV genome displays four major open reading frames (ORFs) that are
organized in a unique and highly condensed way (Block 2007). As shown in Figure
2, all ORFs are in an identical orientation, partially overlap and are encoded by the
negative strand. On the genome, 6 start codons, four promoters and two
transcription-enhancing elements have been identified. The four major ORFs are: I)
the preS/S, encoding the three viral surface proteins; II) the precore/core, encoding
both the core protein, essential for the formation of the nucleocapsid, and the nonstructural pre-core protein, also known as the secreted e-antigen (HBeAg); III) the
pol ORF of the viral polymerase, which possesses reverse transcriptase, DNA
polymerase and RNase H activities, and the terminal protein; and IV) the X ORF,
coding for the small regulatory X protein, which has been shown to be essential in
vivo for viral replication (Zoulim 1994, Lucifora 2011) and is capable of
transactivating numerous cellular and viral genes. Characteristic of the 4 major
HBV ORFs is that they utilize a single common polyadenylation signal motif
(Nassal 2008). Thus, all RNA transcripts are polyadenylated and capped.

68 Hepatology 2012

Figure 2. Genome organization and transcripts of the human hepatitis B virus. The
outer thin lines represent the viral transcripts that initiate at different sites, under the
control of distinct promoters, but are all terminated after a common polyadenylation site.
The RNA signal on the terminally redundant pgRNA is indicated as a hairpin. The thick
lines represent the rcDNA form of the genome as present in infectious virions. The 5’ end
of the minus-strand DNA is covalently linked to the terminal protein of the polymerase.
The 5´ end of the incomplete plus-strand DNA is constituted by an RNA oligo derived from
the 5’ end of pgRNA. DR1 and DR2 indicate the direct repeats. The inner arrows indicate
the open reading frames (adapted from Nassal 2008).

HBV structural and non-structural proteins
The three surface proteins (L, M, and S) are encoded from one open reading frame
(PreS/S) which contains three start codons (one for the large, one for the middle and
one for the small protein) but promotes the transcription of 2 mRNAs of 2.4 and 2.1
Kb, named preS and S RNAs (Glebe 2007). Notably, the preS/S ORF entirely
overlaps with the polymerase open reading frame (Lee 2004). The three HBV
envelope proteins share the C-terminal domain of the S-protein, while the M- and Lprotein display progressive N-terminal extensions of 55 and, genotype-dependent,
107 or 118 amino acids (preS2 and preS1). The small envelope protein contains the
hepatitis B surface antigen (HBsAg). In virions the stoichiometric ratio of L, M and
S is about 1:1:4, while the more abundantly secreted non-infectious subviral
particles (SVPs) contain only traces of L-protein (Bruss 2007). The envelope
proteins are cotranslationally inserted into the ER membrane, where they aggregate,
bud into the ER lumen, and are secreted by the cell, either as 22 nm subviral
envelope particles (SVPs) or as 42 nm infectious virions (Dane particles), after
having enveloped the DNA-containing nucleocapsids. The surface proteins of
mammalian Hepadnaviridae have been shown to be N- and O-glycosylated
(Schildgen 2004, Schmitt 2004). These glycosylations have been shown to be
responsible for proper secretion of progeny viral particles. During synthesis, the
preS1 domain of L is myristoylated and translocated through the ER. This
modification and the integrity of the first 77 amino acids of preS1 have been shown

HBV Virology 69
to be essential for infectivity (Glebe 2005, Nassal 2008) (Schulze 2010). Both
spherical and filamentous SVPs are secreted into the blood of infected individuals in
a 103-106-fold excess relative to the infectious particles. The biological function of
the excess of SVPs in patients is not clear. It was suggested that SVPs might absorb
the neutralizing antibodies produced by the host and hence increase the ability of the
infectious particles to reach the hepatocytes. It has also been suggested that SVPs
contribute to create a state of immune tolerance, which is a precondition for highly
productive persistent infection.
In the cytoplasm, the core protein dimerises and self-assembles to form an
icosahedral nucleocapsid. The full-length core protein is 183 amino acids in length
and consists of an assembly domain and a nucleic acid-binding domain, which plays
an active role in binding and packaging of the pregenomic RNA together with the
viral polymerase, and thus enables the RT-polymerase/RNA complex to initiate
reverse transcription within the newly forming nucleocapsids (Kann 1994, Kann
2007, Kann 1999, Daub 2002). The core protein can be phosphorylated by several
kinases. This step along with the presence of the viral polymerase is important for
the specific packaging of the pgRNA (Kann 1999, Porterfield 2010).
The viral polymerase is the single enzyme encoded by the HBV genome and is an
RNA-dependent DNA polymerase with RNase H activity. The HBV polymerase
consists of three functional domains and a so-called spacer region; the terminal
protein (TP) is located at its N-terminal domain, and serves as a primer for reverse
transcription of the pgRNA into a negative-strand DNA (Zoulim 1994, Nassal
2008). The spacer domain separates the terminal protein from the polymerase
domains (Beck 2007)
Despite the occurrence of nucleotide mutations due to the lack of proofreading
capacity of the HBV polymerase, the peculiar genomic organization of HBV, where
most of the genes overlap, imposes stronger constraints on the amino acid sequence,
which significantly reduces the occurrence of mutations in the absence of strong
selective pressures. Nevertheless, it has been shown that antiviral therapy with
nucleoside analogs can promote the selection of nucleotide mutations within
conserved domains of the reverse transcriptase, which lead to mutations also on the
amino acid sequence of the envelope proteins. Changes on the HBsAg structure may
lead to reduced binding of anti-HBs antibodies, and hence, they may favour the
selection of antibody escape mutants (Harrison 2006).
HBV also produces distinct non-structural proteins whose exact functions are not
fully elucidated. Besides the production of large amounts of empty SVPs, HBV
produces and secretes a non-particulate form of the nucleoprotein, the precore
protein, or HBeAg, which is not required for viral infection or replication, but
appears to act as a decoy for the immune system, and hence, has tolerogenic
functions in promoting viral persistence in the neonates of viremic mothers (Chen
2005, Visvanathan 2006). The precore and core proteins are translated from 2
distinct RNA species that have different 5' initiation sites: the precore RNA and the
pgRNA. Indeed, the precore transcript, which also contains the full core gene,
encodes a signal sequence that directs the precore protein to the lumen of the
endoplasmic reticulum, where it is post-translationally processed. Here, the precore
protein undergoes N- and C-terminal cleavage to produce the mature HBeAg form
(p17), which is then secreted as a monomeric protein. Interestingly, 20 to 30% of
the mature protein is retained in the cytoplasm, where it may antagonise TLR

70 Hepatology 2012
signaling pathways and so contribute to the suppression of the host innate immune
responses (Lang 2011). As an important marker for active viral replication, the
HBeAg is widely used in molecular diagnostics (Chen 2005, Hadziyannis 2006).
The X protein is a multifunctional regulatory protein with transactivating and proapoptotic potential, which can modify several cellular pathways (Bouchard 2004)
and act as a carcinogenic cofactor (Kim 1991, Dandri 1996, Slagle 1996).
Numerous DNA transfection experiments have shown that over-expression of the X
protein (HBx) causes transactivation of a wide range of viral elements and cellular
promoters (Bouchard 2004). The evidence that HBx responsive enhancers/
promoters do not share any common DNA sequence and that HBx does not bind
double-stranded DNA suggested that HBx may exert its transactivating activity
through protein-protein interactions. In vitro studies have shown that HBx can affect
various cytoplasmic signal transduction pathways by activating the Src kinase,
Ras/Raf/MAP kinase, members of the protein kinase C, as well as Jak1/STAT.
Furthermore, in vitro binding studies show that HBx can regulate the proteasome
function, and thus, may control the degradation of cellular and viral proteins (Zhang
2004). It has also been reported that HBx can affect mitochondria function, by
altering its transmembrane potential, as well as that HBx can modulate calcium
homeostasis (Bouchard 2001, Nassal 2008, Yang 2011).
Although the exact role of HBx in the context of HBV infection has not been
clarified, several lines of evidence obtained first using the woodchuck model
(Zoulim 1994) and more recently using uPA/SCID mice (Tsuge 2010) and
HepaRGTM cells (Lucifora 2011), have convincingly shown that HBx is required to
initiate HBV replication and to maintain virion productivity. Notably, these studies
indicated that despite the establishment of comparable cccDNA amounts,
transcription of HBV RNAs was dramatically impaired in cells inoculated with
HBV X, indicating that HBx is essential for viral transcription. These findings are
also in agreement with data showing that HBx is recruited to the cccDNA
minichromosome, where it appears to be involved in epigenetic control of HBV
replication (Belloni 2009, Levrero 2009). In addition, HBx has been shown to
enhance encapsidation of the pgRNA by increasing phosphorylation of the core
protein (Melegari 2005), indicating that HBx may support virion productivity in
various steps of the HBV life cycle.
Most HBV-related HCC show the integration of HBV DNA sequences including
the X gene (Brechot 2004, Pollicino 2011, Lupberger 2007). Although HBV
integrated forms are frequently rearranged and hence not compatible with the
expression of functional proteins, HBx sequences deleted in the C-terminal portion
have been frequently detected in tumoral cells (Iavarone 2003). In virus-associated
cancers, viral proteins have been shown to participate in epigenetic alterations by
disturbing the host DNA methylation system. Interestingly, a study suggested that
the HBV regulatory X protein is a potent epigenetic modifying factor in the human
liver, which can modulate the transcription of DNA methyltransferases required for
normal levels of genomic methylation and maintenance of hypomethylation of
tumor suppressor genes (TSGs) (Park 2007). HBx-promoted hypermethylation of
TSGs suggests a novel mechanism by which this promiscuous transactivating
protein may accelerate hepatocarcinogenesis (Kekule 1993, Dandri 1996).

HBV Virology 71

The HBV replication cycle
During the last 30 years, the generation of various HBV–transfected human
hepatoma cell lines and the use of related HBV viruses, like the duck hepatitis B
virus (DHBV) and the woodchuck hepatitis virus (WHV) have significantly
contributed to elucidate many steps of the hepadnavirus replication cycle (Schultz
2004, Roggendorf 1995, Roggendorf 2007). Nevertheless, the lack of efficient in
vitro infection systems and of easily accessible animal models has significantly
hindered the identification of mechanisms and cellular factors mediating viral entry
and uncoating in human hepatocytes. Although primary hepatocytes remain
permissive in vitro for only a short time after plating, the availability of primary
hepatocytes from tree-shrews (Tupaia belangeri) for infection studies with HBV
and the closely-related woolly monkey hepatitis B virus (WM-HBV) (Kock 2001),
and the discovery of a human hepatoma cell line (HepaRG) able to gain
susceptibility for HBV infection upon induction of differentiation in vitro (Gripon
2002), have lately expanded our possibilities to functionally dissect the HBV entry
process (Glebe 2007, Schulze 2010).
The first step in HBV infection appears to involve a non-cell-type specific
primary attachment to the cell-associated heparan sulfate proteoglycans (Schulze
2007). This first reversible attachment step is then followed by an irreversible
binding of the virus to a specific, but still unknown hepatocyte-specific receptor
(Urban 2010, Glebe 2007). This step probably requires activation of the virus,
resulting in exposure of the myristoylated N-terminus of the L-protein. Important
determinants for infectivity within the HBV envelope proteins were identified using
mutational analyses. These include 75 amino acids of the preS1 domain of the HBV
L‐protein, its myristoylation and the integrity of a region in the antigenic loop of the
S domain (Gripon 2005, Engelke 2006). Potential HBV receptor candidates have
been described in the past, but none of them has been confirmed in a functional
assay (Glebe 2007). Recent studies indicated that cell polarization, in addition to the
differentiation status of the hepatocytes, plays an important role in the infection
process (Schulze 2011).
Upon binding to the cell membrane, two possible entry pathways have been
proposed. Experimental evidence suggests that HBV can be either involved in an
endocytosis process, followed by the release of the nucleocapsid from endocytic
vesicles, or HBV may enter the hepatocytes after fusion of the viral envelope at the
plasma membrane. As soon as the viral nucleocapsids are released into the
cytoplasm, the viral relaxed circular partially double stranded DNA (rcDNA) with
its covalently linked polymerase needs to enter the cell nucleus in order to convert
the rcDNA genome into a covalently closed circular form (cccDNA) (Nassal 2008).
Previous studies indicated that the viral capsids are transported via microtubules to
the nuclear periphery (Rabe 2006). The accumulation of the capsids at the nuclear
envelope would then facilitate interactions with nuclear transport receptors and
adaptor proteins of the nuclear pore complex (Kann 2007). Although immature
capsids may remain trapped within the nuclear baskets by the pore complexes, the
mature capsids eventually disintegrate, permitting the release of both core capsid
subunits and of the polymerase-viral DNA complexes, which diffuse into the
nucleoplasm (Schmitz 2010).

72 Hepatology 2012
Within the infected nuclei the establishment of productive HBV infection requires
the removal of the covalently attached viral polymerase and completion of the
positive-strand by the cellular replicative machinery to form the supercoiled
cccDNA molecule, which is then incorporated into the host chromatin and serves as
the template of viral transcription and replication (Nassal 2008, Newbold 1995). For
the formation of the cccDNA, the terminal protein and one of the redundant
terminal repeats present on the rcDNA need to be removed. So far it is assumed that
cellular ligases and probably other enzymes involved in DNA repair mechanisms
become active and convey the relaxed circular form into the cccDNA (Seeger
2000). Unlike the provirus DNA of retroviruses, the cccDNA does not need to be
incorporated into the host genome. Nevertheless, integrations of HBV DNA
sequences do occur, particularly in the course of hepatocyte turnover and in the
presence of DNA damage, as has been shown in cell culture (Dandri 2002) and in
the woodchuck system (Petersen 1998, Summers 2004, Mason 2005).
Disguised as a stable non-integrated minichromosome (Bock 1994, Bock 2001),
the cccDNA utilizes the cellular transcriptional machinery to produce all viral
RNAs necessary for protein production and viral replication, which takes place in
the cytoplasm after reverse transcription of an over-length pregenomic RNA
(pgRNA) (Figure 3).

cccDNA

AAA
AAA

Figure 3. The HBV lifecycle. Upon hepatocyte infection the nucleocapsid is released into
the cytoplasm and the rcDNA transferred to the cell nucleus where it is converted into the
cccDNA minichromosome. After transcription of the viral RNAs, the pgRNA is
encapsidated and reverse-transcribed by the HBV polymerase. Through Golgi and ER
apparatus the core particles acquire the envelope and are secreted. Via viral entry and
retransporting of the newly synthesized HBV DNA into the cell nucleus, the cccDNA pool
can be amplified.

Experimental DHBV infection studies indicate that the cccDNA can be formed
not only from incoming virions, but also from newly synthesized nucleocapsids,
which instead of being enveloped and secreted into the blood, are rather transported
into the nucleus to ensure accumulation, and later maintenance, of the cccDNA pool
(Zoulim 2005b, Nassal 2008). According to this scenario, multiple rounds of
infection are not needed to establish a cccDNA pool in infected duck hepatocytes.

HBV Virology 73
Moreover, expression of the DHBV viral large surface (LS) protein was shown to
induce a negative-feedback mechanism, whereby the accumulation of the LS protein
would be fundamental to shut off the cccDNA amplification pathway and redirect
the newly synthesized rcDNA-containing nucleocapsids to envelopment and
extracellular secretion (Kock 2010). Although this peculiar nuclear reentry
mechanism has been clearly demonstrated for the duck HBV (Summers 1991,
Nassal 2008, Wu 1990) and a high copy number of cccDNA molecules is generally
detected in chronically infected ducks and woodchucks (1 to 50 copies/cell) (Zhang
2003, Dandri 2000), lower cccDNA intrahepatic loads are generally determined in
human liver biopsies obtained from chronically HBV -infected patients (median 0.1
to 1 cccDNA copy/cell) (Werle-Lapostolle 2004, Wong 2004, Laras 2006, Volz
2007, Wursthorn 2006, Lutgehetmann 2008) and in chronically HBV-infected
human-liver chimeric uPA/SCID mice (Petersen 2008, Lutgehetmann 2011a,
Lutgehetmann 2011b, Lutgehetmann 2010), suggesting that different viral and host
mechanisms may control cccDNA dynamics and cccDNA pool size in human
infected hepatocytes (Levrero 2009). A recent study elegantly showed that HBV
converted the rcDNA into cccDNA less efficiently than DHBV in the same human
cell background (Kock 2010).
Although the formation of the cccDNA minichromosome is essential to establish
productive infection, recent studies performed in uPA/SCID mice indicate that this
step is achieved, initially, only in a minority of human hepatocytes. Indeed, three
weeks post-infection, the intrahepatic cccDNA load is very low (ca. 1 copy/50
human hepatocytes) and only sporadic cells stain HBcAg-positive, while within 8
weeks the majority of human hepatocytes become infected. Thus, several weeks
appear to be necessary for HBV to spread among human hepatocytes in vivo, even
in the absence of adaptive immune responses (Dandri 2011).
HBV polymerase inhibitors do not directly affect cccDNA activity and various in
vitro and in vivo studies support the notion that the cccDNA minichromosome is
very stable in quiescent hepatocytes (Moraleda 1997, Dandri 2000, Dandri 2005,
Lutgehetmann 2010). Thus, the significant decrease in cccDNA levels
(approximately 1 log10 reduction) generally determined after 1 year of therapy with
polymerase inhibitors (Werle-Lapostolle 2004) is imagined to derive from the lack
of sufficient recycling of viral nucleocapsids to the nucleus, due to the strong
inhibition of viral DNA synthesis in the cytoplasm, and less incoming viruses from
the blood. Nevertheless, cccDNA depletion is expected to require many years of
nucleos(t)ide drug administration. Thus, despite the absence of detectable viremia,
the persistence of the cccDNA minichromosome within the infected liver is
responsible for the failure of viral clearance and the relapse of viral activity after
cessation of antiviral therapy with polymerase inhibitors in chronically infected
individuals. Furthermore, if viral suppression is not complete, the selection of
resistant variants escaping antiviral therapy is likely to occur (Zoulim 2005a,
Zoulim 2005b, Zoulim 2009). Resistant HBV genomes can be archived in infected
hepatocytes when nucleocapsids produced in the cytoplasm by reverse transcription
and containing resistant mutants are transported into the nucleus and added to the
cccDNA pool. Under antiviral pressure, these variants will coexist with wild-type
cccDNA molecules and function as templates for the production and possibly
further selection of replication-competent resistant mutants, which will spread to

74 Hepatology 2012
other hepatocytes and, eventually may even replace the wild-type cccDNA
molecules in the liver (Zoulim 2006, Zoulim 2009).
During chronic HBV infection immune-mediated cell injury and compensatory
hepatocyte proliferation may favour cccDNA decline and selection of cccDNA-free
cells (Mason 2005, Zhang 2003, Thermet 2008). Notably, studies with the duck
model show that antiviral therapy with polymerase inhibitors induce a greater
cccDNA reduction in animals displaying higher hepatocyte proliferation rates
(Addison 2002). cccDNA decrease was also determined in chronically WHVinfected woodchuck hepatocytes when cell turnover was induced in vitro by
addition of cellular growth factors and viral replication was suppressed by adefovir
treatment (Dandri 2000). Furthermore, the identification of uninfected cccDNAnegative cell clones containing “traces” of the infection in form of viral integrations
indicate that cccDNA clearance without cell destruction can occur in chronically
infected woodchucks (Mason 2005). Thus, in chronic infection, killing of
hepatocytes may be instrumental not only to eliminate infected cells but also to
induce hepatocyte proliferation which, in turn, may favour cccDNA loss (Dandri
2005, Lutgehetmann 2010). On the other hand, studies have shown that very low
levels of cccDNA can persist indefinitely, possibly explaining lifelong immune
responses to HBV despite clinical resolution of HBV infection (Rehermann 1996).
As mentioned previously, the cccDNA acts chemically and structurally as an
episomal DNA with a plasmid-like structure (Bock 1994, Bock 2001, Newbold
1995), which is organized as a minichromosome by histone and non-histone
proteins. Hence its function is regulated, similarly to the cellular chromatin, by the
activity of various nuclear transcription factors, including transcriptional
coactivators, repressors and chromatin modifying enzymes (Levrero 2009).
Congruent with the fact that HBV infects hepatocytes, nearly all elements regulating
viral transcription have binding sites for liver-specific transcription factors (Levrero
2009, Quasdorff 2008). Nevertheless, although a number of factors regulating viral
transcription are known, the exact molecular mechanisms regulating HBV
transcription are still poorly defined. Both messenger and pregenomic RNAs are
transported into the cytoplasm, where they are respectively translated or used as the
template for progeny genome production. Thus, the transcription of the pgRNA is
the critical step for genome amplification and determines the rate of HBV
replication. Identification of the factors affecting stability and transcriptional
activity of the cccDNA in the course of infection and under antiviral therapy may
assist in the design of new therapeutic strategies aimed at silencing and eventually
depleting the cccDNA reservoir.
The next crucial step in HBV replication is the specific packaging of pgRNA, plus
the reverse transcriptase, into newly forming capsids. The pgRNA bears a secondary
structure – named the ε structure - that is present at both the 5’ and the 3’ ends. The
ε hairpin loops at the 5’ end are first recognized by the viral polymerase and act as
the initial packaging signal (Bartenschlager 1992). Binding of polymerase to the
RNA stem-loop structure ε initiates packaging of one pgRNA molecule and its
reverse transcription. The first product is single-stranded (ss) DNA of minus
polarity; due to its unique protein priming mechanism, its 5′ end remains covalently
linked to the polymerase. The pgRNA is concomitantly degraded, except for its 5′
terminal (~15–18 nucleotides which serve as primer for plus-strand DNA synthesis),
resulting in rcDNA. The heterogeneous lengths of the plus-strand DNAs generated

HBV Virology 75
by capsid-assisted reverse transcription may result from a non-identical supply of
dNTPs inside individual nucleocapsids at the moment of their enclosure by the
dNTP impermeable envelope. This predicts that intracellular cores produced in the
absence of envelopment should contain further extended positive DNAs.
Alternatively, space restrictions in the capsid lumen could prevent plus-strand DNA
completion; in this view, further plus-strand elongation after infection of a new cell
might destabilize the nucleocapsid and thus be involved in genome uncoating (Beck
2007, Nassal 2008)
The final replication step, the assembly and release of HBV Dane particles, is also
not fully understood. The envelopment of the DNA-containing nucleocapsids
requires a balanced coexpression of the S and L proteins in order to recruit the
nucleocapsid to the site of budding. Although the role of the envelope proteins in
regulating the amplification of cccDNA in HBV is not well-characterised, recent
studies indicate that the lack of expression of the envelope proteins increased
cccDNA levels, while coexpression of the envelope proteins not only favours the
secretion of viral particles, but also limits the completion of the plus-strand (Lentz
2011).

Animal models of HBV infection
Because of the narrow host range and the lack of easily accessible and robust in
vitro infection systems the study of HBV biology has been limited. Consequently it
has been attempted by researchers all over the world to establish animal models and
cell culture systems that at least partially reproduce some stages of HBV infection
and can be used, e.g., for the preclinical testing of novel antiviral drugs.
Most of the progress in hepatitis B virus research are based on infection studies
performed with the two most used HBV-related animal viruses: DHBV, which
infects Peking ducks (Mason 1980) and WHV (Summers 1978), which infects the
Eastern American woodchuck (Marmota monax).
One of the major advantages of the DHBV model is that domestic Peking ducks
can be used under normal laboratory conditions and DHBV-permissive primary
hepatocytes from ducklings or embryos are easily accessible. Furthermore, ducks
show very high infectivity rates in vivo (Jilbert 1996) with high levels of DHBV
replication and antigen expression. However, in contrast to mammalian
hepadnaviruses, DHBV infection is cleared within a few days post-infection if the
virus is not transmitted vertically. The DHBV genome is also smaller than that of
the mammalian hepadnaviruses and shares little primary nucleotide sequence
homology (40%) with HBV. Furthermore, DHBV infection is usually not associated
with liver disease and development of hepatocellular carcinoma (HCC).
Nevertheless, the duck model was widely used in preclinical trials (Zimmerman
2008, Reaiche 2010, Chayama 2011) and has contributed substantially to elucidate
the hepadnaviral replication scheme (Mason 1982, Summers 1988, Delmas 2002).
In vitro and in vivo studies with woodchuck hepatitis B virus (WHV) have been
fundamental in the preclinical evaluation of antiviral drugs now in use for treatment
of HBV infection (Moraleda 1997, Tennant 1998, Mason 1998, Block 1998, Dandri
2000, Korba 2004, Menne 2005). This is due to the fact that WHV is more similar
to HBV in terms of genomic organization than the avian hepadnaviruses.
Experimental infection of newborn woodchucks almost invariably leads to chronic

76 Hepatology 2012
infection, whereas most animals infected at older ages develop acute hepatitis that
results in an efficient immune response leading to viral clearance. Since acute and
chronic WHV infections in woodchucks show serological profiles similar to those
of HBV infection in humans, the woodchuck system has provided important
information about factors involved in the establishment of virus infection,
replication and viral persistence (Lu 2001). Virtually all WHV chronic carrier
woodchucks succumb to HCC 2-4 years post infection. Like in human HCC,
regenerative hepatocellular nodules and hepatocellular adenomas are
characteristically observed in WHV-infected woodchuck livers (Korba 2004).
Proto-oncogene activation by WHV DNA integration has been observed frequently
and is thought to play an important role in driving hepatocarcinogenesis in
woodchucks, often activating a member of the myc family by various mechanisms
(Tennant 2004). Viral integration is commonly found in woodchucks even after
resolution of transient infection with WHV (Summers 2003), while its frequency
increases dramatically in chronically infected animals (Mason 2005). Interestingly,
WHV viral integration was used as a genetic marker to follow the fate of infected
hepatocytes during resolution of transient infection in woodchucks (Summers 2003)
and to estimate the amount of cell turnover occurring in the course of chronic
infection (Mason 2005). Experimental infection studies in woodchucks also
demonstrated that WHV mutants that lacked the X gene were unable or severely
impaired to replicate in vivo (Chen 1993, Zoulim 1994, Zhang 2001). The
woodchuck model of viral-induced HCC has been used to test chemoprevention of
HCC using long-term antiviral nucleoside therapy and for the development of new
imaging agents for the detection of hepatic neoplasms by ultrasound and magnetic
resonance imaging (Tennant 2004). One main difference between human and rodent
hepatitis B resides in the absence of associated cirrhosis in woodchuck and squirrel
livers, even after prolonged viral infection (Buendia 1998). It is possible that the
rapid onset of hepatocyte proliferation following liver damage in rodents does
account for this discrepancy. In general, despite important advances achieved in
understanding the pathogenesis of WHV infection, one general disadvantage for
using woodchucks is that they are genetically heterogeneous animals, difficult to
breed in captivity and to handle in a laboratory setting.
Although HBV infects humans exclusively, it can be used to infect chimpanzees
experimentally and, to a certain extent, tupaia, the Asian tree shrew (Baumert
2005). Chimpanzees were the first animals found to be susceptible to HBV infection
(Barker 1973) and play an important role in the development of vaccines and in the
evaluation of the efficacy of therapeutic antibodies (Ogata 1999, Dagan 2003).
Though chimpanzees are not prone to develop chronic liver disease (Gagneux
2004), they provide an ideal model for the analysis of early immunological events
of HBV acute infection and pathogenesis (Guidotti 1999). Infection experiments
with chimpanzees showed that the majority of viral DNA is eliminated from the
liver by non-cytolytic mechanisms that precedes the peek of T cell infiltration
(Guidotti 1999). T cell depletion studies in chimpanzees also indicate that the
absence of CD8-positive cells greatly delay the onset of viral clearance (Thimme
2003). Chimpanzees have been used for preclinical testing of preventive and
therapeutic vaccines (Will 1982, Guidotti 1999, Iwarson 1985, Kim 2008, Murray
2005). Nonetheless, the large size, the strong ethical constraints and the high costs
of chimpanzees severely limit their use for research purposes.

HBV Virology 77
The tree shrew species Tupaia belangeri has been analyzed for the study of HBV
infection both in vitro and in vivo, taking advantage of the adaptability of these nonrodent mammals to the laboratory environment (Baumert 2005, von Weizsacker
2004). Inoculation of tree shrews with HBV-positive human serum was shown to
result in viral DNA replication in their livers, HBsAg secretion into the serum, and
production of antibodies to HBsAg and HBeAg (Walter 1996). Although
experimental infection of tree shrew with HBV infectious serum is not highly
efficient, productive HBV infection was successfully passed through five generations
of tree shrews and was specifically blocked by immunization with hepatitis B vaccine
(Yan 1996a). Interestingly, the development of hepatocellular carcinoma in tree
shrews exposed to hepatitis B virus and/or aflatoxin B1 was reported (Yan 1996b).
Whereas experimental infection of tree shrews causes only a mild, transient infection
with low viral titers in these animals, primary hepatocytes isolated from T. belangeri
turned out to be a valuable alternative source of HBV-permissive cells (von
Weizsacker 2004). More recently, the woolly monkey hepatitis B virus (WMHV)
was isolated from a woolly monkey (Lagothrix lagotricha), an endangered new world
primate (Lanford 1998). Interestingly, it has been shown that primary tupaia
hepatocytes are susceptible to infection with WMHBV (Kock 2001, Dandri 2005a),
providing a useful and more accessible alternative system for studying the early steps
of hepadnaviral infection in vitro (Schulze 2011) and in vivo (Petersen 2008).
Because of the different limitations encountered using chimpanzees and models
based on HBV-related viruses, it is not surprising that recent developments have
focused on using the natural target of HBV infection: the human hepatocyte.
However, primary human hepatocytes are not easy to handle, cannot be propagated
in vitro and their susceptibility to HBV infection is generally low and highly
variable. Furthermore, cultured cells may respond differently to the infection than
hepatocytes in the liver. The generation of mice harboring human chimeric livers
offered new possibilities to overcome some of these limitations. Two major models
are currently available: the urokinase-type plasminogen activator (uPA) transgenic
mouse (Rhim 1994) and the knockout fumarylacetoacetate hydrolase (FAH) mouse
(Azuma 2007). In both systems, the absence of adaptive immune responses permits
the engraftment of transplanted xenogenic hepatocytes, while the presence of
transgene-induced hepatocyte damage creates the space and the regenerative
stimulus necessary for the transplanted cells to repopulate the mouse liver. Both
models permit the establishment of HBV infection, which can then persist for the
life-span of the chimeric mouse (Dandri 2001, Bissig 2010). While mouse
hepatocytes do not support HBV infection, human chimeric mice can be efficiently
infected by injecting infectious serum derived from either patients or chimeric mice.
Furthermore, genetically engineered viruses created in cell culture can be used to
investigate phenotype and in vivo fitness of distinct HBV genotypes and variants
(Tsuge 2005). Within the mouse liver human hepatocytes maintain a functional
innate immune system and respond to stimuli induced by exogenously applied
human IFN α. The lack of an adaptive immune system and the undetectable
responsiveness of mouse liver cells to human IFN α make the model ideal to exploit
the capacities of HBV to interfere with pathways of the innate antiviral response in
human hepatocytes (Lutgehetmann 2011). Chimeric mice can be superinfected or
simultaneously infected with different human hepatotropic viruses to investigate the

78 Hepatology 2012
mechanisms of virus interference and response to antiviral treatment in the setting
of coinfection (Hiraga 2009).

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HCV Virology 85

6. HCV Virology
Bernd Kupfer

History
Hepatitis C virus (HCV) is a major cause of progressive liver disease with
approximately 130-170 million people infected worldwide. HCV induces chronic
infection in up to 80% of infected individuals. The main complications of HCV
infection are severe liver fibrosis and cirrhosis, and 30-50% of individuals with
cirrhosis go on to develop hepatocellular carcinoma (Tong 1995, Poynard 1997).
Until 1975, only two hepatitis viruses had been identified, the “infectious hepatitis
virus” (hepatitis A virus, HAV) and the “serum hepatitis virus” (hepatitis B virus,
HBV). However, other viruses were excluded from being the cause of
approximately 65% of post-transfusion hepatitis. Therefore, these hepatitis cases
were termed “non-A, non-B hepatitis” (NANBH) (Feinstone 1975). Inoculation of
chimpanzees (Pan troglodytes) with blood products derived from humans with
NANB hepatitis led to persistent increases of serum alanine aminotransferase (ALT)
indicating that an infectious agent was the cause of the disease (Alter 1978,
Hollinger 1978). Subsequently, it was demonstrated that the NANBH agent could
be inactivated by chloroform (Feinstone 1983). Moreover, it was reported that the
infectious agent was able to pass through 80 nm membrane filters (Bradley 1985).
Taken together these findings suggested that the NANBH causing agent would be a
small virus with a lipid envelope. However, the lack of a suitable cell culture system
for cultivation of the NANBH agent and the limited availability of chimpanzees
prevented further characterization of the causative agent of NANBH for several
years. In 1989, using a newly developed cloning strategy for nucleic acids derived
from plasma of NANBH infected chimpanzees the genome of the major causative
agent for NANBH was characterized (Choo 1989). cDNA clone 5-1-1 encoded
immunological epitopes that interacted with sera from individuals with NANBH
(Choo 1989, Kuo 1989). The corresponding infectious virus causing the majority of
NANBH was subsequently termed hepatitis C virus (HCV).

86 Hepatology 2012

Taxonomy and genotypes
HCV is a small-enveloped virus with one single-stranded positive-sense RNA
molecule of approximately 9.6 kb. It is a member of the Flaviviridae family. This
viral family contains three genera, flavivirus, pestivirus, and hepacivirus. To date,
only two members of the hepacivirus genus have been identified, HCV and GB
virus B (GBV-B), a virus that had been initially detected together with the thenunclassified virus GB virus A (GBV-A) in a surgeon with active hepatitis (Thiel
2005, Ohba 1996, Simons 1995). However, the natural hosts for GBV-B and GBVC seem to be monkeys of the Saguinus species (tamarins). Analyses of viral
sequences and phylogenetic comparisons support HCV’s membership in a distinct
genus from flavivirus or pestivirus (Choo 1991). The error-prone RNA polymerase
of HCV together with the high replication rate of the virus is responsible for the
large interpatient genetic diversity of HCV strains. Moreover, the extent of viral
diversification of HCV strains within a single HCV-positive individual increases
significantly over time, resulting in the development of quasispecies (Bukh 1995).
Comparisons of HCV nucleotide sequences derived from individuals from
different geographical regions revealed the presence of six major HCV genotypes
with a large number of subtypes within each genotype (Simmonds 2004, Simmonds
2005). Sequence divergence of genotypes and subtypes is 20% and 30%,
respectively. HCV strains belonging to the major genotypes 1, 2, 4, and 5 are found
in sub-Saharan Africa whereas genotypes 3 and 6 are detected with extremely high
diversity in South East Asia. This suggests that these geographical areas could be
the origin of the different HCV genotypes. The emergence of different HCV
genotypes in North America and Europe and other non-tropical countries appears to
represent more recent epidemics introduced from the countries of the original HCV
endemics (Simmonds 2001, Ndjomou 2003). Besides epidemiological aspects,
determination of the HCV genotype plays an important role for the initiation of antiHCV treatment since the response of different genotypes varies significantly with
regard to specific antiviral drug regimens, e.g., genotype 1 is most resistant to the
current therapy of the combination of pegylated interferon α and ribavirin (Manns
2001, Fried 2002).

Viral structure
Structural analyses of HCV virions are very limited since the virus is difficult to
cultivate in cell culture systems, a prerequisite for yielding sufficient virions for
electron microscopy. Moreover, serum-derived virus particles are associated with
serum low-density lipoproteins (Thomssen 1992), which makes it difficult to isolate
virions from serum/plasma of infected subjects by centrifugation. Visualization of
HCV virus-like particles via electron microscopy succeeded only rarely (Kaito
1994, Shimizu 1996a, Prince 1996) and it was a point of controversy if the detected
structures really were HCV virions. Nevertheless, these studies suggest that HCV
has a diameter of 55-65 nm confirming size prediction of the NANBH agent by
ultra-filtration (Bradley 1985). Various forms of HCV virions appear to exist in the
blood of infected individuals: virions bound to very low density lipoproteins
(VLDL), virions bound to low density lipoproteins (LDL), virions complexed with
immunoglobulins, and free circulating virions (Bradley 1991, Thomssen 1992,

HCV Virology 87
Thomssen 1993, Agnello 1999, Andre 2002). The reasons for the close association
of a major portion of circulating virions with LDL and VLDL remain unexplained.
One possible explanation is that HCV theoretically enters hepatocytes via the LDL
receptor (Agnello 1999, Nahmias 2006). Moreover, it is speculated that the
association with LDL and/or VLDL protects the virus against neutralization by
HCV-specific antibodies.
The design and optimization of subgenomic and genomic HCV replicons in the
human hepatoma cell line Huh7 offered for the first time the possibility to
investigate HCV RNA replication in a standardized manner (Lohmann 1999, Ikeda
2002, Blight 2002). However, despite the high level of HCV gene expression, no
infectious viral particles are actually produced. Therefore, it cannot be used for
structural analysis of free virions.
Infectious HCV particles have been achieved in cell culture by using recombinant
systems (Heller 2005, Lindenbach 2005, Wakita 2005, Zhong 2005, Yu 2007).
However, even in these in vitro systems the limited production of viral particles
prevents 3D structural analysis (Yu 2007). It was also shown by cryoelectron
microscopy (cryoEM) and negative-stain transmission electron microscopy that
HCV virions isolated from cell culture have a spherical shape with a diameter of
approximately 50 to 55 nm (Heller 2005, Wakita 2005, Yu 2007) confirming earlier
results that measured the size of putative native HCV particles from the serum of
infected individuals (Prince 1996). The outer surface of the viral envelope seems to
be smooth. Size and morphology are therefore very similar to other members of the
Flaviviridae family such as the dengue virus and the West Nile virus (Yu 2007).
Modifying a baculovirus system (Jeong 2004, Qiao 2004) the same authors were
able to produce large quantities of HCV-like particles (HCV-LP) in insect cells (Yu
2007). Analysing the HCV-LPs by cryoEM it was demonstrated that the HCV E1
protein is present in spikes located on the outer surface of the LPs.
Using 3D modeling of the HCV-LPs together with genomic comparison of HCV
and well-characterized flaviviruses it is assumed that 90 copies of a block of two
heterodimers of HCV proteins E1 and E2 form the outer layer of the virions with a
diameter of approximately 50 nm (Yu 2007). This outer layer surrounds the lipid
bilayer that contains the viral nucleocapsid consisting of several molecules of the
HCV core (C) protein. An inner spherical structure with a diameter of
approximately 30-35 nm has been observed (Wakita 2005) suggesting the
nucleocapsid that harbours the viral genome (Takahashi 1992).

Genome organization
The genome of the hepatitis C virus consists of one 9.6 kb single-stranded RNA
molecule with positive polarity. Similar to other positive-strand RNA viruses, the
genomic RNA of hepatitis C virus serves as messenger RNA (mRNA) for the
translation of viral proteins. The linear molecule contains a single open reading
frame (ORF) coding for a precursor polyprotein of approximately 3000 amino acid
residues (Figure 1). During viral replication the polyprotein is cleaved by viral as
well as host enzymes into three structural proteins (core, E1, E2) and seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B). An additional
protein (termed F [frameshift] or ARF [alternate reading frame]) is predicted as a
result of ribosomal frameshifting during translation within the core region of the

88 Hepatology 2012
genomic RNA (Xu 2001, Walewski 2001, Varaklioti 2002, Branch 2005). Detection
of anti-F protein antibodies in the serum of HCV-positive subjects indicates that the
protein is expressed during infection in vivo (Walewski 2001, Komurian-Pradel
2004).
The structural genes encoding the viral core protein and the viral envelope
proteins E1 and E2 are located at the 5’ terminus of the open reading frame
followed downstream by the coding regions for the non-structural proteins p7, NS2,
NS3, NS4A, NS4B, NS5A, and NS5B (Figure 1). The structural proteins are
essential components of the HCV virions, whereas the non-structural proteins are
not associated with virions but are involved in RNA replication and virion
morphogenesis.
The ORF is flanked by 5’ and 3’ nontranslated regions (NTR; also called
untranslated regions, UTR or noncoding regions, NCR) containing nucleotide
sequences relevant for the regulation of viral replication. Both NTRs harbour highly
conserved regions compared to the protein encoding regions of the HCV genome.
The high grade of conservation of the NTRs makes them candidates i) for improved
molecular diagnostics, ii) as targets for antiviral therapeutics, and iii) as targets for
an anti-HCV vaccine.

Figure 1. Genome organization and polyprotein processing. A) Nucleotide positions
correspond to the HCV strain H77 genotype 1a, accession number NC_004102.
nt, nucleotide; NTR, nontranslated region. B) Cleavage sites within the HCV precursor
polyprotein for the cellular signal peptidase the signal peptide peptidase (SPP) and the viral
proteases NS2-NS3 and NS3-NS4A, respectively.

The 5’NTR is approximately 341 nucleotides long with a complex secondary
structure of four distinct domains (I-IV) (Fukushi 1994, Honda 1999). The first 125
nucleotides of the 5’NTR spanning domains I and II have been shown to be
essential for viral RNA replication (Friebe 2001, Kim 2002). Domains II-IV build
an internal ribosome entry side (IRES) involved in ribosome binding and
subsequent cap-independent initiation of translation (Tsukiyama-Kohara 1992,
Wang 1993).
The 3’NTR consists of three functionally distinct regions: a variable region, a
poly U/UC tract of variable length, and the highly conserved X tail at the 3’
terminus of the HCV genome (Tanaka 1995, Kolykhalov 1996, Blight 1997). The

HCV Virology 89
variable region of approximately 40 nucleotides is not essential for RNA
replication. However, deletion of this sequence led to significantly decreased
replication efficiency (Yanagi 1999, Friebe 2002). The length of the poly U/UC
region varies in different HCV strains ranging from 30 to 80 nucleotides
(Kolykhalov 1996). The minimal length of that region for active RNA replication
has been reported to be 26 homouridine nucleotides in cell culture (Friebe 2002).
The highly conserved 98-nucleotide X tail consists of three stem-loops (SL1-SL3)
(Tanaka 1996, Ito 1997, Blight 1997) and deletions or nucleotide substitutions
within that region are most often lethal (Yanagi 1999, Kolykhalov 2000, Friebe
2002, Yi 2003). Another so-called “kissing-loop” interaction of the 3’X tail SL2 and
a complementary portion of the NS5B encoding region has been described (Friebe
2005). This interaction induces a tertiary RNA structure of the HCV genome that is
essential for HCV replication in cell culture systems (Friebe 2005, You 2008).
Finally, both NTRs appear to work together in a long-range RNA-RNA interaction
possibly resulting in temporary genome circularization (Song 2006).

Genes and proteins
As described above, translation of the HCV polyprotein is initiated through
involvement of some domains in NTRs of the genomic HCV RNA. The resulting
polyprotein consists of ten proteins that are co-translationally or post-translationally
cleaved from the polyprotein. The N-terminal proteins C, E1, E2, and p7 are
processed by a cellular signal peptidase (SP) (Hijikata 1991). The resulting
immature core protein still contains the E1 signal sequence at its C terminus.
Subsequent cleavage of this sequence by a signal peptide peptidase (SPP) leads to
the mature core protein (McLauchlan 2002). The non-structural proteins NS2 to
NS5B of the HCV polyprotein are processed by two virus-encoded proteases (NS2NS3 and NS3) with the NS2-NS3 cysteine protease cleaving at the junction of NS2NS3 (Santolini 1995) and the NS3 serine protease cleaving the remaining functional
proteins (Bartenschlager 1993, Eckart 1993, Grakoui 1993a, Tomei 1993).
The positions of viral nucleotide and amino acid residues correspond to the HCV
strain H77 genotype 1a, accession number NC_004102. Some parameters
characterizing HCV proteins are summarised in Table 1.
Core. The core-encoding sequence starts at codon AUG at nt position 342 of the
H77 genome, the start codon for translation of the entire HCV polyprotein. During
translation the polyprotein is transferred to the endoplasmic reticulum (ER) where
the core protein (aa 191) is excised by a cellular signal peptidase (SP). The C
terminus of the resulting core precursor still contains the signal sequence for ER
membrane translocation of the E1 ectodomain (aa 174-191). This protein region is
further processed by the cellular intramembrane signal peptide peptidase (SPP)
leading to removal of the E1 signal peptide sequence (Hüssy 1996, McLauchlan
2002, Weihofen 2002).
The multifunctional core protein has a molecular weight of 21 kilodalton (kd). In
vivo, the mature core molecules are believed to form homo-multimers located
mainly at the ER membrane (Matsumoto 1996). They have a structural function
since they form the viral capsid that contains the HCV genome. In addition, the core
protein has regulatory functions including particle assembly, viral RNA binding,
and regulation of RNA translation (Ait-Goughoulte 2006, Santolini 1994).

90 Hepatology 2012
Moreover, protein expression analyses indicate that the core protein may be
involved in many other cellular reactions such as cell signalling, apoptosis, lipid
metabolism, and carcinogenesis (Tellinghuisen 2002). However, these preliminary
findings need to be analysed further.
Table 1. Overview of the size of HCV proteins.*
Protein

No. of aa

aa position
in ref. seq.

MW of protein

Core immature
Core mature
F-protein or ARF-protein
E1
E2
p7
NS2
NS3
NS4A
NS4B
NS5A
NS5B

191
174
126-161
192
363
63
217
631
54
261
448
591

1-191
1-174

23 kd
21 kd
~ 16-17 kd
35 kd
70 kd
7 kd
21 kd
70 kd
4 kd
27 kd
56 kd
66 kd

192-383
384-746
747-809
810-1026
1027-1657
1658-1711
1712-1972
1973-2420
2421-3011

* aa, amino acid; MW, molecular weight; kd, kilodalton; ref. seq., reference sequence (HCV
strain H77; accession number NC_004102).

E1 and E2. Downstream of the core coding region of the HCV RNA genome two
envelope glycoproteins are encoded, E1 (gp35, aa 192) and E2 (gp70, aa 363).
During translation at the ER both proteins are cleaved from the precursor
polyprotein by a cellular SP. Inside the lumen of the ER both polypeptides
experience N-linked glycosylation post-translationally (Duvet 2002). Both
glycoproteins E1 and E2 harbour 5 and 11 putative N-glycosylation sites,
respectively.
E1 and E2 are type I transmembrane proteins with a large hydrophilic ectodomain
of approximately 160 and 334 aa and a short transmembrane domain (TMD) of 30
aa. The TMD are responsible for the anchoring of the envelope proteins in the
membrane of the ER and ER retention (Cocquerel 1998, Duvet 1998, Cocquerel
1999, Cocquerel 2001). Moreover, the same domains have been reported to
contribute to the formation of E1-E2 heterodimers (Op de Beeck 2000). The E1-E2
complex is involved in adsorption of the virus to its putative receptors tetraspanin
CD81 and low-density lipoprotein receptor inducing fusion of the viral envelope
with the host cell plasma membrane (Agnello 1999, Flint 1999, Wunschmann
2000). However, the precise mechanism of host cell entry is still not understood
completely. Several other host factors have been identified to be involved in viral
entry. These candidates include the scavenger receptor B type I (Scarselli 2002,
Kapadia 2007), the tight junction proteins claudin-1 (Evans 2007) and occludin
(Ploss 2009), the C-type lectins L-SIGN and DC-SIGN (Gardner 2003, Lozach
2003, Pöhlmann 2003) and heparan sulfate (Barth 2003).
Two hypervariable regions have been identified within the coding region of E2.
These regions termed hypervariable region 1 (HVR1) and 2 (HVR2) differ by up to
80% in their amino acid sequence (Weiner 1991, Kato 2001). The first 27 aa of the
E2 ectodomain represent HVR1, while the HVR2 is formed by a stretch of seven

HCV Virology 91
amino acids (position 91-97). The high variability of the HVRs reflects exposure of
these domains to HCV-specific antibodies. In fact, E2-HVR1 has been shown to be
the most important target for neutralizing antibodies (Farci 1996, Shimizu 1996b).
However, the combination of the mutation of the viral genome with the selective
pressure of the humoral immune response leads to viral escape via epitope
alterations. This makes the development of vaccines that induce neutralizing
antibodies challenging.
The p7 protein. The small p7 protein (63 aa) is located between the E2 and NS2
regions of the polyprotein precursor. During translation the cellular SP cleaves the
E2-p7 as well as the p7-NS2 junction. The functional p7 is a membrane protein
localised in the endoplasmic reticulum where it forms an ion channel (Haqshenas
2007, Pavlovic 2003, Griffin 2003). The p7 protein is not essential for RNA
replication since replicons lacking the p7 gene replicate efficiently (Lohmann 1999,
Blight 2000), however, it has been suggested that p7 plays an essential role for the
formation of infectious virions (Sakai 2003, Haqshenas 2007).
NS2. The non-structural protein 2 (p21, 217 aa) together with the N-terminal
portion of the NS3 protein form the NS2-3 cysteine protease which catalyses
cleavage of the polyprotein precursor between NS2 and NS3 (Grakoui 1993b,
Santolini 1995). The N-terminus of the functional NS2 arises from the cleavage of
the p7-NS2 junction by the cellular SP. Moreover, after cleavage from the NS3 the
protease domain of NS2 seems to play an essential role in the early stage of virion
morphogenesis (Jones 2007).
NS3. The non-structural protein 3 (p70, 631 aa) is cleaved at its N terminus by the
NS2-NS3 protease. The N terminus (189 aa) of the NS3 protein has a serine
protease activity. However, in order to develop full activity of the protease the NS3
protease domain requires a portion of NS4A (Faila 1994, Bartenschlager 1995, Lin
1995, Tanji 1995, Tomei 1996). NS3 together with the NS4A cofactor are
responsible for cleavage of the remaining downstream cleavages of the HCV
polyprotein precursor. Since the NS3 protease function is essential for viral
infectivity it is a promising target in the design of antiviral treatments.
The C-terminal portion of NS3 (442 aa) has ATPase/helicase activity, i.e., it
catalyses the binding and unwinding of the viral RNA genome during viral
replication (Jin 1995, Kim 1995). However, recent findings indicate that other nonstructural HCV proteins such as the viral polymerase NS5B may interact
functionally with the NS3 helicase (Jennings 2008). These interactions need to be
investigated further in order to better understand the mechanisms of HCV
replication.
NS4A. The HCV non-structural protein 4A (p4) is a 54 amino acid polypeptide
that acts as a cofactor of the NS3 serine protease (Faila 1994, Bartenschlager 1995,
Lin 1995, Tanji 1995, Tomei 1996). Moreover, this small protein is involved in the
targeting of NS3 to the endoplasmic reticulum resulting in a significant increase of
NS3 stability (Wölk 2000).
NS4B. The NS4B (p27) consists of 217 amino acids. It is an integral membrane
protein localized in the endoplasmic reticulum. The N-terminal domain of the NS4B
has an amphipathic character that targets the protein to the ER. This domain is
crucial in HCV replication (Elazar 2004, Gretton 2005) and therefore an interesting
target for the development of anti-HCV therapeutics or vaccines. In addition, a
nucleotide-binding motif (aa 129-134) has been identified (Einav 2004). Although

92 Hepatology 2012
the function of NS4B is still unknown, it has been demonstrated that the protein
induces a membranous web that may serve as a platform for HCV RNA replication
(Egger 2002).
NS5A. The NS5A protein (p56; 458 aa) is a membrane-associated phosphoprotein
that appears to have multiple functions in viral replication. It is phosphorylated by
different cellular protein kinases indicating an essential but still not understood role
of NS5A in the HCV replication cycle. In addition, NS5A has been found to be
associated with several other cellular proteins (MacDonald 2004) making it difficult
to determine the exact functions of the protein. One important property of NS5A is
that it contains a domain of 40 amino acids, the so-called IFN-a sensitivitydetermining region (ISDR) that plays a significant role in the response to IFN-abased therapy (Enomoto 1995, Enomoto 1996). An increasing number of mutations
within the ISDR showed positive correlation with sustained virological response to
IFN-a-based treatment.
NS5B. The non-structural protein 5B (p66; 591 aa) represents the RNAdependent RNA polymerase of HCV (Behrens 1996). The hydrophobic domain (21
aa) at the C terminus of NS5B inserts into the membrane of the endoplasmic
reticulum, while the active sites of the polymerase are located in the cytoplasm
(Schmidt-Mende 2001).
The cytosolic domains of the viral enzyme form the typical polymerase righthanded structure with “palm”, “fingers”, and “thumb” subdomains (Ago 1999,
Bressanelli 1999, Lesburg 1999). In contrast to mammalian DNA and RNA
polymerases the fingers and thumb subdomains are connected resulting in a fully
enclosed active site for nucleotide triphosphate binding. This unique structure
makes the HCV NS5B polymerase an attractive target for the development of
antiviral drugs.
Using the genomic HCV RNA as a template, the NS5B promotes the synthesis of
minus-stranded RNA that then serves as a template for the synthesis of genomic
positive-stranded RNA by the polymerase.
Similar to other RNA-dependent polymerases, NS5B is an error-prone enzyme
that incorporates wrong ribonucleotides at a rate of approximately 10-3 per
nucleotide per generation. Unlike cellular polymerases, the viral NS5B lacks a
proof-reading mechanism leading to the conservation of misincorporated
ribonucleotides. These enzyme properties together with the high rate of viral
replication promote a pronounced intra-patient as well as inter-patient HCV
evolution.
F protein, ARFP. In addition to the ten proteins derived from the long HCV
ORF, the F (frameshift) or ARF (alternate reading frame) or core+1 protein has
been reported (Walewski 2001, Xu 2001, Varaklioti 2002). As the designations
indicate the ARFP is the result of a -2/+1 ribosomal frameshift between codons 8
and 11 of the core protein-encoding region. The ARFP length varies from 126 to
161 amino acids depending on the corresponding genotype. In vitro studies have
shown that ARFP is a short-lived protein located in the cytoplasm (Roussel 2003)
primarily associated with the endoplasmic reticulum (Xu 2003). Detection of anti-F
protein antibodies in the serum of HCV-positive subjects indicates that the protein is
expressed during infection in vivo (Walewski 2001, Komurian-Pradel 2004).
However, the functions of ARFP in the viral life cycle are still unknown and remain
to be elucidated.

HCV Virology 93

Viral lifecycle
Due to the absence of a small animal model system and efficient in vitro HCV
replication systems it has been difficult to investigate the viral life cycle of HCV.
The recent development of such systems has offered the opportunity to analyse in
detail the different steps of viral replication.

Figure 2. Current model of the HCV lifecycle. Designations of cellular components are in red.
For a detailed illustration of viral translation and RNA replication, see Pawlotsky 2007.
Abbreviations: HCV +ssRNA, single stranded genomic HCV RNA with positive polarity; rough
ER, rough endoplasmic reticulum; PM, plasma membrane. For other abbreviations see text.

Adsorption and viral entry
The most likely candidate as receptor for HCV is the tetraspanin CD81 (Pileri
1998). CD81 is an ubiquitous 25 kd molecule expressed on the surface of a large
variety of cells including hepatocytes and PBMCs. Experimental binding of antiCD81 antibodies to CD81 were reported to inhibit HCV entry into Huh7 cells and
primary human hepatocytes (Hsu 2003, Bartosch 2003a, Cormier 2004, McKeating
2004, Zhang 2004, Lindenbach 2005, Wakita 2005). Moreover, gene silencing of
CD81 using specific siRNA molecules confirmed the relevance of CD81 in viral
entry (Bartosch 2003b, Cormier 2004, Zhang 2004, Akazawa 2007). Finally,
expression of CD81 in cell lines lacking CD81 made them permissive for HCV
entry (Zhang 2004, Lavillette 2005, Akazawa 2007). However, more recent studies
have shown that CD81 alone is not sufficient for HCV viral entry and that cofactors such as scavenger receptor B type I (SR-BI) are needed (Bartosch 2003b,
Hsu 2003, Scarselli 2002, Kapadia 2007). Moreover, it appears that CD81 is
involved in a post-HCV binding step (Cormier 2004, Koutsoudakis 2006, Bertaud
2006). These findings together with the identification of other host factors involved

94 Hepatology 2012
in HCV cell entry generate the current model for the early steps of HCV infection
(Helle 2008).
Adsorption of HCV to its target cell is the first step of viral entry. Binding is
possibly initiated by the interaction of the HCV E2 envelope glycoprotein and the
glycosaminglycan heparan sulfate on the surface of host cells (Germi 2002, Barth
2003, Basu 2004, Heo 2004). Moreover, it is assumed that HCV initiates hepatocyte
infection via LDL receptor binding (Agnello 1999, Monazahian 1999, Wünschmann
2000, Nahmias 2006, Molina 2007). This process may be mediated by VLDL or
LDL and is reported to be associated with HCV virions in human sera (Bradley
1991, Thomssen 1992, Thomssen 1993). After initial binding the HCV E2
glycoprotein interacts with the SR-BI in cell culture (Scarselli 2002). SR-BI is a
protein expressed on the surface of the majority of mammalian cells. It acts as a
receptor for LDL as well as HDL (Acton 1994, Acton 1996) emphasizing the role of
these compounds for HCV infectivity. Alternative splicing of the SR-BI transcript
leads to the expression of a second isoform of the receptor SR-BII (Webb 1998),
which also may be involved in HCV entry into target cells (Grove 2007). As is the
case for all steps of viral entry the exact mechanism of the HCVE2/SR-BI
interaction remains unknown. In some studies it has been reported that HCV
binding to SR-BI is a prerequisite for the concomitant or subsequent interaction of
the virus with CD81 (Kapadia 2007, Zeisel 2007). The multi-step procedure of
HCV cell entry was shown to be even more complex since a cellular factor termed
claudin-1 (CLDN1) has been newly identified as involved in this process (Evans
2007). CLDN1 is an integral membrane protein that forms a backbone of tight
junctions and is highly expressed in the liver (Furuse 1998). Inhibition assays reveal
that CLDN1 involvement occurs downstream of the HCV-CD81 interaction (Evans
2007). Recent findings suggest that CLDN1 could also act as a compound enabling
cell-to-cell transfer of hepatitis C virus independently of CD81 (Timpe 2007).
Furthermore, it was reported that two other members of the claudin family claudin-6
and claudin-9 may play a role in HCV infection (Zheng 2007, Meertens 2008). The
fact that some human cell lines were not susceptible to HCV infection despite
expressing SR-BI, CD81, and CLDN1 indicates that other cellular factors are
involved in viral entry (Evans 2007). Very recently, a cellular four-transmembrane
domain protein named occludin (OCLN) was identified to represent an additional
cellular factor essential for the susceptibility of cells to HCV infection (Liu 2009,
Ploss 2009). Similar to claudin-1, OCLN is a component of the tight junctions in
hepatocytes. All tested cells expressing SR-BI, CD81, CLDN1, and OCLN were
susceptible to HCV. Although the precise mechanism of HCV uptake in hepatocytes
is still not clarified, these four proteins may represent the complete set of host cell
factors necessary for cell-free HCV entry.
After the complex procedure of binding to the different host membrane factors
HCV enters the cell in a pH-dependent manner indicating that the virus is
internalized via clathrin-mediated endocytosis (Bartosch 2003b, Hsu 2003,
Blanchard 2006, Codran 2006). The acidic environment within the endosomes is
assumed to trigger HCV E1-E2 glycoprotein-mediated fusion of the viral envelope
with the endosome membrane (Blanchard 2006, Meertens 2006, Lavillette 2007).
In summary, HCV adsorption and viral entry into the target cell is a very complex
procedure that is not yet fully understood. Despite having identified several host

HCV Virology 95
factors that probably interact with the viral glycoproteins, the precise mechanisms
of interaction need to continue to be investigated.
Besides the infection of cells through cell-free HCV it has been documented that
HCV can also spread via cell-to-cell transmission (Valli 2006, Valli 2007). This
transmission path may differ significantly with regard to the cellular factors needed
for HCV entry into cells. CD81 is dispensable for cell-to-cell transmission in
cultivated hepatoma cells (Witteveldt 2009). These findings require further
investigation in order to analyze the process of cell-to-cell transmission of HCV
both in vitro and in vivo. Antiviral treatment strategies must account for the cellular
pathways of both cell-free virus and HCV transmitted via cell-to-cell contact.

Translation and posttranslational processes
As a result of the fusion of the viral envelope and the endosomic membrane, the
genomic HCV RNA is released into the cytoplasm of the cell. As described above,
the viral genomic RNA possesses a nontranslated region (NTR) at each terminus.
The 5’NTR consists of four distinct domains, I-IV. Domains II-IV form an internal
ribosome entry side (IRES) involved in ribosome-binding and subsequent capindependent initiation of translation (Fukushi 1994, Honda 1999, TsukiyamaKohara 1992, Wang 1993). The HCV-IRES binds to the 40S ribosomal subunit
complexed with eukaryotic initiation factors 2 and 3 (eIF2 and eIF3), GTP, and the
initiator tRNA resulting in the 48S preinitiation complex (Spahn 2001, Otto 2002,
Sizova 1998, reviewed in Hellen 1999). Subsequently, the 60S ribosomal subunit
associates with that complex leading to the formation of the translational active
complex for HCV polyprotein synthesis at the endoplasmic reticulum. HCV RNA
contains a large ORF encoding a polyprotein precursor. Posttranslational cleavages
lead to 10 functional viral proteins Core, E1, E2, p7, NS2-NS5B. The viral F protein
(or ARF protein) originates from a ribosomal frameshift within the first codons of
the core-encoding genome region (Walewski 2001, Xu 2001, Varaklioti 2002).
Besides several other cellular factors that have been reported to be involved in HCV
RNA translation, various viral proteins and genome regions have been shown to
enhance or inhibit viral protein synthesis (Zhang 2002, Kato 2002, Wang 2005, Kou
2006, Bradrick 2006, Song 2006).
The precursor polyprotein is processed by at least four distinct peptidases. The
cellular signal peptidase (SP) cleaves the N-terminal viral proteins immature core
protein, E1, E2, and p7 (Hijikata 1991), while the cellular signal peptide peptidase
(SPP) is responsible for the cleavage of the E1 signal sequence from the C-terminus
of the immature core protein, resulting in the mature form of the core (McLauchlan
2002). The E1 and E2 proteins remain within the lumen of the ER where they are
subsequently N-glycosylated with E1 having 5 and E2 harbouring 11 putative Nglycosylation sites (Duvet 2002).
In addition to the two cellular peptidases HCV encodes two viral enzymes
responsible for cleavage of the non-structural proteins NS2 to NS5B within the
HCV polyprotein precursor. The zinc-dependent NS2-NS3 cysteine protease
consisting of the NS2 protein and the N-terminal portion of NS3 autocatalytically
cleaves the junction between NS2 and NS3 (Santolini 1995), whereas the NS3
serine protease cleaves the remaining functional proteins (Bartenschlager 1993,
Eckart 1993, Grakoui 1993a, Tomei 1993). However, for its peptidase activity NS3

96 Hepatology 2012
needs NS4A as a cofactor (Failla 1994, Tanji 1995, Bartenschlager 1995, Lin 1995,
Tomei 1996).

HCV RNA replication
The complex process of HCV RNA replication is poorly understood. The key
enzyme for viral RNA replication is NS5B, an RNA-dependent RNA polymerase
(RdRp) of HCV (Behrens 1996). In addition, several cellular as well as viral factors
have been reported to be part of the HCV RNA replication complex. One important
viral factor for the formation of the replication complex appears to be NS4B, which
is able to induce an ER-derived membranous web containing most of the nonstructural HCV proteins including NS5B (Egger 2002). This web could serve as the
platform for the next steps of viral RNA replication. The RdRp uses the previously
released genomic positive-stranded HCV RNA as a template for the synthesis of an
intermediate minus-stranded RNA. Recently it has been reported that the cellular
peptidyl-prolyl isomerases cyclophilin A, B and C (Cyp A, Cyp B, and Cyp C)
could stimulate binding of the RdRp to the viral RNA resulting in increased HCV
RNA synthesis (Watashi 2005, Nakagawa 2005, Yang 2008, Heck 2009). However,
these reports are in part inconsistent and further studies are needed in order to
investigate the involvement of cyclophilins in HCV RNA replication.
After the viral polymerase has bound to its template, the NS3 helicase is assumed
to unwind putative secondary structures of the template RNA in order to facilitate
the synthesis of minus-strand RNA (Jin 1995, Kim 1995). In turn, again with the
assistance of the NS3 helicase, the newly synthesized antisense RNA molecule
serves as the template for the synthesis of numerous plus-stranded RNA. The
resulting sense RNA may be used subsequently as genomic RNA for HCV progeny
as well as for polyprotein translation.

Assembly and release
After the viral proteins, glycoproteins, and the genomic HCV RNA have been
synthesized these single components have to be arranged in order to produce
infectious virions. As is the case for all other steps in the HCV lifecycle viral
assembly is a multi-step procedure involving most viral components along with
many cellular factors. Investigation of viral assembly and particle release is still in
its infancy since the development of in vitro models for the production and release
of infectious HCV occurred only recently. Previously, it was reported that core
protein molecules were able to self-assemble in vitro, yielding nucleocapsid-like
particles. More recent findings suggest that viral assembly takes place within the
endoplasmic reticulum (Gastaminza 2008) and that lipid droplets (LD) are involved
in particle formation (Moradpour 1996, Barba 1997, Miyanari 2007, Shavinskaya
2007, Appel 2008). It appears that LD-associated core protein targets viral nonstructural proteins and the HCV RNA replication complex including positive- and
negative-stranded RNA from the endoplasmic reticulum to the LD (Miyanari 2007).
Beside the core protein, LD-associated NS5A seems to play a key role in the
formation of infectious viral particles (Appel 2008). Moreover, E2 molecules are
detected in close proximity to LD-associated membranes. Finally, spherical viruslike particles associated with membranes can be seen very close to the LD. Using

HCV Virology 97
specific antibodies the virus-like particles were shown to contain core protein as
well as E2 glycoprotein molecules indicating that these structures may represent
infectious HCV (Miyanari 2007). However, the precise mechanisms for the
formation and release of infectious HCV particles are still unknown.

Model systems for HCV research
For a long time HCV research was limited due to a lack of small animal models and
efficient cell culture systems. The development of the first HCV replicon system
(HCV RNA molecule, or region of HCV RNA, that replicates autonomously from a
single origin of replication) 10 years after the identification of the hepatitis C virus
offered the opportunity to investigate the molecular biology of HCV infection in a
standardized manner (Lohmann 1999).
HCV replicon systems. Using total RNA derived from the explanted liver of an
individual chronically infected with HCV genotype 1b, the entire HCV ORF
sequence was amplified and cloned in two overlapping fragments. The flanking
NTRs were amplified and cloned separately and all fragments were assembled into
a modified full-length sequence. Transfection experiments with in vitro transcripts
derived from the full-length clones failed to yield viral replication. For this reason,
two different subgenomic replicons consisting of the 5’IRES, the neomycin
phosphotransferase gene causing resistance to the antibiotic neomycin, the IRES
derived from the encephalomyocarditis virus (EMCV) and the NS2-3’NTR or NS33’NTR sequence, respectively, were generated (Figure 3).

Figure 3. Structure of subgenomic HCV replicons (Lohmann 1999). This figure illustrates
the genetic information of in vitro transcripts used for Huh7 transfection. A) Full-length transcript
derived from the explanted liver of a chronically infected subject. B) Subgenomic replicon
lacking the structural genes and the sequence encoding p7. C) Subgenomic replicon lacking C,
E1, E2, p7, and NS2 genes. neo, neomycin phosphotransferase gene; E-I, IRES of the
encephalomyocarditis virus (EMCV).

98 Hepatology 2012
In vitro transcripts derived from these constructs lacking the genome region
coding for the structural HCV proteins were used to transfect the hepatoma cell line
Huh7 (Lohmann 1999). The transcripts are bicistronic, i.e., the first cistron
containing the HCV IRES enables the translation of the neomycin
phosphotransferase as a tool for efficient selection of successfully transfected cells
and the second cistron containing the EMCV IRES directs translation of the HCVspecific proteins. Only some Huh7 clones can replicate replicon-specific RNA in
titres of approximately 108 positive-stranded RNA copies per microgram total RNA.
Moreover, all encoded HCV proteins are detected predominantly in the cytoplasm
of the transfected Huh7 cells. The development of this replicon is a milestone in
HCV research with regard to the investigation of HCV RNA replication and HCV
protein analyses.
More recently, the methodology has been improved in order to achieve
significantly higher replication efficiency. Enhancement of HCV RNA replication
was achieved by the use of replicons harbouring cell culture-adapted point
mutations or deletions within the NS genes (Blight 2000, Lohmann 2001, Krieger
2001). Further development has led to the generation of selectable full-length HCV
replicons, i.e., genomic replicons that also contain genetic information for the
structural proteins Core, E1, and E2 (Pietschmann 2002, Blight 2002). This
improvement offered the opportunity to investigate the influence of the structural
proteins on HCV replication. Thus it has been possible to analyse the intracellular
localisation of these proteins althoughviral assembly and release has not been
achieved.
Another important milestone was reached when a subgenomic replicon based on
the HCV genotype 2a strain JFH-1 was generated (Kato 2003). This viral strain
derived from a Japanese subject with fulminant hepatitis C (Kato 2001). The
corresponding replicons showed higher RNA replication efficiency than previous
replicons. Moreover, cell lines distinct from Huh7, such as HepG2 or HeLa were
transfected efficiently with transcripts derived from the JFH-1 replicon (Date 2004,
Kato 2005).
HCV pseudotype virus particles (HCVpp). The generation of retroviral
pseudotypes bearing HCV E1 and E2 glycoproteins (HCVpp) offers the opportunity
to investigate E1-E2-dependent HCVpp entry into Huh7 cells and primary human
hepatocytes (Bartosch 2003a, Hsu 2003, Zhang 2004). In contrast to the HCV
replicons where cells were transfected with HCV-specific synthetic RNA
molecules, this method allows a detailed analysis of the early steps in the HCV life
cycle, e.g., adsorption and viral entry.
Infectious HCV particles in cell culture (HCVcc). Transfection of Huh7 and
‘cured’ Huh7.5 cells with full-length JFH-1 replicons led for the first time to the
production of infectious HCV virions (Zhong 2005, Wakita 2005). The construction
of a chimera with the core NS2 region derived from HCV strain J6 (genotype 2a)
and the remaining sequence derived from JFH-1 improved infectivity. Importantly,
the secreted viral particles are infectious in cell culture (HCVcc) (Wakita 2005,
Zhong 2005, Lindenbach 2005) as well as in chimeric mice with human liver grafts
as well as in chimpanzees (Lindenbach 2006).
An alternative strategy for the production of infectious HCV particles was
developed (Heller 2005): a full-length HCV construct (genotype 1b) was placed
between two ribozymes in a plasmid containing a tetracycline-responsive promoter.

HCV Virology 99
Huh7 cells were transfected with those plasmids, resulting in efficient viral
replication with HCV RNA titres of up to 107 copies/ml cell culture supernatant.
The development of cell culture systems that allow the production of infectious
HCV represents a breakthrough for HCV research and it is now possible to
investigate the whole viral life cycle from viral adsorption to virion release. These
studies will help to better understand the mechanisms of HCV pathogenesis and
they should significantly accelerate the development of HCV-specific antiviral
compounds.
Small animal models. Very recently, substantial progress was achieved in
establishing two mouse models for HCV infection viagenetically humanized mice
(Dorner 2011). In this experiment immunocompetent mice were transduced using
viral vectors containing the genetic information of four human proteins involved in
adsorption and entry of HCV into hepatocytes (CD81, SR-BI, CLDN1, OCLN).
This humanisation procedure enabled the authors to infect the transduced mice with
HCV. Although this mouse model does not enable complete HCV replication in
murine hepatocytes it will be useful to investigate the early steps of HCV infection
in vivo. Moreover, the approach should be suitable for the evaluation of HCV entry
inhibitors and vaccine candidates.
A second group of investigators have chosen another promising strategy for
HCV-specific humanisation of mice. After depleting murine hepatocytes human
CD34(+) hematopoietic stem cells and hepatocyte progenitors were cotransplantated
into transgenic mice leading to efficient engraftment of human leukocytes and
hepatocytes, respectively (Washburn 2011). A portion of the humanised mice
became infectable with primary HCV isolates resulting in low-level HCV RNA in
the murine liver. As a consequence HCV infection induced liver inflammation,
hepatitis, and fibrosis. Furthermore, due to the cotransplantation of CD34(+) human
hematopoietic stem cells, an HCV-specific T cell immune response could be
detected.
Both strategies are promising and have already delivered new insights into viral
replication and the pathogenesis of HCV. However, the methods lack some
important aspects and need to be improved. As soon as genetically humanised mice
that are able to replicate HCV completely are created they can be used for the
investigation of HCV pathogenesis and HCV-specific immune responses. The
Washburn method should be improved in order to achieve higher HCV replication
rates. Moreover, reconstitution of functional human B cells would make this mouse
model suitable to study the important HCV-specific antibody response.

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108 Hepatology 2012

7. Prophylaxis and Vaccination
Heiner Wedemeyer

Introduction
Understanding the biology and modes of transmission of hepatitis viruses has
significantly improved over the last decades. Still, prophylactic vaccines are only
available against HAV and HBV. Although an enormous amount of basic and
clinical research has been performed to develop a vaccine against hepatitis C, it is
very unlikely that a prophylactic or therapeutic HCV vaccine will be licensed in the
next few years. A first Phase III vaccine trial against hepatitis E has been successful
in China; nevertheless, it is currently unknown if or when this vaccine will become
available in other countries. Prophylaxis of HCV, HDV (for HBV-infected patients)
and HEV infection therefore must still occur by avoiding all routes of exposure to
the respective hepatitis viruses discussed in detail in Chapters 1-4.

Prophylaxis of hepatitis viruses
Hepatitis A and E
The hepatitis A and E viruses are usually transmitted by oral ingestion of
contaminated food or water. Thus, particular caution is warranted when individuals
from low endemic areas such as western Europe and the US travel to countries with
a high prevalence of HAV and HEV infections. In addition, hepatitis E can also be a
zoonosis. A German case-control study identified 32% of all reported HEV
infections as being autochthonous infections, meaning not associated with travelling
to endemic countries (Wichmann 2008). In these patients consumption of offal and
wild boar meat was independently associated with HEV infection. This may have
significant implications for immunosuppressed patients as cases of chronic hepatitis
E with the development of advanced fibrosis have been described in patients after
organ transplantation (Kamar 2008, Pischke 2010). HEV has frequently been
detected in the meat of pigs; Danish farmers show a higher prevalence of HEV
antibodies. Importantly, zoonotic HEV infection is usually caused by HEV
genotype 3 while HEV genotype 1 can be found in travelling-associated hepatitis E.

Prophylaxis and Vaccination 109
HAV and HEV can also be transmitted by blood transfusion although cases are
extremely rare.

Hepatitis B and D
HBV and HDV were transmitted frequently by blood transfusion before HBsAg
testing of all blood products was introduced in the 1970s. Since then, vertical
transmission and sexual exposure have become the most frequent routes of HBV
infection. Medical procedures still represent a potential source for HBV
transmissions and thus strict and careful application of standard hygienic
precautions for all medical interventions are absolutely mandatory, and not only in
endemic areas. This holds true in particular for immunocompromised individuals
who are highly susceptible to HBV infection as HBV is characterized by a very high
infectivity (Wedemeyer 1998). Moreover, immunosuppressed patients are at risk for
reactivation of occult HBV infection after serological recovery from hepatitis B.
Treatments with high doses of steroids and rituximab have especially been
identified as major risk factors for HBV reactivation (Lalazar 2007, Loomba 2008).
After a new diagnosis of HBV infection, all family members of the patient need to
be tested for their immune status against HBV. Immediate active vaccination is
recommended for all anti-HBc-negative contact persons. HBsAg-positive
individuals should use condoms during sexual intercourse if it is not known if the
partner has been vaccinated. Non-immune individuals who have experienced an
injury and were exposed to HBsAg-positive fluids should undergo passive
immunization with anti-HBs as soon as possible, preferentially within 2-12 hours
(Cornberg 2011).

Hepatitis C
Less than 1% of individuals who are exposed to HCV by an injury with
contaminated needles develop acute HCV infection. At Hannover Medical School,
not a single HCV seroconversion occurred after 166 occupational exposures with
anti-HCV positive blood in a period of 6 years (2000-2005). A systematic review of
the literature identified 22 studies including a total of 6,956 injuries with HCV
contaminated needles. Only 52 individuals (0.75%) became infected. The risk of
acute HCV infection was lower in Europe at 0.42% compared to eastern Asia at
1.5% (Kubitschke 2007). Thus, the risk of acquiring HCV infection after a needlestick injury is lower than frequently reported. Worldwide differences in HCV seroconversion rates may suggest that genetic factors provide some level of natural
resistance against HCV. Factors associated with a higher risk of HCV transmission
are likely to be the level of HCV viremia in the index patient, the amount of
transmitted fluid and the duration between contamination of the respective needle
and injury. Suggested follow-up procedures after needlestick injury are shown in
Figure 1.
Sexual intercourse with HCV-infected persons has clearly been identified as a risk
for HCV infection, as about 10-20% of patients with acute hepatitis C report this as
a potential risk factor (Deterding 2009; Table 1). However, there is also evidence
that the risk of acquiring HCV sexually is extremely low in individuals in stable
partnerships who avoid injuries. Cohort studies including more than 500 HCVinfected patients followed over periods of more than 4 years could not identify any
cases of confirmed HCV transmission. Thus, guidelines generally do not

110 Hepatology 2012
recommend the use of condoms in monogamous relationships (EASL 2011).
However, this does not hold true for HIV-positive homosexual men. Several
outbreaks of acute hepatitis C have been described in this scenario (Fox 2008, Low
2008, van de Laar 2009). Transmitted cases had more sexual partners, increased
levels of high-risk sexual behaviour (in particular, fisting) and were more likely to
have shared drugs via a nasal or anal route than controls (Turner 2006).
Due to the low HCV prevalence in most European countries and due to a
relatively low vertical transmission rate of 1-6%, general screening of pregnant
women for anti-HCV is not recommended. Interestingly, transmission may be
higher for girls than for boys (European Pediatric Hepatitis C Virus Network 2005).
Transmission rates may be higher in HIV-infected women so pregnant women
should be tested for hepatitis C. Other factors possibly associated with high
transmission rates are the level of HCV viremia, maternal intravenous drug use, and
specific HLA types of the children. Cesarean sections are not recommended for
HCV RNA positive mothers as there is no clear evidence that these reduce
transmission rates. Children of HCV-infected mothers should be tested for HCV
RNA after 1 month as maternal anti-HCV antibodies can be detected for several
months after birth. Mothers with chronic hepatitis C can breast-feed their children as
long as they are HIV-negative and do not use intravenous drugs (European Pediatric
Hepatitis C Virus Network 2001, EASL 2011).
The Spanish Acute HCV Study Group has identified hospital admission as a
significant risk factor for acquiring HCV infection in Spain (Martinez-Bauer 2008).
The data are in line with reports from Italy (Santantonio 2006) and the USA (Corey
2006). We have reported data from the German Hep-Net Acute HCV Studies and
found 38 cases (15% of the entire cohort) of acute HCV patients who reported a
medical procedure as the most likely risk factor for having acquired HCV
(Deterding 2008). The majority of those were hospital admissions with surgery in
30 cases; other invasive procedures, including dental treatment, were present in only
4 cases. Medical procedures were significantly more often the probable cause of
infection in patients older than 30 years of age (p=0.002) but not associated with
disease severity or time from exposure to onset of symptoms. Thus, medical
treatment per se still represents a significant risk factor for HCV infection – even in
developed countries. Strict adherence to universal precaution guidelines is urgently
warranted.

Vaccination against hepatitis A
The first active vaccine against HAV was licensed in 1995. The currently available
inactive vaccines are manufactured from cell culture-adapted HAV, grown either in
human fibroblasts or diploid cells (Nothdurft 2008). Two doses of the vaccine are
recommended. The second dose should be given between 6 and 18 months after the
first dose. All vaccines are highly immunogenic and basically all vaccinated healthy
persons develop protective anti-HAV antibodies. Similar vaccine responses are
obtained in both children and adults and no relevant regional differences in response
to HAV vaccination have been observed. The weakest vaccine responses have been
described for young children receiving a 0, 1, 2 months schedule (Hammitt 2008).
Patients with chronic liver disease do respond to vaccination but may display lower
anti-HAV titers (Keeffe 1998). A combined vaccine against HAV and HBV is

Prophylaxis and Vaccination 111
available that needs to be administered three times, on a 0, 1, and 6 months
schedule. More than 80% of healthy individuals have detectable HAV antibodies by
day 21 applying an accelerated vaccine schedule of 0, 7 and 21 days using the
combined HAV/HBV vaccine, and all study subjects were immune against HAV by
2 months (Kallinowski 2003).
HAV vaccines are very well tolerated and no serious adverse events have been
linked with the administration of HAV vaccines (Nothdurft 2008). The vaccine can
safely be given together with other vaccines or immunoglobulins without
compromising the development of protective antibodies.
Vaccination is recommended for non-immune individuals who plan to travel to
endemic countries, medical health professionals, homosexual men, persons in
contact with hepatitis A patients, and individuals with chronic liver diseases. Some
studies have suggested that patients with chronic hepatitis C have a higher risk of
developing fulminant hepatitis A (Vento 1998) although this finding has not been
confirmed by other investigators (Deterding 2006). The implementation of
childhood vaccination programs has led to significant and impressive declines of
HAV infections in several countries, justifying further efforts aiming at controlling
the spread of HAV in endemic countries (Hendrickx 2008). It is important to
highlight that most studies have confirmed that HAV vaccination is cost-effective
(Rein 2008, Hollinger 2007).
Long-term follow-up studies after complete HAV vaccination have been
published. Interestingly, anti-HAV titers sharply decline during the first year after
vaccination but remain detectable in almost all individuals for at least 10-15 years
after vaccination (Van Herck 2011). Based on these studies it was estimated that
protective anti-HAV antibodies should persist for at least 27-30 years after
successful vaccination (Hammitt 2008, Bovier 2010).

Vaccination against hepatitis B
The hepatitis B vaccine is the first vaccine able to reduce the incidence of cancer. In
Taiwan, a significant decline in cases of childhood hepatocellular carcinoma has
been observed since the implementation of programs to vaccinate all infants against
HBV (Chang 1997). This landmark study impressively highlighted the usefulness of
universal vaccination against HBV in endemic countries. Controversial discussions
are ongoing regarding to what extent universal vaccination against HBV may be
cost-effective in low-endemic places such as the UK, the Netherlands or
Scandinavia (Zuckerman 2007). In 1992 the World Health Organization
recommended general vaccination against hepatitis B. It should be possible to
eradicate hepatitis B by worldwide implementation of this recommendation,
because humans are the only epidemiologically relevant host for HBV. 179
countries have introduced a hepatitis B vaccine in their national infant immunization
schedules by the end of 2010, including parts of India and the Sudan (WHO 2011).
The first plasma-derived hepatitis B vaccine was approved by FDA in 1981.
Recombinant vaccines consisting of HBsAg produced in yeast became available in
1986. In the US, two recombinant vaccines are licensed (Recombivax® and
Engerix-B®) while additional vaccines are used in other countries. The vaccines are
administered three times, on a 0, 1, and 6 months schedule.

112 Hepatology 2012
Who should be vaccinated? (The German Guidelines (Cornberg 2011))
− Hepatitis B high-risk persons working in health care settings including trainees,
students, cleaning personnel;
− Personnel in psychiatric facilities or comparable welfare institutions for
cerebrally damaged or disturbed patients; other persons who are at risk because
of blood contact with possibly infected persons dependent on the risk
evaluation, e.g., persons giving first aid professionally or voluntarily,
employees of ambulance services, police officers, social workers, and prison
staff who have contact with drug addicts;
− Patients with chronic kidney disease, dialysis patients, patients with frequent
blood or blood component transfusions (e.g., hemophiliacs), patients prior to
extensive surgery (e.g., before operations using heart-lung machine. The
urgency of the operation and the patient’s wish for vaccination protection are
of primary importance);
− Persons with chronic liver disease including chronic diseases with liver
involvement as well as HIV-positive persons without HBV markers;
− Persons at risk of contact with HBsAg carriers in the family or shared housing,
sexual partners of HBsAg carriers;
− Patients in psychiatric facilities or residents of comparable welfare institutions
for cerebrally damaged or disturbed persons as well as persons in sheltered
workshops;
− Special high-risk groups, e.g., homosexually active men, regular drug users,
sex workers, prisoners serving extended sentences;
− Persons at risk of contacting HBsAg carriers in facilities (kindergarten,
children’s homes, nursing homes, school classes, day care groups);
− Persons travelling to regions with high hepatitis B prevalence for an extended
period of time or with expected close contact with the local population;
− Persons who have been injured by possibly contaminated items, e.g., needle
puncture (see post-exposition prophylaxis);
− Infants of HBsAg-positive mothers or of mothers with unknown HBsAg status
(independent of weight at birth) (see post-exposition prophylaxis).
Routine testing for previous contact with hepatitis B is not necessary before
vaccination unless the person belongs to a risk group and may have acquired
hepatitis B before. Pre-vaccine testing is usually not cost-effective in populations
with anti-HBc prevalence below 20%. Vaccination of an HBsAg-positive individual
can be performed without any danger – however, it is ineffective.

Efficacy of vaccination against hepatitis B
A response to HBV vaccination is determined by the development of anti-HBs
antibodies, detectable in 90-95% of individuals one month after a complete
vaccination schedule (Wedemeyer 2007, Coates 2001). Responses are lower in
elderly people and much weaker in immunocompromised persons such as organ
transplant recipients, patients receiving hemodialysis and HIV-infected individuals.
In case of vaccine non-response, another three courses of vaccine should be
administered and the dose of the vaccine should be increased. Other possibilities to

Prophylaxis and Vaccination 113
increase the immunogenicity of HBV vaccines include intradermal application and
coadministration of adjuvants and cytokines (Cornberg 2011). The response to
vaccination should be controlled in high-risk individuals such as medical health
professionals and immunocompromised persons. Some guidelines also recommend
testing elderly persons after vaccinations as vaccine response does decline more
rapidly in the elderly (Wolters 2003).

Post-exposure prophylaxis
Non-immune persons who have been in contact with HBV-contaminated materials
(e.g., needles) or who have had sexual intercourse with an HBV-infected person
should undergo active-passive immunization (active immunization plus hepatitis B
immunoglobulin) as soon as possible – preferentially within the first 48 hours of
exposure to HBV. Individuals previously vaccinated but who have an anti-HBs titer
of <10 IU/L should also be vaccinated both actively and passively. No action is
required if an anti-HBs titer of >100 IU/l is documented; active vaccination alone is
sufficient for persons with intermediate anti-HBs titers between 10 and 100 IU/L
(Cornberg 2011).

Safety of HBV vaccines
Several hundred million individuals have been vaccinated against hepatitis B. The
vaccine is very well tolerated. Injection site reactions in the first 1-3 days and mild
general reactions are common, although they are usually not long lasting. Whether
there is a causal relationship between the vaccination and the seldomly observed
neurological disorders occurring around the time of vaccination is not clear. In the
majority of these case reports the concomitant events most likely occurred
coincidentally and are independent and not causally related. That hepatitis B
vaccination causes and induces acute episodes of multiple sclerosis or other
demyelating diseases are repeatedly discussed (Geier 2001, Hernan 2004, Girard
2005). However, there are no scientific facts proving such a relationship. Numerous
studies have not been able to find a causal relationship between the postulated
disease and the vaccination (Sadovnick 2000, Monteyne 2000, Ascherio 2001,
Confavreux 2001, Schattner 2005).

Long-term immunogenicity of hepatitis B vaccination
Several studies have been published in recent years investigating the long-term
efficacy of HBV vaccination. After 10-15 years, between one third and two thirds of
vaccinated individuals have completely lost anti-HBs antibodies and only a minority
maintain titers of >100 IU/L. However, in low/intermediate endemic countries such
as Italy, this loss in protective humoral immunity did not lead to many cases of
acute or even chronic HBV infection (Zanetti 2005). To what extent memory B and
T cell responses contribute to a relative protection against HBV in the absence of
anti-HBs remains to be determined. Nevertheless, in high-endemic countries such as
Gambia a significant proportion of infants develop anti-HBc indicating active HBV
infection (18%) and some children develop chronic hepatitis B (van der Sande
2007). Thus, persons at risk should receive booster immunization if HBs antibodies
have been lost.

114 Hepatology 2012

Prevention of vertical HBV transmission
Infants of HBsAg-positive mothers should be immunized actively and passively
within 12 hours of birth. This is very important as the vertical HBV transmission
rate can be reduced from 95% to <5% (Ranger-Rogez 2004). Mothers with high
HBV viremia, of >1 million IU/ml, should receive in addition antiviral therapy with
a potent HBV polymerase inhibitor (European Association For The Study Of The
Liver 2009, Peterson 2011, Han 2011). Tenofovir and telbivudine have been
classified as Category B drugs by the FDA and can therefore be given during
pregnancy as no increased rates of birth defects have been reported. If active/passive
immunization has been performed, there is no need to recommend cesarean section.
Mothers of vaccinated infants can breastfeed unless antiviral medications are being
taken by the mother, which can pass through breast milk.

Vaccination against hepatitis C
No prophylactic or therapeutic vaccine against hepatitis C is available. As reinfections after spontaneous or treatment-induced recovery from hepatitis C virus
infection have frequently been reported, the aim of a prophylactic vaccine will very
likely be not to prevent completely an infection with HCV but rather to modulate
immune responses in such a way that the frequency of evolution to a chronic state
can be reduced (Torresi 2011).
HCV specific T cell responses play an important role in the natural course of
HCV infection. The adaptive T cell response is mediated both by CD4+ helper T
cells and CD8+ killer T cells. Several groups have consistently found an association
between a strong, multispecific and maintained HCV-specific CD4+ and CD8+ T
cell response and the resolution of acute HCV infection. While CD4+ T cells seem
to be present for several years after recovery, there are conflicting data whether
HCV-specific CD8+ T cells responses persist or decline over time (Wiegand 2007).
However, several studies have observed durable HCV-specific T cells in HCVseronegative individuals who were exposed to HCV by occupational exposure or as
household members of HCV-positive partners, but who never became HCV RNA
positive. These observations suggest that HCV-specific T cells may be induced
upon subclinical exposure and may contribute to protection against clinically
apparent HCV infection. T cell responses are usually much weaker in chronic
hepatitis C. The frequency of specific cells is low but also effector function of
HCV-specific T cells is impaired. Different mechanisms are discussed as being
responsible for this impaired T cell function, including higher frequencies of
regulatory T cells (Tregs), altered dendritic cell activity, upregulation of inhibitory
molecules such as PD-1, CTL-A4 or 2B4 on T cells and escape mutations. HCV
proteins can directly or indirectly contribute to altered functions of different
immune cells (Rehermann 2009).
To what extent humoral immune responses against HCV contribute to
spontaneous clearance of acute hepatitis C is less clear. Higher levels of neutralizing
antibodies early during the infection are associated with viral clearance (Pestka
2007). Antibodies with neutralizing properties occur at high levels during chronic
infection, although HCV constantly escapes these neutralizing antibodies (von Hahn
2007). Yet, no completely sterilizing humoural anti-HCV immunity exists in the
long-term after recovery (Rehermann 2009). Attempts to use neutralizing antibodies

Prophylaxis and Vaccination 115
to prevent HCV re-infection after liver transplant have not been successful (Gordon
2011).
Few Phase I vaccine studies based either on vaccination with HCV peptides, HCV
proteins alone or in combination with distinct adjuvants or recombinant viral vectors
expressing HCV proteins have been completed (Torresi 2011). HCV-specific T cells
or antibodies against HCV were induced by these vaccines in healthy individuals.
Studies in chimpanzees have shown that it is very unlikely that a vaccine will be
completely protective against heterologous HCV infections. However, a reasonable
approach might be the development of a vaccine that does not confer 100%
protection against acute infection but prevents progression of acute hepatitis C to
chronic infection. In any case, there are no vaccine programs that have reached
Phase III yet (Halliday 2011). Therapeutic vaccination against hepatitis C has also
been explored (Klade 2008, Wedemeyer 2009, Torresi 2011). These studies show
that induction of HCV-specific humoural or cellular immune responses is possible
even in chronically infected individuals. The first studies showed a modest antiviral
efficacy of HCV vaccination in some patients (Sallberg 2009, Habersetzer 2011,
Wedemeyer 2011). Therapeutic vaccination was also able to enhance responses to
interferon α and ribavirin treatment (Pockros 2010, Wedemeyer 2011). Future
studies will need to explore the potential role of HCV vaccines in combination with
direct acting antivirals against hepatitis C.

Vaccination against hepatitis E
A Phase II vaccine trial performed in Nepal with 200 soldiers showed a vaccine
efficacy of 95% for an HEV recombinant protein (Shrestha 2007). However, the
development of this vaccine has been stopped. Since then, in September 2010, data
from a very large Phase III trial were reported involving about 110,000 individuals
in China (Zhu 2010). The vaccine efficacy of HEV 239 was 100% after three doses
to prevent cases of symptomatic acute hepatitis E. However, it is currently unknown
if this HEV genotype 1 vaccine also prevents against zoonotic HEV genotype 3
infections. Moreover, vaccine efficacy in special risk groups such patients with endstage liver disease, immunocompromised individuals or elderly persons is unknown.
Finally, the duration of protection needs to be determined (Wedemeyer 2011). It is
currently unknown if and when the Chinese vaccine HEV-239 will become
available in other countries. Until then, preventive hygienic measures remain the
only option to avoid HEV infection.

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Hepatitis B: Diagnostic Tests 119

8. Hepatitis B: Diagnostic Tests
Jörg Petersen

Introduction
The diagnosis of hepatitis B virus (HBV) infection was initiated by the discovery of
Australia antigen (hepatitis B surface antigen, HBsAg). During the following
decades, serologic assays were established for HBsAg and other HBV antigens and
antibodies. Advances in molecular biology techniques led to the development of
polymerase chain reaction (PCR) assays for direct determination of hepatitis B virus
DNA (HBV DNA).
Diagnosis of Hepatitis B Virus (HBV) infection tests for a series of serological
markers of HBV and excludes alternative etiological agents such as hepatitis A, C,
and D viruses. Serological tests are used to distinguish acute, self-limited infections
from chronic HBV infections and to monitor vaccine-induced immunity. These tests
are also performed to determine if the patient should be considered for antiviral
therapy. Nucleic acid testing for HBV DNA is used as the standard to quantify HBV
viral load and measure the effectiveness of therapeutic agents.
Other causes of chronic liver disease should be systematically looked for
including coinfection with HCV, HDV or HIV. Cytomegalovirus, Epstein-Barr
virus, enteroviruses, other hepatotoxic drugs, and even herbal medicines should be
considered when appropriate. Moreover, co-morbidities, including alcoholic,
autoimmune, metabolic liver disease with steatosis or steatohepatitis should be
assessed. Finally, vaccination status and previous tests results should be used to
guide appropriate testing.

Serological tests for HBV
Collection and transport
Serological tests for viral antigens can be performed on either serum or plasma
(Yang 2002). The World Health Organization (WHO) has defined an international
standard for normalisation of expression of HBV DNA concentrations (Quint 1990).
Serum HBV DNA levels should be expressed in IU/ml to ensure comparability; the
same assay should be used in the same patient to evaluate antiviral efficacy. Both
HBV antigens and antibody are stable at room temperature for days, at 4°C for
months, and frozen at -20°C to -70°C for many years. Because current testing

120 Hepatology 2012
involves automated enzyme immunoassays that depend on colourimetric or
chemiluminescence signal measurement, care should be taken to avoid hemolysis of
the sample because it may interfere with the ability of the assay to accurately detect
these markers. Care must be taken to avoid the degradation of the viral nucleic acid
in the specimen, which can result in falsely low or no measurable viral load. Serum
should therefore be removed from clotted blood within 4 hrs of collection and stored
at -20°C to -70°C (Krayden 1998). Alternatively, the presence of EDTA in plasma
is known to stabilize viral nucleic acids. EDTA blood can be stored for up to five
days at 4°C without affecting the viral load. Polymerase chain reaction-based tests
that are routinely used as standard can use either serum or plasma. The diagnosis of
HBV infection can also be made by the detection of HBsAg or hepatitis B core
antigen (HBcAg) in liver tissues by immunohistochemical staining and of HBV
DNA by Southern hybridization, in situ hybridisation, or PCR.

Hepatitis B surface antigen and antibody
Hepatitis B surface antigen (HBsAg) is the serologic hallmark of HBV infection. It
can be detected by radioimmunoassays (RIA) or enzyme immunoassays (EIA).
HBsAg appears in serum 1 to 10 weeks after acute exposure to HBV, prior to the
onset of hepatitis and elevation of serum alanine aminotransferase. HBsAg usually
becomes undetectable after four to six months in patients who recover from
hepatitis B. Persistence of HBsAg for more than six months implies chronic
infection. It is estimated that about 5 percent of immunocompetent adult patients
with genuine acute hepatitis B progress to chronic infection (Chu 1989). Among
patients with chronic HBV infection, the rate of clearance of HBsAg is
approximately 0.5 to 1 percent per year (Liaw 1991). The disappearance of HBsAg
is followed by the appearance of hepatitis B surface antibody (anti-HBs). In most
patients, anti-HBs persists for life, thereby conferring long-term immunity.
Coexistence of HBsAg and anti-HBs has been reported in HBsAg positive
individuals (Tsang 1986, Dufour 2000). In most instances, the antibodies are unable
to neutralize the circulating virions. These individuals should therefore be regarded
as carriers of the hepatitis B virus.
In recent years the quantification of HBsAg levels (qHBsAg) has been used to
determine threshold levels to distinguish between patients with active hepatitis B
and inactive carrier state (Brunetto 2010). Furthermore, a continuous decline of
qHBsAg during IFN α therapy has been used as a response marker of therapy
(Marcellin 2009). In contrast, in patients with nucleos(t)ide therapy the
measurement of qHBsAg levels over time have not yielded definite answers yet in
helping to distinguish patients that will clinically resolve chronic hepatitis B
infection with HBsAg loss or seroconversion.

Hepatitis B core antigen and antibody
Hepatitis B core antigen (HBcAg) is an intracellular antigen that is expressed in
infected hepatocytes. It is not detectable in serum. Anti-HBc can be detected
throughout the course of HBV infection in the serum.
During acute infection, anti-HBc is predominantly of IgM class. IgM anti-HBc is
the important marker of HBV infection during the window period between the
disappearance of HBsAg and the appearance of anti-HBs. IgM anti-HBc may
remain detectable up to two years after acute infection. Furthermore, the titer of IgM

Hepatitis B: Diagnostic Tests 121
anti-HBc may increase to detectable levels during exacerbations of chronic hepatitis
B (Maruyama 1994). This can present a diagnostic problem, incorrectly suggesting
acute hepatitis B. Other common causes of acute exacerbation of chronic hepatitis B
are superinfection with hepatitis D virus (delta virus) or hepatitis C virus. IgG antiHBc persists along with anti-HBs in patients who recover from acute hepatitis B. It
also persists in association with HBsAg in those who progress to chronic HBV
infection.
Isolated detection of anti-HBc can occur in three settings: during the window
period of acute hepatitis B when the anti-HBc is predominantly IgM; many years
after recovery from acute hepatitis B when anti-HBs has fallen to undetectable
levels; and after many years of chronic HBV infection when the HBsAg titer has
decreased to below the level of detection. HBV DNA can be detected in the liver of
most persons with isolated anti-HBc. Transmission of HBV infection has been
reported from blood and organ donors with isolated anti-HBc. There are, in a small
percentage of cases, false-positive isolated anti-HBc test results.
The evaluation of individuals with isolated anti-HBc should include repeat testing
for anti-HBc, HBsAg, anti-HBe, and anti-HBs. Those who remain isolated anti-HBc
positive should be tested for the presence of IgM anti-HBc to rule out recent HBV
infection. Individuals with evidence of chronic liver disease should be tested for
HBV DNA to exclude low-level chronic HBV infection.

Hepatitis B e antigen and antibody
Hepatitis B e antigen (HBeAg) is a secretory protein processed from the precore
protein. It is generally considered to be a marker of HBV replication and infectivity.
The presence of HBeAg is usually associated with high levels of HBV DNA in
serum and higher rates of transmission of HBV infection. HBeAg to anti-HBe
seroconversion occurs early in patients with acute infection, prior to HBsAg to antiHBs seroconversion. However, HBeAg seroconversion may be delayed for years to
decades in patients with chronic HBV infection. In such patients, the presence of
HBeAg is usually associated with the detection of high levels of HBV DNA in
serum and active liver disease. However, HBeAg-positive patients with perinatally
acquired HBV infection may have normal serum ALT concentrations and minimal
inflammation in the liver (Chang 1988).
Seroconversion from HBeAg to anti-HBe is usually associated with a decrease in
serum HBV DNA and remission of liver disease. However, some patients continue
to have active liver disease after HBeAg seroconversion. Such individuals may have
low levels of wild type HBV or HBV variants with a stop codon in the precore or
dual nucleotide substitutions in the core promoter region that prevent or decrease
the production of HBeAg (Carman 1989).

Serum HBV DNA assays
Qualitative and quantitative tests for HBV DNA in serum have been developed to
assess HBV replication. Currently, most HBV DNA assays use real-time PCR
techniques, report results in IU/mL, have a lower limit of detection of around 20
IU/mL and a range of linearity up to 8 log10 IU/mL.
Recovery from acute hepatitis B is usually accompanied by the disappearance of
HBV DNA in serum. However, HBV DNA may remain detectable in serum for

122 Hepatology 2012
many years if tested by PCR assays (Cornberg 2011) suggesting that the virus
persists but is controlled by the immune system.
In patients with spontaneous or treatment-induced HBeAg seroconversion in
chronic hepatitis B, PCR assays usually remain positive except in patients with
HBsAg seroconversion. By contrast, most patients who develop HBeAg
seroconversion during nucleos(t)ide analog therapy have undetectable serum HBV
DNA. In fact, many patients receiving nucleos(t)ide analog therapy remain HBeAg
positive despite having undetectable serum HBV DNA for months or years. The
explanation for this phenomenon is unclear but is likely related to the lack of direct
effect of nucleos(t)ide analogs on covalently closed circular HBV DNA (ccc DNA)
and viral RNA transcription and viral protein expression.
HBV DNA levels are also detectable in patients with HBeAg negative chronic
hepatitis, although levels are generally lower than in patients with HBeAg positive
chronic hepatitis. Because of the fluctuations in HBV DNA levels there is no
absolute cutoff level that is reliable for differentiating patients in the inactive carrier
state from those with HBeAg negative chronic hepatitis B (Chu 2002).

HBV genotypes
HBV can be classified into eight genotypes and four major serotypes. There have
been reports about differing therapeutic responses with nucleos(t)ide analogs and
interferon α with respect to different genotypes. Furthermore, some genotypes, such
as B and C, may have a greater risk for the development of hepatocellular
carcinomas. Nevertheless, in the clinical setting in contrast to hepatitis C, the
diagnosis of HBV genotypes is not part of clinical routine (Thursz 2011).

Antiviral resistance testing
Drug-resistant hepatitis B virus (HBV) mutants frequently arise, leading sometimes
to treatment failure and progression to liver disease. There has been much research
time invested into the mechanisms of resistance to nucleos(t)ides and the selection
of mutants. The genes that encode the polymerase and envelope proteins of HBV
overlap, so resistance mutations in the polymerase usually affect the hepatitis B
surface antigen; these alterations affect infectivity, vaccine efficacy, pathogenesis of
liver disease, and transmission throughout the population (see Chapter 2).
Associations between HBV genotype and resistance phenotype have allowed crossresistance profiles to be determined for many commonly detected mutants, so
genotyping assays can be used to adapt therapy. In vitro phenotyping procedures are
established in a rather small number of HBV laboratories and are not commercially
available. Known mutations can be detected by commercially available tests with a
threshold of about 5% (line probe assays, Inno-Lipa®) whereas determination of
novel mutations remain to be detected by research oriented laboratories with full
length sequencing methods. Novel ultra-deep pyrosequencing techniques are much
more sensitive in order to detect many more viral variants but are a tool only for
specialised research laboratories and not part of clinical routine (Zoulim 2009,
Margeridon-Thermet 2009).

Hepatitis B: Diagnostic Tests 123

Assessment of liver disease
As a first step, the causal relationship between HBV infection and liver disease has
to be established and an assessment of the severity of liver disease needs to be
performed. Not all patients with chronic hepatitis B virus infection have persistently
elevated aminotransferases. Patients in the immune tolerant phase have persistently
normal ALT levels and a proportion of patients with HBeAg-negative chronic
hepatitis B may have intermittently normal ALT levels. Therefore appropriate,
longitudinal long-term follow-up is crucial.
The assessment of the severity of liver disease should include: biochemical
markers, including aspartate aminotransferase (AST) and ALT, gammaglutamyl
transpeptidase (GGT), alkaline phosphatase, prothrombin time and serum albumin;
blood counts; and hepatic ultrasound. Usually, ALT levels are higher than AST.
However, when the disease progresses to cirrhosis, the ratio may be reversed. A
progressive decline in serum albumin concentrations and prolongation of the
prothrombin time, often accompanied by a drop in platelet counts, are
characteristically observed once cirrhosis has developed (EASL 2009).

Acute HBV infection
The diagnosis of acute hepatitis B is based upon the detection of HBsAg and IgM
anti-HBc. During the initial phase of infection, markers of HBV replication, HBeAg
and HBV DNA, are also present. Recovery is accompanied by the disappearance of
HBV DNA, HBeAg to anti-HBe seroconversion, and subsequently HBsAg to antiHBs seroconversion.
The differential diagnosis of HBsAg-positive acute hepatitis includes acute
hepatitis B, exacerbations of chronic hepatitis B, reactivation of chronic hepatitis B,
superinfection of a hepatitis B carrier with hepatitis C or D virus (Tassopoulos
1987), and acute hepatitis due to drugs or other toxins in a hepatitis B carrier.

Past HBV infection
Previous HBV infection is characterized by the presence of anti-HBs and IgG antiHBc. Immunity to HBV infection after vaccination is indicated by the presence of
anti-HBs only.
HBsAg
− If negative, acute HBV infection is ruled out (Dufour 2000).
− If positive, the patient is infected with HBV. A repeat test six months later will
determine if the infection has resolved or is chronic.
Anti-HBs
− If negative, the patient has no apparent immunity to HBV
− If positive, the patient is considered immune to HBV (either because of
resolved infection or vaccination).
Anti-HBc-immunoglobulin M
In rare cases, anti-HBc immunoglobulin (Ig) M may be the only HBV marker
detected during the early convalescence or 'window period' when the HBsAg and
anti-HBs tests are negative. Because current tests for HBsAg are very sensitive, an
anti-HBc IgM that is typically positive with acute HBV infection is not generally

124 Hepatology 2012
required to diagnose active infection. Because some chronic HBV carriers remain
anti-HBc IgM positive for years, epidemiological information is necessary to
confirm that the infection is indeed acute. A negative anti-HBc IgM in the presence
of a positive HBsAg suggests that the infection is likely chronic. For these reasons,
routine testing for anti-HBc IgM is not generally recommended to screen for acutely
infected patients.

Chronic HBV infection
Chronic HBV infection is defined by the continued presence of HBsAg in the blood
for longer than six months. Additional tests for HBV replication, HBeAg and serum
HBV DNA, should be performed to determine if the patient should be considered
for antiviral therapy. All patients with chronic HBV infection should be regularly
monitored because HBV DNA and ALT levels vary during the course of infection
to monitor for progression of liver disease. In addition, patients who are not
candidates for treatment at the time of presentation may become candidates for
treatment during follow-up.
HBeAg-negative patients who have normal serum ALT and low (<2000 IU/mL)
or undetectable HBV DNA are considered to be in an inactive carrier state. These
patients generally have a good prognosis and antiviral treatment is not indicated.
However, serial tests are necessary to accurately differentiate them from patients
with HBeAg-negative chronic hepatitis who have fluctuating ALT and/or HBV
DNA levels (Lok 2007). Patients who are truly inactive carriers should continue to
be monitored but at less frequent intervals. HBeAg-negative patients with elevated
serum ALT concentrations should be tested for serum HBV DNA to determine if
the liver disease is related to persistent HBV replication.
HBsAg
− If negative, chronic HBV infection is typically ruled out.
− If positive, the patient is considered HBV-infected. Chronic infection is
diagnosed when the HBsAg remains detectable for longer than six months.
Antibody to hepatitis B core protein
− If negative, past infection with HBV is typically ruled out.
− If positive, the patient has been infected with HBV. Infection may be resolved
(HBsAg-negative) or ongoing (HBsAg-positive). If the infection is resolved,
the person is considered naturally immune to HBV infection.
Antibody to hepatitis B surface protein
− If negative, the patient has no apparent immunity to HBV
− If positive, the patient is considered immune to HBV (either because of
resolved infection or as the result of prior vaccination). Very rarely (less than
1%) chronic carriers can be positive for HBsAg and antibody to hepatitis B
surface protein (anti-HBs) at the same time (Tsang 1986, Dufour 2000). In
such cases, the patient is considered infectious.

Serum transaminases
Once an individual has been diagnosed with chronic HBV infection, follow-up
testing must be performed for alanine aminotransferase (ALT), a marker of liver cell

Hepatitis B: Diagnostic Tests 125
inflammation. Repeat periodic testing is indicated because the ALT levels can
fluctuate (e.g., from less than the upper limit of normal to intermittently or
consistently elevated). Sustained and intermittent elevations in ALT beyond the
upper limit of normal are indicative of hepatic inflammation and correlate with an
increased risk of progressive liver disease. It must be noted that the normal ALT
ranges are both age and sex dependent and, occasionally, individuals with severe
liver disease may not manifest elevated ALT (Cornberg 2011, EASL 2009).

Occult HBV infection
This is defined as the presence of detectable HBV DNA by PCR in patients who are
negative for HBsAg. Most of these patients have very low or undetectable serum
HBV DNA levels accounting for the failure to detect HBsAg. Infections with HBV
variants that decrease HBsAg production or have mutations in the S gene with
altered S epitopes that evade detection in serology assays for HBsAg are
uncommon. HBV DNA is often detected in the liver and transplantation of livers
from these persons can result in de novo HBV infection (Margeridon-Thermet
2009).

Assessment of HBV immunity
Immunity to HBV is acquired from a resolved infection or from vaccination. The
HBV vaccine has been shown to induce protective immunity in 90% to 95% of
vaccinees. Most vaccinees will have protective levels of anti-HBs for 5-10 years
after vaccination, although the exact duration of immunity remains undefined.
Anti-HBs
− If the anti-HBs level is less than 10 mIU/mL, this implies that the person is
nonimmune to HBV. In individuals who have received a complete course of
HBV vaccine, the level of anti-HBs may drop to less than 10 mIU/mL after
five to 10 years, but these individuals are generally considered to be immune
based on their vaccination history (Maruyama 1994).
− If the anti-HBs result is greater than 10 mIU/mL, the person is considered to be
immune. Immunity may be due to immunization or resolved natural infection.
These two states can be distinguished by testing for antibody to hepatitis B
core protein (anti-HBc), which is present in subjects that have had HBV
infection but absent in vaccinees (see below).
Anti-HBc
− If the anti-HBc total test is positive, this is compatible with current or resolved
HBV infection. A negative HBsAg confirms a resolved infection. HBV
vaccination does not induce anti-HBc total.

Liver biopsy and noninvasive liver transient elastography
A liver biopsy is recommended for determining the degree of necroinflammation
and fibrosis in patients with either increased ALT or HBV DNA levels >2000 IU/ml
(or both) since hepatic morphology can assist the decision to start treatment. Biopsy
is also useful for evaluating other possible causes of liver disease such as steatosis
or steatohepatitis. Although liver biopsy is an invasive procedure, the risk of severe
complications is low. It is important that the size of the needle biopsy specimen be

126 Hepatology 2012
large enough to precisely analyse the degree of liver injury and fibrosis. A liver
biopsy is usually not required in patients with clinical evidence of cirrhosis or in
those in whom treatment is indicated irrespective of the grade of activity or the
stage of fibrosis.
There is growing interest in the use of noninvasive methods, including serum
markers and transient elastography, to assess hepatic fibrosis to complement or
avoid a liver biopsy (Cornberg 2011, EASL 2009).

References
Brunetto MR, Oliveri F, Colombatto P, et al. Hepatitis B surface antigen serum levels help to
distinguish active from inactive hepatitis B virus genotype D carriers.
Gastroenterology 2010;139:483-90. (Abstract)
Carman WF, Jacyna MR, Hadziyannis S, et al. Mutation preventing formation of hepatitis B e
antigen in patients with chronic hepatitis B infection. Lancet 1989;2:588. (Abstract)
Chang MH, Hwang LY, Hsu HC, et al. Prospective study of asymptomatic HBsAg carrier
children infected in the perinatal period: clinical and liver histologic studies.
Hepatology 1988;8:374 (Abstract)
Chu CJ, Hussain M, Lok AS. Quantitative serum HBV DNA levels during different stages of
chronic hepatitis B infection. Hepatology 2002;36:1408. (Abstract)
Chu CM, Liaw YF, Pao CC, Huang MJ. The etiology of acute hepatitissuperimposed upon
previously unrecognized asymptomatic HBsAg carriers. Hepatology 1989;9:452.
(Abstract)
Cornberg, M, Protzer U, Petersen J, et al. Aktualisierung der S3 Leitliniezur Prophylaxe,
Diagnostik und Therapie der Hepatitis-B-Virusinfektion. Z Gastroenterol 2011;49:871930
Dufour DR, Lott JA, Nolte FS, Gretch DR, Koff RS, Seeff LB. Diagnosis and monitoring of
hepatic injury. I. Performance characteristics of laboratory tests. Clin Chem
2000;46:2027-49. (Abstract)
EASL Clinical Practice Guidelines: Management of chronic Hepatitis B. J Hepatology
2009;50:227-42
Hepatitis B virus resistance to nucleos(t)ide analogues. Zoulim F, Locarnini S. Gastroenterology
2009;137:1593-608 (Abstract)
Krajden M, Comanor L, Rifkin O, Grigoriew A, Minor JM, Kapke GF. Assessment of hepatitis B
virus DNA stability in serum . J Clin Microbiol 1998;36:382-6.
Liaw YF, Sheen IS, Chen TJ, et al. Incidence, determinants and significance of delayed
clearance of serum HBsAg in chronic hepatitis B virus infection: a prospective study.
Hepatology 1991;13:627. (Abstract)
Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 2007;45:507. (Abstract)
Marcellin P, Bonino F, Lau GK, et al. Sustained response of hepatitis B e antigen-negative
patients 3 years after treatment with peginterferon alpha-2a. Gastroenterology
2009;136:2169-79. (Abstract)
Margeridon-Thermet S, Shulman NS, Ahmed A, et al. Ultra-deep pyrosequencing of hepatitis B
virus quasispecies from nucleoside and nucleotide reverse-transcriptase inhibitor
(NRTI)-treated patients and NRTI-naive patients. J Infect Dis 2009;199:1275-85
(Abstract)
Maruyama T, Schödel F, Iino S, et al. Distinguishing between acute and symptomatic chronic
hepatitis B virus infection. Gastroenterology 1994;106:1006. (Abstract)
Quint WG, de Bruijn I, Kruining H, Heijtink RA. HBV-DNA detection by gene amplification in
acute hepatitis B. Hepatology 1990;12:653-6. (Abstract)
Raimondo G, Allain JP, Brunetto MR, et al. Statements from the Taormina expert meeting on
occult hepatitis B virus infection. J Hepatol 2008;49:652. (Abstract)
Tassopoulos NC, Papaevangelou GJ, Sjogren MH, et al. Natural history of acute hepatitis B
surface antigen-positive hepatitis in Greek adults. Gastroenterology 1987;92:1844.
(Abstract)
Thursz M, Yee L, Khakoo S Understanding the host genetics of chronic hepatitis B and C.
Semin Liver Dis 2011;31:115-127. (Abstract)

Hepatitis B: Diagnostic Tests 127
Tsang TK, Blei AT, O'Reilly DJ, Decker R. Clinical significance of concurrent hepatitis B surface
antigen and antibody positivity. Dig Dis Sci 1986;31:620. (Abstract)
Yang HI, Lu SN, Liaw YF, et al. Hepatitis B e antigen and the risk of hepatocellular carcinoma.
N Engl J Med 2002;347:168-74. (Abstract)

128 Hepatology 2012

9. Hepatitis B Treatment
Florian van Bömmel, Johannes Wiegand, Thomas Berg

Introduction
Individuals with HBV infection carry a significantly increased risk of lifethreatening complications such as hepatic decompensation, liver cirrhosis and hepatocellular carcinoma (HCC) (Beasley 1988). Recent studies have shown that the
level of serum HBV DNA correlates higher with the risk of developing cirrhosis and
HCC as compared to other baseline or virologic parameters (Chen 2006, Iloeje
2006) (Figure 1). Thus, suppressing the replication of HBV to undetectable levels is
now a major goal in HBV treatment (Liaw 2008, Lok 2007, EASL 2009, Cornberg
2011). Moreover, it has now become clear that continuous suppression of HBV
replication can revert liver fibrosis or even cirrhosis in most patients (Marcellin
2011, Schiff 2011). HBeAg seroconversion is another endpoint, provided that HBV
replication remains durably suppressed to low levels. The ultimate treatment goal,
however, the loss of HBsAg or HBsAg seroconversion, remains difficult to achieve.
The level of hepatitis B surface antigen (HBsAg) before and during interferon-based
treatment is becoming a marker for response to interferon based treatment.
There are two drug classes available for the treatment of chronic HBV infection:
the immune modulator interferon α (standard or pegylated (PEG)-INF α) and
nucleoside or nucleotide analogs, which act as reverse transcriptase inhibitors of the
HBV polymerase. Currently, the nucleoside analogs lamivudine (LAM), telbivudine
(LdT), entecavir (ETV) and the acyclic nucleotide analogs adefovir dipivoxil
(ADV) and tenofovir disoproxil fumarate (TDF) are available. Due to this broad
spectrum of therapeutic options disease progression and complications can be
prevented if the infection is diagnosed early and treated effectively. The early
diagnosis of chronic hepatitis B by HBsAg screening in high-risk groups and in
patients with elevated transaminases plays a crucial role in the management of HBV
infection.

Indication for antiviral therapy
Acute hepatitis B
Acute hepatitis resolves spontaneously in 95-99% of cases (McMahon 1985, Liaw
2009). Therefore, treatment with the currently available drugs is generally not

Hepatitis B Treatment 129
indicated. However, in a recent trial comparing treatment with LAM 100 mg/day
versus no treatment in 80 Chinese patients with fulminant hepatitis B, a reduced
mortality of 7.5% was found in patients receiving LAM treatment compared to 25%
in the control group (p=0.03) (Yu 2010). These observations are supported by a
placebo-controlled trial investigating the use of LAM in 71 patients with fulminant
hepatitis B in India (Kumar 2007). Several case reports from Europe also revealed
that patients with severe and fulminate hepatitis B may benefit from early antiviral
therapy with LAM or other nucleos(t)ide analogs by reducing the need for highurgency liver transplantation (Tillmann 2006). As a result, treatment for fulminant
hepatitis B with LAM is recommended by EASL and with LAM or LdT by AASLD
(EASL 2009, Lok 2009). Interferon therapy is contraindicated in patients with acute
HBV infection because of risk of increasing hepatitis. The endpoint of treatment of
acute HBV infections is HBsAg clearance (EASL 2009, Lok 2007).

Figure 1. Cumulative incidence of liver cirrhosis in untreated HBV-infected individuals
within a mean observation period of 11.4 years (REVEAL Study). The incidence of liver
cirrhosis increases over time depending on baseline HBV DNA levels (Iloeje 2006). The relative
risk for developing HCC was 1.4 in patients with HBV DNA levels of 300 to 1,000 and increased
to 2.4 in patients with 1,000-10,000 to 5.4 in patients with 10,000 to 100,000 and to 6.7 in
patients with HBV DNA levels >1 million copies/ml. A similar association between HBV DNA
levels and the risk of HCC development was shown (Chen 2006).

Chronic hepatitis B
All patients with HBsAg positive chronic hepatitis should be considered as possible
candidates for antiviral therapy especially in situations when there is a significant
level of HBV replication (Chen 2006, Iloeje 2006). Differentiation between
HBeAg-positive and HBeAg-negative chronic hepatitis B is not necessary anymore
for treatment indication, although with respect to the choice of the appropriate
antiviral drug these criteria may be still useful.

130 Hepatology 2012
Table 1. Key guideline recommendations for indication for antiviral treatment of
HBV infection.
AASLD
(Lok 2007, Lok
2009)

APASL
(Liaw 2008)

EASL
(EASL 2009)
Belgian
(Colle 2007)

Dutch
(Buster 2008)

German
(Cornberg 2011)
Italian
(Carosi 2008)

Turkish TASL
(Akarca 2008)

Consider treatment:
• HBeAg(+): HBV DNA >20,000 IU/ml + ALT ≤2x ULN + biopsy shows
moderate/severe inflammation or significant fibrosis
• HBeAg(+): HBV DNA >20,000 IU/ml + ALT >2x ULN. Observe for 3-6
months and treat if no spontaneous HBeAg loss
• HBeAg(-): HBV DNA >20,000 IU/ml + ALT >2x ULN
Consider biopsy:
• HBeAg(+): HBV DNA >20,000 IU/ml + ALT >2x ULN + compensated
• HBeAg(+): HBV DNA >20,000 IU/ml + ALT 1-2x ULN + age >40 years
or family history of HCC
• HBeAg(-): HBV DNA >2,000-20,000 IU/ml + ALT 1-2x ULN
Consider treatment:
• All patients: HBV DNA detectable + advanced fibrosis/cirrhosis
• HBeAg(+): HBV DNA >20,000 IU/ml + ALT >2x ULN + impending/
overt decompensation
• HBeAg(-): HBV DNA > 2,000 + ALT >2x ULN + impending/ overt
decompensation
Consider treatment:
• HBV DNA >20,000 IU/ml + ALT >2x ULN + moderate to severe
necroinflammation
Consider treatment:
• HBeAg(+): HBV DNA >20,000 IU/ml + ALT >2x ULN (or
moderate/severe hepatitis on biopsy)
• HBeAg(-): HBV DNA ≥2,000 IU/ml and elevated ALT
Consider biopsy:
• Fluctuating or minimally elevated ALT (especially in those older than
35-40 years)
Consider treatment:
• HBeAg(+) and HBeAg(-): HBV DNA ≥20,000 IU/ml and ALT ≥2x ULN
or active necrotic inflammation
• HBeAg(-): HBV DNA ≥2,000–20,000 IU/ml and ALT ≥2x ULN (and
absence of any other cause of hepatitis)
Consider treatment:
• HBV DNA >2,000 IU/ml + minimal inflammation/low fibrosis or ALT
elevation
Consider treatment:
• HBeAg(+): HBV DNA >20,000 IU/ml + ALT >2x ULN
• HBeAg(-): HBV DNA >2,000 IU/ml + abnormal ALT and or fibrosis
(Ishak ≥S2)
Consider biopsy:
• HBeAg(-): HBV DNA >2,000 IU/ml + borderline ALT, or if DNA 2,000–
20,000 IU/ml + high ALT
Consider treatment:
• HBV DNA >2,000 IU/ml + histological fibrosis >2
• HBV DNA >20,000 IU/ml + any histological finding + ALT >2x ULN

Current recommendations of the different national and international societies are
shown in Table 1 (Akarca 2008, Carosi 2008, Colle 2007, Cornberg 2011, EASL
2009, Janssen 2008, Juszczyk 2008, Keeffe 2007, Liaw 2008, Lok 2009, Waked
2008). In most of these guidelines, the most relevant factor for a decision to initiate
treatment has shifted from histological proven disease activity to the level of HBV

Hepatitis B Treatment 131
DNA. Thus, most of the recently published guidelines now recommend antiviral
treatment for patients with HBV DNA levels >2,000 IU/mL (corresponding to
>10,000 copies/mL) in association with a sign of ongoing hepatitis which can either
be ALT levels greater than 2 times the upper limit of normal or significant fibrosis
demonstrated by liver histology greater than A1/F1. The recently updated German
treatment guidelines emphasise the importance of suppression of HBV replication
by recommending treatment in patients with HBV DNA levels >2,000 IU/mL and
any elevation of ALT levels or signs of fibrosis (Cornberg 2011).
All patients with liver cirrhosis or high-grade liver fibrosis and any measurable
HBV DNA should be considered for antiviral therapy (EASL 2009, Lok 2007,
Cornberg 2011). The indication for antiviral treatment according to the recent
German guidelines is depicted in Figure 2 (Cornberg 2011). In patients with
decompensated cirrhosis Child-Pugh score B or C, INF α is contraindicated.

Figure 2. Indication for antiviral treatment according to the German guidelines for the
treatment of chronic HBV infection. Treatment should be considered if HBV DNA levels
4
exceed 10 copies/ml and if ALT are elevated or if liver histology is abnormal. Of note,
asymptomatic carriers with family history of HCC should receive treatment even if signs of
hepatitis are absent (Cornberg 2011).

Inactive chronic HBsAg carriers, characterised by negative HBeAg and positive
anti-HBeAg, low HBV DNA levels (<2,000 IU/ml) and serum aminotransferases
within normal levels do not have an indication for antiviral therapy (Cornberg 2011,
Brunettto 2011). However, differentiation between inactive HBsAg carriers and
patients with chronic HBeAg-negative hepatitis may be difficult in some cases.
Elevated transaminases are no reliable parameter for assessing the stage of liver
fibrosis and long-term prognosis of HBV-infected patients. Even in patients with
normal or slightly elevated aminotransferases there can be a significant risk for the
development of HBV-associated complications (Chen 2006, Iloeje 2006, Kumar
2008). It is reasonable to perform a liver biopsy in these individuals and to control

132 Hepatology 2012
the level of HBV DNA at three-month intervals. However, a liver biopsy is not
mandatory to initiate treatment for the majority of patients (Table 1).
HBV immunotolerant patients are mostly under 30 years old and can be recognised by their high HBV DNA levels, positive HBeAg, normal ALT levels and
minimal or absence of significant histological changes. According to most practice
guidelines immediate therapy is not required (Akarca 2007, Balik 2008, Carosi
2008, Colle 2007, Cornberg 2011, EASL 2009, Buster 2008, Juszczyk 2008, Keeffe
2007, Liaw 2008, Lok 2009, Waked 2008). However, patients with elevated risk for
HCC development, such as those with a positive family history, and patients from
high endemic areas like Asia or Africa may perhaps benefit from early antiviral
therapy (Cornberg 2011). Studies are under way to further clarify this issue,
especially to answer the question whether early intervention with antiviral therapy
will positively influence the long-term risk for HCC.

Summary of treatment indications in the German Guidelines of 2011
–

–
–
–
–

–

All patients with chronic hepatitis B should be evaluated for treatment.
Indication for treatment initiation depends on the level of viral replication
(HBV DNA ≥2,000 IU/mL, corresponding to ml ≥10,000 copies/mL), any
elevation of serum aminotransferases and the histological grading and
staging.
Patients with advanced fibrosis or cirrhosis and detectable viremia need
consistent antiviral therapy.
Reactivation of HBV replication due to immunosuppression should be
avoided by preventive therapy.
Alcohol and drug consumption are not a contraindication for treatment with
nucleos(t)ide analogs.
Therapy with nucleos(t)ide analogs during pregnancy may be considered if
the benefit outweighs the risk. A running treatment with LAM or TDF can be
continued during pregnancy.
Occupational and social aspects and extrahepatic complications may justify
therapy in individual cases.

Endpoints of antiviral treatment
Due to persistence of episomal covalently closed circular DNA (cccDNA), a
template of the HBV genome located in the nucleus of infected hepatocytes, a
complete eradication of HBV infection is currently impossible (Rehermann 1996).
Reactivation of an HBV infection can occur in certain circumstances from these
nuclear reservoirs even decades after HBsAg loss, for instance during
immunosuppressive therapy. The aim of treatment of chronic hepatitis B is to
reduce complications such as liver failure and HCC and to increase survival (EASL
2009, Lok 2009, Cornberg 2011). To determine the success of antiviral therapy
surrogate markers are used during and after treatment. These parameters include
virologic (HBeAg and HBsAg status, HBsAg levels, HBV DNA level) and patientrelated parameters (aminotransferases, liver histology).
Suppression of HBV replication. In two recent studies a close correlation between baseline HBV DNA levels and progression of the disease was demonstrated.
In the REVEAL study, 3774 untreated HBV-infected individuals were followed

Hepatitis B Treatment 133
over a mean time period of 11.4 years in Taiwan (Chen 2006, Iloeje 2006). HBV
DNA levels at baseline were the strongest predictors of cirrhosis and HCC development (Figure 1). In multivariate models, the relative risk of cirrhosis increased
when HBV DNA reached levels greater than 300 copies/mL, independent of
whether patients were negative or positive for HBeAg. In addition, individuals with
HBV DNA levels ≥104 copies/mL (or ≥2,000 IU/mL) were found to have a 3-15
fold greater incidence of HCC as compared to those with a viral load <104
copies/mL. According to these results, a meta-analysis covering 26 prospective
studies revealed a statistically significant and consistent correlation between viral
load levels and histologic, biochemical, or serologic surrogate markers (MommejaMarin 2003). It can therefore be concluded that the complete and persistent
suppression of HBV replication is a reliable endpoint for the treatment of chronic
HBV infection.
Induction of HBeAg seroconversion. In HBeAg-positive patients,
seroconversion from HBeAg to anti-HBe was found to be a reliable surrogate
marker for prognosis of chronic HBV infection leading in many cases to an inactive
HBsAg carrier state (Figure 3). In these patients, HBsAg remains detectable but
HBV replication continues at low or even undetectable levels and transaminases are
generally within normal ranges.

Figure 3. Possible endpoints of treatment of chronic HBV infection. After achieving
HBeAg or HBsAg seroconversion, antiviral treatment can be stopped. However, it is
recommended to maintain treatment for a period of 6-12 months after HBeAg or HBsAg
seroconversion.

134 Hepatology 2012
Long-term observations reveal, however, that HBeAg seroconversion cannot
always be taken as a guarantee of long-term remission. A reactivation of the disease
with “seroreversion” (HBeAg becoming detectable again) as well as a transition to
HBeAg-negative chronic hepatitis B with increased, often fluctuating, HBV DNA
levels, can occur in up to 30% of patients (Hadziyannis 1995, Hadziyannis 2001,
Hadziyannis 2006). Therefore, HBeAg seroconversion should be regarded as a
stable treatment endpoint only in conjunction with durable and complete
suppression of HBV replication.
In the natural course of HBV infection, the time point of HBeAg seroconversion
is important regarding the probability of long-term complications. In a recent longterm observational study in 483 HBeAg-positive patients achieving spontaneous
HBeAg seroconversion, it was shown that for 15 years after HBeAg seroconversion
the incidence of cirrhosis and HCC was lower for patients who had achieved
HBeAg seroconversion at an age <30 years old compared to patients achieving
seroconversion at an age >40 years old (Chen 2010). This observation raises the
question of whether HBeAg seroconversions during antiviral treatment in patients
older than 40 years are also associated with a higher risk of complications compared
to patients who achieve HBeAg seroconversion at a younger age.
Sustained response and “immune control”. The endpoint of therapy for patients
with HBeAg-negative disease is more difficult to assess. Long-term suppression of
HBV replication and ALT normalization are the only practical parameters of
response to therapy. Once antiviral therapy is stopped, durability of response is not
guaranteed due to the fluctuating course of HBeAg-negative chronic hepatitis B.
For treatment with PEG-IFN α in both, HBeAg-positive and -negative patients,
the inducing of a so-called ‘immune control’ status, characterized by persistent
suppression of viral replication with HBV DNA levels <2,000 IU/ml and
normalisation of ALT levels was recently defined as another, combined treatment
endpoint (Marcellin 2009). If this condition is maintained over time, it increases the
probability of HBsAg loss and reduces the development of liver fibrosis and HCC.
Late relapse beyond 6 months post-treatment has been described, but a sustained
response at 1 year post-treatment appears to be durable through long-term followup (EASL 2009, Marcellin 2009). However, the immune control status needs to be
regularly monitored, and treatment needs to be re-introduced in case of increase of
HBV replication. For patients presenting any signs of liver fibrosis or family
history of HCC, immune control should not be regarded as the treatment endpoint
but rather the complete suppression of HBV replication.
Induction of HBsAg loss. The ultimate goal of antiviral treatment is HBsAg loss
or even seroconversion to anti-HBs. Because HBsAg loss or seroconversion is
associated with a complete and definitive remission of the activity of chronic
hepatitis B and an improved long-term outcome, it is regarded as a cure from
chronic hepatitis B. However, HBsAg loss or seroconversion can be induced in
only a limited number of patients after short-term treatment (<5%). Interestingly, in
recent follow-up studies in PEG-INF α as well as nucleoside/nucleotide analog
treated patients an increase of the rates of HBsAg loss during long-term studies was
shown (Marcellin 2009, Marcellin 2011). However, as the probability of HBsAg
seroclearance during therapy with nucleoside or nucleotide analogs is linked to the
decrease of HBsAg levels during the early treatment period, it seems questionable

Hepatitis B Treatment 135
if after a treatment duration of 4-5 years significantly higher rates of HBsAg loss
can be expected (Marcellin 2011).
Reversion of liver fibrosis. With long-term treatment with different nucleoside
and nucleotide analogs it has been demonstrated that liver fibrosis and even
cirrhosis can be reverted in the majority of patients. This was recently impressively
shown in a subgroup of 59 patients from a rollover study including two Phase III
trials of the efficacy of ETV in treatment-naïve patients. Liver biopsies from
baseline and after a median treatment duration of 6 years (range, 3-7 years) found
an histologic improvement, defined as a decrease of 2 points or greater in the
Knodell necroinflammatory score in absence of worsening of the Knodell fibrosis
score, in 96% of patients. In addition, an improvement of more than 1 point in the
Ishak fibrosis score was seen in 88%, including all 10 patients who had advanced
fibrosis or cirrhosis when they entered the Phase 3 studies (Chang 2010a). More
recently, in a subanalysis of the tenofovir Trials 102 and 103 evaluating 348
patients who underwent biopsies before and after five years of therapy, 88%
experienced an improvement in overall liver histology as measured by an
improvement of at least two points in the Knodell score of HAI (histologic activity
index) (Figure 4). Of the 94 patients who had cirrhosis at the start of therapy, 73%
experienced regression of cirrhosis, and 72% had at least a two-point reduction in
fibrosis scoring (Marcellin 2011).

Figure 4. Changes in liver histology after five years of TDF treatment. In a study looking at
348 patients with paired liver biopsies, regression of liver fibrosis and even liver cirrhosis (Ishak
score 5 and 6) was found in the majority of patients.

136 Hepatology 2012

Criteria for treatment response
Virologic response
–
sustained decrease of HBV DNA, to at least <2,000 IU/mL (corresponding to
<10,000 copies/mL), ideally to <60 IU/mL (<300 copies/mL).
–
sustained HBe seroconversion in HBeAg positive patients
–
ideally, loss of HBsAg
Biochemical response
–
sustained ALT normalization
Histologic response
–
reduction of fibrosis (histological staging)
–
reduction of inflammatory activity (histological grading)
Potential long-term effects
–
avoidance of cirrhosis, hepatocellular carcinoma (HCC), transplantation, and
death

How to treat
Therapy of chronic hepatitis B is possible with PEG-INF α in order to induce an
immunologic long-term control by finite treatment or with nucleos(t)ide analogs by
long-term inhibition of HBV replication (Figure 5) (Table 2).
At first, the option of interferon therapy should be evaluated. However, if a
patient does not fulfil the criteria for a higher likelihood for treatment success with
PEG-INF α, has contraindications, or is intolerant, long-term therapy with
nucleos(t)ide analogs is recommended (Figure 5). If a nucleos(t)ide analog is
chosen, several parameters have to be considered prior to therapy: the antiviral
efficacy of the drug, the durability of response, the resistance barrier, and the stage
of liver disease.
If the initial viral load is low and liver cirrhosis has been excluded, any approved
drug may be used. The use of LAM, however, should be restricted to patients with
mild fibrosis and HBV DNA levels <105 copies/ml. For patients with high-level
HBV replication (>109 copies/ml) only drugs with a high genetic barrier should be
used (i.e., ETV or TDF) (Table 3).

Treatment options
Because of a limited tolerability due to adverse events, duration with PEG-IFN α is
limited for a period of 6-12 months (maximum 24 months). Nucleoside and
nucleotide analogs have a good tolerability and are used in long-term treatment.
However, the efficacy of these oral agents can be hampered by emergence of
resistance. Two interferons and five oral HBV polymerase inhibitors are currently
approved for the treatment of chronic HBV infections: standard IFN α-2b and PEGIFN α-2a, lamivudine (LAM), adefovir dipivoxil (ADV), telbivudine (LdT),
entecavir (ETV) and tenofovir disoproxil fumarate (TDF) (Table 2). The efficacy of
the available drugs after one year of treatment, assessed by the proportion of
individuals with HBV DNA below the limit of detection, normalised transaminases
and HBeAg seroconversion is shown in Figure 6.

Hepatitis B Treatment 137

Figure 5. Treatment algorithm for chronic HBV infection according to the German
Guidelines (Cornberg 2011). The indication for interferon therapy should always be
considered. For treatment with nucleoside or nucleotide analogs, agents with high genetic
barrier against resistance such as entecavir or tenofovir should be preferred.

Table 2. Overview of interferons and oral antiviral drugs currently approved for the
treatment of HBV infection.
Drug

Name

Interferon α
Standard Interferon α-2a

Roferon

Standard Interferon α-2b
Pegylated Interferon α-2a
Nucleoside analogs
Lamivudine
Telbivudine
Entecavir

Dose
®

®

Intron A
®
Pegasys
®

Zeffix
®
Sebivo
®
Baraclude

Nucleotide analogs
®
Adefovir dipivoxil
Hepsera
®
Tenofovir disoproxil fumarate Viread
* see Figure 6

Duration
2

2.5-5 mio. U/m body surface 4-6 months
3x/week
5-10 mio. IU 3x/week
4-6 months
180 µg/week
48 weeks
100 mg/day
600 mg/day
0.5 mg/day
1 mg/day for patients with
lamivudine resistance

long-term*
long-term*
long-term*
long-term*

10 mg/day
300 mg/day

long-term*
long-term*

138 Hepatology 2012
Table 3. Recommendations for the use of nucleos(t)ide analogs in clinical practice.
Drug

Advantage

Disadvantage

Recommendation

Lamivudine
(LAM)

• Low treatment costs
• Oral solution
available for children
or individual dosage
in case of renal
impairment

• High risk of
resistance in longterm monotherapy
• Cross-resistance to
ETV and LdT

Adefovir
dipivoxil
(ADV)

• Experience in
• Moderate antiviral
combination with LAM
activity
• No cross-resistance
• Primary nonto LAM
response in 10-20%
of cases
• Slow viral kinetics
during therapy
• Risk of viral
resistance in longterm monotherapy
• Nephrotoxicity
• High antiviral efficacy • Moderate risk for viral
resistance in long• Potentially no crossterm monotherapy
resistance to
entecavir
• Neuropathy and
myopathy
• High antiviral efficacy • In LAM-experienced
patients high risk for
• Low risk for viral
the development of
resistance in longviral resistance and
term monotherapy in
virologic failure in
lamivudine-naïve
long-term
patients
monotherapy
• Combination therapy

• Use as first-line
therapy only in
selected patients with
low viral load
• Prevention of
exacerbation in
HBsAg+, HBV DNApatients with
immunosuppression
• Preemptive therapy in
case of HBsAgnegative, anti-HBc
positive patients with
immunosuppression
• Use in pregnancy
possible
• Not to be used as
first-line therapy

Telbivudine
(LdT)

Entecavir
(ETV)

• First-line therapy
• Can be combined
with TDF

• First-line therapy
• Can be combined
with TDF

with TDF as rescue
therapy
• Oral solution
available for
individual dosage in
case of renal
impairment
Tenofovir
• High antiviral efficacy • Nephrotoxicity
• First line therapy
disoproxil
• Low risk for viral
• Decrease in bone
• Can be combined
fumarate
resistance in longmineral density
with ETV, LdT or LAM
(TDF)
term monotherapy
if needed
* in HBV-monoinfected patients no renal toxicity was observed in 5 years of TDF treatment

Hepatitis B Treatment 139

Interferons
INF α is a natural occurring cytokine with immunomodulatory, antiproliferative and
antiviral activity. During treatment, the therapeutic efficacy of INF α can often be
clinically recognised by an increase of ALT levels to at least twice the baseline
levels. These ALT flares often precede virologic response.
The main aim of INF α treatment is to induce a long-term remission by finite
treatment duration. Overall a long-term response defined by either HBeAg
seroconversion or durable suppression of HBV DNA to low or undetectable levels
can be achieved in approximately 30% of treated patients. In these responders the
chance for HBsAg loss in the long-term is relatively high.

Figure 6. One-year efficacy of medications currently approved for the treatment of
chronic HBV infection (Lok 2009). Treatment efficacy is expressed as suppression of HBV
DNA below the limit of detection, ALT normalisation and rates of HBeAg seroconversion. As no
head-to-head trials comparing the substances have been undertaken, differences in antiviral
efficacy have to be interpreted with caution.

Standard INF α. Standard IFN α was approved for treatment of chronic hepatitis
B in 1992. IFN α is applied in dosages ranging from 5 million units (MU) to 10 MU
every other day or thrice weekly. In a meta-analysis, a significant improvement in
endpoints was shown in patients with HBeAg-positive chronic hepatitis B being
treated with standard IFN compared to untreated patients (Craxí 2003). Complete
remission of fibrotic changes was observed in some patients and the loss of HBsAg
occurred comparatively often. Furthermore, there was a trend towards reduction of
hepatic decompensation (treated 8.9% vs. untreated 13.3%), hepatocellular
carcinoma (1.9 vs. 3.2%), and liver associated deaths (4.9 vs. 8.7%) (Craxí 2003).
A significant decrease in ALT and in HBV DNA serum levels was also shown for
standard IFN α in the treatment of HBeAg-negative chronic hepatitis B (Brunetto
2003). However, a high percentage (25-89%) of these patients relapses after the end
of treatment showing elevation of ALT levels and a return of HBV DNA levels. The
relapse rate seems to be higher when treatment duration is short (16 to 24 weeks)

140 Hepatology 2012
compared to longer treatment (12 to 24 months). A retrospective comparison of IFN
therapies lasting from 5 to 12 months showed that with longer treatment the chance
of a long-term response was 1.6 times higher (normalization of ALT, HBV DNA
<1x106 copies/ml 1-7 years after end of therapy). The overall response rates were
54% at the end of therapy, 24% at 1 year after therapy, and 18% 7 years after
therapy (Manesis 2001).
Patients with long-term response to treatment have a more favourable course than
patients who were untreated, unresponsive, or who had a relapse interferon α
therapy with respect to progression to liver cirrhosis, liver associated deaths, and
development of hepatocellular carcinoma (Brunetto 2003, Lampertico 2003).
However, due to higher antiviral efficacy PEG-IFN α should be preferred to
standard IFN α.
PEG-INF α. The addition of a polyethylene glycol molecule to the IFN resulted
in a significant increase in half-life, thereby allowing administration once weekly.
Two types of subcutaneously administered PEG-IFN α were developed: PEG-IFN
α-2a and PEG-IFN α-2b, of which PEG-IFN α-2a was licensed for the treatment of
chronic HBV infections in a weekly dose of 180 µg for 48 weeks in both HBeAgpositive and HBeAg-negative patients. However, PEG-IFN α-2b shows similar
efficacy. After one year on treatment with PEG-IFN α-2a and α-2b, 22% to 27% of
patients were reported to achieve HBeAg seroconversion (Janssen 2005, Lau 2005).
The safety profiles of standard IFN α and PEG-IFN α are similar. Following
therapy termination a relatively high relapse rate is to be expected (>50%). The dose
of 180 µg per week applied for 48 weeks was recently shown to exert a stronger
antiviral efficacy compared to administration for 24 weeks or to administration of
90 µg per week (Liaw 2011). In a small Italian study it was shown that prolongation
of 48 weeks of treatment with 180 µg PEG-IFN α per week by another 48 weeks of
135 µg PEG-IFN α-2a may enhance antiviral efficacy and increase the rate of
patients achieving HBsAg loss, at least in HBeAg-negative patients with HBV
genotype D (Lampertico 2010). However, the optimal treatment duration for PEGIFN α has not been defined yet and treatment beyond 48 weeks is not recommended
by current guidelines.
PEG-IFN α in HBeAg-positive patients. Four randomized, controlled studies
investigating the efficacy of PEG-IFN α in HBeAg-positive patients have been
conducted (Crespo 1994, Chan 2005, Janssen 2005, Lau 2005). These studies
compared 180 µg PEG-INF α per week to standard IFN, LAM, and/or a
combination treatment with PEG-INF α + LAM for 48 weeks. Sustained HBeAg
seroconversion at the end of follow-up (week 72) was significantly higher in
patients treated with PEG-IFN α-2a alone or in combination with LAM than in
patients treated with LAM alone (32% and 27% versus 19%) (Marcellin 2004).
Importantly, it was recently shown that PEG-IFN α can induce
immunomodulatory effects which persist beyond the end of therapy leading to high
HBsAg clearance rates in the follow-up period. In a recent study, 97 patients with
chronic HBV infection who had received treatment with standard IFN α were
retrospectively analyzed for a median period of 14 (range, 5-20) years. During the
observation period, 28 patients (29%) of this cohort lost HBsAg (Moucari 2009).
PEG-IFN α in HBeAg-negative patients. The efficacy and safety of 48 weeks
treatment with 180 µg PEG-IFN α-2a once weekly + placebo, + 100 mg LAM daily,
or LAM alone was compared in 177, 179, and 181 HBeAg-negative patients,

Hepatitis B Treatment 141
respectively. After 24 weeks of follow-up, the percentage of patients with
normalisation of ALT levels or HBV DNA levels below 20,000 copies/ml was
significantly higher with PEG-IFN α-2a monotherapy (59% and 43%, respectively)
and PEG-IFN α-2a plus LAM (60% and 44%) than with LAM monotherapy (44%
and 29%); the rates of sustained suppression of HBV DNA below 400 copies/ml
were 19% with PEG-IFN α-2a monotherapy, 20% with combination therapy, and
7% with LAM alone.
Also in HBeAg-negative patients HBsAg loss can be induced in some patients by
PEG-IFN α treatment. In a study in 315 patients who were treated with either PEGIFN α-2a, LAM 100 mg or a combination of both drugs for 48 weeks, three years
after the end of treatment, the rate of HBsAg loss was 8.7% in those who had been
treated with PEG-IFN α-2a alone or in combination with LAM while no patient
treated with LAM as monotherapy cleared HBsAg (Marcellin 2009a). Of the patients who had received a PEG-IFN α-2a and who still had undetectable HBV DNA
three years after treatment, 44% had lost HBsAg.
Prolongation of PEG-IFN α treatment beyond 48 weeks may increase sustained
response rates. This was found in an Italian study in 128 mainly genotype D–
infected HBeAg-negative patients who were randomized to either treatment with
180 µg/week PEG-IFN α-2a for 48 weeks or a continuing treatment with PEG-IFN
α-2a at 135µg/week. Additionally, in a third arm patients received combination
treatment with PEG-IFN α-2a 180µg/week and LAM 100 mg/day, followed by 48
weeks of PEG-IFN α-2a in the dosage of 135 µg/week. As a result, 48 weeks after
the end of treatment 26% of patients who had received 96 weeks of PEG-IFN
treatment showed HBV DNA levels <2,000 IU/mL compared to only 12% of the
patients who had received PEG-IFN for 48 weeks. Combination with LAM showed
no additional effect (Lampertico 2010a).

Nucleoside and nucleotide analogs
Nucleoside and nucleotide analogs inhibit HBV replication by competing with the
natural substrate deoxyadenosine triphosphate (dATP) and causing terminating of
the HBV DNA chain prolongation. They represent two different subclasses of reverse transcriptase inhibitors: while both are based on purines or pyrimidines,
acyclic nucleotide analogs have an open (acyclic) ribose ring that confers greater
binding capacity to resistant HBV polymerase strains.
Treatment duration for nucleos(t)ide analogs is not well-defined but a short-term
application of these agents for 48 weeks is associated with prompt relapse in
viremia and they should be administered for longer periods. Treatment efficacy of
nucleoside and nucleotide analogs implies complete suppression of HBV DNA
levels in serum. This should be achieved within six months if agents with high risk
for resistance development as LAM, ADV, and LdT are used.
Effective long-term control of HBV replication with nucleoside or nucleotide analogs is associated with a reduction of long-term complications such as HCC and
development of liver cirrhosis (Toy 2009). Studies with different nucleoside and
nucleotide analogs have demonstrated that suppression of HBV replication is
associated with a significant decrease in histologic inflammatory activity and fibrosis, including partial reversion of liver cirrhosis (Chen 2006, Iloeje 2006, Mommeja-Marin 2003, Chen 2010, Marcellin 2011, Schiff 2011). With increasing treatment duration HBeAg seroconversion rates increase (Liaw 2000, Lok 2000). Most

142 Hepatology 2012
importantly, there is also evidence that effective inhibition of HBV replication can
reduce HBV cccDNA, possibly running parallel to the decline in serum HBsAg
levels (Werle-Lapostolle 2004, Wursthorn 2006). These findings may indicate that
long-term antiviral therapy may lead to a complete response in a significant number
of patients.
A central aspect of HBV polymerase inhibitor treatment is the prevention and
management of HBV resistance to these drugs (see Chapter 10). Resistance against
nucleoside or nucleotide analogs can occur during suboptimal treatment and often
leads to aggravation of liver disease. Because of cross resistance between several
nucleoside and nucleotide analogs, nucleoside-naïve and nucleoside-experienced
patients have to be distinguished and prior nucleoside experience should be taken
into account when choosing a second line therapy. However, highly potent substances such as ETV and TDF show minimal or even no resistance development in
treatment-naïve patients over 5-6 years (Snow-Lampert 2011).
Lamivudine (LAM). LAM, a (-) enantiomer of 2’ -3’ dideoxy-3’-thiacytidine, is
a nucleoside analog that was approved for the treatment of chronic HBV infection in
1988 with a daily dose of 100 mg. This dose was chosen based on a preliminary trial
that randomly assigned 32 patients to receive 25, 100, or 300 mg of LAM daily for a
total of 12 weeks (Dienstag 1995). In this study the dose of 100 mg was more
effective than 25 mg and was similar to 300 mg in reducing HBV DNA levels.
LAM exerts its therapeutic action in its phosphorylated form. By inhibiting both the
RNA- and DNA-dependent DNA polymerase activities, the synthesis of both the
first strand and the second strand of HBV DNA are interrupted.
Long-term LAM treatment is associated with an increasing rate of antiviral drug
resistance reaching approximately 70% after 5 years in patients with HBeAgpositive HBV infections. Therefore, in many guidelines LAM is not considered a
first-line agent in the treatment of chronic HBV infection any more. However, LAM
still may play a role in combination regimens or in patients with mild chronic
hepatitis B expressing low levels of HBV DNA (<105 copies/ml). An early and
complete virologic response to LAM within 6 months of therapy (<400 copies/mL)
constitutes a prerequisite for long-term control of HBV infection without the risk of
developing resistance.
Adefovir dipivoxil (ADV). Adefovir dipivoxil was approved for treatment of
chronic hepatitis B in the US in 2002 and in Europe in 2003. It is an oral diester
prodrug of adefovir, an acyclic nucleotide adenosine analog that is active in its
diphosphate form. Because the acyclic nucleotide already contains a phosphatemimetic group, it needs only two, instead of three, phosphorylation steps to reach
the active metabolite stage. ADV was the first substance with simultaneous activity
against wild type, pre-core, and LAM-resistant HBV variants. It is active in vitro
against a number of DNA viruses other than HBV and retroviruses (i.e., HIV). The
dose of 10 mg per day was derived from a study comparing 10 mg versus 30 mg/d.
The higher dosage leads to stronger suppression of HBV DNA levels but also to
renal toxicity with an increase of creatinine levels (Hadziyannis 2003).
ADV was the first acyclic nucleotide that was widely used in the treatment of
LAM-resistant HBV infections. However, the antiviral effect of ADV in the
licensed dosage of 10 mg/day is rather low as compared to other available antivirals
(Figure 4); this disadvantage makes ADV vulnerable to HBV resistance

Hepatitis B Treatment 143
(Hadziyannis 2006a). Now that TDF is approved, ADV should not be used as firstline monotherapy.
Telbivudine (LdT). Telbivudine is a thymidine analog which is active against
HBV but at least in vitro not active against other viruses, including HIV and
hepatitis C virus (HCV). LdT at 600 mg/day expresses higher antiviral activity
compared to either LAM at 100 mg/day or ADV at 10 mg/day (Figure 4). More
patients achieved HBeAg loss within 48 weeks as compared to other nucleos(t)ides.
LdT was reported to be non-mutagenic, non-carcinogenic, non-teratogenic, and to
cause no mitochondrial toxicity. A favourable safety profile at a daily dose of 600
mg was demonstrated (Hou 2008, Lai 2007). However, CK elevations were
observed more often as compared to the group treated with LAM and neurotoxicity
may be an issue when LdT is administered in combination with PEG-INF α
(Fleischer 2009). Thus, in the GLOBE trial, during a period of 104 weeks grades 3/4
elevations in CK levels were observed in 88 of 680 (12.9%) patients who received
LdT and in 28 of 687 (4.1%) patients who received LAM (p<0.001) (Liaw 2009).
However, rhabdomyolysis was not observed. Peripheral neuropathy was described
in 9 of 48 (18.75%) patients who received combination therapy of PEG-INF αnd
LdT and only in 10 of 3500 (0.28%) patients who received LdT monotherapy
(Goncalves 2009).
Resistance to LdT has been found to occur in up to 21% after 2 years of treatment
(Tenney 2009), predominantly in patients who did not achieve undetectable HBV
DNA level after 24 weeks of treatment (Zeuzem 2009). LdT shows cross-resistance
to LAM and ETV. As a consequence LdT should not be used in LAM or ETV
refractory patients.
Entecavir (ETV). Entecavir, a cyclopentyl guanosine nucleoside analog, is a
selective inhibitor of HBV replication and was licensed in 2006. Entecavir blocks
all three polymerase steps involved in the replication process of the hepatitis B
virus: first, base priming; second, reverse transcription of the negative strand from
the pregenomic messenger RNA; third, synthesis of the positive strand of HBV
DNA. In comparison to all other nucleoside and nucleotide analogs, ETV is more
efficiently phosphorylated to its active triphosphate compound by cellular kinases. It
is a potent inhibitor of wild-type HBV but is less effective against LAM-resistant
HBV mutants. Therefore, ETV was approved at a dose of 0.5 mg per day for
treating naïve HBeAg-positive and -negative patients at the dose of 1 mg per day for
patients with prior treatment with LAM (Lai 2005, Sherman 2008). ETV and LAM
are the only nucleoside analogs available as a tablet and an oral solution.
Treatment-naïve HBeAg-positive patients achieved undetectable HBV DNA
levels in 67% and 74% after one and two years of ETV treatment, reaching 94%
after five years, respectively (Figure 4, Figure 7) (Chang 2010). Long-term studies
in ETV responder patients demonstrated that response can be maintained in nearly
all patients over an observation period of up to six years. So far, the rate of
resistance at six years of treatment is estimated to be approximately 1.2% for
treatment-naïve patients (Tenney 2009). Loss of HBsAg occurs in 5% of treatmentnaïve individuals after two years of ETV therapy (Gish 2010). A non-randomised
Italian study in a mixed population of predominantly HBeAg-negative patients
could demonstrate undetectable HBV DNA levels in 91% and 97% of patients at 1
and 2 years of ETV treatment, respectively (Lampertico 2010).

144 Hepatology 2012
In LAM-resistant patients ETV is less potent. Only 19% and 40% of these
patients achieved undetectable HBV DNA after one and two years, respectively,
despite an increased dose of 1 mg/day (Gish 2007, Sherman 2008). Due to crossresistance up to 45% of patients with LAM resistance develop resistance against
ETV after 5 years of treatment (Tenney 2009).
ETV has a favourable tolerability profile and can be easily adjusted to renal
function. However, ETV may cause severe lactic acidosis in patients with impaired
liver function and a MELD score of >20 points (Lange 2009).

Figure 7. Percentage of patients achieving HBV DNA levels <400 copies/ml during longterm treatment with 1 mg ETV per day (Chang 2010). The long-term cohort ETV-901
consists of HBeAg-positive patients initially treated in the study ETV-022 (ETV 0.5 mg/day),
which was designed for a duration of one year.

Tenofovir (TDF). Tenofovir disoproxil fumarate, an ester prodrug form of
tenofovir (PMPA; (R)-9-(2-phosphonylmethoxypropyl)), is an acyclic nucleoside
phosphonate, or nucleotide analog closely related to ADV. TDF has selective
activity against retroviruses and hepadnaviruses and is currently approved for the
treatment of HIV infection and of chronic hepatitis B. TDF showed marked antiviral
efficacy over five years with complete virologic response rates (HBV DNA <400
copies/ml) reaching nearly 100% in treatment-naïve HBeAg-negative and -positive
patients (Figure 8). In HBeAg-positive patients, 11% of patients experienced
HBsAg loss (Marcellin 2011). Other clinical studies showing a high efficacy of
TDF in LAM-resistant HBV infections irrespective of the mutation mediating LAM
resistance (van Bömmel 2010, Levrero 2010). Due to possibly existing cross-

Hepatitis B Treatment 145
resistance to ADV, the efficacy of TDF might be hampered by the presence of ADV
resistance in patients with high HBV viremia; however, a breakthrough of HBV
DNA during TDF treatment in patients with previous ADV failure or in treatmentnaïve patients has not been observed (van Bömmel 2010, Levrero 2010, SnowLampert 2011).

Figure 8. Percentage of patients achieving HBV DNA levels <400 copies/mL during longterm treatment with 300 mg TDF per day (Marcellin 2010). Patients were originally
randomised to treatment with 300 mg TDF or 10 mg ADV per day. After one year, patients
receiving ADV were switched to TDF. Please note that the on-treatment analysis excluding the
missing patients showed undetectable HBV DNA in 96% of the TDF-TDF group and in 100% of
the ADV-TDF group.

TDF is generally well-tolerated and not associated with severe side effects. For
HBV-monoinfected, treatment-naïve patients, renal safety during TDF monotherapy
was investigated in three studies. In a randomized study comprising HBeAgnegative patients, none of 212 patients treated with TDF for three years and none of
112 patients who were treated with ADV for one year and then switched to TDF for
two years had a decrease in GFR to levels of <50 ml/min or an increase of serum
creatinine levels to >0.5 mg/dl (Marcellin 2009). In a similar study in HBeAgpositive patients, of 130 patients treated with TDF for 3 years and of 76 patients
treated with ADV for one year and consecutively with TDF for 2 years, only one
patients showed an increase in serum creatinine levels >0.5 mg/dl starting at year
two (Heathcote 2011). In a sub-analysis of both studies in 152 HBeAg-positive and
-negative Asian patients, no increase of serum creatinine >0.5 mg/dl or of eGFR
<50 ml/min was found in up to 3 years of TDF treatment (Liaw 2009a). In contrast,
in a recent study a benefit in renal function could be found in treated patients when
compared to untreated patients with HBV infection, which might reflect a lower
incidence of glomerulonephritis caused by HBsAg-induced immune complexes in
treated patients (Mauss 2011).
The use of tenofovir in HIV-coinfected patients is discussed in detail in Chapter
17.
Combination therapy as first-line treatment. As of now, first-line combination
treatments with nucleoside and nucleotide analogs or PEG-IFN α + nucleos(t)ide
analogs are not indicated. There is only one study comparing a combination therapy

146 Hepatology 2012
with LAM and ADV to LAM monotherapy in untreated patients (Sung 2008). In
this study, there was no difference in the virologic and biochemical response
between both groups. The rate of LAM resistance was much lower in the
combination group. However, the development of resistance could not be
completely avoided even with the use of an additional dose of ADV. Another study
analyzing the combination of LAM with LdT also showed no benefit for
combination therapy (Lai 2005).
Especially in patients with liver cirrhosis, a fast and complete suppression of
HBV replication is desirable. A monotherapy with ETV was found to be as safe and
effective as monotherapy with TDF, and an addition of emtricitabine to TDF
showed no improvement in response. Therefore, in these patients as well,
combination treatment is currently not recommended (Liaw 2011).
Combination treatment with LdT and PEG-INF α should not happen. In a recent
study, peripheral neuropathy was described in 9 of 48 (18.8%) patients who
received combination therapy of PEG-INF α and LdT and only in 10 of 3,500
(0.28%) patients who received LdT monotherapy (Goncalves 2009). Although
combination of LAM plus PEG-IFN α failed to demonstrate benefit when evaluated
at the end of follow-up in most studies, a more pronounced on-treatment virologic
response (week 48) was observed with combination therapy as compared to LAM or
PEG-IFN α alone. This more profound HBV DNA suppression induced by the
combination regimen was associated with a lower incidence of LAM resistance
(presence of resistance mutations in 1% vs. 18% at the end of therapy).
However, combination therapies between PEG-IFN α and more potent
nucleos(t)ide analogs may be attractive. Recently, a combination treatment of ETV
and PEG-IFN α after 4 years of complete response to ETV was superior to
continuation of ETV treatment by HBeAg and HBsAg loss and seroconversion rates
(Ning 2011). Similar studies are currently being undertaken investigating
combination treatment of PEG-IFN α and TDF. However due to the preliminary
character of the results a combination treatment of nucleos(t)ide analogs plus PEGINF α is still not recommended.

Choosing the right treatment option
One can choose either to treat with PEG-IFN α in order to induce a long-term
control by finite treatment or with nucleos(t)ide analogs to inhibit HBV replication
in the long-term (Figure 5).
At first, interferon therapy should be evaluated. However, if a patient does not
fulfil the criteria for PEG-IFN α, has contraindications, or is intolerant, long-term
therapy with nucleos(t)ide analogs is recommended. If a nucleos(t)ide analog is
chosen several parameters have to be considered prior to therapy: the antiviral
efficacy of the drug, the durability of response, the resistance barrier, and the stage
of liver disease.
If the initial viral load is low and liver cirrhosis has been excluded, any approved
drug may be used. The use of LAM, however, should be restricted to patients with
mild fibrosis and HBV DNA levels <2,000 IU/mL (or <105 copies/mL). For patients
with high-level HBV replication (>2 x 108 IU/mL or >109 copies/mL) only drugs
with a high genetic barrier should be used (i.e., ETV or TDF) (Table 3).

Hepatitis B Treatment 147

Prognostic factors for treatment response
Several factors are positively associated with long-term remission and may help to
guide treatment decisions. Pretreatment factors predictive of HBeAg seroconversion
are low viral load, high ALT levels (above 2-5 x ULN) and high histological
grading (Flink 2006, Hadziyannis 2006a, Lai 2007, Perrillo 1990, Perrillo 2002,
Wong 1993, Yuen 2007, Zoulim 2008). These general baseline predictors are
relevant especially for treatment regimens with PEG-IFN α but may in part be
relevant also for nucleos(t)ide analogs (Table 4).
A pooled analysis from the two largest trials using PEG-IFN α-2a or -2b in
chronic hepatitis B tried to calculate a score predicting successful interferon therapy
based on an individual patient’s characteristics (viral load, ALT level, HBV
genotype, age, gender). However, this approach may only be feasible in HBeAgpositive patients (Buster 2009).
Table 4. Predictors of response to antiviral therapy.
Peg-interferon α

Nucleos(t)ide analogs
Before treatment

Low viral load (HBV DNA ≤10 IU/mL), high serum ALT levels (above 3
times ULN), high activity scores on liver biopsy (at least A2)

During treatment

Undetectable HBV DNA in a
real-time PCR assay at 24 or 48
weeks is associated with HBeAg
seroconversion in HBeAgpositive patients and lower
incidence of resistance

7

HBeAg decrease
HBV genotype

HBV DNA decrease <20,000 IU/ml
at 12 weeks is associated with 50%
chance of HBeAg seroconversion
in HBeAg-positive patients and with
a 50% chance of sustained
response in HBeAg-negative
patients
HBeAg decrease at week 24 may
predict HBeAg seroconversion

HBV genotype shows no
influence on suppression of HBV
DNA levels.
HBsAg seroconversions only
observed for genotypes A and D

Association with HBV genotype A
and B and response to IFN α is
higher than with genotypes C and
D, however the association is weak
and HBV genotype should not be
the only argument for treatment
decision

HBV genotypes and treatment response. HBV genotypes have been shown to
be associated with IFN α treatment success. Patients with HBV genotype A,
prevalent in northern Europe and the US, show a much higher rate of HBeAg and
HBsAg seroconversion than patients with HBV genotype D, prevalent in the south
of Europe, or the HBV genotypes B or C originating from Asia (Keeffe 2007,
Wiegand 2008). During treatment with nucleos(t)ide analogs, suppression of HBV
replication and induction of HBeAg loss can be achieved regardless of the present
genotype. However, HBsAg loss was almost exclusively observed in patients with
genotypes A or D.
HBV DNA levels and treatment response. During antiviral therapy, the
decrease of HBV DNA levels from baseline is the most important tool in monitoring
treatment efficacy. Complete response to antiviral therapy is defined as suppression
of HBV DNA to below the limit of detection as measured by a sensitive real time
PCR assay (Figure 9). Incomplete suppression is characterized by persistent HBV

148 Hepatology 2012
replication despite antiviral therapy. Ongoing HBV replication should be avoided to
prevent the selection of resistant HBV strains by replication of the virus in the
presence of drug in the so-called “plateau phases”. An HBV DNA breakthrough
despite continuous antiviral therapy is often caused by viral resistance. Measuring
of HBV DNA kinetics early during therapy will help to guide antiviral treatment
and to establish early stopping rules or add-on strategies to avoid antiviral failure
(Figure 9).

Figure 9. Possible courses of HBV DNA levels during treatment with nucleoside or
nucleotide analogs. Incomplete suppression of HBV DNA results in either a “plateau phase” or
in a continuous slow decline. A plateau phase represents a high risk for selection of resistant
HBV variants, therefore treatment should be changed to a more effective agent or combination
therapy. A continuous slow decline should induce a treatment change after 6 months if drugs
with a low genetic barrier like LAM or LdT are used. If drugs with a high genetic barrier like ETV
or TDF are applied, a continuous slow decline can be monitored for at least 12 months without
increased risk of consecutive HBV resistance.

Incomplete or partial virologic response to oral nucleoside or nucleotide analogs
is defined as a decrease of HBV DNA >1 log10 but remaining measurable
(Lavanchy 2004) (Figure 9). The definition of partial response depends on the type
of treatment; thus, for agents with a high genetic barrier against resistance like ETV
or TDF partial response is defined after 12 months and for substances with a low
genetic barrier like LAM or LdT, after 6 months of monotherapy. In case of partial
response to a drug with a low genetic barrier, an appropriate rescue therapy should
be initiated. By current guidelines, a combination treatment with a nucleotide analog
is recommended for these patients. However, it was recently shown that patients
with partial response to LAM or to ADV have a high probability of responding to
TDF monotherapy, without risking the development of resistance (Heathcote 2011,
Marcellin 2011b, van Bömmel 2010, Berg 2010). Patients with a partial response to
ADV were also shown to have a high probability of responding to a subsequent

Hepatitis B Treatment 149
monotherapy with ETV, irrespective of the presence of mutations associated with
HBV resistance to ADV (Leung 2009, Leung 2009a).
For patients with partial response to a drug with a high genetic barrier as ETV or
TDF, current guidelines also recommend the initiation of a combination treatment.
Recently published long-term studies have shown that the continuation of a new
monotherapy in these patients does increase the percentage of patients with
undetectable HBV DNA without increasing the risk of development of resistance
(Chang 2010, Marcellin 2011b, Snow-Lampert 2011) (Figure 7, Figure 8). Thus,
during monotherapy with TDF in HBeAg-positive and HBeAg-negative patients, an
increase of patients with complete suppression of HBV DNA between the end of the
first and the end of the fifth year of treatment from 81% and 90% to 100% was
shown.
For monotherapy with ETV at 1 mg/day, an increase from 55% to 91% and 94%
after the fourth and fifth years was demonstrated (Chang 2010). In case of
incomplete viral suppression at week 48, a continuation of monotherapy with TDF
or ETV 1 mg is advisable as long as HBV DNA levels decrease continuously.
However, the debate on whether switching or adding a second drug as optimal
management is still unanswered.
Even though prolongation of monotherapy with ETV or TDF will probably lead
to undetectable HBV DNA in the long term in most patients, a fast suppression of
HBV replication is mandatory in some patients (e.g., those with liver cirrhosis) to
stop the progression of liver disease. For these patients, no definite therapeutic
strategies have been evaluated yet. Preliminary results of a study assessing the
efficacy of a rescue combination therapy with ETV and TDF have recently been
able to induce suppression to undetectable levels in most patients with partial
response; however, data on long-term efficacy and safety are not available (Petersen
2011).
In any case of treatment failure, adherence to therapy should be evaluated prior to
treatment modification. Elimination of HBV DNA during TDF-based therapeutic
regimes can drop from 87% to 71% of cases if adherence is not ensured, which is
also important in preventing drug resistance (Berg 2010).
Since only 30-35% of all patients treated with PEG-IFN α reach HBeAg
seroconversion after 48 weeks, studies have been conducted recently to predict the
probability of seroconversion in relation to viral kinetics. In one retrospective
analysis early prediction of stable seroconversion was possible by week 12 of
therapy if HBV DNA had reached levels below 5 log10 UI/mL within this short
treatment period (Fried 2005). In 53% of these patients, HBeAg seroconversion was
observed while patients with HBV DNA levels of 5 to 9 log10 copies/ml or levels
above 9 log10 IU/mL achieved HBeAg seroconversion in only 17% and 14%,
respectively.
Timepoint of HBeAg loss. In one study with 172 patients who were treated with
PEG-IFN α-2b as monotherapy or in combination with LAM, the loss of HBeAg
within the first 32 weeks of treatment was shown to be an on-treatment predictor for
HBsAg loss during a mean period of 3.5 years after the end of treatment. HBsAg
loss was found in 36% of the patients with early HBeAg loss and only in 4% of the
patients with HBeAg loss after 32 weeks of treatment (Buster 2009).

150 Hepatology 2012
HBsAg levels and treatment response. Response of HBeAg-positive and
HBeAg-negative patients to PEG-IFN treatment can be predicted by measuring
HBsAg levels before and changes of HBsAg levels during treatment (Figure 10).

Figure 10. On-treatment prediction of treatment response by HBsAg levels. In different
trials, an association of the decline in HBsAg levels within the first 12 weeks of PEG-IFN α
treatment and treatment response defined as HBV DNA levels <2,000 copies/mL six months
after treatment was found (Zonneveld 2010, Piratvis-uth 2011, Lau 2009, Gane 2011,
Rijckborst 2010, Moucari 2009). Patients showing no decline in HBsAg levels at week 12 had
only a very small chance of long-term response.

During PEG-IFN treatment for HBeAg-positive chronic HBV infection, an
absence of a decline in HBsAg levels at week 12 of treatment reduces the
probability of response to <5% in one study (Sonnefeld 2010). In the NEPTUNE
trial investigating the predictive value of HBsAg levels in 114 HBeAg-positive
patients receiving PEG-IFN α2a over 48 weeks, it was shown that in patients
achieving suppression of HBsAg to levels <1,500 IU/mL after 12 weeks of
treatment, the chance of reaching HBeAg seroconversion, suppression of HBV
DNA to levels <2000 IU/mL and HBsAg loss 6 months after treatment was 58%,
52% and 10%, compared to 42%, 31% and 0% in patients with HBsAg levels
between 1500-20,000. In this study, patients still showing HBsAg levels >20,000
IU/mL after 12 weeks of treatment achieved none of the endpoints (Gane 2011).
Beyond that, the probability of HBeAg loss rose to 68% in patients with elevation of
ALT levels >2 x the upper limit of normal at treatment initiation (Figure 11).

Hepatitis B Treatment 151

Figure 11. The level of HBsAg levels after 12 weeks of treatment with PEG-IFN α-2a is
predictive for HBeAg seroconversion six months after treatment. A combination of ALT
levels and HBsAg decline improves positive predictive value in these patients (Gane 2011).

Also in HBeAg-negative patients the decrease of HBsAg after 12 weeks of PEGIFN α treatment can predict long-term response. This prediction can be made even
more precise regarding the kinetics of both HBsAg and HBV DNA. In another
study comprising 48 patients who were treated with PEG-IFN α-2a, a decrease in
serum HBsAg levels of 0.5 and 1 log10 IU/mL at weeks 12 and 24 of therapy was
associated with a positive predictive value for HBsAg loss of 90% and 97% at week
96 after treatment, respectively (Moucari 2009).

Monitoring before and during antiviral therapy
Before therapy, HBV DNA levels should be measured with a highly sensitive assay.
These results should be confirmed 1-2 months after initiation of therapy. In
addition, ALT levels reflecting the inflammatory activity as well as creatinine levels
should be determined. HBV genotyping is only recommended in patients who are
considered candidates for treatment with interferon. HBV resistance testing can be
useful in patients with prior failure to more than one nucleoside/nucleotide analog,
but this is not yet a standard diagnostic approach. HBV resistance has to be
expected when an increase of HBV DNA of >1 log10 during antiviral treatment is
observed. In cases of primary treatment failure an appropriate second line treatment
can be chosen without resistance testing.
During therapy, HBV DNA, ALT and creatinine levels should be measured
initially, after 4 to 6 weeks and then every 3 months. The early identification of viral
resistance and an early adjustment of therapy are crucial. Patients with suppression
of HBV replication to <300 copies/ml (60 IU/ml) for at least 2 years may perhaps

152 Hepatology 2012
be scheduled at 6 month intervals (Table 5). However, no studies have been
performed that support this procedure.
In HBeAg-positive patients, HBeAg and anti-HBe as well as HBsAg and antiHBs should be also measured if HBV DNA levels become undetectable to identify
seroconversion as an endpoint of HBV therapy (Table 5).
Because the risk for HCC development remains increased even in patients with
complete suppression during long-term treatment with nucleos(t)ide analogs, these
patients should still regularly receive ultrasound examinations (Figure 12)
(Papatheodoridis 2011).
Table 5. Recommendation for laboratory tests for monitoring antiviral therapy.
Tests before antiviral treatment
HBV DNA quantitative
HBeAg, anti-HBe
HBsAg quantitative
HBV genotype
ALT level
Creatinine level
Other chemistry tests

Interval

If IFN-based treatment is planned
If IFN-based treatment is planned

Tests during antiviral treatment

Interval

HBV DNA quantitative

After 4-6 weeks, after 12 weeks, then every 3-6
months
3-6 months, if HBV DNA is undetectable
3-6 months, in HBeAg-positive patients after HBeAg
seroconversion in and HBeAg-negative patients if
DNA is undetectable
If HBV DNA increases >1 log during antiviral
treatment and pretreatment history is not tractable,
but first check for treatment adherence!
Initially every month, than every 3-6 months
Every 3-6 months
Every 3-6 months

HBeAg, anti-HBe
HBsAg, anti-HBs
HBV
HBV resistance test

ALT level
Creatinine level*
Other chemistry tests

* Patients treated with TDF should initially be monitored every 4 weeks to watch for decrease of
kidney function

Treatment duration and stopping rules
In HBeAg-positive patients continuous treatment with nucleos(t)ide analogs is
necessary as long as HBeAg seroconversion is not achieved. Even after
seroconversion antiviral therapy should be continued for at least another 12 months
to avoid the risk of “seroreversion” upon stopping the nucleos(t)ide analog therapy.
Criteria for optimal treatment duration with nucleos(t)ide analogs are still lacking
in patients with HBeAg-negative chronic hepatitis B, therefore currently unlimited
treatment with nucleos(t)ide analogs is recommended.
PEG-IFN α should be administered for 48 weeks in HBeAg-positive and negative patients.
Recently, the effect of stopping therapy after a long-term ADV treatment of 4 to 5
years with complete viral suppression was recently evaluated (Hadziyannis 2008).
Despite the fact that all patients suffered a slight virologic relapse within 3 months
of stopping therapy, most patients went below detection over the following 4 years

Hepatitis B Treatment 153
without any therapy. Moreover, 28% of the patients lost HBsAg. But final
recommendations about the treatment period with defined stopping rules do not
exist for HBeAg negative patients.
In patients with liver cirrhosis oral antiviral treatment should not be discontinued
at any time point because of the risk of liver decompensation during a virologic
rebound.

Figure 12. Cumulative incidence of hepatocellular carcinoma (HCC) in 818 patients with
HBeAg-negative chronic hepatitis B (CHB) treated with nucleos(t)ide analogs. Virological
remission defined as durable suppression of HBV DNA to levels <200 IU/mL did not
significantly affect the HCC incidence in the long term (p=0.38) (Papatheodoridis 2011).

Treatment of HBV infection in special populations
Pregnancy. For a neonate born to a mother with high levels of HBV DNA (>8
log10 copies/mL) the risk of perinatal transmission is elevated. Therefore, antiviral
treatment is principally recommended in these women. PEG-IFN α is not indicated
in pregnant women, but most nucleos(t)ide analogs can be used. The risk of
teratogenicity of nucleos(t)ide analogs is assessed by a classification based on data
gathered in clinical trials as well as through the FDA Pregnancy Registry. TDF and
LdT are listed as pregnancy category B drugs and LAM, whereas ADV and ETV as
category C drugs.
In pregnant women with high levels of HBV DNA, LAM treatment during the last
trimester of pregnancy was reported to reduce the risk of intrauterine and perinatal
transmission of HBV if given in addition to passive and active vaccination by HBIg
and HBV (van Zonneveld 2003). During treatment with TDF, the birth defect
prevalence was recently shown to be as high as during treatment with LAM (Brown
2009). Finally, LdT administered for an average of 15 weeks at the end of
pregnancy plus active-passive immunization to neonates reduced vertical
transmission rates from 23% to 4% over immunization alone (Han 2011). However,

154 Hepatology 2012
treatment with nucleos(t)ide analogs during pregnancy should be carefully
monitored and limited to the second and third trimester. As exacerbations of chronic
hepatitis B may occur, women with HBV should be monitored closely after delivery
(ter Borg 2008).
Immunosuppression. During immunosuppressive treatment, a reactivation of an
asymptomatic or inactive HBV infection can occur in 20% to 50% of patients (Lok
2009). Reactivations can occur in HBsAg carriers, but also in HBsAg-negative but
anti–hepatitis B core antibody (HBc)–positive patients. These reactivations are
characterised by increase in HBV replication followed by increase in liver
inflammation during immune reconstitution resulting in liver damage or even liver
failure in some patients (Feld 2010, Roche 2011).
HBV reactivation was especially frequently observed during treatment with
corticosteroids and antitumor necrosis factor therapies (i.e., infliximab, etanercept,
adalimumab), anti-CD20 therapies (i.e, rituximab-containing chemotherapeutics),
intra-arterial chemoembolisation for HCC (Vassilopoulos 2007, Moses 2006, Park
2005, Rutgeerts 2009). Reactivations during chemotherapy tend to appear
predominantly in men as well as in those undergoing treatments for breast cancer or
lymphoma.
Prior to initiating immunosuppressive therapies, screening for HBV infection is
recommended (Lok 2009, EASL 2009). Patients with baseline HBV DNA levels
<2,000 IU/mL should continue antiviral therapy for 6-12 months after the
discontinuation of chemotherapy/immunosuppression, while patients with baseline
HBV DNA levels >2,000 IU/mL should continue HBV therapy until they reach a
treatment endpoint.

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160 Hepatology 2012

10. Management of Resistance in HBV
Therapy
Stefan Mauss and Heiner Wedemeyer

Introduction
Interferon monotherapy has been the standard of care for chronic hepatitis B since
the mid-1990s. Primary resistance to interferon presents as lack of HBe or HBs
antigen loss or seroconversion. Interferon-induced immune control of HBV is less
frequently reported for HBV genotypes B, C and D than for HBV genotype A
(Erhardt 2005, Flink 2006). However, the development of resistance mutations to
interferon while on therapy has not been reported to date. Recently, in patients with
chronic hepatitis C, a genetic polymorphism at locus IL28B has been identified as a
host factor associated with response to interferon-based therapy (Ge 2009). If
similar host factors exist for response to interferon therapy in chronic hepatitis B,
they are not known. However, a recent paper did demonstrate an association of the
natural history of hepatitis B infection and genetic variants in the HLA-DP locus
(Kamatami 2009).
Since the introduction of lamivudine, treatment of chronic hepatitis B has been
characterised by a rapid increase in the number of available antiviral drugs, all
belonging to the class of HBV polymerase inhibitors (Figure 1). Due to better
tolerance and more convenient administration compared to interferon, HBV
polymerase inhibitors today account for the vast majority of prescribed therapies for
chronic hepatitis B in Western countries. However, due to the slow kinetics of
immune control, long-term suppression of HBV is needed, particularly in HBeAgnegative patients harbouring the precore mutant. This is due to the high relapse rate
after discontinuation of antiviral therapy in patients with precore mutants in the
absence of HBs antigen seroconversion. HBs antigen seroconversion is a rare event
in the first years of treatment.
For this reason, the understanding of resistance and cross-resistance of HBV
polymerase inhibitors is relevant in long-term treatment strategies. Suboptimal
antiviral therapy resulting in the development of early resistance will harm future
treatment options and lead to progressive liver disease, especially in those with

Management of Resistance in HBV Therapy 161
limited treatment options (Brunelle 2005, Kurashige 2009). In addition, some HBV
polymerase variants may interact with immunologically relevant epitopes of the
envelope resulting in immune escape mutants. These mutants may be able to
successfully infect vaccinated individuals. Although this finding is currently an in
vitro observation, any confirmation of this phenomenon in patients will result in a
serious public health concern, particularly in countries with a high prevalence of
hepatitis B.

Figure 1. Proportion of patients with undetectable HBV DNA after 48 or 52 weeks of
treatment. Data does not represent “head-to-head” trials (based on Heathcote 2007, Lai 2006,
Liaw 2009, Marcellin 2003, Marcellin 2007).

Antiviral HBV therapy – how to avoid resistance
Treatment of HBV is relatively safe and easy compared to hepatitis C treatment or
HIV therapy. But avoiding the induction of resistance is one of the critical efforts
that need to be made by physicians and patients. They need to choose the right
therapy and monitoring schedule, and pay close attention to good adherence.
Entecavir and tenofovir have proven efficacy and very little or no resistance in
treatment-naïve patients in the first years of therapy (Heathcote 2011,Yuen 2011).
In patients with limited HBV replication, telbivudine has also shown good results,
although in patients with high viral load treatment results can be compromised by
the development of resistance, also true for adefovir and lamivudine (Zeuzem
2009).
As previously stated, treating patients for longer periods with HBV polymerase
inhibitors can result in the development of viral resistance – particularly in patients
with less than optimal viral suppression (Lai 2006). In particular, lamivudine and
telbivudine are prone to developing resistance rapidly. Therapy with HBV
polymerase inhibitors needs to fully suppress viral replication (HBV DNA <300
copies/ml). HBV DNA should be monitored after the first 4-6 weeks of therapy to
assess adherence and then every 3-6 months while on therapy. If complete viral

162 Hepatology 2012
suppression determined by an ultrasensitive assay is not achieved on monotherapy
within the first 6 months on lamivudine, telbivudine or adefovir, treatment should
be switched to tenofovir or entecavir. In patients on either tenofovir or entecavir,
combination therapy with non-cross-resistant HBV polymerase inhibitors may be
considered after 12 months in case a plateau of viral replication is reached. There is
only one study to date showing a stronger efficacy of combination therapy in
patients with high viral load comparing entecavir monotherapy with entecavir plus
tenofovir (Lok 2011). For tenofovir the benefit of adding a second drug has not been
assessed prospectively.
Resistance to nucleoside polymerase inhibitors, i.e., lamivudine, telbivudine,
emtricitabine or entecavir, eliminates or markedly reduces antiviral efficacy of all
other nucleosides and may affect even nucleotide polymerase inhibitors due to
cross-resistance.
Resistance can also be associated with significant flares of hepatitis and has been
associated with a higher rate of clinical complications in one Asian study (Liaw
2004) and with a lower overall survival in an Italian cohort (DiMarco 2004).
Therefore, resistance needs to be avoided, particularly in patients with liver
cirrhosis. Based on these severe consequences of treatment failure, we would
recommend selecting a drug with a high genetic barrier for antiviral resistance in
cirrhotic individuals.

Treatment endpoints
In HBe antigen-positive patients infected with wild-type HBV strains HBeAg
seroconversion has been shown to be associated with a reduction in liver-associated
morbidity and increased survival (Niederau 1996). Thus, HBe antigen
seroconversion is considered a clinical endpoint in this patient population and
discontinuation of HBV polymerase inhibitors is recommended 6-12 months after
HBe antigen seroconversion in those who have not developed liver cirrhosis
(Cornberg 2007). HBe antigen loss is reported in up to 50% of patients treated with
HBV polymerase inhibitors after prolonged periods – several years - of therapy
(Hadziyannis 2006). Recent cohort data sheds some doubt on the durability of HBe
antigen seroconversion via therapy with polymerase inhibitors, with reported
relapse rates of about 50%, which is considerably higher than with interferoninduced HBe antigen seroconversion (Reijinders 2010).
Treatment with pegylated interferon alfa-2a for 48 weeks results in HBe antigen
seroconversion and a very low relapse rate in about a third of patients (Lau 2005).
Discontinuation of HBV polymerase inhibitor therapy in patients without HBe
antigen seroconversion usually results in relapse of chronic hepatitis B. With
interferon, the situation may become more complex and is at least partially
dependent on the HBV genotype in addition to the HBe antigen status (Erhardt
2005, Erhardt 2010).
HBV polymerase inhibitors treatment endpoints in HBe antigen-negative hepatitis
B in most cases are restricted to sustained normalisation of ALT levels, suppression
of HBV DNA and improvement in liver histology, as HBs antigen seroconversion is
rare with current treatment options. Consequently, treatment duration and endpoints
are more difficult to define in these patients. Reappearance of HBV DNA after
stopping HBV polymerase inhibitor treatment is observed in almost all patients,

Management of Resistance in HBV Therapy 163
even after fully suppressive treatment for multiple years (Marcellin 2004, Petersen
2011). Most guidelines therefore recommend indefinite treatment of HBe antigennegative patients without HBs antigen seroconversion.
PEG-IFN α-2a has also been studied in HBe antigen-negative hepatitis B leading
to a 6-month off-treatment response (HBV DNA <400 copies/ml) in up to 20% of
patients (Marcellin 2004). HBs antigen seroconversion happens in about 5% of
patients after a year of treatment with PEG-IFN. In addition, about 20% of patients
reach a low replicative status of their chronic hepatitis B, at least temporarily, after
interferon discontinuation (Bonino 2007). After an observational period of five
years after one year of interferon-based therapy, the seroconversion rate increases to
12% (Marcellin 2009). For HBV polymerase inhibitors HBs antigen seroconversion
has been reported for HBe antigen negative patients in less than 5% of patients in
published prospective studies.

Resistance patterns of HBV polymerase inhibitors
Lamivudine was the first approved HBV polymerase inhibitor. It is characterized by
good clinical tolerability, moderate antiviral efficacy and rather quick development
of resistance in cases of not fully suppressive antiviral therapy (Figure 2). Within
the first year of therapy up to 20% of patients may develop mutations in the YMDD
motif associated with loss of activity against HBV. About 70-80% of patients
without HBe antigen seroconversion develop lamivudine-resistant variants after
four or more years of therapy (Figure 2).

Figure 2. Cumulative incidence of HBV polymerase inhibitor resistance. These numbers
are average estimates based on numerous studies. Resistance rates differ between trials
and cohorts. Overall, resistance rates have been higher in HBe antigen-positive patients than in
HBe antigen-negative patients. Long-term data for adefovir has only been reported for HBe
antigen-negative patients and thus resistance rates may be even higher for HBe antigenpositive individuals. Data for entecavir is biased since both patients with best responses (e.g.,
HBe antigen seroconversion) and patients with suboptimal virological responses (>700,000
copies/ml after one year of treatment) were withdrawn from the study.

164 Hepatology 2012
Lamivudine mutations confer cross-resistance to telbivudine, emtricitabine and
entecavir. Preliminary data indicate that the development of multiple lamivudineassociated mutations may even reduce the efficacy of tenofovir therapy (Lada
2008).
Emtricitabine has comparable antiviral properties and a similar resistance profile
to lamivudine (Lim 2006). However it is only approved as an antiretroviral
medication for HIV, not for treatment of chronic hepatitis B. In HBV, its use is
mainly limited as part of combination therapy with tenofovir in HIV-coinfected
patients with an indication for antiretroviral therapy.
Telbivudine has shown superior antiviral efficacy compared to lamivudine in HBe
antigen-positive and -negative patients. However, development of resistance is
considerable in naïve patients with highly replicative hepatitis B and the resistance
pattern is essentially the same as that of lamivudine, resulting in complete crossresistance of the two compounds (Liaw 2009, Zeuzem 2009) (Table 1). Outcomes
are better and antiviral efficacy more sustained in patients with an HBV DNA of
less than 106 IU/ml (Zeuzem 2009). Combination therapy of telbivudine and
lamivudine does not improve the antiviral efficacy nor does it delay the
development of resistance compared to telbivudine monotherapy (Lai 2005).

Figure 3. Resistance patterns of different antiviral drugs used for the treatment of
chronic hepatitis B. The numbers indicate the respective amino acid position in the HBV
polymerase gene. For entecavir, resistance at positions 204/180 plus an additional mutation at
position 184, 202 or 250 is required to lead to clinically significant drug resistance. Most but not
all variants have been shown to be associated with drug resistance both in vitro and in vivo.

Adefovir was the second approved HBV polymerase inhibitor. It has full activity
in lamivudine-resistant patients. However, its antiviral potency is limited by its
nephrotoxicitiy. Due to tubular damage of the kidney, the approved dose is limited
to 10 mg/day, although 30 mg/day showed superior antiviral efficacy (Marcellin
2003). The reduced antiviral potency is counterbalanced, however, by a favourable
resistance profile. Development of resistance occurs later and to a lesser extent
compared to lamivudine or telbivudine (Figure 3), although resistance to adefovir

Management of Resistance in HBV Therapy 165
may occur more often in patients with pre-existing lamivudine resistance (Lee
2006). No association of response to treatment with HBV genotypes was evident in
the registrational trials (Westland 2003).
Table 1. Recommendations in secondary treatment failure of HBV polymerase
inhibitors.
Resistance against
nucleoside analogs

Recommended therapeutic option

Lamivudine
Telbivudine
Entecavir

Tenofovir, adefovir*
Tenofovir, adefovir*
Tenofovir, adefovir*

Resistance against
nucleotide analogs

Recommended therapeutic option

Adefovir (LAM-naïve)
Adefovir (LAM-resistant)
Tenofovir (no in vivo data available)
*in case tenofovir is not available

Entecavir, tenofovir, (telbivudine), (lamivudine)
tenofovir
Entecavir, (telbivudine), (lamivudine)

Adefovir-resistant or non-responding HBV strains seem to respond to tenofovir
with a slower viral decline, but without signs of true cross-resistance (Berg 2008,
Van Bömmel 2010). Adefovir resistant strains respond fully to entecavir therapy
(Reijinders 2010).
The combination of adefovir plus lamivudine in the presence of lamivudine
resistance delays the development of adefovir resistance considerably compared to
switching to adefovir monotherapy (Lampertico 2006, Lampertico 2007).
Entecavir is an HBV nucleoside polymerase inhibitor with good antiviral efficacy
and slow development of resistance in treatment-naïve patients (Chang 2006, Lai
2006, Lampertico 2009). This is due to the fact that more than one mutation in the
HBV polymerase gene is required to confer resistance to entecavir. However,
entecavir shares some resistance mutations with lamivudine and telbivudine. The
presence of lamivudine resistance mutations at L180M, M204I, L180M + M204V
facilitates the development of resistance to entecavir because only one additional
mutation is required for the development of full resistance. As a result, in contrast to
treatment of naïve patients where entecavir is clearly superior to lamivudine, its
antiviral potency is markedly reduced in patients with lamivudine resistance and up
to 40% of lamivudine-resistant patients develop full entecavir resistance after 3
years of treatment (Tenney 2007, Colonno 2007).
Patients with resistance only to adefovir have favourable treatment results with
entecavir, while patients with combined adefovir and lamivudine resistance do not
respond well to entecavir monotherapy (Reijnders 2007, Nguyen 2009, Chloe 2009,
Shim 2009).
Tenofovir is approved for the treatment of HIV and HBV. Early data from HBV/
HIV-coinfected patients showed a strong antiviral potency and slow development of
resistance (Núñez 2002, Nelson 2003, van Bommel 2004). In its registrational trials,
tenofovir was superior to adefovir resulting in substantially higher rates of full viral
suppression in HBe antigen-positive (tenofovir 69% vs. adefovir 9%, HBV DNA
<40 IU/ ml) and HBe antigen-negative patients (tenofovir 91% vs. adefovir 56%
HBV DNA <40 IU/ml) at 52 weeks of therapy (Heathcote 2009, Marcellin 2008). In

166 Hepatology 2012
HIV-positive patients, anecdotal cases of renotubular dysfunction were reported.
Otherwise tenofovir is well-tolerated. It is active in lamivudine-resistant patients
(Schmutz 2006, Manns 2009). So far, no obvious resistance patterns to tenofovir
associated with antiviral failure in trials and cohorts have been observed (SnowLampart 2010).
The acquisition of adefovir resistance mutations and multiple lamivudine
resistance mutations may impair the activity of tenofovir (Fung 2005, Lada 2008,
van Bömmel 2010), although even in these situations tenofovir retains activity
against HBV (Berg 2008, Petersen 2009).

Combination therapy of chronic hepatitis B to
delay development of resistance
Combination therapy is thought to be superior to monotherapy, particularly in
patients with highly replicative hepatitis B (HBV DNA >109 copies/ml). However,
so far the response rate in trials assessing the long-term efficacy of tenofovir and
entecavir show a long-acting antiviral effect even in patients with high viral load
and little to no development of resistance (Snow-Lampart 2011). Trials assessing de
novo combination therapy versus monotherapy are limited. The experience with
combining telbivudine and lamivudine suggests that combinations of two nucleoside
analogs with an overlapping resistance profile do not have an additive antiviral
effect (Lai 2005). In contrast, combining a nucleoside with a nucleotide polymerase
inhibitor with different resistance profiles may be of benefit (Sung 2008, Lok 2011).
Trials that will provide more evidence on how to best use the current antiviral
options are currently underway or are being designed. However, these trials may
require larger patient numbers than currently included and may need longer
observational periods due to agents like entecavir and tenofovir having such
considerable efficacy as monotherapy. However, it should be remembered that – in
contrast to HIV – immune control of HBV is possible, limiting the duration of
therapy in particular in HBe antigen-positive patients. With the availability of HBV
polymerase inhibitors with high resistance barriers, even treatment-naïve patients
with high levels of HBV replication should be treated initially with one drug. In
patients with considerable viral replication despite good adherence a possible option
is to add a non-cross-resistant drug in order to maximise viral suppression and to
avoid development of resistance.

Management of drug resistance
Primary and secondary treatment failure has to be distinguished in the treatment of
hepatitis B. A clinically sufficient primary response after 6 months is defined by a
reduction of HBV DNA to at least <103 copies/ml (200 IU/ml) or by a continuous
drop of HBV DNA through month 12. In contrast, if a rise in HBV DNA by one log
or more is observed while on antiviral therapy, a secondary resistance or nonadherence is very likely to be present. HBV resistance usually arises several months
before biochemical relapse with elevation of transaminases, thus regular HBV DNA
monitoring is required during antiviral therapy (e.g., every 3 months) (Cornberg
2007). Testing for variants associated with resistance might be useful if HBV DNA
levels rise during treatment.

Management of Resistance in HBV Therapy 167
Most viral breakthroughs in treatment-naïve patients on entecavir or tenofovir are
the result of adherence issues. Therefore, patient adherence should be assessed
before genotypic resistance testing is done.
Additional compensatory mutations can develop if monotherapy is continued
despite HBV resistance, thereby broadening the possibilities of cross-resistance
(Locarnini 2004). Knowledge of the antiviral efficacy, the resistance barrier, and the
resistance profile of each available oral antiviral is a prerequisite for the rational use
of nucleos(t)ide analogs for hepatitis B. In the case of resistance to a nucleoside
analog (lamivudine, telbivudine, emtricitabine, entecavir), early replacement by
tenofovir or add-on treatment with adefovir (if tenofovir is not available) is
recommended. In the opposite scenario, a nucleoside addition to current nucleotide
treatment should happen if adefovir or tenofovir treatment failure begins to occur
(Figure 4). In the case of adefovir, switching from adefovir to tenofovir should be
assessed as an additional measure.
Historically, most data generated has been from patients with lamivudine
resistance. In this setting the advantage of adding adefovir rather than switching to
adefovir is well-established (Lampertico 2005, Lampertico 2007). Moreover,
adefovir should be added early at low HBV DNA levels, when a rise in HBV DNA
has been confirmed but before a biochemical relapse has occurred. Today, the most
appropriate strategy may be a switch to tenofovir with or without continuation of
lamivudine (Manns 2009).

Figure 4. Mutations in the HBV polymerase. Due to the overlapping reading frame between
HBV polymerase and envelope sequences, mutations in the HBV polymerase, in particular at
codons 173, 180 and 204, may lead to changes in the conformation of immunodominant
domains of the HBV envelope.

168 Hepatology 2012

Special considerations in HIV/HBV coinfection
In patients with chronic hepatitis B and HIV coinfection, the first question to ask is
if there is an indication for antiretroviral therapy. In patients with no such indication
interferon or an HBV polymerase inhibitor without HIV activity are options. The
initially recommended monotherapy with entecavir is now considered obsolete – the
anti-HIV activity of entecavir has been described (M184V) in anecdotal cases
(MacMahon 2007). Currently, adefovir and telbivudine are recommended, based on
limited in vivo data for adefovir or in vitro data and some anecdotal case reports for
telbivudine (Delaugerre 2002, Sheldon 2005, Avilla 2009, Milazzo 2009). As both
drugs have limitations in the setting of HBV-monoinfected patients the initiation of
antiretroviral therapy allowing the use of tenofovir plus lamivudine/emtricitabine
should be considered, in particular in patients with advanced liver fibrosis.
In patients with an indication for antiretroviral therapy, a regimen containing
tenofovir with or without lamivudine or emtricitabine is favored in order to avoid
development of lamivudine or emtricitabine resistance in HBV. The incidence of
HBV resistance in patients treated with lamivudine after two years is about 50% in
HIV/HBV-coinfected patients (Benhamou 1999). In patients who have already
developed lamivudine-resistant HBV, tenofovir should be added to or replace
lamivudine for HBV treatment (Schmutz 2006). Whether entecavir should be added
in patients on tenofovir +/– emtricitabine/lamivudine or replace tenofovir in case of
renal impairment should be decided on an individual basis (Ratcliffe 2011).
A change of antiretroviral regimen in HBV/HIV-coinfected patients due to the
development of HIV resistance must take the HBV infection into consideration, as
the chronic hepatitis B may be exacerbated in the absence of an active HBV
polymerase inhibitor.
More information on this topic can be found in Chapter 17.

Immune escape and polymerase inhibitor
resistance
Another relevant but unexpected consequence of lamivudine resistance is the
induction of conformational changes in the HBs antigen due to an overlapping
reading frame in the genetic sequence of the HBV polymerase and the HBs antigen
(Figure 4). Because of this, mutations in the HBV polymerase may induce changes
in the envelope of the virus resulting in an altered immunogenicity. This may result
in vaccine escape mutants. In vitro and ex vivo studies support this hypothesis,
which may have important public health implications (Mathews 2006, Sheldon
2007). Studies in chimpanzees have indeed confirmed that infections with druginduced HBV variants are possible despite the presence of high anti-HBs levels that
were considered protective in the vaccinated host (Kamili 2009).
In addition to humoral escape, lamivudine resistance may also affect cellular
immunity against HBV. As mentioned earlier, suboptimal antiviral therapy, e.g.,
with lamivudine, especially in high prevalence countries, could undermine the
success of vaccination efforts leading to a spread of HBV vaccine escape mutants.
The YMDD motif is also part of an MHC class I restricted CTL epitope. YMDDspecific cytotoxic T lymphocytes may partially cross-react with YVDD and YIDD
variants (Lin 2005) and thereby contribute to a prevention of emergence of

Management of Resistance in HBV Therapy 169
resistance. However, more studies are needed to explore in detail the consequences
of the development of viral resistance to polymerase inhibitors for T cell immunity
against HBV.

Conclusion
In summary, therapy with HBV polymerase inhibitors to date is limited to two
active subclasses with different resistance profiles. Resistance due to suboptimal
treatment, i.e., on only one drug, can eliminate or reduce the effect of other drugs
due to partial or complete cross-resistance. This sequence is well documented for
lamivudine, telbivudine and entecavir. The superiority of de novo combination
therapy for HBV over sequential monotherapy may be likely for patients with very
high HBV viremia, but still has to be confirmed in well-designed prospective
clinical trials. In patients with low or intermediate viremia, the risk for development
of resistance is rather low when using drugs with a high genetic barrier and when a
rapid suppression of HBV replication is achieved (Figure 5).
The choice of first-line treatment strategy will determine future treatment options;
being judicious is paramount, as suboptimal therapeutic approaches can result in a
rapid exhaustion of options within just a few years.

Figure 5. Antiviral potency and genetic resistance barrier of currently approved HBV
polymerase inhibitors.

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174 Hepatology 2012

11. Hepatitis D – Diagnosis and Treatment
Heiner Wedemeyer

Introduction
Hepatitis delta is considered the most severe form of viral hepatitis in humans. The
hepatitis delta virus (HDV) is a defective RNA virus which requires the hepatitis B
virus (HBV) surface antigen (HBsAg) for complete replication and transmission,
while the full extent of the HBV helper function is unexplored (Rizzetto 1983,
Taylor 2006). Hence, hepatitis delta occurs only in HBsAg-positive individuals
either as acute coinfection or as superinfection in patients with chronic hepatitis B
(Wedemeyer 2010) (Figure 1). Several studies have shown that chronic HDV
infection leads to more severe liver disease than chronic HBV monoinfection with
an accelerated course of fibrosis progression, possibly a slightly increased risk of
hepatocellular carcinoma and early decompensation in the setting of established
cirrhosis (Hughes 2011, Fattovich 2000, Fattovich 1987). Simultaneous HBV and
HDV infection has also been shown to be more severe than infection with HBV
alone in chimpanzees (Dienes 1990). An easy to apply clinical score has been
suggested to predict the likelihood of experiencing a clinical event for patients with
hepatitis delta, the baseline-event-anticipation (BEA) score (Calle Serrano 2011). So
far, only (pegylated) interferon α treatment has been shown to exert some antiviral
activity against HDV and has been linked to improve the long-term outcome. Data
on the use of pegylated interferon confirm earlier findings, PEG-IFN leads to
sustained virological response rates in about one quarter of patients. Alternative
treatment options including HBV entry inhibitors and prenylation inhibitors are
currently in clinical development.

Hepatitis D – Diagnosis and Treatment 175

Figure 1. Courses of hepatitis delta.

Virology of hepatitis delta
The HDV virion is approximately 36 nm large, containing HDV RNA and delta
antigen. HDV RNA is single-stranded, highly base-paired, circular and by far the
smallest known genome of any animal virus, containing close to 1700 nucleotides
(Taylor 2006). It is coated with the envelope protein derived from the pre-S and S
antigens of the hepatitis B virus. The HDV RNA has six open reading frames
(ORFs), three on the genomic and three on the antigenomic strand. One ORF codes
for the hepatitis delta antigen (HDAg), while the other ORFs do not appear to be
actively transcribed. Two HDAgs exist: the small HDAg (24 kD) is 155 amino acids
long and the large HDAg (27 kD) is 214 amino acids long. A single nucleotide
change (A-G) in the small HDAg sequence leads to the synthesis of the large
HDAg. The small HDAg accelerates genome synthesis, while the large HDAg that
inhibits HDV RNA synthesis is necessary for virion morphogenesis (Taylor 2006).
Replication of HDV RNA occurs through a ‘double rolling circle model’ in which
the genomic strand is replicated by a host RNA polymerase to yield a multimeric
linear structure that is then autocatalytically cleaved to linear monomers and ligated
into the circular HDV RNA viral progeny.
Genetic analysis has revealed the presence of at least eight HDV genotypes
(Hughes 2011) (Figure 2). Genotype 1 is the most frequently seen genotype and is
distributed throughout the world, especially in Europe, the Middle East, North
America and North Africa. Genotype 2 is seen in East Asia and the Yakutia region
of Russia, and genotype 3 is seen exclusively in the northern part of South America,
especially in the Amazon Basin. Genotype 4 is seen in Taiwan and Japan and
genotypes 5-8 in African countries. Genotype 1 is associated with both severe and
mild disease whereas genotype 2 causes a milder disease over a long-term course
(Su 2006). All patients who have been included in the large European HIDT-I

176 Hepatology 2012
treatment trial in Germany, Turkey and Greece were infected with HDV genotype I
(Zachou 2010).

Figure 2. Prevalence of HDV genotypes.

Epidemiology of hepatitis delta
Hepatitis delta is not an uncommon disease. Being linked to HBV, HDV is spread in
the same way as HBV, mainly through parenteral exposure (Niro 1999). It is highly
endemic in Mediterranean countries, the Middle East, Central Africa, and northern
parts of South America (Hughes 2011) (Figure 2). In Western countries, high antiHDV prevalence is found in HBsAg-positive intravenous drug users both in Europe
(Wedemeyer 2007, Gaeta 2000) and North America (Kurcirka 2010). Worldwide,
more than 350 million people are chronically infected with HBV and 15-20 million
of those are estimated to be anti-HDV positive (Wedemeyer 2010). Delta hepatitis
was endemic in Southern Europe. Several studies performed in the 1980s and 1990s
showed a prevalence of anti-HDV among HBsAg-positive individuals of more than
20%. As a result of the implementation of HBV vaccination programs, the incidence
of HDV infections significantly decreased in Southern Europe in the 1990s (Gaeta
2000) (Figure 3). In Turkey, HDV prevalence in HBsAg-positive patients range
from <5% in western Turkey to >27% in southeast Turkey (Degertekin 2008). Other
countries with a particularly high prevalence of hepatitis delta are Mongolia with up
to one third of chronic hepatitis cases being caused by HDV infection (Tsatsralt-Od
2005), some Central Asian republics, northwestern states of Brazil, and some
Polynesian islands (Hughes 2011). Of note, prevalence rates of HBV and HDV are
not linked - for example, HDV infections are rather rare in most parts of mainland
China despite very high frequencies of hepatitis B.

Hepatitis D – Diagnosis and Treatment 177

Figure 3. Prevalence of hepatitis D virus in Italy and Germany.
a)
b)

Chronic Hepatitis D: a vanishing disease. From Gaeta GB, Hepatology 2000.
Hepatitis D virus infection - Not a vanishing disease in Europe! From Wedemeyer,
Hepatology 2007.

Chronic delta hepatitis still represents a significant health burden in Central
Europe – in particular due to immigration from highly endemic areas (Wedemeyer
2007, Erhardt 2003) (Figure 4, Table 1). In our experience at a referral center for
liver disease, about 8-10% of HBsAg-positive patients still test positive for antiHDV (Figure 3). More than three quarters of our delta hepatitis patients were not
born in Germany. However, the geographical origin of our patients has changed
during the last decade. While until the mid-1990s the majority of HDV-positive
patients were born in Turkey, the proportion of Eastern European patients has
significantly increased in recent years (Wedemeyer 2007) (Table 1). Similarly, high
HDV prevalence in immigrant populations has been described in clinics in the UK
(Cross 2008), France and Italy (Le Gal 2007, Mele 2007). HDV can also be found
in high frequencies in HBsAg-positive HIV-infected individuals with about 14.6%
in different European regions (Soriano 2011).

178 Hepatology 2012

Figure 4. Diagnostic steps in delta hepatitis.

Table 1. Hepatitis D virus (HDV) detection in Germany between 1992–1996 vs 1997–
2006 compared to the country of birth of patients.
Origin of patients

HDV diagnosis*
1992–1996
n=45

HDV diagnosis*
1997–2006
n=100

p-value

Germany

20.9 (n=9)

19.2 (n=19)

n.s.

Turkey

42.0 (n=18)

20.2 (n=20)

0.009

Eastern Europe/NIS

14.0 (n=6)

37.3 (n=37)

0.005

* in %. From Heidrich, Journal of Viral Hepatitis, 2009; n.s., not significant; NIS, Newly Independent
States (ex-USSR)

Pathogenesis of HDV infection

Knowledge about the pathogenesis of delta hepatitis infection is limited. Clinical
observations have provided examples of mostly an immune-mediated process in
delta hepatitis disease. However, patterns suggesting a cytopathic viral disease have
occasionally been observed. A typical example of the latter were outbreaks of
severe hepatitis in the northern part of South America (Nakano 2001). These mostly
fulminant hepatitis cases were induced by genotype 3 delta virus. However, in the
usual case of delta hepatitis the liver histology is not different from a patient with
hepatitis B or hepatitis C with accompanying necroinflammatory lesions.
Importantly, HDV viremia is not directly associated with the stage of liver disease
(Zachou 2010). Cellular immune responses against the hepatitis D virus have been
described (Nisini 1997, Aslan 2003, Huang 2004, Grabowski 2011) suggesting that
the quantity and quality of T cell responses may be associated with some control of
the infection. Some data from our group indicate that the frequency of cytotoxic
CD4+ T cells is higher in delta hepatitis patients than in individuals with HBV or

Hepatitis D – Diagnosis and Treatment 179
HCV infection (Aslan 2006) and that HDV-specific IFN gamma and IL-2 responses
are more frequent in patients with low HDV viremia (Grabowski 2011). This still
limited information suggests that HDV is mainly an immune-mediated disease, at
least in HDV genotype 1 infection. Ideally, antiviral therapies should therefore also
aim to enhance anti-HDV immunity to confer long-term control of the infection.
Still, sterilizing immunity against HDV has not been demonstrated yet. Of note,
chimpanzees that have recovered from HDV infection were successfully reinfected
with HDV in one study performed in the 1980s (Negro 1988). Coinfections with
multiple hepatitis viruses are associated with diverse patterns of reciprocal
inhibition of viral replication (Raimondo 2006, Wedemeyer 2010). HDV has
frequently been shown to suppress HBV replication (Jardi 2001, Sagnelli 2000).
Between 70% and 90% of delta hepatitis patients are HBeAg-negative with low
levels of HBV DNA. Humanized HBsAg-positve mice that become superinfected
HDV also show a decrease in HBV replication (Lutgehetmann 2011). A molecular
explanation for the suppression of HBV replication by HDV has been suggested as
HDV proteins p24 and p27 may repress HBV enhancers (Williams 2009). However,
viral dominance may change over time (Wedemeyer 2010) and about half of the
hepatitis delta patients showed significant HBV replication in one study (Schaper
2010).
There is increasing evidence that HDV not only suppresses HBV replication but
also HCV replication in triple-infected patients. In our experience, less than one
fifth of anti-HCV/HBsAg/anti-HDV-positive individuals are positive for HCV RNA
(Heidrich 2009). We even observed a case where acute HDV/HBV superinfection
led to clearance of chronic hepatitis C infection (Deterding 2009). It is not clear how
many anti-HCV-positive/HCV RNA-negative patients recover from HCV infection
and how many patients just show a suppressed HCV replication in the context of
viral coinfections.

Clinical course of delta hepatitis
Acute HBV/HDV coinfection
Acute HBV/HDV coinfection leads to recovery in more than 90% of cases but
frequently causes severe acute hepatitis with a high risk for developing a fulminant
course (Rizzetto 2009). In contrast, HDV is cleared spontaneously only in a
minority of patients with HDV superinfection of chronic HBsAg carriers (Figure 1).
The observation that histopathology of simultaneous HBV and HDV infection is
more severe than in infection with HBV alone has also been documented in
experiments with chimpanzees (Dienes 1990). Several outbreaks of very severe
courses of acute delta hepatitis in patients have been described in different regions
of the world (Casey 1996, Flodgren 2000, Tsatsralt-Od 2006). Fortunately, acute
delta hepatitis has become rather infrequent over the last two decades in Western
countries due to the introduction of vaccination programs.

Chronic delta hepatitis
Several studies have shown that chronic HDV infection leads to more severe liver
disease than chronic HBV monoinfection, with an accelerated course of fibrosis
progression, and early decompensation in the presence of cirrhosis (Fattovich 1987,
Jardi 2001, Sagnelli 2000, Rizzetto 2000, Uzunalimoglu 2001). HDV accounts for

180 Hepatology 2012
almost half of all cases of liver cirrhosis and hepatocellular carcinoma in southeast
Turkey (Degertekin 2008, Uzunalimoglu 2001, Yurdaydin 2006a). An observational
study from Taiwan has reported a cumulative survival of HDV genotype 1-infected
patients of as low as 50% after 15 years (Su 2006). Long-term follow-up data from
Italy confirm the particularly severe course of hepatitis delta (Romeo 2009; Niro
2010). HDV infection has also been associated with a higher risk of developing
liver cirrhosis in HIV-coinfected patients as 66% of HIV/HBV/HCV/HDV-infected
patients but only 6% of HBV/HCV/HIV-infected patients present with liver
cirrhosis in a Spanish cohort (Castellares 2008). Similarly, delta hepatitis was
associated with poorer survival in HIV-infected patients in Taiwan (Sheng 2007).
An easy to apply clinical score, the baseline-event anticipation (BEA) score, has
been suggested to predict the risk of developing liver-related morbidity and
mortality (Calle Serrano 2011). Factors associated with a poor long-term outcome
included age above 40, male sex, low platelet counts, high bilirubin and INR values
and southeast Mediterranean origin.

Diagnosis of delta hepatitis
We recommend that every HBsAg-positive patient be tested for anti-HDV
antibodies at least once. There is currently no evidence that direct testing for HDV
RNA in the absence of anti-HDV is of any use. A positive result for anti-HDV does
not necessarily indicate “active” delta hepatitis, as HDV RNA can become negative
indicating recovery from HDV infection. Over the long term as well, anti-HDV
antibodies can be lost after recovery. However, anti-HDV may persist for years
even when the patient has experienced HBsAg seroconversion (Wedemeyer 2007).
“Active” replicative delta hepatitis should be confirmed by the detection of HDV
RNA. If HDV RNA is positive, subsequent evaluation of grading and staging of
liver disease, surveillance for hepatocellular carcinoma and consideration of
antiviral treatment is indicated. HDV RNA quantification is offered by some
laboratories. However, so far there is no evidence that HDV RNA levels correlate
with any clinical marker of liver disease (Zachou 2010). HDV RNA quantification
is useful in particular if antiviral treatment is indicated. Stopping rules during
antiviral treatment depending on the level of antiviral decline are currently being
evaluated. Patients with less than a 3 log10 decline of HDV RNA after 24 weeks of
treatment will not benefit from antiviral treatment with PEG-IFN α-2b (Erhardt
2006). There is currently no WHO standard for HDV. Various PCR assays have
been presented recently, however, the performance of these assays may differ
significantly and detection rates for rare HDV genotypes may be low (Mederacke
2010, Niro 2011).
HDV genotyping is performed by some research labs and may help to identify
patients with a higher or lower risk of developing end-stage liver disease (Su 2006).
In Western countries almost all patients are infected with HDV-genotype 1, thus
genotyping may be considered only in immigrants or populations with mixed
genotype prevalence.
In the 1980s and 1990s the diagnosis of active delta hepatitis was dependent on
anti-HDV IgM testing. Anti-HDV IgM testing might still be useful in patients who
test HDV RNA negative but have evidence of liver disease, which cannot be
explained by other reasons. Due to the variability of the HDV genome and the lack

Hepatitis D – Diagnosis and Treatment 181
of standardization of HDV RNA assays, HDV RNA may test false negative or be
under the detection limit of the assay in the case of fluctuating viral load. In these
cases, HDV RNA testing should be repeated and anti-HDV IgM testing might be
performed, if available. Anti-HDV IgM levels also correlate with disease activity
and may be predictive for response to IFN α-based antiviral therapy (Mederacke
2012, in press).
As delta hepatitis only occurs in the context of HBV coinfection, a solid work-up
of HBV infection including HBV DNA quantification and HBeAg/anti-HBe
determination is warranted. Most hepatitis delta patients in Europe are infected with
HBV genotype D but infection with genotype A can also occur (Soriano 2011)
which may have significant implications for treatment decisions as HBV genotype
A shows a better responses to interferon α therapy (Janssen 2005). Similarly, testing
for anti-HCV and anti-HIV is mandatory. In our experience, up to one third of antiHDV-positive patients also test positive for anti-HCV (Heidrich 2009).

Treatment of delta hepatitis
Nucleoside and nucleotide analogs
Several nucleoside and nucleotide analogs used for the treatment of HBV infection
have been shown to be ineffective against HDV (Table 2). Famciclovir, used in the
1990s to treat HBV infection (Wedemeyer 1999), had no significant antiviral
activity against HDV in a Turkish trial (Yurdaydin 2002). Similarly, lamivudine
was ineffective in trials of delta hepatitis (Wolters 2000, Niro 2005a, Yurdaydin
2008, Lau 1999b). Ribavirin alone or in combination with interferon also did not
lead to increased rates of HDV RNA clearance (Niro 2005a, Gunsar 2005, Garripoli
1994). However, a long-term observational study of HIV-infected individuals
receiving HAART followed HBV/HDV/HIV-coinfected individuals for a median of
more than 6 years; over this time, a decline of HDV RNA from 7 log10 to 5.8 log10
was observed and 3 out of 16 patients became HDV RNA-negative (Sheldon 2008).
Thus, very long treatment with HBV polymerase inhibitors may lead to beneficial
effects in delta hepatitis possibly due to a reduction of HBsAg levels. Future longterm trials will need to confirm these data in triple-infected individuals.
Table 2. Treatment options in delta hepatitis.
Nucleos(t)ide Analogs
Famciclovir ineffective

Yurdaydin 2002

Lamivudine ineffective

Wolters 2000, Lau 1999, Niro 2005a,
Niro 2008, Yurdaydin 2008
Niro 2006, Garripoli 1994, Gunsar 2005

Ribavirin ineffective

Interferon α
Sustained biochemical responses in 0-36% of patients Farci 1994, Di Marco 1996, Niro 2005b,
Yurdaydin 2008
Few studies with virological endpoints
Treatment >12 months may be required
Higher IFN doses were associated with better survival Farci 2004
in small study cohort

Another promising and surprising alternative to the currently approved HBV
polymerase inhibitors may be clevudine. Clevudine, a nucleoside analog currently

182 Hepatology 2012
in development for the treatment of hepatitis B, has recently been shown to inhibit
delta virus viremia in woodchucks (Casey 2005). However, a first pilot trial showed
no significant HDV RNA declines (Yakut 2010).

Recombinant interferon α
Interferon α has been used for the treatment of delta hepatitis since the mid 1980s
(Rizzetto 1986). Since then, many trials have explored different durations and doses
of interferon α in HDV-infected patients. However, data are difficult to compare as
endpoints are different in the trials and few studies have followed HDV RNA levels
over time (Niro 2005b).
One randomized Italian study on the use of high dose interferon α associated a
beneficial long-term outcome in delta hepatitis patients with high dose interferon
treatment (Farci 1994, Farci 2004). Some studies have used extended doses of
interferon treatment and it seems that two years of treatment is superior in terms of
HDV RNA clearance (Niro 2005b). In one NIH case report, 12 years of interferon
treatment led finally to resolution of both HDV infection and HBsAg clearance (Lau
1999a). High doses of interferon and extended treatment are tolerated by only a
minority of patients and treatment options are very limited for the majority (Manns
2006).

Pegylated interferon α
Pegylated interferon has been used in small trials to treat delta hepatitis, with
sustained virological response rates of about 20% (Castelnau 2006, Niro 2006,
Erhardt 2006) (Table 3).
Table 3. Pegylated interferon in delta hepatitis.
Study

Outcome

Castelnau, Hepatology
2006

12 months of PEG-IFN α-2b (n=14)

SVR in 6 patients (43%)

Niro, Hepatology 2006

72 weeks of PEG-IFN α-2b (n=38)
– Monotherapy: n=16
– PEG-INF + ribavirin during first 48
weeks: n=22

SVR in 8 patients (21%)
Ribavirin had no
additional effect

Erhardt, Liver Int 2006

48 weeks of PEG-IFN α-2b (n=12)

SVR in 2 patients (17%)

Wedemeyer, NEJM 2011

a) 48 weeks PEG-IFN α-2a
+ adefovir (n=31) or
b) PEG-IFN α-2a + placebo (n=29) or
c) Adefovir (n=30)

HDV RNA-negative
Group a) 23%
Group b) 24%
Group c) 0%

Results of the Hep-Net International Delta hepatitis Intervention Trial (HIDIT-1)
were published in 2011 (Wedemeyer 2011). 90 patients (42 in Germany, 39 in
Turkey and 9 in Greece) with chronic HDV infection and compensated liver disease
were randomized to receive either 180 µg PEG-IFN α-2a QW plus 10 mg adefovir
dipivoxil QD (group A, N=31), 180 µg PEG-IFN α-2a QW plus placebo (group B,
N = 29) or 10 mg adefovir dipivoxil qd alone (group C, N=30) for 48 weeks. HBV
DNA and HDV RNA were investigated by real-time PCR. Ten patients did not
complete 48 weeks of therapy because of disease progression (N=6) or interferon-

Hepatitis D – Diagnosis and Treatment 183
associated side effects (N=4). Both PEG-IFN groups showed a significantly higher
reduction in mean HDV RNA levels than the adefovir monotherapy group by week
48. HDV RNA was negative 24 weeks after the end of treatment in 28% of patients
receiving PEG-IFN but in none of patients treated with adefovir alone. While
patients receiving PEG-IFN α-2a alone or adefovir monotherapy had similar mean
HBsAg levels at week 0 and week 48, the PEG-IFN α-2a/adefovir combination
group showed a 1.1 log10 IU/ml decline of HBsAg levels by week 48 (p <0.001)
with 10/30 patients achieving a decline in HBsAg of more than 1 log (10) IU/ml.
These data are in line with a report from Greece of a significant decline in HbsAg
levels in delta hepatitis patients receiving long-term treatment with interferon α
(Manesis 2007).
Overall the HIDIT-1 study showed that (i) PEG-IFN α-2a displays a significant
antiviral efficacy against HDV in more than 40% of patients with 24% becoming
HDV RNA negative after 48 weeks; (ii) adefovir dipivoxil has little efficacy in
terms of HDV RNA reduction but may be considered for patients with significant
HBV replication; (iii) combination therapy of PEG-IFN α-2a plus adefovir has no
advantages for HBV DNA or HDV RNA reduction; (iv) a combination therapy of
pegylated interferon with adefovir was superior to either monotherapy in reducing
HBsAg levels in HBV-infected patients (Wedemeyer 2011). However, adefovir
treatment was associated with a decline in glomerular filtration rates (Mederacke
2012, in press) and thus PEG-IFN α/adefovir combination treatment cannot be
recommended as the first-line treatment for all patients with hepatitis delta.
Currently, additional trials are ongoing to investigate the efficacy of PEG-IFN α2a in combination with tenofovir for the treatment of delta hepatitis. First data of the
HIDIT-2 trial are expected in 2012. Moreover, alternative treatment options are
currently being explored. Among these, prenylation inhibitors may be promising
(Bordier 2003). HDV replication depends on a prenylation step and prenylation
inhibitors have already been developed for the treatment of malignancies. The HBV
entry inhibitor Myrcludex-B is also being developed for hepatitis delta. MyrcludexB is a lipopeptide derived from the preS1 domain of the HBV envelope (Petersen
2008) and has been shown to hinder HDV infection in uPA/SCID mice transplanted
with human hepatocytes (Lütgehetmann 2011).

Liver transplantation for hepatitis delta
Liver transplantation remains the ultimate treatment option for many hepatitis delta
patients with end-stage liver disease. Hepatitis delta patients have lower risk for
reinfection after transplantation than HBV monoinfected patients (Samuel 1993). If
prophylaxis by passive immunization with anti-HBs antibodies and administration
of HBV polymerase is applied, HBV/HDV reinfection can be prevented in all
individuals (Rosenau 2007) leading to an excellent long-term outcome after
transplantation. HDV RNA levels rapidly decline during the first days after
transplantation (Mederacke 2011) but HDAg may persist in the transplanted liver
for several years (Smedile 1998, Mederacke 2011). Long-term prophylaxis to
prevent HBV reinfection should be recommended in patients transplanted for
hepatitis delta patients as reinfection may lead to HDV reactivation for which
treatment options are very limited.
More information on hepatitis delta for physicians and patients can be found on
the website of the Heptitis Delta International Network: www.hepatitis-delta.org

184 Hepatology 2012

Figure 5. Treatment algorithm for hepatitis delta.

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Hepatitis C: Diagnostic Tests 189

12. Hepatitis C: Diagnostic Tests
Christian Lange and Christoph Sarrazin

Common symptoms of hepatitis C like fatigue, muscle ache, loss of appetite or
nausea are unspecific and, in many cases, mild or not present. Consequently,
hepatitis C is often diagnosed accidentally and, unfortunately, remains heavily
under-diagnosed. It is estimated that only 30-50% of individuals infected with HCV
are aware of their disease and can take advantage of treatment options and avoid the
risk of further transmission of the virus (Deuffic-Burban 2010). Untreated hepatitis
C advances to a chronic state in up to 80% of people, which leads to liver cirrhosis
in 20-40% with an accompanying risk of hepatic decompensation, hepatocellular
carcinoma and death (McHutchison 2004). In light of these facts, HCV diagnostics
should be performed thoroughly in all patients presenting with increased
aminotransferase levels, with chronic liver disease of unclear etiology and with a
history of enhanced risk of HCV transmission (i.e., past IV or nasal drug
dependency, transmission of blood or blood products before the year 1990, major
surgery before 1990, needle stick injuries, non-sterile tattoos or piercings, enhanced
risk of sexual transmission).
For the diagnosis of hepatitis C both serologic and nucleic acid-based molecular
assays are available (Scott 2007). Serologic tests are sufficient when chronic
hepatitis C is expected, with a sensitivity of more than 99% in the 3rd generation
assays. Positive serologic results require HCV RNA measurement in order to
differentiate between chronic hepatitis C and resolved HCV infection from the past.
When acute hepatitis C is considered, serologic screening alone is insufficient
because anti-HCV antibodies may develop late after transmission of the virus. In
contrast, HCV RNA is detectable within a few days of infection, making nucleic
acid-based tests mandatory in diagnosing acute hepatitis C. HCV RNA
measurement is furthermore essential in the determination of treatment indication,
duration and success (Terrault 2005). The latter has to be confirmed at clearly
defined times during treatment to decide whether therapy should be continued or
not. It should be repeated 24 weeks after treatment completion to assess whether a
sustained virologic response (SVR) has been achieved. Both qualitative and
quantitative HCV RNA detection assays are available. Qualitative tests are highly
sensitive and are used for diagnosing hepatitis C for the first time, for the screening
of blood and organ donations and for confirming SVR after treatment completion.
Quantitative HCV RNA detection assays offer the possibility of measuring the viral
load exactly and are essential in treatment monitoring. Qualitative and quantitative

190 Hepatology 2012
HCV RNA assays are now being widely replaced by real-time PCR-based assays
that can detect HCV RNA over a very wide range, from low levels of approximately
10 IU/ml up to 10 million IU/ml.
After diagnosing hepatitis C, the HCV genotype should be determined by nucleic
acid-based techniques in every patient considered for HCV therapy, because the
currently recommended treatment schedules and durations as well as the specific
ribavirin doses differ among the genotypes.
Morphological methods like immunohistochemistry, in situ hybridization or PCR
from liver specimens play no relevant role in the diagnosis of hepatitis C because of
their low sensitivity, poor specificity and low efficacy compared to serologic and
nucleic acid-based approaches.

Serologic assays
In current clinical practice, antibodies against multiple HCV epitopes are detected
by commercially available 2nd and 3rd generation enzyme-linked immunoassays
(EIAs). In these tests, HCV-specific antibodies from serum samples are captured by
recombinant HCV proteins and are then detected by secondary antibodies against
IgG or IgM. These secondary antibodies are labelled with enzymes that catalyse the
production of coloured, measurable compounds.
The first applied EIAs for the detection of HCV-specific antibodies were based on
epitopes derived from the NS4 region (C-100) and had a sensitivity of 70–80% and
a poor specificity (Scott 2007). C-100-directed antibodies occur approximately 16
weeks after viral transmission. 2nd generation EIAs additionally detect antibodies
against epitopes derived from the core region (C-22), NS3 region (C-33) and NS4
region (C-100), which leads to an increased sensitivity of approximately 95% and to
a lower rate of false-positive results. With these assays HCV-specific antibodies can
be detected approximately 10 weeks after HCV infection (Pawlotsky 2003). To
narrow the diagnostic window from viral transmission to positive serological
results, a 3rd generation EIA has been completed by an antigen from the NS5 region
and/or the substitution of a highly immunogenic NS3 epitope. This innovation
allows the detection of anti-HCV antibodies approximately four to six weeks after
infection with a sensitivity of more than 99% (Colin 2001). Anti-HCV IgM
measurement can narrow the diagnostic window in only a minority of patients. AntiHCV IgM detection is also not sufficient to discriminate between acute and chronic
hepatitis C because some chronically infected patients produce anti-HCV IgM
intermittently and not all patients respond to acute HCV infection by producing
anti-HCV IgM.
The specificity of serologic HCV diagnostics is difficult to define since an
appropriate gold standard is lacking. It is evident, however, that false-positive
results are more frequent in patients with rheuma-factors and in populations with a
low hepatitis C prevalence, for example in blood and organ donors. Although
several immunoblots for the confirmation of positive HCV EIA results are
available, these tests have lost their clinical importance since the development of
highly sensitive methods for HCV RNA detection. Immunoblots are mandatory to
make the exact identification of serologically false-positive-tested individuals
possible. Importantly, the sensitivity of immunoblotting is lower compared to EIAs,
which bears the risk of the false-negatively-classifying of HCV-infected individuals.

Hepatitis C: Diagnostic Tests 191
False-negative HCV antibody testing may occur in patients on hemodialysis or in
severely immunosuppressed patients like in HIV infection or in hematological
malignancies.

HCV core antigen assays
In principle, detection of the HCV core antigen in serum could be a cheaper
alternative to nucleic acid testing for the diagnosis and management of hepatitis C.
However, the introduction of a reliable and sensitive HCV core antigen assay is
burdened with a number of difficulties like the development of specific monoclonal
antibodies recognizing all different HCV subtypes and the need for accumulation
and dissociation of HCV particles from immune complexes to increase sensitivity.
The first HCV core antigen detection system (trak-C, Ortho Clinical Diagnostics)
became commercially available in the US and Europe several years ago. This HCV
core antigen assay proved highly specific (99.5%), genotype independent, and had a
low inter- and intra-assay variability (coefficient of variation 5–9%) (Veillon 2003).
HCV core antigen is measurable 1–2 days after HCV RNA becomes detectable. The
limit of detection is 1.5 pg/ml (approximately 10,000–50,000 IU/ml HCV RNA). In
a study of anti-HCV antibody and HCV RNA- positive patients presenting in an
outpatient clinic, 6/139 people (4%) were HCV core antigen negative. In these
patients, HCV RNA concentrations were 1300–58,000 IU/ml highlighting the
limitations of the HCV core antigen assay as confirmation of ongoing hepatitis C in
anti-HCV-positive patients. As a consequence, this first HCV core antigen assay
was withdrawn from the market.
More recently, another quantitative HCV core antigen assay (Architect HCV Ag,
Abbott Diagnostics), a further development of the previous assay, was approved by
the EMA. This assay comprises 5 different antibodies to detect HCV core antigen, is
highly specific (99.8%), equally effective for different HCV genotypes, and shows a
relatively low sensitivity for determination of chronic hepatitis C (corresponding to
600-1000 IU/ml HCV RNA). However, HCV core antigen correlated well but not
fully linearly with HCV RNA serum levels, and false-negative results were obtained
in patients with impaired immunity (Mederacke 2009, Medici 2011). Taken
together, the new HCV core antigen assay could be a cheaper, though somewhat
less sensitive alternative for nucleic acid testing. For careful monitoring of treatment
with standard dual combination therapies or directly acting antiviral agents,
prospective studies have to be performed to determine proper rules and time points
for response-guided treatment algorithms.

Nucleic acid testing for HCV
Until 1997, HCV quantitative results from various HCV RNA detection systems did
not represent the same concentration of HCV RNA in a clinical sample. Because of
the importance of an exact HCV RNA load determination for management of
patients, the World Health Organization (WHO) established the HCV RNA
international standard based on international units (IU) which is used in all
clinically applied HCV RNA tests. Other limitations of earlier HCV RNA detection
assays were false-negative results due to polymerase inhibition, for example by drug
interference, false-positive results due to sample contamination because the reaction
tubes had to be opened frequently, or due to under- and over-quantification of

192 Hepatology 2012
samples of certain HCV genotypes (Morishima 2004, Pawlotsky 2003, Pawlotsky
1999). Currently, several HCV RNA assays are commercially available (Table 1).
Table 1. Commercially available HCV RNA detection assays.
Assay

Distributor

Technology

Approval status

Qualitative HCV RNA detection assays
Amplicor™ HCV 2.0

Roche Molecular Systems

PCR

FDA, CE

Versant™ HCV

Siemens Medical
Solutions Diagnostics

TMA

FDA, CE

Amplicor™ HCV Monitor 2.0 Roche Molecular Systems

PCR

CE

HCV SuperQuant™

National Genetics Institute

PCR

Versant™ HCV RNA 3.0

Siemens Medical
Solutions Diagnostics

bDNA

Quantitative HCV RNA detection assays

FDA, CE

Cobas Ampliprep/ High pure Roche Molecular Systems
system /Cobas TaqMan

Real-time PCR FDA, CE

Abbott RealTime™ HCV

Real-time PCR FDA, CE

Abbott Diagnostics

Qualitative assays for HCV RNA detection
Until recently, qualitative assays for HCV RNA had substantially lower limits of
detection in comparison with quantitative HCV RNA assays. The costs of a
qualitative assay are also lower compared to a quantitative assay. Therefore,
qualitative HCV RNA tests are used for the first diagnosis of acute hepatitis C, in
which HCV RNA concentrations are fluctuating and may be very low, as well as for
confirmation of chronic hepatitis C infection in patients with positive HCV
antibodies. In addition, they are used for the confirmation of virologic response
during, at the end of, and after antiviral therapy, as well as in screening blood and
organ donations for presence of HCV.

Qualitative RT-PCR
In reverse transcriptase-PCR- (RT-PCR-) based assays, HCV RNA is used as a
matrix for the synthesis of a single-stranded complementary cDNA by reverse
transcriptase. The cDNA is then amplified by a DNA polymerase into multiple
double-stranded DNA copies. Qualitative RT-PCR assays are expected to detect 50
HCV RNA IU/ml or less with equal sensitivity for all genotypes.
The Amplicor™ HCV 2.0 is an FDA- and CE-approved RT-PCR system for
qualitative HCV RNA testing that allows detection of HCV RNA concentrations
down to 50 IU/ml of all genotypes (Table 1) (Nolte 2001). The DNA polymerase of
Thermus thermophilus used in this assay provides both DNA polymerase and
reverse transcriptase activity and allows HCV RNA amplification and detection in a
single-step, single-tube procedure.

Transcription-mediated amplification (TMA) of HCV RNA
TMA-based qualitative HCV RNA detection has a very high sensitivity (Hendricks
2003, Sarrazin 2002). TMA is performed in a single tube in three steps: target
capture, target amplification and specific detection of target amplicons by a
hybridization protection assay. Two primers, one of which contains a T7 promoter,

Hepatitis C: Diagnostic Tests 193
one T7 RNA polymerase and one reverse transcriptase, are necessary for this
procedure. After RNA extraction from 500µl serum, the T7 promoter-containing
primer hybridises the viral RNA with the result of reverse transcriptase-mediated
cDNA synthesis. The reverse transcriptase also provides an RNase activity that
degrades the RNA of the resulting RNA/DNA hybrid strand. The second primer
then binds to the cDNA that already contains the T7 promoter sequence from the
first primer, and a DNA/DNA double-strand is synthesised by the reverse
transcriptase. Next, the RNA polymerase recognizes the T7 promoter and produces
100-1000 RNA transcripts, which are subsequently returned to the TMA cycle
leading to exponential amplification of the target RNA. Within one hour,
approximately 10 billion amplicons are produced. The RNA amplicons are detected
by a hybridisation protection assay with amplicon-specific labelled DNA probes.
The unhybridised DNA probes are degraded during a selection step and the labelled
DNA is detected by chemiluminescence.
A commercially available TMA assay is the Versant™ HCV RNA Qualitative
Assay. This system is accredited by the FDA and CE and provides an extremely
high sensitivity, superior to RT-PCR-based qualitative HCV RNA detection assays
(Hofmann 2005, Sarrazin 2001, Sarrazin 2000). The lower detection limit is 5-10
IU/ml with a sensitivity of 96-100%, and a specificity of more than 99.5%,
independent of the HCV genotype.

Quantitative HCV RNA detection
HCV RNA quantification can be achieved either by target amplification techniques
(competitive and real-time PCR) or by signal amplification techniques (branched
DNA (bDNA) assay) (Table 1). Several FDA- and CE-approved standardised
systems are commercially available. The Cobas Amplicor™ HCV Monitor is based
on a competitive PCR technique whereas the Versant™ HCV RNA Assay is based
on a bDNA technique. More recently, the Cobas® TaqMan® assay and the Abbott
RealTime™ HCV test, both based on real-time PCR technology, have been
introduced. The technical characteristics, detection limits and linear dynamic
detection ranges of these systems are summarized below. Due to their very low
detection limit and their broad and linear dynamic detection range, they have
already widely replaced the previously used qualitative and quantitative HCV RNA
assays.

Competitive PCR: Cobas® Amplicor HCV 2.0 monitor
The Cobas® Amplicor HCV 2.0 monitor is a semi-automated quantitative detection
assay based on a competitive PCR technique. Quantification is achieved by the
amplification of two templates in a single reaction tube, the target and the internal
standard. The latter is an internal control RNA with nearly the same sequence as the
target RNA with a clearly defined initial concentration. The internal control is
amplified by the same primers as the HCV RNA. Comparison of the final amounts
of both templates allows calculation of the initial amount of HCV RNA. The
dynamic range of the Amplicor HCV 2.0 monitor assay is 500 to approximately
500,000 IU/ml with a specificity of almost 100%, independent of the HCV
genotype (Konnick 2002, Lee 2000). For higher HCV RNA concentrations predilution of the original sample is required.

194 Hepatology 2012

Branched DNA hybridisation assay (Versant® HCV RNA 3.0
quantitative assay)
Branched DNA hybridisation assay is based on signal amplification technology.
After reverse transcription of the HCV RNA, the resulting single-stranded
complementary DNA strands bind to immobilised captured oligonucleotides with a
specific sequence from conserved regions of the HCV genome. In a second step,
multiple oligonucleotides bind to the free ends of the bound DNA strands and are
subsequently hybridised by multiple copies of an alkaline phosphatase-labelled
DNA probe. Detection is achieved by incubating the alkaline phosphatase-bound
complex with a chemiluminescent substrate (Sarrazin 2002). The Versant HCV
RNA assay is at present the only FDA- and CE-approved HCV RNA quantification
system based on a branched DNA technique. The lower detection limit of the
current version 3.0 is 615 IU/ml and linear quantification is ensured between 615–
8,000,000 IU/ml, independent of the HCV genotype (Morishima 2004). The bDNA
assay only requires 50 µl serum for HCV RNA quantification and is currently the
assay with the lowest sample input.

Real-time PCR-based HCV RNA detection assays
Real-time PCR technology provides optimal features for both HCV RNA detection
and quantification because of its very low detection limit and broad dynamic range
of linear amplification (Sarrazin 2006) (Figure 1). Distinctive for real-time PCR
technology is the ability to simultaneously amplify and detect the target nucleic
acid, allowing direct monitoring of the PCR process. RNA templates are first
reverse-transcribed to generate complementary cDNA strands followed by a DNA
polymerase-mediated cDNA amplification.

Figure 1. Detection limits and linear dynamic ranges of commercially available HCV RNA
detection assays.

DNA detection simultaneous to amplification is preferentially achieved by the use
of target sequence-specific oligonucleotides linked to two different molecules, a
fluorescent reporter molecule and a quenching molecule. These probes bind the

Hepatitis C: Diagnostic Tests 195
target cDNA between the two PCR primers and are degraded or released by the
DNA polymerase during DNA synthesis. In case of degradation the reporter and
quencher molecules are released and separated, which results in the emission of an
increased fluorescence signal from the reporter. Different variations of this principle
of reporter and quencher are used by the different commercially available assays.
The fluorescence signal, intensified during each round of amplification, is
proportional to the amount of RNA in the starting sample. Quantification in absolute
numbers is achieved by comparing the kinetics of the target amplification with the
amplification kinetics of an internal control of a defined initial concentration.
Highly effective and almost completely automated real-time PCR-based systems
for HCV RNA measurement have been introduced. For replacement of the
qualitative TMA and the quantitative bDNA-based assays, a real-time based PCR
test (Versant® kPCR Molecular System) is being developed.
All commercially available HCV RNA assays are calibrated to the WHO standard
based on HCV genotype 1. Significant differences between different RT-PCR
assays and other quantitative HCV RNA tests have been reported - in the case of the
real-time PCR-based assays a slight under-quantification by one assay and a slight
over-quantification by the other, in comparison to the WHO standard by Cobas®
TaqMan®. In addition, it has been shown that results may vary significantly between
assays with different HCV genotypes despite standardisation to IU (Chevaliez
2007, Vehrmeren 2008).

Cobas® TaqMan® HCV test
The FDA- and CE-accredited Cobas® TaqMan® (CTM) assay uses reporter- and
quencher-carrying oligonucleotides specific to the 5’UTR of the HCV genome and
to the template of the internal control, a synthetic RNA for binding the same primers
as for HCV RNA. Reverse transcription and cDNA amplification is performed by
the Z05 DNA polymerase. For HCV RNA extraction from serum or plasma
samples, a Cobas® TaqMan® assay was developed either in combination with the
fully automated Cobas® Ampliprep (CAP) instrument using magnetic particles, or in
combination with manual HCV RNA extraction with glass fiber columns using the
high pure system (HPS) viral nucleic acid kit. The current versions of both
combinations have a lower detection limit of approximately 10 IU/ml and a linear
amplification range of HCV RNA from approximately 40 to 10,000,000 IU/ml.
Samples from HCV genotypes 2-5 have been shown to be under-quantified by the
first version of the HPS-based Cobas® TaqMan® assay. The recently released
second version of this assay has now demonstrated equal quantification of all HCV
genotypes (Colucci 2007). For the Cobas® Ampliprep/ Cobas® TaqMan®
(CAP/CTM) assay significant under-quantification of HCV genotype 4 samples has
been shown. However, a prototype second version of this assay was able to
accurately quantify HCV RNA samples from patients infected with all HCV
genotypes, including HCV genotype 4 transcripts with rare sequence variants that
had been under-quantified by the first generation assay (Vermehren 2011). The
second version CAP/CTM assay will be commercially available in 2012. Taken
together, the Cobas® TaqMan® assay makes both highly sensitive qualitative and
linear quantitative HCV RNA detection feasible with excellent performance in one
system with complete automation.

196 Hepatology 2012

RealTime HCV test
The CE-accredited RealTime HCV Test uses reporter- and quencher-carrying
oligonucleotides specific for the 5’UTR as well. HCV RNA concentrations are
quantified by comparison with the amplification curves of a cDNA from the
hydroxypyruvate reductase gene from the pumpkin plant Curcurbita pepo, which is
used as an internal standard. This internal standard is amplified with different
primers from those of the HCV RNA, which may be the reason for the linear
quantification of very low HCV RNA concentrations. The RealTime HCV Test
provides a lower detection limit of approximately 10 IU/ml, a specificity of more
than 99.5% and a linear amplification range from 12 to 10,000,000 IU/ml
independent of the HCV genotype (Vehrmeren 2008; Michelin 2007; Sabato 2007).
In a recent multi-centre study, its clinical utility to monitor antiviral therapy of
patients infected with HCV genotypes 1, 2 and 3 has been proven and the FDA
consequently approved the RealTime HCV Test (Vermehren 2011). In this study,
highly concordant baseline HCV RNA levels as well as highly concordant data on
rapid and early virologic response were obtained compared to reference tests for
quantitative and qualitative HCV RNA measurement, the Versant® HCV
Quantitative 3.0 branched DNA hybridization assay and the Versant® HCV RNA
Qualitative assay, respectively.

HCV genotyping
HCV is heterogeneous with an enormous genomic sequence variability, developed
due to a rapid replication cycle with the production of 1012 virions per day and the
low fidelity of the HCV RNA polymerase. Six genotypes (1-6), multiple subtypes
(a, b, c…) and most recently a seventh HCV genotype have been characterized.
These genotypes vary in approximately 30% of their RNA sequence with a median
variability of approximately 33%. HCV subtypes are defined by differences in their
RNA sequence of approximately 10%. Within one subtype, numerous quasispecies
exist and may emerge during treatment with specific antivirals. These quasispecies
are defined by a sequence variability of less than 10% (Simmonds 2005). Because
the currently recommended treatment durations and ribavirin doses depend on the
HCV genotype, HCV genotyping is mandatory in every patient who considers
antiviral therapy (Bowden 2006). For triple therapies with HCV protease inhibitors
and future multiple direct acting antiviral combination therapies, determination of
HCV subtypes is of importance because of significant different barriers to resistance
on the HCV subtype level.
Both direct sequence analysis and reverse hybridisation technology allow HCV
genotyping. Initial assays were designed to analyse exclusively the 5’ untranslated
region (5’UTR), which is burdened with a high rate of misclassification especially
on the subtype level. Current assays were improved by additionally analyzing the
coding regions, in particular the genes encoding the non-structural protein NS5B
and core protein, both of which provide non-overlapping sequence differences
between the genotypes and subtypes (Bowden 2006).

Hepatitis C: Diagnostic Tests 197

Reverse hybridising assay (Versant® HCV Genotype 2.0
System (LiPA))
In reverse hybridising, biotinylated cDNA clones from HCV RNA are produced by
reverse transcriptase and then transferred and hybridised to immobilised
oligonucleotides specific to different genotypes and subtypes. After removing
unbound DNA by a washing step, the biotinylated DNA fragments can be detected
by chemical linkage to coloured probes.
The Versant® HCV Genotype 2.0 System is suitable for indentifying genotypes 16 and more than 15 different subtypes and is currently the preferentially used assay
for HCV genotyping. By simultaneous analyses of the 5’UTR and core region, a
high specificity is achieved especially to differentiate the genotype 1 subtypes. In a
study evaluating the specificity of the Versant® HCV Genotype 2.0 System, 96.8%
of all genotype 1 samples and 64.7% of all genotype samples were correctly
subtyped. No misclassifications at the genotype level were observed. Difficulties in
subtyping occurred in particular in genotypes 2 and 4. Importantly, none of the
misclassifications would have had clinical consequences, which qualifies the
Versant® HCV Genotype 2.0 System as highly suitable for clinical decision-making
(Bouchardeau 2007).

Direct sequence analysis (Trugene® HCV 5’NC genotyping
kit)
The TruGene® assay determines the HCV genotype and subtype by direct analysis
of the nucleotide sequence of the 5’UTR region. Incorrect genotyping rarely occurs
with this assay. However, the accuracy of subtyping is poor because of the exclusive
analyses of the 5’UTR (Pawlotsky 2003).

Real-time PCR technology (RealTime™ HCV Genotype II
assay)
The current RealTime HCV Genotype II assay is based on real-time PCR
technology, which is less time consuming than direct sequencing. Preliminary data
revealed a 96% concordance at the genotype level and a 93% concordance on the
genotype 1 subtype level when compared to direct sequencing of the NS5B and
5’UTR regions. Nevertheless, single genotype 2, 3, 4, and 6 isolates were
misclassified at the genotype level, indicating a need for assay optimization (Ciotti
2010).

Implications for diagnosing and managing acute
and chronic hepatitis C
Diagnosing acute hepatitis C
When acute hepatitis C is suspected, the presence of both anti-HCV antibodies and
HCV RNA should be tested. For HCV RNA detection, sensitive qualitative
techniques with a lower detection limit of 50 IU/ml or less are required, for example
TMA, qualitative RT-PCR or the newly developed real-time PCR systems. Testing
for anti-HCV alone is insufficient for the diagnosis of acute hepatitis C because
HCV specific antibodies appear only weeks after viral transmission. In contrast,

198 Hepatology 2012
measurable HCV RNA serum concentrations emerge within the first days after
infection. However, HCV RNA may fluctuate during acute hepatitis C, making a
second HCV RNA test necessary several weeks later in all negatively tested patients
with a suspicion of acute hepatitis C. When HCV RNA is detected in seronegative
patients, acute hepatitis C is very likely. When patients are positive for both antiHCV antibodies and HCV RNA, it may be difficult to discriminate between acute
and acutely exacerbated chronic hepatitis C. Anti-HCV IgM detection will not
clarify because its presence is common in both situations.

Diagnosing chronic hepatitis C
Chronic hepatitis C should be considered in every patient presenting with clinical,
morphological or biological signs of chronic liver disease. When chronic hepatitis C
is suspected, screening for HCV antibodies by 2nd or 3rd generation EIAs is
adequate because their sensitivity is >99%. False-negative results may occur rarely
in immunosuppressed patients (i.e., HIV) and in patients on dialysis. When antiHCV antibodies are detected, the presence of HCV RNA has to be determined in
order to discriminate between chronic hepatitis C and resolved HCV infection. The
latter cannot be distinguished by HCV antibody tests from rarely occurring falsepositive serological results, the exact incidence of which is unknown. Serological
false-positive results can be identified by the additional performance of an
immunoblot assay. Many years after disease resolution, anti-HCV antibodies may
become undetectable via commercial assays in some patients.

Diagnostic tests in the management of hepatitis C therapy
The current treatment recommendations for acute and chronic hepatitis C are based
on HCV genotyping and on HCV RNA load determination before, during and after
antiviral therapy. When HCV RNA has been detected, exact genotyping and HCV
RNA load determination is necessary in every patient considered for antiviral
therapy. Exact subtyping might gain increased importance during therapies with
directly acting antiviral (DAA) agents because some subtypes behave differently
regarding the development of resistance. Low HCV RNA concentration (<600,000–
800,000 IU/ml) is a positive predictor of SVR. Genotyping is mandatory for the
selection of the optimal treatment regimen and duration of therapy, since many
DAA agents are selectively effective for only some HCV genotypes (Lange 2010),
and since treatment durations generally can be shorter for patients infected with
HCV genotypes 2 or 3 compared to patients infected with genotypes 1 or 4 (Manns
2006).
Dual combination therapy (PEG-IFN + ribavirin)
For HCV genotype 1 (and 4) treatment can be shortened to 24 weeks in patients
with low baseline viral load (<600,000–800,000 IU/ml) and rapid virologic response
(RVR) with undetectable HCV RNA at week 4 of treatment. In slow responders
with a 2 log10 decline but still detectable HCV RNA levels at week 12 and
undetectable HCV RNA at week 24, treatment should be extended to 72 weeks. In
patients with complete early virologic response with undetectable HCV RNA at
week 12 (cEVR), standard treatment is continued to 48 weeks. Genotypes 5 and 6
are treated the same as genotype 1 infected patients due to the lack of adequate
clinical trials, whereas genotypes 2 and 3 generally allow treatment duration of 24
weeks, which may be shortened to 16 weeks (depending on RVR and [low] baseline

Hepatitis C: Diagnostic Tests 199
viral load) or extended to 36-48 weeks depending on the initial viral decline
(Manns 2006, Layden-Almer 2006).
Independent of the HCV genotype, proof of HCV RNA decrease is necessary to
identify patients with little chance of achieving SVR. HCV RNA needs to be
quantified before and 12 weeks after treatment initiation and antiviral therapy
should be discontinued if a decrease of less than 2 log10 HCV RNA levels is
observed (negative predictive value 88-100%). In a second step, HCV RNA should
be tested with highly sensitive assays after 24 weeks of treatment because patients
with detectable HCV RNA at this time point only have a 1-2% chance of achieving
SVR.
Triple therapy (telaprevir or boceprevir + peginterferon/ribavirin)
Complex treatment algorithms have been introduced with the approval of tripletherapies that now include a HCV NS3 protease inhibitor for chronic HCV genotype
1 infection. These algorithms are different for treatment with telaprevir or
boceprevir.
Shortening of treatment duration with boceprevir triple therapy to 28 weeks is
possible in treatment-naïve patients who achieve an extended rapid virologic
response (eRVR) defined by undetectable HCV RNA levels at weeks 8 and 24 of
treatment. Stopping rules are based on detectable HCV RNA concentrations above
100 IU/ml at week 12 or detectable HCV RNA by a sensitive assay at week 24.
For telaprevir triple therapy, shortening of treatment duration to 24 weeks in
treatment-naïve and relapser patients is based on undetectable HCV RNA at weeks
4 and 12 of treatment. Stopping rules include detectable HCV RNA >1000 IU/ml at
week 4 or 12 and detectable HCV RNA at week 24.
Measurement of HCV RNA at additional time points during boceprevir- or
telaprevir-based triple therapies is recommended to identify viral breakthrough due
to the emergence of HCV variants resistant to DAA agents.
SVR, defined as the absence of detectable HCV RNA 24 weeks after treatment
completion, should be assessed by an HCV RNA detection assay with a lower limit
of 50 IU/ml or less to evaluate long-lasting treatment success.
Due to the differences in HCV RNA concentrations of up to a factor of 4 between
the different commercially available assays, despite standardisation of the results to
IU, and due to intra- and interassay variability of up to a factor of 2, it is
recommended to always use the same assay in a given patient before, during and
after treatment and to repeat HCV RNA measurements at baseline in cases with
HCV RNA concentrations between 400,000 and 1,000,000 IU/ml. Furthermore, the
new stopping rules for boceprevir and telaprevir triple therapies based on viral cutoffs of 100 and 1000 IU/ml respectively, were assessed by the Cobas® TaqMan®
assay and no comparative data with other HCV RNA assays are available yet.

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202 Hepatology 2012

13. Standard Therapy of Chronic Hepatitis C
Virus Infection
Markus Cornberg, Svenja Hardtke, Kerstin Port, Michael P. Manns, Heiner
Wedemeyer

Goal of antiviral therapy
Globally, there are approximately 130-170 million people chronically infected with
hepatitis C virus (HCV), in Europe 8-11 million (Cornberg 2011, Shepard 2005).
Despite the implementation of blood-donor screening in the early ‘90s, there is still
an anticipated increase of HCV-related cirrhosis, hepatic decompensation, and
hepatocellular carcinoma (HCC) over the course of the next decade (Davis 2010).
The goal of antiviral therapy is to cure hepatitis C via a sustained elimination of the
virus (2011). A sustained elimination of HCV is achieved if the HCV RNA remains
negative six months after the end of treatment (sustained virological response, SVR)
(Table 1). Follow-up studies document that more than 99% of patients who achieve
an SVR remain HCV RNA negative 4-5 years after the end of treatment and no
signs of hepatitis have been documented (Manns 2008, Swain 2010). Importantly,
long-term benefits of SVR are the reduction of HCV-related hepatocellular
carcinoma and overall mortality (Backus 2011, Veldt 2007). In 2011, the FDA
accepted SVR-12 as endpoint for future trials because HCV relapse usually occurs
within the first 12 weeks after the end of treatment.
In addition to liver disease, several extra hepatic manifestations, such as
cryoglobulinemia,
non-Hodgkin
lymphoma,
membranoproliferative
glomerulonephritis or porphyria cutanea tarda have been reported in the natural
history of hepatitis C virus infection (HCV). Antiviral treatment may improve
symptoms even without achieving an SVR. On the other hand, antiviral therapy may
worsen extrahepatic manifestations (Pischke 2008, Zignego 2007).

Basic therapeutic concepts and medication
Before the identification of HCV as the infectious agent for non-A, non-B
hepatitis (Choo 1989), interferon α (IFN) led to a normalisation of transaminases
and an improvement of liver histology in some patients (Hoofnagle 1986). After the
identification of HCV it became possible to measure success of therapy as a long-

Standard Therapy of Chronic Hepatitis C Virus Infection 203
lasting disappearance of HCV RNA from serum, the SVR. Since then, SVR rates
have increased from 5-20% with IFN monotherapy up to 40-50% with the
combination of IFN + ribavirin (RBV) (McHutchison 1998, Poynard 1998).
Different HCV genotypes (HCV G) show different SVR rates. Patients with the
most frequent HCV G1 require a longer treatment duration and still get a lower
SVR compared to HCV G2 and HCV G3 (Figure 1). The development of pegylated
interferon α (PEG-IFN) improved the pharmacokinetics of IFN, allowing more
convenient dosing intervals and resulting in higher SVR, especially for HCV G1.
Two PEG-IFNs are available; PEG-IFN α-2b (PEG-Intron®, Merck) and PEG-IFN
α-2a (PEGASYS®, Roche). Although smaller trials from southern Europe have
suggested slightly higher SVR rates in patients treated with PEG-IFN α-2a (Ascione
2010, Rumi 2010), a large US multicentre study did not detect any significant
difference between the two PEG-IFNs + RBV regarding SVR (McHutchison
2009b).
The two PEG-IFNs do have different pharmacokinetic profiles due to their
different polyethylene glycol moieties. PEG-IFN α-2b is bound to a single linear 12kDa polyethylene glycol molecule, whereas PEG-IFN α-2a is covalently attached to
a 40-kDa branched chain polyethylene glycol moiety. The distinct sizes of the PEGIFN influence the volume of distribution. PEG-IFN α-2b is dosed according to body
weight (1.5 µg/kg once weekly), while the larger PEG-IFN α-2a is given in a fixed
dose of 180 µg once weekly (reviewed in (Cornberg 2002)) (Table 2). PEG-IFN α2b may also be dosed at 1.0 µg/kg once patients become negative for HCV RNA
without major declines in SVR rates (Manns 2011a, McHutchison 2009b). RBV
should be administered according to the bodyweight of the patient. A retrospective
analysis of the large PEG-IFN α-2b + RBV pivotal trial revealed that the optimal
dose of RBV (Rebetol®, Merck) is at least 11 mg/kg (Manns 2001). A prospective,
multicentre, open-label, investigator-initiated study confirmed that PEG-IFN α-2b
plus weight-based RBV is more effective than flat-dose ribavirin, particularly in
HCV G1 patients (Jacobson 2007). A RBV dose of 15 mg/kg would be ideal,
although higher doses are associated with higher rates of anemia (Snoeck 2006).
When combined with PEG-IFN α-2a, a RBV dose of 1000 mg if <75 kg or 1200 mg
if ≥75 kg is recommended for HCV G1 patients while a flat dose of 800 mg RBV
was initially suggested for patients with HCV G2 and 3 (Hadziyannis 2004).
However, a weight-based dose of ribavirin (12-15 mg/kg) may be preferred,
especially in difficult to treat patients and in Response-guided Therapy (RGT)
treatment approaches (EASL 2011, Sarrazin 2010). In 2011, the first direct antiviral
agents (DAA) were approved for patients with HCV G1. Two inhibitors of the HCV
protease (PI), boceprevir (Victrelis®, Merck) and telaprevir (Incivek®, Vertex;
Incivo®, Johnson & Johnson), improve SVR rates up to 75% in naïve HCV G1
patients (Jacobson 2011b, Poordad 2011b) and 29-88% in treatment experienced
HCV G1 (Bacon 2011, Zeuzem 2011). However, both PIs require combination with
PEG-IFN + RBV because monotherapy would result in rapid emergence of drug
resistance. Both boceprevir (BOC) and telaprevir (TLV) can be combined with
PEG-IFN α-2a or PEG-IFN α-2b.

204 Hepatology 2012
Table 1. Relevant definitions for HCV treatment.
Abbreviation Term
SVR
SVR-12
RVR
eRVR (BOC)

eRVR (TLV)

EVR
cEVR

Description

Sustained Virological
HCV RNA negative 6 months after the end of
Response
therapy
Sustained Virological
HCV RNA negative 12 weeks after the end of
Response
therapy; FDA-accepted endpoint for future trials
Rapid Virological Response HCV RNA negative after 4 weeks of therapy
Extended Rapid Virological HCV RNA negative (LLD not LLQ) between
Response (for boceprevir) week 8 and week 24 of BOC therapy: RGT
criterion for BOC
Extended Rapid Virological HCV RNA negative (LLD not LLQ) between
Response (for telaprevir)
week 4 and week 12 of TLV therapy: RGT
criterion for TLV
Early Virological Response HCV RNA decline ≥2 log10 at week 12

NR (BOC)

Complete Early Virological
Response
Nonresponse (boceprevir)

NR (TLV)

Nonresponse (telaprevir)

BT

Breakthrough

RL

Relapse

PR

Partial Response

NULR

Null response

LI

Lead-In

HCV RNA negative at week 12
HCV RNA ≥100 IU/mL at week 12; or HCV
RNA positive at week 24; futility rule for BOC
HCV RNA ≥1000 IU/mL at week 4 or week 12;
or HCV RNA positive at week 12: Futility rule
for TLV
HCV RNA was LLD but increased to ≥100
IU/mL or increase of HCV RNA ≥ 1log10 during
therapy
HCV RNA negative at EOT and recurrence of
HCV RNA during the follow-up of 6 months.
HCV RNA decline ≥2 log10 at week 12 but
positive at week 24 during PEG-IFN/RBV
HCV RNA decline <2 log10 at week 12 during
PEG-IFN/RBV
4 weeks PEG-IFN/RBV before adding a PI

LLD, lower limit of detection (<10-15 IU/mL; here indicated as negative); LLQ, lower limit of
quantification; EOT, end of treatment; RGT, response-guided therapy

Standard Therapy of Chronic Hepatitis C Virus Infection 205

Figure 1. Development of chronic hepatitis C therapy. The sustained virologic response
rates have improved from around 5% with interferon monotherapy in the early 90s to >70%
today with triple therapy of PEG-IFN + ribavirin + PI.

Table 2. Approved drugs for the treatment of chronic hepatitis C (2011).
Medication

Dosing

Type I interferons

Subcutaneous injection

Pegylated Interferon α-2a
(Pegasys®)
Pegylated Interferon α-2b (PEGIntron®)
Interferon α-2a (Roferon®)

180 µg once weekly

Interferon α-2b (Intron A®)

3 Mill I.U. three times weekly

Consensus Interferon (Infergen®)

9 µg three times weekly

Ribavirin

Oral tablets or capsules

Ribavirin (Copegus®)

800 - 1200 mg daily (200 mg or 400 mg tablets)

Ribavirin (Rebetol®)

600 - 1400 mg daily (200 mg capsules or solution)

HCV protease inhibitors

Oral tablets or capsules

Boceprevir (Victrelis®)

800 mg (4 x 200 mg capsules) every 7-9 hours

Telaprevir (Incivek®, Incivo®)

750 mg (2 x 375 mg tablets) every 7-9 hours

1.5 µg/kg once weekly
3 - 4.5 Mill I.U. three times weekly

206 Hepatology 2012

Predictors of treatment response
During the last decade, tailoring treatment duration and dosing according to
individual parameters associated with response have improved SVR. Predicting
SVR before the start of antiviral treatment helps in making treatment decisions.
Important baseline factors associated with SVR to PEG-IFN/RBV are the HCV
genotype, the degree of liver fibrosis and steatosis, baseline viral load, presence of
insulin resistance, age, gender, body mass index, ethnicity, and HIV co-infection
(Berg 2011, McHutchison 2009b). Many of these factors may have less relevance
for triple therapy, i.e., insulin resistance seems not to impact SVR to PEGIFN/RBV/PI (Berg 2011, Serfaty 2010) whereas low-density lipoprotein (LDL) was
associated with SVR (at least for TLV) (Berg 2011).
On the other hand, new parameters seem to be more important such as HCV
subtype 1a and 1b. Patients with HCV G1a have a higher risk of developing
resistance during PI-based therapy compared to HCV G1b because HCV G1a
requires an exchange of only one nucleotide versus two for HCV G1b in position
155 to develop resistance (reviewed in Sarrazin and Zeuzem 2010b).
During treatment, the kinetics of the HCV RNA decline is a strong predictor of
response. HCV RNA measurements at week 4, 12 and 24 are important for a
response-guided treatment approach for PEG-IFN/RBV but also for the new triple
therapy including BOC and TLV. Definitions of response and futility rules are
summarized in Table 1. (Futility rules means that if at these time points, the viral
load threshold is exceeded or detected in serum, therapy should be stopped.)
Recently, genome-wide association studies have identified host genetic
polymorphisms (i.e., rs12979860, rs8099917) located on chromosome 19 located
upstream to the region coding for IL28B (or IFN λ3) associated with SVR to
treatment with PEG-IFN/RBV in HCV G1 patients (Ge 2009, Rauch 2010, Suppiah
2009, Tanaka 2009) but also to a lesser extent for HCV G2/3 (Mangia 2010c,
Sarrazin 2011b). Data on IL28B explain the different responses to PEG-IFN/RBV
between different ethnic groups, i.e., low SVR in African Americans and high SVR
in Asian patients. However, the negative predictive value is not strong enough to
recommend general testing (EASL 2011). Viral kinetics, especially response at
week 4, have a higher predictive value (Sarrazin 2011a) and the relevance of IL28B
as a predictive marker for the success of triple therapy with PEG-IFN/RBV/PI is
less significant (Jacobson 2011a, Pol 2011a, Poordad 2011a). However, IL28B
testing may be useful to determine the IFN responsiveness and the likelihood of
achieving RVR with PEG-IFN/RBV before starting triple therapy. It may be of
relevance to discuss treatment options with the individual patient (see below).
Additional predictive markers are being evaluated. For example, low serum levels
of interferon γ inducible protein 10 (IP 10) are associated with SVR and may
improve the predictive value for discrimination between SVR and nonresponse
(Darling 2011, Fattovich 2011).

Antiviral resistance
The development of direct antiviral agents leads to the emerging problem of drug
resistance due to so-called resistant-associated amino acid variants (RAVs) of the
virus. Patients who received monotherapy with BOC or TLV develop resistance

Standard Therapy of Chronic Hepatitis C Virus Infection 207
within a few days (Sarrazin 2007). RAVs associated with resistance to BOC and
TLV are listed in Table 3. Due to their overlapping resistance profiles, one PI
cannot substitute the other in the case of viral breakthrough. Also, a combination of
the two PIs is not rationale. As mentioned above, combination with PEG-IFN/RBV
is mandatory for the usage of BOC or TLV and RAVs to BOC and TLV have not
been associated with less sensitivity to PEG-IFN/RBV (Kieffer 2007). Importantly,
if patients have a decreased PEG-IFN/RBV response, the risk of developing
significant RAVs is higher. Measures for the prevention of drug resistance are
adherence to the dose of the medications (most importantly to the PI) and
compliance with the futility rules (see below). If RAVs emerge, it is not completely
known for how long they persist and if this has any significant consequences for
future therapies. Some studies suggest that the majority of resistant variants revert to
wild type 1-2 years after the end of therapy (Sarrazin 2007, Sherman 2011b). At this
stage there is no rationale to routinely analyse HCV sequences either before therapy
or during treatment because it has no practical consequence. Dominant RAVs
before treatment have been documented (Kuntzen 2008) but the influence of
treatment response is not well characterised.
Table 3. Resistant-associated amino acid variants of HCV NS3 protease to
boceprevir and telaprevir (adapted from Sarrazin 2012).
V36A
/M

T54S
/A

V55A

BOC

X

X

X

TLV

X

X

Q80R
/K

R155K
/T/Q

A156S

A156T
/V

X

X

X

X

X

X

D168A
/E/G/H
/T/Y

V170A
/T
X

Treatment of HCV genotype 1
Treatment of naïve patients
Untreated patients with HCV genotype 1 (HCV G1) have various treatment options.
Triple therapy with PEG-IFN+RBV+PI increases the overall SVR by 25-31%
(Table 4). Many patients qualify for response-guided therapy (RGT) based on viral
kinetics. In 44-65% of patients with eRVR treatment duration can be reduced to 2428 weeks (Figures 2A, 2B), some 4-6 times more than with PEG-IFN/RBV.
However, in patients with favourable predictors for SVR (low baseline HCV RNA,
IL28CC, no advanced fibrosis), dual therapy with PEG-IFN/RBV may still be an
option. In those patients, a lead-in of 4 weeks PEG-IFN/RBV can identify patients
with RVR who achieve high SVR without adding a PI. Patients with low viral load
at baseline who achieve RVR have demonstrated 78-100% SVR with 24 weeks
PEG-IFN/RBV dual therapy alone (Berg 2009, Ferenci 2008, Jensen 2006, Sarrazin
2011a, Zeuzem 2006) (Table 5). Not adding BOC or TLV will reduce costs and
adverse events, two factors that can lead to treatment discontinuation. The number
of patients who qualify for dual therapy may vary depending on the distribution of
IL28B polymorphisms. On the other hand, a lead-in therapy may identify patients
with a poor response to IFN with a high chance of developing resistance. Only 2931% of patients who have <1 log10 reduction of HCV RNA after 4 weeks PEGIFN/RBV go on to achieve SVR when they add BOC. Other negative predictors

208 Hepatology 2012
(HCV G1a, cirrhosis) together with the lead-in concept may increase the negative
predictive value of achieving SVR. In that case a wait-and-see strategy may be
considered. The 4-week lead-in strategy also proved useful in assessing compliance,
tolerability and safety before initiating the PI. The lead-in concept was developed in
the BOC studies with the hypothesis of reducing resistance and improving SVR
(Kwo 2010). However, the lead-in phase seems to have no significant effect on the
SVR or on the development of antiviral resistance (Kwo 2010, Zeuzem 2011).
Lead-in has also been evaluated for TLV but only in treatment-experienced patients
(Zeuzem 2011). It is recommended to discuss the lead-in option and the
consequences with the patient before initiation of treatment.
Table 4. Phase III studies with BOC or TLV treatment regimens in treatment naïve
patients with HCV genotype 1. Studies are no head-to-head studies and SVR between
different studies are difficult to compare because they had significant differences in
genetic and socioeconomic backgrounds.
Study

Dosing

eRVR, SVR

1.5 µg/kg PEG-IFN α-2b, 600-1400 mg
a) eRVR: 40/363 (11%) / NB: 12%
RBV 48 weeks
SVR: 137/363 (38%) / NB: 40%
44 weeks Placebo (wk 4-48)
b) 1.5 µg/kg PEG-IFN α-2b, 600-1400 mg
b) eRVR: 156/368 (42%) / NB: 45%
RBV 28*-48 weeks
SVR: 233/368 (63%) / NB: 67%
24 weeks 800 mg tid BOC (wk 4-28)
c) eRVR: 155/366 (42%) / NB: 44%
c) 1.5 µg/kg PEG-IFN α-2b, 600-1400 mg
SVR: 242/366 (66%) / NB: 68%
RBV 48 weeks
44 weeks 800 mg tid BOC (wk 4-48)
ADVANCE
a) 180 µg PEG-IFN α-2a, 1000-1200 mg
a) eRVR: 210/363 (58%)
(Jacobson 2011b)
RBV 24*-48 weeks,
SVR: 271/363 (75%)**
12 weeks 750 mg tid TLV (wk 0-12)
N=1088
(T12PR)
*24 weeks if
b) 180 µg PEG-IFN α-2a, 1000-1200 mg
eRVR TLV
b) eRVR: 207/363 (57%)
RBV 24*-48 weeks,
SVR: 250/364 (69%)
8 weeks 750 mg tid TLV, 4 weeks
Placebo (wk 0-12)
c) 180 µg PEG-IFN α-2a, 1000-1200 mg
c) SVR: 158/361 (44%)
RBV 48 weeks,
12 weeks Placebo (wk 0-12)
ILLUMINATE
a) eRVR: 180 µg PEG-IFN α-2a, 1000a)
SVR: 149/162 (92%)
(Sherman 2011a)
1200 mg RBV 24 weeks, 12 weeks 750
mg tid TLV (wk 0-12)
N=540
b) eRVR: 180 µg PEG-IFN α-2a, 1000N=352 (65%)
b)
SVR: 140/160 (88%)
1200 mg RBV 48 weeks, 12 weeks 750
eRVR
mg tid TLV (wk 0-12)
N=322
c) no eRVR: 180 µg PEG-IFN α-2a, 1000randomised
c)
SVR: 76/118 (64%)
1200 mg RBV 48 weeks, 12 weeks 750
mg tid TLV (wk 0-12)
** numbers from the published data are different from the numbers accepted by the FDA, i.e. 79% SVR for
telaprevir 12 weeks, PEG-IFN/RBV
SPRINT-2
(Poordad 2011b)
N=938 nonblack
(NB)
N=159 black
*28 weeks if
eRVR BOC

a)

Treatment regimens with boceprevir
Boceprevir (BOC) is a linear peptidomimetic ketoamide serine protease inhibitor
that binds reversibly to the HCV nonstructural 3 (NS3) active site. BOC results in a
significant decline of HCV RNA but given as monotherapy it leads to rapid
emergence of viral resistance (Sarrazin and Zeuzem 2010b). Thus, combination
with PEG-IFN/RBV is still necessary (Mederacke 2009). 800 mg BOC is given as
200 mg capsules every 7-9 hours together with food in combination with the
optimal dose of PEG-IFN/RBV (Table 2). In all Phase III trials BOC was added

Standard Therapy of Chronic Hepatitis C Virus Infection 209
after the 4-week lead-in period as described above. In SPRINT-2 (serine protease
inhibitor therapy 2), the Phase III study with 1097 treatment-naïve HCV G1
patients, safety and efficacy of two regimens of BOC added to PEG-IFN α-2b/RBV
after a 4-week lead-in with PEG-IFN/RBV were compared to PEGIFN/RBV/placebo (Table 4) for 44 weeks. The two groups receiving BOC were
treated with an RGT concept or a fixed duration of BOC. Patients in the RGT group
received 24 weeks triple combination after the lead-in period. Treatment with PEGIFN/RBV was continued through week 48 only if the criteria for eRVR were not
met (HCV RNA levels undetectable from week 8 through week 24). Patients in the
fixed therapy duration group received PEG-IFN/RBV/BOC for 44 weeks following
the 4-week lead-in phase. Based on published data for response rates being lower
for African-American patients, black and non-black patients were analysed as two
different pre-defined cohorts of the SPRINT-2 study. Overall, adding BOC to PEGIFN/RBV could significantly improve SVR in previously untreated patients with
HCV genotype 1 leading to approval in 2011 (FDA: May; EMA: July). Non-black
patients achieved 27-28% higher SVR, black patients increased SVR by 19-30%.
Table 5. High SVR in naïve patients with HCV genotype 1 and low baseline viral
load treated with 24 weeks of PEG-IFN/RBV.
Study

Treatment

Subgroups
(fast responder)

Weeks

SVR

(Zeuzem
2006)
N=235
(Berg
2009)
N=433

1.5 µg/kg PEG-IFN α-2b
800-1400 mg ribavirin

<600,000 IU/ml TW0
<600,000 IU/ml TW0 &
<29 IU/ml TW4 (RVR)
<5.3 IU/ml TW4 (RVR)
<800,000 IU/ml TW0 &
<5.3 IU/ml TW4 (RVR)

24
24

50%
89%

18-24
18-24

80%
100%

(Sarrazin
2011a)
N=398

1.5 µg/kg PEG-IFN α-2b
800-1400 mg ribavirin

<800,000 IU/ml TW0 & 24
<5-10 IU/ml TW4 (RVR)

88%

(Jensen
2006)
N=216

180 µg PEG-IFN α-2a or
800 mg or 1000-1200 mg
ribavirin

<50 IU/ml TW4 (RVR)
>50 IU/ml TW4 (RVR)

24
24

89%
19%

(Ferenci
2008)
N=120

180 µg PEG-IFN α-2a or
1000-1200 mg ribavirin

<50 IU/ml TW4 (RVR)

24

74% ITT
79% PP

1.5 µg/kg PEG-IFN α-2b
800-1400 mg ribavirin

* SVR, sustained viral response; RVR, rapid virologic response.

The responsiveness to PEG-IFN/RBV is very important for the success of
treatment with BOC. This is emphasized by the fact that the HCV RNA decline at
week 4 is highly predictive of SVR. Patients with more than 1 log10 HCV RNA
decrease after the 4-week lead-in phase demonstrated an SVR of about 80% if
treated with BOC but only 28-38% responded if HCV RNA declined less than 1
log10. Thus, the lead-in phase can be valuable to predict the responsiveness to PEGIFN/RBV for further individualization of therapy as discussed above (Figure 3).
Importantly, the overall SVR rates between the RGT group and the fixed 48-week
therapy group were comparable (Table 4). Patients achieving eRVR were eligible

210 Hepatology 2012
for a 28-week total therapy duration and almost all patients (96%) went on to
achieve SVR (Poordad 2011b). Of note, HCV RNA negative means below the limit
of detection (LLD) and not below limit of quantification (LLQ). This is important
because SVR is diminished in patients with LLQ at weeks 8-24 who were treated
for a shorter duration (Harrington 2011).
FDA and EMA have approved RGT for treatment naïve patients except for
patients with liver cirrhosis (Figure 2A) but the accepted treatment duration for
BOC-RGT is different to the study design of the Phase III study (32 vs 24 weeks
BOC for patients without eRVR) (Figure 2A). In addition, a retrospective analysis
led to the futility rule of HCV RNA >100 IU/mL at week 12. The predictive value
for nonresponse was 100%. BOC was initially combined with PEG-IFN α-2b.
Recently, a study in therapy-experienced patients including relapsers and partial
responders showed similar results with PEG-IFN α-2a/RBV (Flamm 2011). Thus,
both PEG-IFNs can be combined with BOC.
Treatment regimens with telaprevir
Telaprevir (TLV) is also an orally administered reversible, selective,
peptidomimetic NS3/4A serine protease inhibitor, which leads to a significant
decline of HCV RNA although viral resistance emerges rapidly if given as
monotherapy (Sarrazin 2007). Thus, 750 mg TLV given as 375 mg tablets every 7-9
hours together with food (ideally >20 g fat) requires combination with optimal
PEG-IFN/RBV. Telaprevir was administered for a maximum of 12 weeks in the
Phase III trials; longer treatment duration is associated with increasing adverse
events (McHutchison 2010). Two large Phase III studies (ADVANCE and
ILLUMINATE) with a total of 1628 treatment-naïve HCV G1 patients showed that
PEG-IFN/RBV/TLV significantly improved SVR compared to PEG-IFN/RBV and
RGT is possible (Jacobson 2011b, Sherman 2011a). TLV was approved for the
treatment of HCV G1 in 2011 (FDA: May; EMA: September). In the ADVANCE
trial, 3 treatment groups were assessed for efficacy and safety using RGT in
treatment-naïve patients (Jacobson 2011b). 12 weeks of TLV versus 8 weeks of
TLV in combination with 24-48 weeks PEG-IFN/RBV were compared to 48 weeks
PEG-IFN/RBV dual therapy. Patients who achieved eRVR qualified for 24 weeks
of therapy (Table 4). SVR was significantly higher among those receiving TLV
compared to the placebo group; 12 weeks TLV resulted in the highest SVR (Table
4). In all treatment groups, more than 80% of patients who achieved eRVR had
SVR (89%, 83%, and 97%, respectively) (Jacobson 2011b).
To validate RGT, telaprevir 750 mg every 8 hours for 12 weeks was evaluated in
an open-label study (ILLUMINATE trial) to prospectively assess 24 vs 48 weeks of
treatment for HCV G1 patients who achieved eRVR. If HCV RNA levels were
undetectable at weeks 4 and 12, patients were randomly assigned to continue with
PEG-IFN/RBV for an additional 24 or 48 weeks. If eRVR was not attained, patients
received PEG-IFN/RBV for up to 48 weeks. Of the 540 subjects, 389 (72%)
achieved HCV RNA levels LLD at week 4 and 352 (65%) achieved eRVR. Patients
who achieved eRVR and were randomized to the 24-week cohort experienced 92%
SVR versus 88% who were treated for 48 weeks (Table 4) (Sherman 2011a).
Importantly, patients with liver cirrhosis showed higher relapse rates with shorter
treatment, therefore RGT for TLV has only been approved for naïve HCV G1
patients without liver cirrhosis. Also, retrospective analysis of the data showed that

Standard Therapy of Chronic Hepatitis C Virus Infection 211
early HCV RNA measurement at week 4 is predictive of nonresponse to TLV.
Patients with HCV RNA values >1000 IU/mL after 4 weeks PEG-IFN/RBV/TLV
did not achieve SVR. Therefore, therapy must be stopped.

Figure 2A. Treatment with BOC/PEG-IFN/RBV: Approved treatment algorithm for HCV G1
patients. *, RGT if eRVR (HCV RNA LLD week 8-24); #, EMA did not approve RGT for BOC
regimens in previously treated patients.

Figure 2B. Treatment with TLV/PEG-IFN/RBV: Approved treatment algorithm for HCV G1
patients. *RGT if eRVR (HCV RNA LLD week 4-12).
*** If patients have contraindications for BOC or TLV, dual therapy with PEG-IFN/RBV should
be given for 24-72 weeks according to the HCV RNA decline at week 4 and week 12 (Sarrazin,
Berg, Cornberg 2010 S3-Leitlinie). The treatment algorithm is similar to Figure 6.

212 Hepatology 2012

Figure 3. Suggestion to use the lead-in strategy for individualisation of treatment in
patients with HCV genotype 1. **The number of patients with low baseline HCV RNA and
RVR may vary between different countries due to IL28B differences.

Treatment of patients with prior antiviral treatment failure
As more patients have been treated, the size of the population of patients who have
failed to achieve SVR with PEG-IFN/RBV has expanded. Many nonresponder
patients have advanced liver disease and successful treatment may extend life
expectancy (Backus 2011, Veldt 2007). Retreatment of patients with previous
treatment failure is one of the most important current topics in the treatment of
chronic hepatitis C.
Definition of treatment failure
Definition of response to or failure on antiviral therapy is very important when
considering retreating patients with chronic hepatitis C because the success of BOCor TLV-based regimens depends on the IFN responsiveness. Patients may have been
treated with different treatment regimens and compliance during the previous
therapy was probably very varied. Most importantly, HCV RNA kinetics and the
response profile during the previous therapy have to be taken into account before
starting a new treatment. It is crucial to screen the patient’s records and check
treatment duration, drug dosing and HCV RNA of the previous therapy. Nonresponse is the failure of a patient to clear HCV RNA at any point during treatment.
Definitions used for trials assessing novel therapy approaches have generally
defined non-response as the failure to achieve EVR, which is ≥2 log10 reduction of
HCV RNA after 12 weeks. Classifications of non-response include null response,
partial response, relapse, and breakthrough (see Table 1, Figure 4).

Standard Therapy of Chronic Hepatitis C Virus Infection 213

Figure 4. Different scenarios of treatment failure to antiviral therapy in chronic hepatitis
C.

Retreatment of HCV G1 patients with relapse after PEG-IFN/RBV
Retreatment with PEG-IFN/RBV of relapse patients after IFN- or PEG-IFN-based
combination therapy with ribavirin resulted in an SVR of 24-34% (Bacon 2011,
Poynard 2009, Zeuzem 2011). Triple therapy with PEG-IFN/RBV/PI increases SVR
dramatically to 69-88% (Bacon 2011, Zeuzem 2011) (Table 6). Relapse patients are
the ideal patients for retreatment with a triple therapy regimen. Patients have already
proven to respond to PEG-IFN and RBV. Thus, the backbone to prevent PI
resistance is effective and a lead-in strategy may not be as important as in other
situations. Although RGT was not evaluated in the Phase III REALIZE trial with
TLV, a rollover study including relapse patients from Phase II studies has
demonstrated that shorter treatment is effective in patients with eRVR (Muir 2011).
Therefore, RGT is possible with BOC and TLV regimes (Figures 2A, 2B) if
cirrhosis is excluded (Ghany 2011, Sarrazin 2012). In contrast, BOC RGT has only
been approved by the FDA and not by the EMA because SVR was slightly lower in
the RESPOND-2 RGT group (Table 6).
Retreatment of HCV G1 patients with partial response to PEG-IFN/RBV
Patients who are partial responders (PR) to standard PEG-IFN/RBV combination
therapy have demonstrated SVRs ranging between 7% and 15% with a standard
PEG-IFN/RBV retreatment (Bacon 2011, Zeuzem 2011). Retreatment with triple
therapy increases SVR to 40%-59% (Bacon 2011, Zeuzem 2011) (Table 6). FDA
but not EMA approved RGT for BOC (Figures 2A, 2B). Treatment duration for
PEG-IFN/RBV/TLV is 48 weeks for all PR patients (Figure 2B). The 4-7-fold
increase justifies retreatment. However, SVR decreases significantly in patients with
cirrhosis (34% with TLV) and other negative response factors (Pol 2011b).

214 Hepatology 2012
Retreatment of HCV G1 patients with null response to PEG-IFN/RBV
Patients who are null responders (NULR) to standard PEG-IFN/RBV combination
therapy have demonstrated SVRs ranging between 5% and 16% with an optimised
PEG-IFN/RBV retreatment (Jensen 2009, Poynard 2009, Zeuzem 2011).
Retreatment with PEG-IFN/RBV/PI did increase SVR more than 6-fold in the
REALIZE trial (Zeuzem 2011). However, overall SVR with triple therapy is limited
to 29-38% (Vierling 2011, Zeuzem 2011) (Table 6). If further negative predictive
factors are present, SVR decreases to 27% in HCV G1a patients and to 14% in
cirrhotic patients (not significantly different from PEG-IFN/RBV) (Figure 7A). This
may justify the lead-in concept to decide if treatment with a PI is beneficial. Patients
who do not acheive a 1 log10 decline of HCV RNA after 4 weeks demonstrate only
15% SVR (Zeuzem 2011). Futility rules are the same for treatment-experienced
patients as for treatment-naïve patients (Figures 2A, 2B).
Table 6. Phase III studies with BOC or TLV treatment regimens in treatmentexperienced patients infected with HCV genotype 1. Studies are not head-to-head and
SVR between studies are difficult to compare because they had significant differences in
genetic and socioeconomic backgrounds.
Study

Dosing

RESPOND-2
(Bacon 2011)
n=403

a)

1.5 µg/kg PEG-IFN α-2b, 600-1400 mg
RBV 48 weeks
44 weeks Placebo (wk 4-48)
1.5 µg/kg PEG-IFN α-2b, 600-1400 mg
RBV 36*-48 weeks
32 weeks 800 mg tid BOC (wk 4-36)
1.5 µg/kg PEG-IFN α-2b, 600-1400 mg
RBV 48 weeks
44 weeks 800 mg tid BOC (wk 4-48)
180 µg PEG-IFN α-2a, 1000-1200 mg
RBV 48 weeks
44 weeks Placebo (wk 4-48)
180 µg PEG-IFN α-2a, 1000-1200 mg
RBV 48 weeks
44 weeks 800 mg tid BOC (wk 4-48)
1.5 µg/kg PEG-IFN α-2b, 600-1400 mg
RBV 48 weeks
44 weeks 800 mg tid BOC (wk 4-48)

a)

REL: 29%
PR: 7%

All: 21%

b)

REL: 69%
PR: 40%

All: 59%

c)

REL: 75%
PR: 52%

All: 66%

a)

REL, PR: 21%

b)

REL, PR: 64%

180 µg PEG-IFN α-2a, 1000-1200 mg
RBV 48 weeks,
12 weeks Placebo (wk 0-12)

a)

REL: 24%
PR: 15%
NULR: 5%

180 µg PEG-IFN α-2a, 1000-1200 mg
RBV 48 weeks,
4 weeks Placebo (wk 0-4), 12 weeks 750
mg tid TLV (wk 4-16)
 Lead-in cohort

b)

REL: 88%
PR: 54%
NULR: 33%

180 µg PEG-IFN α-2a, 1000-1200 mg
RBV 48 weeks,
12 weeks 750 mg tid TLV (wk 0-12), 4
weeks Placebo (wk 12-16),

c)

REL: 83%
PR: 59%
NULR: 29%

b)
*36 weeks
if eRVR BOC
c)

(Flamm 2011)
n=201

a)

b)

PROVIDE
(Vierling 2011)
n=48
(42 available)
REALIZE
(Zeuzem 2011)
n=663

SVR

a)

b)

c)

38% (16/42)

Standard Therapy of Chronic Hepatitis C Virus Infection 215

PEG-IFN maintenance therapy
There has been much interest concerning the use of low-dose PEG-IFN maintenance
therapy in patients with a null response since data has suggested that IFN may halt
progression of liver disease (Nishiguchi 1995). There are two major published trials
that have analysed if maintenance treatment with IFN alters the natural course of
chronic hepatitis C. In the EPIC3 trial, nonresponders to IFN/RBV with
compensated cirrhosis and no evidence of HCC received 0.5 µg/kg PEG-IFN α-2b
or no treatment for a maximum period of 5 years or until patients developed clinical
events (hepatic decompensation, HCC, death, or liver transplantation). The study
revealed no significant difference in time to first clinical event among patients who
received PEG-IFN compared with controls (Bruix 2011).
The HALT-C trial, a long-term maintenance study supported by the National
Institutes of Health evaluated a large cohort of chronic HCV-infected patients who
had failed previous IFN-based therapy and had METAVIR stage F2-F4. Patients
received 90 µg PEG-IFN α-2a maintenance treatment if they did not respond during
the first 20 weeks with standard therapy. Despite the fact that there were greater
reductions in viremia, decreases in alanine aminotransferase, and
necroinflammation in the patients who received PEG-IFN, none of the important
clinical outcomes (rates of death, decompensation, hepatocellular carcinoma, and
increase in fibrosis) were favourably affected by PEG-IFN therapy (Di Bisceglie
2008). In conclusion, long-term treatment with low-dose PEG-IFN cannot be
recommended (Sarrazin 2010a).

Treatment of HCV genotypes 2 and 3
Naïve patients
TLV shows antiviral efficacy against HCV G2 but is not effective against HCV G3
(Foster 2011). Data for BOC have only been presented in abstract form for 400 mg
TID in a small number of patients (Silva 2011). Importantly, both PIs are approved
only for the treatment of HCV G1. Thus, SOC for HCV G2/3 infection remains the
combination of PEG-IFN/RBV. Although a fixed duration of treatment (24 weeks)
has been advocated, the optimal results are likely to be achieved when the duration
of therapy is adjusted based on viral kinetics. Many studies have investigated the
reduction of treatment duration for HCV G2/3 to 16, 14, or even 12 weeks. Overall,
reducing the treatment duration to less than 24 weeks increases the number of
relapses (Andriulli 2008, Dalgard 2008, Mangia 2005, Manns 2011a, Shiffman
2007b). However, some HCV G2/3 patients may indeed be treatable for 12-16
weeks if certain prerequisites are fulfilled, especially the rapid virologic response
(RVR) by week 4 of therapy (Slavenburg 2009). Only patients with RVR have high
SVR rates after 16 weeks (Manns 2011a, von Wagner 2005), 14 weeks (Dalgard
2008), or even 12 weeks of therapy (Mangia 2005) (Table 7).
In addition to the RVR, the specific HCV genotype and the baseline viral load are
associated with response. Patients with HCV G2 respond better to PEG-IFN/RBV
therapy than those infected with HCV G3 (Zeuzem 2004b). Furthermore, the shorter
treatment schedules reveal that HCV G3 patients with low baseline viremia (<400800,000 IU/ml) had a much better chance of responding than those with high viral
load (>400-800,000 IU/ml) (Shiffman 2007b, von Wagner 2005). Patients with

216 Hepatology 2012
HCV G3 plus low viral load who achieve RVR can be treated for less than 24
weeks. However, reducing treatment duration is not recommended in patients with
advanced liver fibrosis or cirrhosis, insulin resistance, diabetes mellitus, hepatic
steatosis or BMI >30 kg/m2 (Aghemo 2006, Sarrazin 2010a, Sarrazin 2011).
Patients treated with a response-guided approach should be started on high-dose
ribavirin, which appears to increase the rate of RVR in patients with HCV G2/3
undergoing short treatment (Mangia 2010b).
In contrast, HCV G2/3 patients who do not achieve RVR (especially HCV G3 and
high viral load) may be treated for longer than 24 weeks (i.e., 36-48 weeks) (Figure
5). However, most data are retrospective (Willems 2007). A prospective study from
Italy showed a numerically significant benefit of 36 weeks versus 24 weeks (75%
vs. 62%) (Mangia 2010a). Further prospective studies investigating treatment
extension to 36 or 48 weeks are ongoing. Depending on the assay used to determine
RVR, around 25-30% of HCV G2/3 patients belong to this difficult-to-treat
population not achieving RVR (Table 8). Tailoring treatments individually for
patients with HCV G2/3 will reduce costs and side effects and further optimise the
response rates.

Figure 5. Recommendation for treatment of HCV genotypes 2 and 3. Sensitive HCV RNA
assays (limit of detection 12-15 IU/ml or 50 IU/ml) at weeks 4 and 12 may determine treatment
duration. Reducing treatment duration is not recommended in patients with liver cirrhosis,
insulin resistance, diabetes mellitus or hepatic steatosis.

Standard Therapy of Chronic Hepatitis C Virus Infection 217
Table 7. Response-guided therapy for patients with HCV genotypes 2 and 3.
Study

Treatment

Subgroups

(von Wagner 180 µg PEG-IFN α-2a >600 IU/ml TW4
2005)
800-1200 mg ribavirin <600 IU/ml TW4
n=153
<600 IU/ml TW4
(Shiffman
2007b)
n=1469

(Mangia
2005)
n=283

(Dalgard
2008)
n=428

(Manns
2011a)
n=682

1.0 µg PEG-IFN α-2b
1.5 µg PEG-IFN α-2b
800-1400 mg ribavirin

24
24

SVR*

24
16
24
16
24
16

36%
80%, 84% if HCV
RNA<800,000 IU/ml
82%, 93% if HCV
RNA<800,000 IU/ml
70%
62%
85%
79%
81%
82%

24

76%

Standard group

24

91% if TW4 HCV RNA
<50 IU/ml

>50 IU/ml TW4 (no RVR)

24

64%

<50 IU/ml TW4 (RVR)

12

85%

<50 IU/ml TW4 (RVR)

24

91% ITT, 93% with F24
HCV RNA test

<50 IU/ml TW4 (RVR)

14

81% ITT, 86% with F24
HCV RNA test

>50 IU/ml TW4 (no-RVR)

24

55% ITT, 59% with F24
HCV RNA test

180 µg PEG-IFN α-2a All patients
800 mg ribavirin
All patients
<50IU/ml TW4 (RVR)
<50IU/ml TW4 (RVR)
<400,000IU/ml TW0
(LVL)
<400,000IU/ml TW0
(LVL)
1.0 µg PEG-IFN α-2b Standard group
1000-1200 mg ribavirin

1.5 µg PEG-IFN α-2b
800-1400 mg ribavirin

Therapy
weeks

16

All patients

24 (1.5) 67% ITT, 82% as treated

All patients

24 (1.0) 64% ITT, 80% as treated

All patients

16 (1.5) 57% ITT, 68% as treated

* SVR, sustained viral response; RVR, rapid virologic response; LVL, low baseline viral
load.

218 Hepatology 2012
Table 8. SVR of patients with HCV genotypes 2 or 3 not achieving RVR.
Study

Frequency of patients
without RVR

(von Wagner 2005)
7%
180 µg PEG-IFN
(HCV RNA >600 IU/ml TW4)
α-2a 800-1200 mg ribavirin

SVR without RVR
(24 wks therapy)
36%

(Shiffman 2007b)
180 µg PEG-IFN α-2a
800 mg ribavirin

36%
45%
(HCV RNA >50 IU/ml TW4) (24 wk group)

(Mangia 2005)
1.0 µg/kg PEG-IFN α-2b
1000-1200 mg ribavirin

36%-38%
(HCV RNA >50 IU/ml TW4)

48%-64%

(Dalgard 2004)
1.5 µg/kg PEG-IFN α-2b
800-1400 mg ribavirin

22%
(HCV RNA >50 IU/ml TW4/TW8)

56%

(Dalgard 2008)
1.5 µg/kg PEG-IFN α-2b
800-1400 mg ribavirin

29%
(HCV RNA >50 IU/ml TW4)

55%

Treatment of HCV G2/3 patients with prior antiviral
treatment failure
Patients with relapse after a short course of PEG-IFN/RBV show adequate SVR
after retreatment for 24 weeks (Mangia 2009). In patients with unfavourable
predictors, longer treatment duration for 48 weeks is advisable (EASL 2011).
Nonresponders can be retreated with an additional course of PEG-IFN/RBV. It is
important to optimise dose and duration of treatment. HCV G2 nonresponders may
benefit from retreatment with PEG-IFN/RBV/PI (so far only data for TLV). Triple
therapy is off-label but may be considered in difficult to treat HCV G2 patients with
an urgent treatment indication. Future DAAs will be pan-genotypic and therefore
also effective for HCV G3 (see Chapter 14). Nonresponder patients with mild
fibrosis may therefore wait for new treatment options, but it is important to
understand that fibrosis progression is faster in patients with HCV G3 (Bochud
2009).

Treatment of HCV genotypes 4, 5, and 6
BOC and TLV have hardly been tested in patients with HCV G4, 5, or 6. Neither PI
is approved for the treatment of HCV G4, 5, or 6. Thus, SOC remains the
combination of PEG-IFN/RBV. In general, treatment duration of 48 weeks is
recommended based on the results of the large, randomized Phase III trials (Fried
2002, Hadziyannis 2004, Manns 2001). However, these trials included few patients
with HCV G4, 5, and 6 and further large, prospective randomized studies with RGT
are rare. Importantly, HCV G4, 5, and 6 are very common in areas where chronic
hepatitis C is highly prevalent. For example, HCV G4 is most prevalent in the
Middle East and Egypt where it accounts for >80% of all HCV cases
(approximately 34 million patients) (Khattab 2011). HCV G5 is most prevalent in
South Africa, and genotype 6 in Southeast Asia (Nguyen 2005). The available study

Standard Therapy of Chronic Hepatitis C Virus Infection 219
results, although limited, suggest that patients with HCV G4, 5 and 6 may show
different clinical courses and treatment outcomes. Ethnicity-related factors (i.e.,
IL28B, regional aspects) may contribute to these findings. Overall, data from
smaller studies suggest that HCV G4, 5 and 6 appear easier-to-treat compared to
HCV G1 but the optimal treatment duration is not clear (Antaki 2010, Nguyen
2005) (Table 9). Although some studies show SVR on the same order as for HCV
G2/3 patients, a fixed duration of 24 weeks of treatment as for HCV G2/3 is not
advisable, even for patients with HCV G6, which appears to show the best SVR
(Lam 2010, Nguyen 2008). RGT based on early viral kinetics should be possible.
Patients who achieve RVR are candidates for a short treatment regimen of 24 weeks
if they don’t have predictors of poor response (see above). Based on data for HCV
G1 (Berg 2006, Sanchez-Tapias 2006), patients without RVR and/or partial
response may be considered for 72 weeks. This has been proposed for HCV G4 by
an international expert panel (Khattab 2011), but the evidence is limited. The
proposed algorithm is shown in Figure 6. We suggest treating HCV G5 and 6 also
according to this algorithm. Patients with treatment failure may be considered for
retreatment, especially if the previous therapy was suboptimal. It is important to
optimise dose and duration of treatment during retreatment.

Figure 6. Suggestion for treatment of HCV genotypes 4, 5, and 6. This algorithm was
initially proposed for HCV G4 (adapted Khattab 2011). Sensitive HCV RNA assays (limit of
detection 12-15 IU/ml or 50 IU/ml) at weeks 4 and 12 may determine treatment duration.
Reducing treatment duration is not recommended in patients with predictors of poor response
(liver cirrhosis, insulin resistance, diabetes mellitus or hepatic steatosis, high baseline viral load
>800,000 IU/mL).

220 Hepatology 2012
Table 9. Efficacy of antiviral treatment with PEG-IFN plus ribavirin in patients with
chronic hepatitis C infected with genotypes 4, 5, and 6. Selected trials.
Study

Treatment

HCV genotype/Duration

SVR

(Diago 2004)
n=49

180 µg PEG-IFN α-2a
800/1000/1200 mg ribavirin

G4

24 weeks
24 weeks
48 weeks
48 weeks

0% (if low RBV)
67% (if high RBV)
63% (if low RBV)
79% (if high RBV)

(Hasan 2004)
n=66

1.5µg/kg PEG-IFN α-2b
1000/1200 mg ribavirin

G4

48 weeks
48 weeks
48 weeks

68%
55% (if HVL)
86% (if LVL)

(Kamal 2005)
n=287

1.5µg/kg PEG-IFN α-2b
1000/1200 mg ribavirin

G4

24 weeks
36 weeks
48 weeks

29%
66%
69%

(Kamal 2007)
n=358

1.5µg/kg PEG-IFN α-2b
10.6 mg/kg ribavirin
RGT

G4

24 weeks RGT
36 weeks RGT
48 weeks RGT
48 weeks

86% (if RVR)
76% (if cEVR)
56% (if EVR)
58%

(Ferenci
2008)
n=66

180 µg PEG-IFN α-2a
1000/1200 mg ribavirin
RGT

G4

24 weeks RGT

87% (if RVR)

(Bonny 2006)
n=59

PEG-IFN α-2a or b
800-1200 mg ribavirin

G5

48 weeks

58%

(Lam 2010)
n=60

PEG-IFN α-2a
800-1200 mg ribavirin

G6

24 weeks
48 weeks

70%
79%

(Nguyen
2008)
n=35

PEG-IFN α-2a or b
800-1200 mg ribavirin

G6

24 weeks
48 weeks

39%
75%

RBV, ribavirin; LVL, low baseline viral load; HVL, high baseline viral load; RGT, responseguided therapy

Optimisation of HCV treatment
Adherence to therapy
Adherence to therapy is one of the most important factors associated with the
success of antiviral treatment (McHutchison 2002). The definition of adherence
used here is the “80/80 rule”, that is, patients who receive more than 80% of the
medication and are treated for more than 80% of the planned duration of treatment
are considered adherent. One of the first studies investigating the effect of
adherence in PEG-IFN/RBV treatment demonstrated that patients who fulfilled the

Standard Therapy of Chronic Hepatitis C Virus Infection 221
80/80 rule had a 63% SVR compared to 52% of those with less than 80% adherence
(McHutchison 2002). Another study showed that a cumulative ribavirin dose of
more than 60% is important to achieve an SVR (Reddy 2007). For the new triple
therapy, adherence to the PI becomes even more important as mentioned above. The
three-times-daily regimen necessitates highly motivated and compliant patients.
BOC and TLV have to be taken every 7-9 hours together with food. Reduction of
the PI or irregular intake bears the risk for rapid emergence of drug resistance. Dose
reduction of the PI is associated with significantly diminished SVR (Gordon 2011)
and is therefore not an option to manage side effects. An optimal management of
PEG-IFN/RBV side effects therefore is essential in order to optimise treatment
responses. In the case of anemia, dose reduction of ribavirin is possible and not
associated with impaired SVR to triple therapy (Roberts 2011). Another important
and new issue is drug interactions that can diminish the effectiveness of the PI or
induce toxicity of concomitant medications, which may lead to discontinuation of
all drugs. Knowledge about drug interactions is therefore important for the optimal
management of patients receiving PEG-IFN/RBV/PI.

Management of side effects and complications
Severe side effects may reduce adherence to therapy and may result in dose
modifications that result in a less-than-optimal response. IFN, ribavirin and the new
protease inhibitors induce side effects that have to be managed with the patient
(Table 10). The IFN-related side effects can be divided into IFN-induced bone
marrow suppression, flu-like symptoms, neuropsychiatric disorders, and
autoimmune syndromes. The main problem of ribavirin is hemolytic anemia.
Boceprevir and telaprevir are associated with additional side effects such as rash or
dysgeusia and additionally an increase of anemia (Table 10) (Jacobson 2011b,
Manns 2011b, Vertex 2011, Zeuzem 2011). Overall, side effects result in premature
withdrawals from therapy (5-17% during triple therapy depending on the duration of
therapy (Jacobson 2011b)) and additional patients require dose modifications during
treatment. The frequency of treatment discontinuations and dose modifications are
lower in recent studies, suggesting an improved understanding and management of
adverse events (Manns 2006). Similar developments are expected also for treatment
with PIs. For example, the reported cases of rash decreased from 60% in Phase II
(McHutchison 2009a) to 36% in the Phase III trial (Jacobson 2011b). However, the
frequency of adverse events that occurred in registration trials with carefully
selected patients may differ from general clinical practice, where patients with, e.g.,
history of psychiatric disorders or advanced liver disease are being treated. For
example, factors significantly associated with developing anemia on TVR were
older age and advanced fibrosis (Roberts 2011).
IFN side effects
The effect of IFN on bone marrow results in decreased granulocytes and
thrombocytes during treatment. These are usually moderate if normal counts are
initially present. However, dose modifications are necessary, especially in patients
with initially low counts (Manns 2006). This limits the use of IFN in patients with
advanced liver cirrhosis who often have low platelets and are also more vulnerable
to infections. Therapeutic concepts in order to raise platelet levels safely would have

222 Hepatology 2012
a significant effect on the effective management of patients, especially those with
advanced liver disease.
A promising novel agent is the oral thrombopoietin receptor agonist eltrombopag
that has been tested in patients with chronic hepatitis C and liver cirrhosis
(McHutchison 2007). Eltrombopag was able to increase platelet levels in 75-95% of
patients depending on the dose, and antiviral therapy was then initiated. Twelve
weeks of antiviral therapy were then taken by 36-65% of patients receiving 30-75
mg of eltrombopag vs only 6% of patients in the placebo group (McHutchison
2007). A recent Phase III study including patients with platelets <75 K/µl has shown
that eltrombopag pretreatment for 9 weeks and later combination with PEGIFN/RBV could significantly increase SVR in comparison to eltrombopag
pretreatment and later PEG-IFN/RBV/placebo (Afdhal 2011). Neutropenia is
another of the most common reasons for dose modification.
Granulocyte macrophage colony stimulating factor (GM-CSF, Filgrastim) could
potentially be used to stabilize neutrophil counts during IFN therapy (Shiffman
1998, Younossi 2008). While administration of GM-CSF may enable patients to
remain on treatment, a systematic review documented only weak evidence that this
improves the likelihood of SVR compared to dose reduction (Tandon 2011). The
economic evaluation was inconclusive, therefore further cost-benefit analyses and
trials are required to recommend routine use of these agents. However, IFN-induced
neutropenia is generally not associated with a significant increased risk for bacterial
infections (Soza 2002).
Flu-like symptoms usually occur during the first weeks of treatment and severity
declines over time. These symptoms include fever, chills, headache, arthralgia, and
myalgia. Antipyretic drugs such as paracetamol can help to prevent or reduce these
side effects (Manns 2006).
Neuropsychiatric side effects such as irritability, severe fatigue, and apathy are
frequent (>50%) and pose a great problem for many patients and their family
members. Severe depression can occur and suicide has been reported (Janssen
1994). Psychiatric care and the use of antidepressants, especially serotonin uptake
inhibitors (SSRIs) may help reduce IFN-induced depression and consequently
improve adherence to hepatitis C therapy. A double-blind placebo-controlled study
in 100 patients with chronic hepatitis C was terminated prematurely due to
significant superiority of SSRIs over placebo in terms of decreasing scores on the
Hospital Anxiety and Depression Scale (HADS). All SSRI-treated patients were
able to complete IFN treatment (Kraus 2008). SSRI treatment is highly effective in
HCV patients during IFN-based therapies, when starting early after the onset of
clinically relevant depression. Of note, citalopram should no longer be used at doses
greater than 40 mg per day because it can cause prolongation of the QT interval on
the electrocardiogram. This may be of relevance during triple therapy.
IFN has immunomodulatory properties, and treatment can induce autoimmune
phenomena (Wesche 2001). The most frequent problem is the development of
autoimmune thyroiditis. In most cases thyroiditis starts with hyperthyroidism that
later turns into hypothyroidism. Autoimmune thyroiditis has been reported in up to
20% of patients during IFN-based therapies (Costelloe 2010). However, only a few
patients develop thyroid disease that requires ongoing therapy (Costelloe 2010, Tran
2011). Patients with preexisting thyroid antibodies may have a higher risk and it is

Standard Therapy of Chronic Hepatitis C Virus Infection 223
possible that hepatitis C itself may be a cause of autoimmune thyroiditis (GanneCarrie 2000).
Other autoimmune diseases can also be aggravated by IFN therapy (e.g., diabetes
or autoimmune hepatitis). Patients with documented HCV infection may get worse
during IFN treatment if an underlying autoimmune hepatitis is present. This has
been observed particularly in LKM antibody-positive individuals. These patients
require careful monitoring if IFN is considered as first-line treatment. However, IFN
therapy seems to be safe in most HCV/anti-LKM-1-positive patients (Dalekos 1999,
Todros 1995).
Ribavirin side effects
The main side effect of ribavirin is hemolytic anemia that frequently results in
ribavirin dose reduction or even discontinuation, which may significantly affect the
SVR with PEG-IFN/RBV alone (Reddy 2007). Treatment with erythropoietin
(EPO) can reverse ribavirin-associated anemia and allow full adherence to ribavirin
therapy (Afdhal 2004). Although the use of EPO can reduce the incidence and
severity of ribavirin induced anemia, there is limited evidence that EPO has an
effect on SVR. A prospective, randomized, controlled trial has evaluated the effect
of EPO on SVR. Patients receiving PEG-IFN α-2b plus 13.3 mg/kg/day ribavirin
were compared to patients receiving PEG-IFN α-2b, 13.3 mg/kg/day ribavirin and
40,000 U/week EPO. Although there were significantly fewer ribavirin dose
reductions in those patients who received EPO, no improvement in SVR was
shown. A third group received a higher starting dose of 15.2 mg/kg/day in
combination with EPO and they did show a significantly higher SVR but there was
no control group in this trial (Shiffman 2007a). A placebo- controlled trial
investigated the use of 30,000 U/week EPO or placebo in addition to PEGIFN/RBV when hemoglobin reduced to ≤12 g/dL in men and ≤11 g/dL in women.
SVR was not significantly improved with EPO. Overall, EPO may improve quality
of life, and in some individuals it may also improve the chance of achieving an SVR
(in those requiring high doses of RBV). However, EPO use is off-label. Alternative
ribavirin-like drugs with less toxicity and/or higher antiviral efficacy have failed
(Benhamou 2009, Marcellin 2010). Drug monitoring of ribavirin could be an option
to optimise the ribavirin dose without losing efficacy (Svensson 2000). The
pharmacokinetics of ribavirin suggests that not only body weight but also renal
function (glomerular filtration rate) should be considered when selecting the
ribavirin dose (Bruchfeld 2002). Importantly, RBV is considered as teratogenic and
therefore contraindicated during and 6 months after pregnancy. Effective birth
control measures are necessary (Sarrazin 2010a).
Protease inhibitor side effects
Both BOC and TLV were associated with more frequent and severe anemia.
Approximately 1-1.5 g/dL additional decrease of hemoglobin can be expected. The
different treatment duration of both drugs results in different grades and duration of
anemia. In the BOC trials, approximately 50% of patients receiving BOC
experienced anemia, compared to 29% of participants who did not receive the drug
(Poordad 2011b). Of note, treatment with EPO was allowed in the BOC trials.
Participants in the TLV trial were not permitted to receive EPO and anemia rates
were 36% and 17% for patients who did or did not receive the drug, respectively

224 Hepatology 2012
(Jacobson 2011b) (Table 10). Around 3% of participants taking TLV discontinued
due to anemia, versus less than 1% of participants taking only PEG-IFN/RBV.
Frequent monitoring during the first weeks of PI treatment is important. Significant
anemia occurs after 4 weeks of treatment. In the case of anemia, RBV should be
reduced. Importantly RBV dose reduction was not associated with diminished SVR
(Roberts 2011). In the BOC trials, SVR was similar in anemic patients who were
managed with RBV dose reduction versus EPO (Sulkowski 2011). A prospective
trial is on going that compare EPO usage versus reduction of RBV in the
management of anemia. Blood transfusion is an option to rapidly increase
hemoglobin in patients with severe anemia. A total of 5% of patients in the
telaprevir groups received blood transfusions compared to 2% of patients on
standard therapy (Jacobson 2011b). It has to be considered that older patients with
more advanced liver disease may have higher rates of anemia (Roberts 2011). The
last option is to discontinue the PI. Importantly, the dose of BOC or TLV must not
be reduced during therapy or restarted after discontinuation. Besides anemia, BOC
and TLV may also increase neutropenia (Table 10).
Another important new side effect specific for TLV is the development of skin
rash. A majority of patients receiving TLV (56%) reported the development of rash,
typically pruritic and eczematous, usually involving <30% of the body surface area
(Vertex 2011). Rash develops usually after 3-4 weeks but can occur at any time
during TLV treatment. Rash can be mild (37%), moderate (14%) or severe (5%)
(Incivo 2011). Serious, life-threatening skin reactions such as Stevens Johnson
Syndrome or Drug Rash with Eosinophilia and Systematic Symptoms (DRESS)
have been reported in 0.1-0.4%, respectively (Incivo 2011). Patients with
developing rash must be closely monitored for progression. In the case of severe
rash (generalized rash >50% of body surface area, mucous membrane ulceration,
target lesions, epidermal detachment), consultation with a specialist in dermatology
is recommended and treatment with TLV should be discontinued. Rash is usually
reversible but it may still worsen for a few days after discontinuation of TLV and
may take weeks for complete resolution. PEG-IFN/RBV may be continued if the
rash improves within 7 days. Early management of rash is important to prevent
progression. Topical corticosteroids and oral antihistamines (consider drug
interactions) help to relieve symptoms. Systemic corticosteroids are not
recommended during the early management.
Further specific adverse events concerning TLV are anal discomfort, anal pruritus
and hemorrhoids, which was documented in 29% of study participants (Vertex
2011) (Table 10). In our experience, almost every patient reports symptoms if
specifically asked. For most patients these symptoms were mild or moderate and do
not lead to discontinuation of the drug.
A more specific adverse event for BOC is reversible dysgeusia (metallic taste),
which occurred in 37% in the BOC trials (Table 10). Patients treated with TLV also
experience dysgeusia, reported as a “sticky taste”.
Because BOC and TLV must be used in combination with PEG-IFN/RBV,
contraindications and warnings concerning PEG-IFN/RBV are also applicable to
BOC and TLV. Therefore, intense care must be taken to prevent pregnancy in
female patients and in female partners of male patients. Of note, oral hormonal
contraception is theoretically less effective due to drug interaction with BOC or
TLV. In case of patient pregnancy all drugs must be discontinued. Pregnancies

Standard Therapy of Chronic Hepatitis C Virus Infection 225
should
be
reported
to
the
(http://ribavirinpregnancyregistry.com).

ribavirin

pregnancy

registry

Drug interactions
BOC and TLV undergo extensive hepatic metabolism especially by the cytochrome
P450 CYP3A pathway. Thus, both PIs are target as well as perpetrator of drug
interactions. As inhibitors of CYP3A, both PIs can result in increased plasma
concentrations of concomitant drugs that are metabolized via the same route,
leading to prolonged therapeutic effects and/or toxicity. In contrast, concomitant
drugs that induce CYP3A may result in decreased plasma concentrations of BOC or
TLV, which can reduce the therapeutic effect. TLV is also an inhibitor of PGP
transport. Coadministration of TLV with drugs that are substrates for PGP transport
may result in increased plasma concentrations of such drugs, which could increase
adverse reactions. Based on in vitro experiments BOC also has the potential to
inhibit PGP. Importantly, BOC is metabolized not only by cytochrome P450mediated oxidation but also significantly by ketone reduction via aldo-keto
reductase (AKR). Because the biotransformation and clearance of BOC involves
two different enzymatic pathways, drug interactions with BOC may be less likely
compared to TLV. For the optimal management of triple therapy, it is essential to
specifically ask patients about concomitant medications and investigate if those
drugs may interact with the PI. Even herbals and food have to be considered as St.
John’s Wort is a potent inducer of CYP3A and naringin, an ingredient of grapefruit,
an inhibitor. A list of drug interactions is given in the prescribing information.
Supportive online tools or apps for mobile devices are available. One example is the
comprehensive drug interaction resource provided by the University of Liverpool
(http://www.hep-druginteractions.org). The website provides clinically useful and
evidence-based information which is updated when new drug interactions are
analysed and published. Drug interactions are considered significant if the area
under the plasma concentration time curve (AUC) is changed by more than 30%.

226 Hepatology 2012
Table 10. Common side effects (>5% of patients) recorded in the PEG-IFN/RBV/PI
trials. The incidence of side effects between different studies is difficult to compare
because there were significant differences in genetic and socioeconomic backgrounds.
There were methodological differences in assessing side effects as well. Patients were
selected on the basis of well-defined inclusion and exclusion criteria. Important
differences between PEG-IFN/RBV and PEG-IFN/RBV/PI are highlighted in bold.
Side effects

Incidence with PEGIFN/RBV

Incidence with
PEG-IFN/RBV/BOC

Incidence with PEGIFN/RBV/TLV

Fatigue

50%†, 57%*

57%*

56%†

Insomnia

31%‡,*

32%*

32%‡

Headache

39%‡, 43%*

44%*

41%-43%‡

Pyrexia

24%‡, 31%*

31%*

26%-30%‡

Nausea

31%‡, 40%*

45%*

40-43%‡

Diarrhea

17%†, 18%*

23%*

26%†

Alopecia

25%*

26%*

n.a.

Depression

20%*

20%*

No difference‡

Anemia

17%†, 29% §,*

49%§*

36%†

Neutropenia

18%*,**

23%*

23%**

Dysgeusia

3%†, 15%*

37%*

10%†

Rash

17%*, 34%†

16%*

56%†

Pruritus

23%*, 28%†

21%*

47%†

Anorectal
discomfort

1%*, 3%†

1%*

11%†

Anal pruritus

1%†

n.a.

6%†

Hemorrhoids

3%*,†

4%*

12%†

* Manns 2011b, † Vertex 2011, ‡ Jacobson 2011b, § EPO was allowed,
** Zeuzem 2011

Treatment of hepatitis C in special populations
Patients with acute hepatitis C
The goal of acute hepatitis C treatment is the prevention of persistent HCV
infection. The natural rate of HCV evolution to a chronic state is 50-90%. As a
vaccine is not yet available, early treatment of acute HCV infection with IFN is the
only option to prevent persistent HCV infection; however, the diagnosis of acute
primary HCV infection may be difficult and its distinction from exacerbation of an
underlying unrecognized chronic HCV infection may be difficult (Sarrazin 2010a).
The immediate treatment of patients with symptomatic acute hepatitis C with
recombinant IFN or PEG-IFN monotherapy for 24 weeks can prevent the
development of chronic hepatitis C in approximately 90% of cases (Broers 2005,
Jaeckel 2001, Santantonio 2005, Vogel 1996, Wiegand 2006). However, good
patient adherence to therapy is necessary to achieve these response rates (Wiegand

Standard Therapy of Chronic Hepatitis C Virus Infection 227
2006). Coadministration with ribavirin does not seem to be necessary. This may be
different in patients with HIV coinfection (Grebely 2011a) (see Chapter 18). TLV
and BOC have not been tested in patients with acute HCV infection. Symptomatic
patients also have a good chance of clearing HCV spontaneously (Gerlach 2003,
Hofer 2003), occurring usually in the first 12 weeks after the onset of symptoms.
As for patients with treatment-induced SVR, spontaneous clearance of HCV is
also associated with IL28B polymorphisms and IP-10 (Beinhardt 2012, Grebely
2010, Thomas 2009, Tillmann 2010), which may be useful for decision-making.
The treatment of only those patients who remain HCV RNA-positive 12 weeks after
the onset of symptoms results in an overall SVR (self-limited and treatmentinduced) in 91% of patients (Gerlach 2003). Asymptomatic patients, however,
should probably be treated immediately since these patients have a higher risk for
evolution to a chronic state. However, early treatment of acute HCV infection to
prevent chronic disease does have its limitations. A main problem is that primary
HCV infection is usually asymptomatic and most patients cannot be identified in
this early stage of disease. Another reason is that a number of patients have medical
contraindications for treatment with IFN or are not ready for therapy because they
are still active intravenous drug users (IDU).
There are two concerns in treating active IDU with IFN. In case of successful
therapy there is a risk of reinfection with HCV (Grebely 2011b). The second
concern is the side effect profile of IFN, especially the neuropsychiatric problems
that may result in a worsening of addictive behaviour (Wiegand 2006). In addition,
it has been shown that the acceptance of and adherence to antiviral therapy by these
patients can be low due to the side effects of IFN (Broers 2005).
There are open questions on the treatment of acute hepatitis C. For example, a
study coordinated by the German Competence Network for Viral Hepatitis (HepNet) tested if the wait-and-see strategy for 12 weeks is as effective as immediate
treatment. The first preliminary data from this trial were presented at the 2009
EASL Annual Meeting. Early treatment was superior in the intent-to-treat analysis,
although this was mainly due to higher drop out rates in the delayed treatment arm.
Of note, all patients who started treatment later and who completed treatment and
follow-up responded to treatment. The trial also showed for the first time that early
treatment is as effective in patients with asymptomatic acute hepatitis C (Deterding
2009). Thus, the decision to wait for 12 weeks after diagnosis or to treat
immediately may be made individually. Importantly, the compliance of the patient
should be assessed. Host genetics (IL28B), other markers (IP-10), or HCV RNA
kinetics during the first weeks may help to decide when to treat. For example,
asymptomatic patients with IL28B rs12979860-CT or TT may be treated
immediately. In the next five to ten years we will presumably have new DAAs that
will allow IFN-free therapies.

Patients with normal aminotransferase levels
Approximately 30% of patients with chronic hepatitis C maintain persistently
normal alanine aminotransferase (ALT) levels despite having detectable HCV RNA
in serum. These patients have generally mild liver disease and show a slow
progression to cirrhosis. However, up to one third of patients with normal ALT can
present with significant liver fibrosis necessitating an effective treatment (Bacon
2002, Zeuzem 2004a). In current guidelines, ALT elevation is not a prerequisite to

228 Hepatology 2012
start antiviral therapy and the assessment of liver disease severity should be made
regardless of ALT (EASL 2011). In general, the efficacy and safety of PEGIFN/RBV therapy in patients with persistently normal ALT levels seems to be
comparable to that seen in patients with elevated ALT levels. 48 weeks of PEG-IFN
α-2a plus ribavirin has been shown to lead to an SVR of 52% in patients with
chronic hepatitis C and persistently normal ALT levels. Treatment-related flares in
ALT activity were not observed (Zeuzem 2004a). This has been not analysed in
detail for triple therapy regimes.

Patients with compensated liver cirrhosis
Successful therapy of patients with advanced fibrosis and liver cirrhosis is
associated with decreased incidence of HCC, decompensation and liver-related
mortality (Morgan 2010, Veldt 2007). In addition, in patients awaiting liver
transplantation, successful therapy prevents graft rejection (Everson 2005, Forns
2003). Thus, patients should be considered for therapy if no contraindications are
present. However, SVR is diminished in patients with cirrhosis, for the new triple
therapy as well (Pol 2011b) (Figure 7A, 7B). Patients with liver cirrhosis must be
treated for a fixed duration of 48 weeks. Thus, exposure to drugs associated with
side effects is still long in the new era of PIs. Treatment of patients with liver
cirrhosis requires a close monitoring of patients. Hematological adverse events are
more frequent than in non-cirrhotic patients (EASL 2011). Of note, patients with
advanced cirrhosis (i.e., platelets <75K/µl) have not been treated with BOC or TLV
because they were excluded from the large Phase II and III studies. In general,
treatment should be limited to patients with Child-Pugh A cirrhosis. In patients with
Child-Pugh B cirrhosis, therapy may be only considered in individual cases in
experienced centres. If ascites is present, antibiotic prophylaxis should be given. If
patients with cirrhosis achieve SVR, it is important to perform HCC surveillance
because cirrhosis remains and HCC development is reduced but not abolished
(EASL 2011).

Standard Therapy of Chronic Hepatitis C Virus Infection 229
Figure 7A. SVR of TLV-based regimens in patients with HCV genotype 1 according to
fibrosis stage. Subanaylsis of the REALIZE Phase III trial (Pol 2011b).

Figure 7B. SVR of BOC-based regimens in patients with HCV genotype 1 with advanced
fibrosis and cirrhosis (F3-F4). Subanaylsis of the SPRINT-2 and RESPOND-2 trials (Bruno
2011).

Patients after liver transplantation
HCV reinfection occurs in almost all patients after liver transplantation. While the
course of hepatitis C in liver transplant recipients was believed to be rather benign
in the late ‘80s and early ‘90s (Boker 1997), HCV has led to a more rapid
progression post-transplant in recent years (Berenguer 2005, Neumann 2004) with
cirrhosis within the first 5-10 years in 20-30% of patients. HCV definitely takes a
more rapid course post-transplant than in immunocompetent individuals and
treatment needs are obvious. Antiviral therapy of HCV may be started before
transplant to prevent reinfection of the graft. If this approach is successful,
reinfection can be prevented in two-thirds of patients (Forns 2003). However,
treatment with IFN/RBV is poorly tolerated in individuals with decompensated
cirrhosis with a high risk for infections and this approach is feasible in only a
minority of patients (Everson 2005, Forns 2003). Preemptive treatment within the
first 4-6 weeks post transplant has been disappointing with SVR between 5% and
33% for different regimens including IFN monotherapy and IFN/RBV (Terrault
2003). Studies using PEG-IFN/RBV reported an SVR of 26-48% (Carrion 2007,
Dumortier 2004, Neff 2004, Roche 2008, Rodriguez-Luna 2004). Treatment
duration should be at least similar to non-transplanted patients considering early
viral kinetics and the HCV genotype. However, bone marrow toxicity, depression,
and rejection are limiting factors that require aggressive management (e.g., growth
factors) (Rodriguez-Luna 2004). The ribavirin dose may have to be adjusted since
many patients have some degree of renal insufficiency. Interestingly, the risk for
IFN-induced graft rejection seems to be higher if ribavirin is not used. BOC and
TLV are currently being evaluated in patients after liver transplantation. Besides
adverse events (anemia, neutropenia), drug interactions with immunosuppressive
drugs need to be considered. For example, TLV increases levels of tacrolimus by
approximately 70-fold (Garg 2011). In conclusion, patients with established graft
hepatitis should be treated with PEG-IFN/RBV, but at this stage, BOC and TLV
should not be used in transplant patients outside clinical trials. Whether reinfection
can be prevented by currently developed DAAs will have to be addressed in future
studies.

230 Hepatology 2012

Hemodialysis patients
Treatment needs for dialysis patients with hepatitis C are obvious especially if
patients are considered for kidney transplantation. The outcome of HCV postkidney transplantation is worse than for HCV-negative patients after renal
transplantation. However, IFN-based therapies are contraindicated posttransplantation since they may induce rejection. Thus, if possible, HCV should be
eliminated before transplantation. There have been several smaller reports on the
treatment of HCV with IFN monotherapy in patients with end-stage renal disease
(Fabrizi 2002). Surprisingly, the results for IFN monotherapy on dialysis were better
than in patients not undergoing dialysis, with SVR results of 21-64%. Data on
combination with ribavirin are limited since ribavirin is contraindicated in this
setting. However, ribavirin can be given at lower doses in dialysis patients, usually
at 200-400 mg daily (Bruchfeld 2001). It has to be considered that there may be
significant differences between the two pegylated interferons in the setting of
dialysis since PEG-IFN α-2a is eliminated mainly by the liver while PEG-IFN α-2b
is cleared via the kidney (reviewed in Cornberg 2002). Thus, only PEG-IFN α-2a
should be used in this setting. The efficacy of BOC and TLV has not been tested in
HCV patients with end stage renal disease or in patients undergoing hemodialysis.
Theoretically, BOC and TLV can be administered in patients with compensated
renal insufficiency and dose adjustment is not necessary because both drugs are
metabolized through the liver and mainly eliminated via the feces with minimal
urinary excretion (Ghany 2011).

Drug abuse and patients on stable maintenance
substitution
Treatment of patients with active drug use is an individual approach and should only
be performed in an experienced multidisciplinary setting including hepatologists,
psychiatrists and addictologists. Drug interactions with BOC and TLV need to be
considered.

Patients with coinfections
Due to the similar routes of transmission, patients with chronic hepatitis C are
frequently coinfected with hepatitis B virus, hepatitis D virus or human
immunodeficiency virus. This important patient group is discussed in Chapters 11,
18 and 19.

Patients with hemophilia
Due to contaminated clotting factor concentrates many patients with hemophilia are
infected with HCV and/or HIV. Studies investigating PEG-IFN/RBV in hemophilia
patients are limited and often include small numbers of patients. Review of
available data suggest that treatment success of HCV-infected hemophiliacs is
similar to that achieved in the general HCV-infected population (Franchini 2008).

Patients with extrahepatic manifestations
More than 50% of HCV-infected patients suffer from extrahepatic manifestations
ranging from fatigue to severe symptoms of mixed cryoglobulinemia (Cacoub 1999)
(see Chapter 16). The primary goal of treatment is HCV eradication, which is
associated with improvement of clinical symptoms, especially in patients with

Standard Therapy of Chronic Hepatitis C Virus Infection 231
mixed cryoglobulinemia (Cresta 1999, Pischke 2008, Zignego 2007). Insulin
resistance can be improved in HCV G1 patients with SVR (Thompson 2012). In
patients with severe symptoms of mixed cryoglobulinemia, treatment with
rituximab may be considered (Cacoub 2008). Recent studies have also tested the
combination of PEG-IFN/RBV and rituximab. The clinical response may be
achieved faster and SVR is not diminished in patients who receive rituximab
(Dammacco 2010, Saadoun 2010). Exacerbation of certain extrahepatic
manifestations may occur with IFN-based therapy or IFN may be contraindicated
(Zignego 2007). BOC and TLV have not been investigated in patients with
extrahepatic manifestations.

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Hepatitis C: New Drugs 239

14. Hepatitis C: New Drugs
Christian Lange, Christoph Sarrazin

Introduction
Combination therapy with pegylated interferon-α plus weight-based ribavirin leads
to sustained virologic response (SVR, defined by undetectable HCV RNA 24 weeks
after treatment completion) in approximately 50% of all HCV genotype 1-infected
patients, compared to 70-90% of patients infected with HCV genotypes 2 and 3
(Zeuzem 2009). The limited treatment success of this treatment especially in HCV
genotype 1 patients, the long treatment durations (up to 72 weeks), as well as the
numerous side-effects of PEG-IFN α and ribavirin therapy, and an exploding
knowledge of the HCV life cycle and of structural features of the HCV proteins, has
spurred the development of many promising directly acting antiviral agents (DAA)
(Kim 1996, Lindenbach 2005, Lohmann 1999, Lorenz 2006, Wakita 2005). In
principle, each of the four HCV structural and six non-structural proteins, HCVspecific RNA structures such as the IRES, as well as host factors on which HCV
depends, are suitable targets for DAA agents. In the following section, DAA
compounds currently in clinical development are presented (Table 1, Figure 1).
Table 1. Selected directly acting antiviral agents in the pipeline.
Drug name

Company

NS3-4A protease inhibitors
Ciluprevir (BILN 2061)
Boehringer Ingelheim
Telaprevir (VX-950)
Vertex / Janssen
Boceprevir (SCH503034)
Merck (S-P)
Simeprevir (TMC435350)
Janssen / Medivir
Danoprevir (R7227)
Roche / InterMune
Vaniprevir (MK-7009)
Merck
MK-5172
Merck
BI201335
Boehringer Ingelheim
Narlaprevir (SCH900518)
Schering-Plough
Asunaprevir (BMS-650032) Bristol-Myers Squibb
PHX1766
Phenomix
GS-9256
Gilead

Target / Active site

Phase

Active site / macrocyclic
Active site / linear
Active site / linear
Active site / macrocyclic
Active site / macrocyclic
Active site / macrocyclic
Active site / macrocyclic
Active site / linear
Active site / linear
Active site
Active site
Active site

Stopped
IV
IV
III
II
Halted/II
II
III
Halted
II
I
II

240 Hepatology 2012
Drug name

Company

GS-9451
Gilead
ABT450
Abbott
IDX320
Idenix
ACH-1625
Achillion
Nucleoside analog NS5B polymerase inhibitors
Valopicitabine (NM283)
Idenix / Novartis
Mericitabine (R7128)
Roche / Pharmasset
R1626
Roche
PSI-7977
Pharmasset
PSI-938
Pharmasset
IDX184
Idenix
Non-nucleoside NS5B polymerase inhibitors (NNI)
BILB 1941
Boehringer Ingelheim
BI207127
Boehringer Ingelheim
MK-3281
Merck
TMC647055
Janssen
Filibuvir (PF-00868554)
Pfizer
VCH759
ViroChem Pharma
VCH916
ViroChem Pharma
VCH222
ViroChem Pharma
ANA598
Anadys
ABT-072
Abbott
ABT-333
Abbott
HCV-796
ViroPharma / Wyeth
GS-9190
Gilead
IDX375
Idenix
NS5A inhibitor
Daclatasvir (BMS-790052)
Bristol-Myers Squibb
BMS-824393
Bristol-Myers Squibb
PPI-461
Presidio Pharmaceuticals
GS-5885
Gilead
Indirect inhibitors / unknown mechanism of action
Alisporivir (Debio-025)
Debiopharm
NIM811
Novartis
SCY-635
Scynexis
Nitazoxanide
Miravirsen
Santaris
Celgosivir
Migenix

Target / Active site
Active site
Active site
Active site
Active site / macrocyclic?

Phase
I
II
II
II

Active site
Active site
Active site
Active site
Active site
Active site

Stopped
II
Stopped
II
II
II

NNI site 1 / thumb 1
NNI site 1 / thumb 1
NNI site 1 / thumb 1
NNI site 1 / thumb 1
NNI site 2 / thumb 2
NNI site 2 / thumb 2
NNI site 2 / thumb 2
NNI site 2 / thumb 2
NNI site 3 / palm 1
NNI site 3 / palm 1
NNI site 3 / palm 1
NNI site 4 / palm 2
NNI site 4 / palm 2
NNI site 4 / palm 2

Stopped
II
II
II
II
II
II
II
II
II
Stopped
II
II

NS5A domain 1 inhibitor
NS5A protein
NS5A protein
NS5A protein

II
I
I
I

Cyclophilin inhibitor
Cyclophilin inhibitor
Cyclophilin inhibitor
PKR induction (?)
miRNA122 antisense RNA
Α-glucosidase inhibitor

III
I
II
II
II
II

HCV life cycle and treatment targets
HCV is a positive-sense single-stranded RNA virus of approximately 9600
nucleotides. The HCV genome contains a single large open reading frame encoding
for a polyprotein of about 3100 amino acids. From this initially translated
polyprotein, the structural HCV protein core (C) and envelope glycoproteins 1 and 2
(E1, E2); p7; and the six non-structural HCV proteins NS2, NS3, NS4A, NS4B,
NS5A and NS5B, are processed by both viral and host proteases. The core protein
forms the viral nucleocapsid carrying E1 and E2, which are receptors for viral
attachment and host cell entry. The non-structural proteins are multifunctional

Hepatitis C: New Drugs 241
proteins essential for the HCV life cycle (Bartenschlager 2004). P7 is a small
hydrophobic protein that oligomerises into a circular hexamer, most likely serving
as an ion channel through the viral lipid membrane. The large translated section of
the HCV genome is flanked by the strongly conserved HCV 3´ and 5´ untranslated
regions (UTR). The 5´UTR is comprised of four highly structured domains forming
the internal ribosome entry site (IRES), which plays an important role in HCV
replication (Figure 2).

Figure 1. HCV life cycle and targets for directly acting antiviral (DAA) agents.

NS3-4A protease inhibitors
Molecular biology
After receptor-mediated endocytosis, the fusion of HCV with cellular membranes,
and uncoating the viral nucleocapsid, the single-stranded positive-sense RNA
genome of the virus is released into the cytoplasm to serve as a messenger RNA for
the HCV polyprotein precursor. HCV mRNA translation is under the control of the
internal ribosome entry site (IRES), formed by domains II-IV of the HCV 5´UTR
(Moradpour 2007). IRES mediates HCV polyprotein translation by forming a stable
complex with the 40S ribosomal subunit, eukaryotic initiation factors and viral
proteins.

242 Hepatology 2012

Figure 2. Genomic organisation of HCV.

From the initially translated HCV polyprotein the three structural and seven nonstructural HCV proteins are processed by both host and viral proteases (Moradpour
2007). NS2 is a metalloproteinase that cleaves itself from the NS2/NS3 protein,
leading to its own loss of function and to the release of the NS3 protein (Lorenz
2006). NS3 provides a serine protease activity and a helicase/NTPase activity. The
serine protease domain comprises two b-barrels and four α-helices. The serine
protease catalytic triad – histidine 57, asparagine 81 and serine 139 – is located in a
small groove between the two b-barrels (Kim 1996, Kim 1998). NS3 forms a tight,
non-covalent complex with its obligatory cofactor and enhancer NS4A, which is
essential for proper protein folding (Figure 3). The NS3-4A protease cleaves the
junctions between NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B.
Besides its essential role in protein processing, NS3 is integrated into the HCV
RNA replication complex, supporting the unwinding of viral RNA by its helicase
activity. Moreover, NS3 might play an important role in HCV persistence via
blocking TRIF-mediated toll-like receptor signalling and Cardif-mediated RIG-I
signalling, subsequently resulting in impaired induction of type I interferons
(Meylan 2005). Thus, pharmacologic NS3 inhibition might support viral clearance
by restoring the innate immune response.

Hepatitis C: New Drugs 243

Figure 3. Molecular structure of the HCV NS3-4A protease.

The active site of the NS3-4A protease is located in a shallow groove between the
two b-barrels of the protease making the design of compound inhibitors relatively
difficult. Nevertheless, many NS3-4A protease inhibitors have been developed
which can be divided into two classes, the macrocyclic inhibitors and linear tetrapeptide α-ketoamide derivatives. In general, NS3-4A protease inhibitors have been
shown to strongly inhibit HCV replication during monotherapy, but also may cause
the selection of resistant mutants, which is followed by viral breakthrough. The
additional administration of pegylated interferon and ribavirin, however, was shown
to reduce the frequency of development of resistance. Future strategies aim for
combination therapies with different antiviral drugs to prevent the development of
resistance. The most advanced compounds are telaprevir and boceprevir, both
approved in 2011.

Ciluprevir (BILN 2061)
The first clinically tested NS3-4A inhibitor was ciluprevir (BILN 2061), an orally
bioavailable, peptidomimetic, macrocyclic drug binding non-covalently to the active
center of the enzyme (Lamarre 2003) (Figure 4). Ciluprevir monotherapy was
evaluated in a double-blind, placebo-controlled pilot study in treatment-naïve
genotype 1 patients with liver fibrosis and compensated liver cirrhosis (Hinrichsen
2004). Ciluprevir, administered twice daily for two days at doses ranging from 25 to
500 mg, led to a mean 2-3 log10 decrease of HCV RNA serum levels in most
patients. Importantly, the stage of disease did not affect the antiviral efficacy of
ciluprevir. The tolerability and efficacy of ciluprevir in HCV genotype 2- and 3infected individuals was examined in an equivalent study design, but compared to
genotype 1 patients, the antiviral activity was less pronounced and more variable in
these patients (Reiser 2005). Ciluprevir development has been halted.

244 Hepatology 2012

Figure 4. Molecular structure of selected NS3-4A inhibitors.

Telaprevir (Incivek/Incivo®) and boceprevir (Victrelis®)
Telaprevir and boceprevir were approved for the treatment of chronic hepatitis C
virus genotype 1 infection by the FDA, EMA and several other countries in 2011.
Both telaprevir and boceprevir are orally bioavailable, peptidomimetic NS3-4A
protease inhibitors belonging to the class of α-ketoamid derivatives (Figure 4). Like
other NS3-4A inhibitors, telaprevir and boceprevir are characterized by a
remarkable antiviral activity against HCV genotype 1. However, monotherapy with
these agents results in the rapid selection of drug-resistant variants followed by viral
breakthrough (Reesink 2006, Sarrazin 2007). Phase II and III clinical studies have
shown that the addition of pegylated interferon α plus ribavirin leads to a
substantially reduced frequency of resistant mutants and viral breakthrough, and to
significantly higher SVR rates in both treatment-naïve and treatment-experienced
HCV genotype 1 patients compared to treatment with pegylated interferon α and
ribavirin alone (Bacon 2011, Jacobson 2011, Poordad 2011, Sherman 2011, Zeuzem
2011). Therefore, telaprevir- and boceprevir-based triple therapy can be considered
the novel standard of care for HCV genotype 1 patients. Results of the Phase III
telaprevir and boceprevir approval studies are summarized in Figure 5.

Hepatitis C: New Drugs 245
A)
ADVANCE

80

75

72

69

60

REALIZE

92 88

100

% SVR

% SVR

100

ILLUMINATE

(response-guided)

44

40

86

80
57

60
40

20

20

0

0

31

24

15
5

relapser

B)

67 69
53

60
40

40

100

relapser

80

75

Black

42
23

% SVR

% SVR

80

Caucasian

nullresponder

RESPOND-2

SPRINT-2
100

part. nonresponder

69

part. nonresponder

52

60
40

20

20

0

0

40
29
7

Figure 5. SVR rates in Phase III clinical trials evaluating telaprevir (A) or boceprevir
(B) in combination with PEG-IFN α and ribavirin. ADVANCE, ILLUMINATE and
SPRINT-2 enrolled treatment-naïve patients, REALIZE and RESPOND-2 enrolled
treatment-experienced patients. Telaprevir was administered for 8 or 12 weeks in
combination with PEG-IFN α-2a and ribavirin, followed by 12-40 weeks of PEG-IFN α-2a
and ribavirin alone. Boceprevir was administered over the whole treatment period of 28 or
48 weeks in combination with PEG-INF α-2b and ribavirin, except of the first 4 weeks of
lead-in therapy of PEG-IFN α-2b and ribavirin only. eRVR, extended early virologic
response; SOC, standard of care; LI, lead-in (4 weeks of PEG-INF α plus ribavirin only).

Other NS3 protease inhibitors
Other NS3 protease inhibitors are currently in various phases of development
(danoprevir (R7227/ITMN191), vaniprevir (MK-7009), BI201335, simeprevir
(TMC435350), narlaprevir (SCH900518), asunaprevir (BMS-650032), PHX1766,
ACH-1625, IDX320, ABT-450, MK-5172, GS-9256, GS-9451) and will
significantly increase treatment options for chronic hepatitis C in the near future. In
general, comparable antiviral activities to telaprevir and boceprevir in HCV
genotype 1 infected patients were observed during mono- (and triple-) therapy
studies (Brainard 2010, Manns 2011, Reesink 2010). Potential advantages of these
second and third generation protease inhibitors might be improved tolerability,
broader genotypic activity, different resistance profiles, and/or improved
pharmacokinetics for once-daily dosage (e.g., TMC435, BI201335). Different

246 Hepatology 2012
resistance profiles between linear tetrapeptide and macrocyclic inhibitors binding to
the active site of the NS3 protease have been revealed. However, R155 is the main
overlapping position for resistance and different mutations at this amino acid site
within the NS3 protease confer resistance to nearly all protease inhibitors currently
in advanced clinical development (Sarrazin 2010). An exception is MK-5172, which
exhibits potent antiviral activity against variants carrying mutations at position
R155. In addition, MK-5172 had potent antiviral activity against both HCV
genotype 1 and 3 isolates (Brainard 2010).

Resistance to NS3-4A inhibitors
Because of the high replication rate of HCV and the poor fidelity of its RNAdependent RNA polymerase, numerous variants (quasispecies) are continuously
produced during HCV replication. Among them, variants carrying mutations
altering the conformation of the binding sites of DAA compounds can develop.
During treatment with specific antivirals, these preexisting drug-resistant variants
have a fitness advantage and can be selected to become the dominant viral
quasispecies. Many of these resistant mutants exhibit an attenuated replication with
the consequence that, after termination of exposure to specific antivirals, the wild
type may displace the resistant variants (Sarrazin 2007). Nevertheless, HCV
quasispecies resistant to NS3-4A protease inhibitors or non-nucleoside polymerase
inhibitors can be detected at low levels in some patients (approx. 1%) who have
never been treated with these specific antivirals before (Gaudieri 2009). The
clinical relevance of these pre-existing mutants is not completely understood,
although there is evidence that they may reduce the chance of achieving an SVR
with DAA-based triple therapies if the patient’s individual sensitivity to pegylated
interferon α + ribavirin is low.
More recently, the Q80R/K variant has been described as conferring low-level
resistance to simeprevir (TMC435), a macrocyclic protease-inhibitor. Interestingly,
the Q80K variant can be detected in approximately 10% of HCV genotype 1infected patients (typically in subtype 1a isolates) and a slower viral decline during
simeprevir-based triple therapy was observed (Lenz 2011). Table 2 summarizes the
resistance profile of selected NS3-4A inhibitors. Although the resistance profiles
differ significantly, R155 is an overlapping position for resistance development and
different mutations at this position confer resistance to nearly all protease inhibitors
(not MK-5172) currently in advanced clinical development (Sarrazin 2010).
Importantly, many resistance mutations could be detected in vivo only by clonal
sequencing. For example, mutations at four positions conferring telaprevir
resistance have been characterized so far (V36A/M/L, T54A, R155K/M/S/T and
A156S/T), but only A156 could be identified initially in vitro in the replicon system
(Lin 2005). These mutations, alone or as double mutations, conferred low
(V36A/M, T54A, R155K/T, A156S) to high (A156T/V, V36M + R155K, V36M +
156T) levels of resistance to telaprevir (Sarrazin 2007). It is thought that the
resulting amino acid changes of these mutations alter the confirmation of the
catalytic pocket of the protease, which impedes binding of the protease inhibitor
(Welsch 2008).

Hepatitis C: New Drugs 247
Table 2. Resistance mutations to HCV NS3 protease inhibitors.
36

54

55

80

155

156A

156B

168

170

Telaprevir*
(linear)
Boceprevir*
(linear)
SCH900518*
(linear)
BI-201335*
(linear?)
BILN-2061 **
(macrocyclic)
Danoprevir*
(macrocyclic)
MK-7009*
(macrocyclic)
TMC435*
(macrocyclic)
BMS-650032*
(macrocyclic)
GS-9451*
(macrocyclic)
ABT450*
(macrocyclic)
IDX320**
(macrocyclic)
ACH1625**
(macrocyclic)
MK-5172***
(macrocyclic)
36: V36A/M; 54: T54S/A; 55: V55A; 80: Q80R/K; 155: R155K/T/Q; 156A: A156S; 156B:
A156T/V; 168: D168A/V/T/H; 170: V170A/T
* mutations associated with resistance in patients
** mutations associated with resistance in vitro
*** no viral break-through during 7 days monotherapy
# Q80 variants have been observed in approximately 10% of treatment-naïve patients and was
associated with slower viral decline during simeprevir (TMC435) triple therapy

As shown for other NS3-4A protease inhibitors as well (e.g., danoprevir), the
genetic barrier to telaprevir resistance differs significantly between HCV subtypes.
In all clinical studies of telaprevir alone or in combination with PEG-IFN α and
ribavirin, viral resistance and breakthrough occurred much more frequently in
patients infected with HCV genotype 1a compared to genotype 1b. This difference
was shown to result from nucleotide differences at position 155 in HCV subtype 1a
(aga, encodes R) versus 1b (cga, also encodes R). The mutation most frequently
associated with resistance to telaprevir is R155K; changing R to K at position 155
requires 1 nucleotide change in HCV subtype 1a, and 2 nucleotide changes in
subtype 1b isolates (McCown 2009).
It will be important to define whether treatment failure due to the development of
variants resistant to DAA agents has a negative impact on re-treatment with the
same or a different DAA treatment regimen. Follow-up studies of telaprevir and

248 Hepatology 2012
boceprevir Phase III studies have revealed a rapid decline of resistant variants below
the limit of detection (>20% of quasispecies) of population sequencing techniques
(Barnard 2011, Sherman 2011). However, telaprevir- and boceprevir-resistant
variants were detectable by a clonal sequencing approach several years after
treatment in single patients who had been treated with telaprevir or boceprevir
within smaller Phase Ib studies (Susser 2011).

NS5B polymerase inhibitors
Molecular biology
HCV replication is initiated by the formation of the replication complex, a highly
structured association of viral proteins and RNA, of cellular proteins and cofactors,
and of rearranged intracellular lipid membranes derived from the endoplasmic
reticulum (Moradpour 2007). The key enzyme in HCV RNA replication is NS5B,
an RNA-dependent RNA polymerase that catalyzes the synthesis of a
complementary negative-strand RNA by using the positive-strand RNA genome as
a template (Lesburg 1999) (Figure 6). From this newly synthesized negative-strand
RNA, numerous RNA strands of positive polarity are produced by NS5B activity
that serve as templates for further replication and polyprotein translation. Because
of poor fidelity leading to a high rate of errors in its RNA sequencing, numerous
different isolates are generated during HCV replication in a given patient, termed
HCV quasispecies. It is reasoned that due to the lack of proofreading of the NS5B
polymerase together with the high replication of HCV, every possible mutation is
generated each day.
NS5B RNA polymerase inhibitors can be divided into two distinct categories.
Nucleoside analog inhibitors (NIs) like valopicitabine (NM283), mericitabine
(R7128), R1626, PSI-7977, PSI-938 or IDX184 mimic the natural substrates of the
polymerase and are incorporated into the growing RNA chain, thus causing direct
chain termination by tackling the active site of NS5B (Koch 2006). Because the
active centre of NS5B is a highly conserved region of the HCV genome, NIs are
potentially effective against different genotypes. Single amino acid substitutions in
every position of the active centre may result in loss of function or in extremely
impaired replicative fitness. Thus, there is a relatively high barrier in the
development of resistances to NIs.
In contrast to NIs, the heterogeneous class of non-nucleoside inhibitors (NNIs)
achieves NS5B inhibition by binding to different allosteric enzyme sites, which
results in conformational protein change before the elongation complex is formed
(Beaulieu 2007). For allosteric NS5B inhibition high chemical affinity is required.
NS5B is structurally organized in a characteristic “right hand motif”, containing
finger, palm and thumb domains, and offers at least four NNI-binding sites, a
benzimidazole-(thumb 1)-, thiophene-(thumb 2)-, benzothiadiazine-(palm 1)- and
benzofuran-(palm 2)-binding site (Lesburg 1999) (Figure 6). Because of their
distinct binding sites, different polymerase inhibitors can theoretically be used in
combination or in sequence to manage the development of resistance. Because NNIs
bind distantly to the active centre of NS5B, their application may rapidly lead to the
development of resistant mutants in vitro and in vivo. Moreover, mutations at the
NNI binding sites do not necessarily lead to impaired function of the enzyme.
Figure 7 shows the structure of selected nucleoside and non-nucleoside inhibitors.

Hepatitis C: New Drugs 249

Figure 6. Structure of the HCV NS5B RNA polymerase and binding sites.

Figure 7. Molecular structure of selected NS5B polymerase inhibitors.

Nucleoside analogs
Valopicitabine (NM283, 2'-C-methylcytidine/NM107), the first nucleoside inhibitor
investigated in patients with chronic hepatitis C, showed a low antiviral activity

250 Hepatology 2012
(Afdhal 2007). Due to gastrointestinal side effects the clinical development of
NM283 was stopped.
The second nucleoside inhibitor to be reported in patients with chronic hepatitis C
was R1626 (4'-azidocytidine/PSI-6130). A Phase 1 study in genotype 1-infected
patients observed a high antiviral activity at high doses of R1626 in genotype 1infected patients (Pockros 2008). No viral breakthrough with selection of resistant
variants was reported from monotherapy or combination studies with pegylated
interferon ± ribavirin (Pockros 2008). Due to severe lymphopenia and infectious
disease adverse events further development of R1626 was stopped.
Mericitabine (RG7128) is still in development and the most advanced nucleoside
polymerase inhibitor. Mericitabine is safe and well-tolerated, effective against all
HCV genotypes, and thus far no viral resistance against mericitabine has been
observed in clinical studies. Interim results of current Phase II clinical trials in HCV
genotype 1-, 2-, 3-infected patients of R7128 in combination with pegylated
interferon and ribavirin revealed superior SVR rates of mericitabine-based triple
therapy compared to PEG-IFN α alone (Pockros 2011). In an all oral regimen,
administration of R7128 in combination with the protease inhibitor
R7227/ITMN191 for 14 days, a synergistic antiviral activity of both drugs was
observed (Gane 2010). No viral breakthrough with selection of resistant variants has
been reported.
Very promising clinical data have been published recently for PSI-7977, a
nucleoside analog NS5B inhibitor effective against all HCV genotypes. In HCV
genotype 2- and 3- infected patients, PSI-7977 (400 mg once daily) in combination
with ribavirin for 12 weeks + PEG-IFN α for 4-12 weeks resulted in 100% RVR and
100% week 12 SVR rates (Gane 2011). No PSI-7977-associated side effects have
been reported, and no virologic breakthrough has been observed. A second study
evaluated PSI-7977-based triple therapy in treatment-naïve HCV genotype 1infected patients. In this study, PSI-7977 was administered for 12 weeks, together
with PEG-IFN α and ribavirin for 24 or 48 weeks in total, according to whether
HCV RNA was below the limit of detection at treatment weeks 4 and 12 or not,
respectively (Lawitz 2011). Most patients were eligible for the shortened treatment
duration of 24 weeks, and SVR was achieved in approximately 90% of all patients.
Other nucleoside analogs (e.g., PSI-938 and IDX184) are at earlier stages of
clinical development (Sarrazin 2010).
Overall, the newer nucleoside analogs (PSI-7977, PSI-938) also demonstrate high
antiviral activities that, together with their high genetic barrier to resistance, suggest
that they are optimal candidates for all-oral combination therapies (see below).

Non-nucleoside analogs
At least 4 different allosteric binding sites have been identified for inhibition of the
NS5B polymerase by non-nucleoside inhibitors. Currently, numerous nonnucleoside inhibitors are in Phase I and II clinical evaluation (e.g., NNI site 1
inhibitor BI207127; NNI site 2 inhibitors filibuvir (PF-00868554), VCH-759, VCH916 and VCH-222; NNI site 3 inhibitor ANA598, NNI site 4 inhibitors HCV-796,
and ABT-333) (Ali 2008, Cooper 2007, Erhardt 2009, Kneteman 2009). In general,
these non-nucleoside analogs display a low to medium antiviral activity and a low
genetic barrier to resistance, evidenced by frequent viral breakthrough during
monotherapy studies and selection of resistance mutations at variable sites of the

Hepatitis C: New Drugs 251
enzyme. In line with these experiences in Phase I studies, a Phase II triple therapy
study with filibuvir in combination with pegylated interferon and ribavirin showed
high relapse and relative low SVR rates (Jacobson 2010). In contrast to nucleosideanalogs, non-nucleoside analogs in general do not display antiviral activity against
different HCV genotypes (Sarrazin 2010). Due to their low antiviral efficacy and
low genetic barrier to resistance, non-nucleoside analogs will probably not be
developed as part of triple therapy but rather as components of quadruple or all-oral
regimens (see below).

NS5A inhibitors
The HCV NS5A protein seems to play a manifold role in HCV replication,
assembly and release (Moradpour 2007). It was shown that NS5A is involved in the
early formation of the replication complex by interacting with intracellular lipid
membranes, and it initiates viral assembly at the surface of lipid droplets together
with the HCV core (Shi 2002). NS5A may also serve as a channel that helps to
protect and direct viral RNA within the membranes of the replication complex
(Tellinghuisen 2005). Moreover, it was demonstrated that NS5A is able to interact
with NS5B, which results in an enhanced activity of the HCV RNA polymerase.
Besides its regulatory impact on HCV replication, NS5A has been shown to
modulate host cell signaling pathways, which has been associated with interferon
resistance (Wohnsland 2007). Furthermore, mutations within the NS5A protein have
been clinically associated with resistance / sensitivity to IFN-based antiviral therapy
(Wohnsland 2007).
BMS-790052 was the first NS5A inhibitor to be clinically evaluated. Even low
doses of BMS-790052 display high antiviral efficacy against all HCV genotypes in
vitro. Monotherapy with BMS-790052 led to a sharp initial decline of HCV RNA
concentrations, though its genetic barrier to resistance is relatively low (Gao 2010).
According to an interim analysis of a Phase IIb clinical trial in treatment-naïve HCV
genotype 1 and 4 patients, treatment with 20 or 60 mg BMS-790052 once daily in
combination with PEG-IFN α and ribavirin for 24 or 28 weeks, 54% of all patients
achieved an extended RVR, compared to 13% in the control group (Hezode 2011).
SVR rates of this study are awaited.
During monotherapy, rapid selection of variants resistant to BMS-790052
occurred (Nettles 2011). The most common resistance mutations in HCV genotype
1a patients were observed at residues M28, Q30, L31, and Y93 of NS5A. In HCV
genotype 1b patients, resistance mutations were observed less frequently,
predominantly at positions L31 and Y93. These resistance mutations increased the
EC50 to BMS-790052 moderately to strongly (Fridell 2011). However, no crossresistance between BMS-790052 and other DAA agents has been reported.
Collectively, BMS-790052 is a highly promising agent for both triple therapy as
well as all-DAA combination therapy approaches.
Other NS5A inhibitors (e.g., BMS-824393, PPI-461, GS-5885) are in early
clinical development.

252 Hepatology 2012

Compounds targeting viral attachment and entry
The tetraspanin protein CD81, claudin-1, occludine, scavenger receptor class B type
1 (SR-B1), the low-density lipoprotein (LDL) receptor, glycosaminoglycans and the
dendritic cell- /lymph node-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN/L-SIGN) have been identified as putative ligands for E1 and E2
in the viral attachment and entry steps (Moradpour 2007). HCV entry inhibition
might enrich future hepatitis C treatment opportunities, in particular in the
prevention of HCV liver graft reinfection. HCV entry inhibition can be theoretically
achieved by the use of specific antibodies or small molecule compounds either
blocking E1 and E2 or their cellular receptors. So far, only results from clinical
trials using polyclonal (e.g., civacir) (Davis 2005) or monoclonal (e.g., HCV-AB
68) (Schiano 2006) HCV-specific antibodies are available. The clinical benefit of
these antibodies has been poor, however. The development of small molecule entry
inhibitors is in a preclinical stage and is complicated by difficulties in the
crystallographic characterization of HCV envelope proteins.

Host factors as targets for treatment
Cyclophilin B inhibitors
HCV depends on various host factors throughout its life cycle. Cyclophilin B is
expressed in many human tissues and provides a cis-trans isomerase activity, which
supports the folding and function of many proteins. Cyclophilin B enhances HCV
replication by incompletely understood mechanisms, like the modulation of NS5B
activity. Debio-025 (alisporivir) is an orally bioavailable cyclophilin B inhibitor
exerting an antiviral impact on both HCV and HIV replication. In clinical trials in
HIV- and HCV-coinfected patients, treatment with 1200 mg Debio-025 twice daily
for two weeks led to a mean maximal log10 reduction of HCV RNA of 3.6 and of
HIV DNA of 1.0 (Flisiak 2008). Debio-025 was well-tolerated and no viral
breakthrough occurred during the 14 days of treatment.
Combination therapy of Debio-025 200 mg, 600 mg or 1000 mg and PEG-IFN α2a was evaluated in a double-blind placebo-controlled Phase II trial in treatmentnaïve patients monoinfected with HCV genotypes 1, 2, 3 or 4. Treatment was
administered for 29 days. Mean log10 reductions in HCV RNA at day 29 were 4.75
(1000 mg), 4.61 (600 mg) and 1.8 (200 mg) in the combination therapy groups
compared to 2.49 (PEG-IFN α-2a alone) and 2.2 (1000 mg Debio-025 alone) in the
monotherapy groups. No differences in antiviral activity were observed between
individuals infected with the different genotypes. Debio-025 was safe and well
tolerated but led to a reversible bilirubin increase in some of the patients treated
with 1000 mg Debio-025 daily (Flisiak 2009). A high genetic barrier to resistance of
Debio-025 and a broad HCV genotypic activity highlight the potential of drugs
targeting host proteins.
In a Phase II clinical trial in treatment-naïve HCV genotype 1 patients,
combination therapy with Debio-025, PEG-IFN α-2a and ribavirin for 24-48 weeks
resulted in SVR rates of 69-76% compared to 55% in the control group (Flisiak
2011).

Hepatitis C: New Drugs 253

Nitazoxanide
Nitazoxanide with its active metabolite tizoxanide is a thiazolide antiprotozoal
approved for the treatment of Giardia lamblia and Cryptosporidium parvum
infections. In vitro studies have revealed an essential inhibitory impact on HCV and
HBV replication by still unknown mechanisms.
Results of two Phase 2 studies evaluating 500 mg nitazoxanide twice daily for 12
weeks followed by nitazoxanide, PEG-IFN α-2a ± RBV for 36 weeks yielded
conflicting results with SVR rates of 79% in treatment-naïve genotype 4 patients,
but of only 44% in HCV genotype 1 patients (Rossignol 2009). Additional studies
are warranted to determine the role of nitazoxanide in the treatment of chronic
hepatitis C.

Silibinin
Silymarin, an extract of milk thistle (Silybum marianum) with antioxidant activity,
has been empirically used to treat chronic hepatitis C and other liver diseases.
Silibinin is one of the six major flavonolignans in silymarin. Surprisingly, recent
reports demonstrated that silibinin inhibits HCV at various steps of its life cycle
(Ahmed-Belkacem 2010, Wagoner 2010). In addition, intravenous silibinin in nonresponders to prior IFN-based antiviral therapy led to a decline in HCV RNA
between 0.55 to 3.02 log10 IU/ml after 7 days and a further decrease after an
additional 7 days in combination with PEG-IFN α-2a/RBV in the range of 1.63 and
4.85 log10 IU/ml (Ferenci 2008). Ongoing studies will clarify the role of silibinin in
the treatment of chronic hepatitis C, including HCV liver graft reinfection.

Miravirsen
MicroRNA-122 (miRNA-122) is a liver-specific microRNA that has been shown to
be a critical host factor for HCV (Landford 2010). MiRNA-122 binds to the 5´NTR
region of the HCV genome, which appears to be vital in the HCV replication
process. Miravirsen is a modified antisense oligonucleotide that targets miRNA-122
and thereby prevents binding of miRNA-122 to the HCV genome. In a Phase IIa
proof-of-principle study, weekly subcutaneous injections of miravirsen led to a
reduction of HCV RNA serum concentration of up to 2.7 log10 IU/mL, indicating
that an antisense oligonucleotide-based approach of miRNA-122 inhibition could be
a promising modality for antiviral therapy (Janssen 2010). No relevant side effects
were seen in this study.

Newer combination therapies
The approval of the HCV protease inhibitors telaprevir and boceprevir in 2011
constitutes a milestone in the treatment of chronic HCV genotype 1 infection.
Nevertheless, telaprevir- or boceprevir-based triple therapy has certain limitations.
In particular, treatment success still depends on the interferon-sensitivity of
individual patients because a slow decline of HCV viral load during triple therapy is
associated with a high risk of antiviral resistance development. Consequently, viral
breakthrough of drug resistant variants was observed in a significant number of
patients with partial or null response to previous treatment with PEG-IFN α and
ribavirin, in patients with limited decline of HCV viral load during lead-in treatment
with PEG-IFN α and ribavirin alone, or in difficult to cure populations like Blacks

254 Hepatology 2012
or patients with advanced liver fibrosis. In addition, triple therapy is not an option
for patients with contraindications to PEG-IFN α or ribavirin, such as patients with
decompensated liver cirrhosis or liver transplant failure.
To overcome these limitations, numerous trials have been initiated to investigate
the potential of combination therapies with different DAA agents alone (Table 3).
As is well established in the treatment of HIV infection, combining DAA agents
with different antiviral resistance profiles should result in a substantially decreased
risk of viral breakthrough of resistant variants. Nucleoside analog NS5B inhibitors
plus drugs targeting host factors such as the cyclophilin inhibitor alisporivir display
a high genetic barrier to resistance development and may therefore be key agents for
effective DAA combination therapies (Sarrazin 2010). In contrast, NS3-4A and
NS5A inhibitors display a low genetic barrier to resistance development, but in view
of their high antiviral efficacy they appear to be promising combination partners for
nucleoside analogs or cyclophilin inhibitors. Due to their low antiviral efficacy and
low genetic barrier to resistance development, the role of non-nucleoside analog
NS5B inhibitors is currently less clear. A potential advantage of non-nucleoside
analogs is their binding to multiple target sites that may allow simultaneous
treatment with several non-nucleoside analogs.
Currently, DAA combination treatment regimens can be classified according to
the usage of PEG-IFN α into quadruple therapy regimens and all-oral therapy
regimens. Quadruple therapy approaches are based on therapy of PEG-IFN α and
ribavirin plus combination of two DAA agents from different classes. In contrast,
all-oral treatment comprises interferon-free regimens including combinations of
various DAA compounds with or without ribavirin.

Quadruple therapy
Preliminary SVR data of a small but highly informative trial serves as a proof-ofconcept for the potential of quadruple therapy approach for patients with previous
null response to PEG-IFN α + ribavirin (Lok 2011). In this Phase II study, 11 HCV
genotype 1 patients with prior null response were treated with a combination of the
NS5A inhibitor BMS-790052 and the protease inhibitor BMS-650032 together with
PEG-IFN α and ribavirin for 24 weeks. Quadruple therapy resulted in 100% SVR 12
weeks after treatment completion in both HCV genotype 1a- and 1b-infected
patients. Even though the number of patients included in this trial was very limited,
this high SVR rate after quadruple therapy seems impressive compared to SVR rates
of ~30% that were achieved with telaprevir-based triple therapy in prior null
responders (Zeuzem 2011).
A Phase II clinical trial assessed quadruple therapy with the non-nucleoside NS5B
inhibitor tegobuvir in combination with the NS3-4A inhibitor GS-9256 + PEG-IFN
α and ribavirin for 28 days in treatment-naïve HCV genotype 1 patients (Zeuzem
2011). The primary endpoint of this study was rapid virologic response (RVR),
which was achieved in 100% of patients. After 28 days of quadruple therapy,
treatment with PEG-IFN α and ribavirin was continued, which led to complete early
virologic reponse (cEVR) in 94% of patients (Zeuzem 2011).
Another Phase II clinical trial investigated a response-guided approach during
quadruple therapy containing the non-nucleoside NS5B inhibitor VX-222 (100 mg
or 400 mg) in combination with the NS3-4A inhibitor telaprevir + PEG-IFN α and
ribavirin in treatment-naïve HCV genotype 1 patients (Nelson 2011). Quadruple

Hepatitis C: New Drugs 255
treatment was administered for 12 weeks. All treatment was stopped after 12 weeks
in patients who were HCV RNA-negative at treatment weeks 2 and 8. Patients in
whom HCV RNA was detectable at treatment week 2 or 8 received an additional 12
weeks of PEG-IFN α and ribavirin alone. Up to 50% of patients met the criteria for
the 12-week treatment duration. Of those, 82-93% achieved an SVR 12 weeks after
treatment completion. In patients who were treated with an additional 12 weeks of
PEG-IFN α and ribavirin, the end-of-treatment response was 100%.
Collectively, the quadruple therapy approach appears to be highly promising in
patients with limited sensitivity to interferon-α, even in patients with HCV subtype
1a.

All-oral therapy without ribavirin
A first interferon-free clinical trial (the INFORM-1 study) evaluated the
combination of a polymerase inhibitor (R7128) and an NS3 inhibitor
(R7227/ITMN191). In this proof of principle study, patients were treated with both
compounds for up to 2 weeks (Gane 2010). HCV RNA concentrations decreased by
up to 5.2 log10 IU/ml, viral breakthrough was observed in only one patient (although
no resistant HCV variants were identified), and HCV RNA was undetectable at the
end of dosing in up to 63% of treatment-naïve patients. However, the fundamental
question of whether an SVR can be achieved with combination therapies of
different DAA compounds without PEG-IFN α and ribavirin was not answered by
this trial.
SVR data are available for a Phase II clinical trial investigating therapy with the
NS5A inhibitor BMS-790052 in combination with the NS3-4A protease inhibitor
BMS-60032 for 24 weeks in 10 HCV genotype 1 patients with a previous null
response to PEG-IFN α and ribavirin (Lok 2011). 36% of patients achieved an SVR
24 weeks after treatment completion. All patients with viral breakthrough were
infected with HCV genotype 1a, and in all of them HCV variants with resistance
mutations against both agents were detected. Although data of longer follow-up
periods are needed, this trial constitutes a proof-of-principle that SVR can be
achieved via all-oral regimens, even in patients infected with HCV subtype 1b. This
was confirmed with a 100% SVR rate in a small study evaluating the same agents
(BMS-790052 and BMS-60032) in Japanese HCV genotype 1b previous null
responders (Chayama 2011).
Another trial has investigated 12 weeks of PSI-7977 monotherapy (400 mg once
daily) in HCV genotype 2- and 3-infected patients (n=10). 100% of patients
achieved an RVR and EOTR, which translated into an SVR in 60% of patients
(Gane 2011).

All-oral therapy with ribavirin
Two trials evaluated all-oral DAA combination therapies with ribavirin. In one of
them, combination therapy of the NS3-4A inhibitor BI-201335, the non-nucleoside
NS5B inhibitor BI-207127 (400 or 600 mg TID) and ribavirin for 4 weeks was
assessed (Zeuzem 2011). Virologic response rates in patients treated with 600 mg
TID of BI-207127 were 82%, 100% and 100% at treatment days 15, 22, and 29,
respectively (Zeuzem 2011). In patients who received the lower dose of BI-207127,
virologic response rates were significantly lower, and in these patients lower

256 Hepatology 2012
virologic response rates were observed for patients infected with HCV subtype 1a
compared to subtype 1b.
Another trial compared tegobuvir (a non-nucleoside NS5B inhibitor) + GS-9256
(a NS3-4A inhibitor) with or without ribavirin in treatment-naïve HCV genotype 1
patients (Zeuzem 2011). Importantly, tegobuvir + GS-9256 + ribavirin led to a
higher HCV RNA decline after 28 days of treatment compared to tegobuvir + GS9256 alone (-5.1 log10 vs. -4.1 log10, respectively), indicating that ribavirin might be
an important component of interferon-free DAA combination therapies. SVR data
of these and additional combination therapy regimens are expected in the near
future.
Additional trials investigated all-oral combination regimens with ribavirin in
HCV genotype 2 and 3 patients. 12 weeks of PSI-7977 plus ribavirin resulted in
100% RVR, EOTR, and SVR rates in a small number of treatment-naïve patients
(n=10) (Gane 2011). In contrast, during treatment with the cyclophilin A inhibitor
alisporivir in combination with ribavirin, only approximately 50% of HCV genotype
2 and 3 patients became HCV RNA-negative at treatment week 6 (Pawlotsky 2011).
Nevertheless, these data highlight the impressive potential of all-oral regimens,
when agents with little risk of antiviral resistance development such as nucleoside
analog NS5B inhibitors are used in combination with ribavirin.
Table 3. Selected trials evaluating DAA combination therapies.
DAAs combined

Additional medication

BMS-650032 (NS3-4A inhibitor)
+ BMS-790052 (N5A inhibitor)
BI-201335 (NS3-4A inhibitor)
+ BI-207127 (non-nuc. NS5B inhibitor)
GS-9190 (non-nuc. NS5B inhibitor)
+ GS-92568 (NS3-4A inhibitor)
Danoprevir (NS3-4A inhibitor)
+ RG-7128 (nuc. NS5B inhibitor)
Telaprevir (NS3-4A inhibitor)
+ VX-222 (non-nuc. NS5B inhibitor)
PSI-938 (purine nuc. NS5B inhibitor)
+ PSI-7977 (pyrimidine nuc. NS5B inhibitor)

+ / - PEG-IFN α
and ribavirin
+ ribavirin
+ / - PEG-IFN α
+ / - ribavirin
+ / - PEG-IFN α
followed by PEG-IFN α
and ribavirin
+ / - ribavirin
+ / - PEG-IFN α
-

Phase
II
II
II
II
II
II

Novel interferons
Over the last years, attempts have been made to reduce side effects and treatment
discomfort of PEG-IFN α. However, interferons with longer half-life and sustained
plasma concentrations (e.g., albinterferon, a fusion protein of IFN α 2b with human
albumin) have so far shown no overall benefit with respect to SVR rates (Zeuzem
2010). Still promising is the development of pegylated interferon lambda 1 (PEGIFN lambda 1). Like other type 3 interferons, IFN lambda 1, which is also called
interleukin-29 (IL-29), binds to a different receptor than IFN α, but downstream
signaling pathways of IFN lambda and IFN α are largely comparable. The IFN
lambda receptor is predominantly expressed in hepatocytes. Thus, interferon-related
side effects may be less frequent during PEG-IFN lambda treatment. A Phase I
clinical trial evaluating pegylated interferon lambda with or without ribavirin was
completed (Muir 2010). Interferon lambda was well-tolerated and the majority of

Hepatitis C: New Drugs 257
patients achieved a greater than 2 log10 decline of HCV RNA by 4 weeks.
According to an interim analysis of a subsequent Phase II clinical trial, PEG-IFN
lambda (240 ug, 180 ug, or 120 ug once weekly) was compared to PEG-IFN α-2a.
PEG-IFN lambda at doses of 240 or 180 ug resulted in approximately 10% higher
RVR and approximately 20% higher cEVR rates, a lower frequency of flu-like
symptoms, but with more frequent aminotransferase and bilirubin elevations than
PEG-IFN α-2a (Zeuzem 2011).

Conclusions
Telaprevir- and boceprevir-based triple therapy of treatment-naïve and treatmentexperienced HCV genotype 1 patients results in substantially increased SVR rates
compared to PEG-INF-α and ribavirin alone. The approval of these agents
represents a major breakthrough in the treatment of chronic hepatitis C. However,
successful use of these drugs will require a precise classification of response
patterns to previous treatment, careful on-treatment monitoring of HCV viral load
and emergence of antiviral resistance as well as of additional side effects and
numerous possible drug-drug interactions. Next-generation NS3-4A protease
inhibitors and NS5A inhibitors may have even more favorable properties than
telaprevir and boceprevir in terms of HCV genotype coverage, safety profiles, less
pronounced drug-drug interactions, or possible once-daily administration. However,
the triple therapy approach has several limitations. First of all, concomitant IFN α
and ribavirin are necessary to avoid the development of antiviral resistance.
Consequently, the efficacy of triple therapy was limited in prior null responders to
PEG-IFN α and ribavirin, and triple therapy cannot be administered to patients with
contraindications to PEG-IFN α or ribavirin. Recent data indicate that the
development of DAA combination therapies in all-oral or quadruple treatment
regimens will likely be a very potent option for these patients. In such DAA
combination regimens, the inclusion of drugs with a high genetic barrier to
resistance such as nucleoside NS5B inhibitors or drugs targeting host factors such as
alisporivir may be important.

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262 Hepatology 2012

15. Management of Adverse Drug Reactions
Martin Schaefer and Stefan Mauss

Introduction
Good adherence is a key factor for success in the treatment of hepatitis C. However,
almost all patients on treatment with interferon and ribavirin will experience adverse
events that can threaten good adherence. Therefore, proactive clinical management
is crucial to avoid suboptimal therapy and treatment discontinuations.
The most common adverse events in patients on treatment with pegylated
interferon plus ribavirin are flu-like symptoms, myalgia, sleep disturbances,
asthenia, gastrointestinal disorders and depressive episodes (Table 1).
Table 1. Incidence of most reported IFN α-induced psychiatric side effects. Data
from Outpatient Department, Essen-Mitte Clinics, Essen.
Psychiatric side effects

Incidence

Fatigue
Sleep disturbances
Irritability
Cognitive disturbances with impairments of concentration and memory
Depressive episodes
Mild
Moderate
Severe
Delirium, psychosis
Suicidal syndrome

50-80%
45-65%
60-85%
45-60%
20-60%
30-60%
20-30%
5-10%
1-6%
<1%

For most adverse events, clinical trials with dose adjustment have not been done,
and because of this, recommendations in this review are necessarily partially based
on clinical experience.

Flu-like symptoms, fever, arthralgia and myalgia
Flu-like symptoms, fever, arthralgia and myalgia appear a few hours after the PEGIFN injection and may last for up to three days. One common approach is the use of

Management of Adverse Drug Reactions 263
paracetamol or other NSAIDs immediately before or after the injection of
interferon. Flu-like symptoms usually diminish spontaneously over the first weeks
of treatment (Figure 1).
Low platelets are a contraindication for the use of acetylsalicylic acid, diclofenac
or ibuprofen because of the inhibition of platelet aggregation. High doses of
paracetamol may result in liver toxicity. Doses exceeding 2 g/day of paracetamol
are not recommended.

Figure 1. Time course of interferon-associated adverse events.

Gastrointestinal disorders
Nausea can be mitigated by prokinetic agents such as metoclopramide or
domperidone taken before the ribavirin. This may also positively influence the
frequently observed loss of appetite.
Dry mouth has been reported as a result of inhibition of saliva production, a
frequent complication of ribavirin, which may continue after discontinuation of
therapy.

Weight loss
The average weight loss in interferon-based controlled studies is around 6-10% for a
treatment period of 48 weeks (Seyam 2004). This may be predominantly due to loss
of appetite and reduction in calorie intake. The weight loss is rapidly reversible
upon discontinuation of therapy.

Asthenia and fatigue
Asthenia and fatigue are frequent complaints of patients that usually increase slowly
in intensity over the first couple weeks of therapy (Figure 1). In patients with
marked anemia these symptoms can be improved by raising low hemoglobin with

264 Hepatology 2012
the use of erythropoietin, reduction of ribavirin or red blood cell transfusion
(Pockros 2004). Asthenia is also reported by patients without marked anemia. In
these patients hypothyroidism may be the explanation. Symptomatic treatment of
asthenia and fatigue in patients without an underlying complication such as anemia,
depression or hypothyroidism is difficult.
Chronic fatigue has been successfully treated in individual cases with
antidepressants or tryptophan (Sammut 2002; Schaefer 2008). A first prospective
randomised controlled trial showed superior effects of the 5-HT3 receptor
antagonist ondansetron compared to placebo (Piche 2005). However, currently
available data does not offer specific treatment recommendations.

Cough and dyspnea
Cough while on therapy is frequently reported and is most probably due to edema of
the mucosa of the respiratory system. Therefore, advanced, not well-controlled
asthma bronchiale may be a contraindication for hepatitis C therapy. Dyspnea is
another frequent complaint with a more complex etiology involving mucosa
swelling, anemia and asthenia.

Disorders of the thyroid gland
Hypothyroidism while on interferon-based therapy is reported with an incidence of
3-10% (Bini 2004, Tran 2005). Hyperthyroidism is less frequently observed with an
incidence of 1-3% (Bini 2004, Tran 2005). Interferon-induced thyroiditis or the
induction of thyroid antibodies is reported as an underlying mechanism.
Hypothyroidism is treated via substitution of thyroid hormone whereas clinical
symptomatic hyperthyroidism may be treated with ß-blockers or carbimazole.
Premature termination of interferon-based therapy is usually not necessary. About
half of the cases of hypothyroidism are reversible upon discontinuation of
interferon-based therapy, although some cases may need prolonged periods of
thyroid hormone replacement therapy.

Psychiatric adverse events
Incidence and profile of psychiatric adverse events
The most commonly emerging IFN α-induced psychiatric adverse events are
outlined in Table 1. However, data on the frequency of psychiatric side effects
differs depending on the design of the trial. Most hepatological trials are only
monitored for depression as a single symptom without using depression scales or
diagnostic instruments, leading to an underreporting of mild to moderate depressive
episodes. Most psychiatric trials used self-rating scales (e.g., SDS-scale, BDI-Scale)
or monitor patients via expert rating scales (Hamilton Depression Scale [HAMDS]
or Montgomery Asperg Depression Scale [MADRS]) to detect depressive
syndromes and treatment-related mood changes even if total scores do not fulfil
DSM-IV criteria for major depression. Regarding these more sensitive psychiatric
rating methods, over 50% of patients suffer from sleep disorders, chronic fatigue,
irritability or cognitive disturbances (Schaefer 2007, Schaefer 2002, Dieperink
2000, Renault 1987). Anxiety occurs in 30-45%, especially during the first 2 months

Management of Adverse Drug Reactions 265
of treatment. Mild depression with symptoms like reduced self-esteem, anhedonia,
loss of interest, rumination, a diminished libido and spontaneous crying can be
observed in 30-60% of the patients. 20-30% of treated patients develop moderate to
severe depressive episodes (Bonnaccorso 2002, Dieperink 2000, Renault 1987,
Schaefer 2002, Malaguarnera 2002). Major depression has been reported in 15-55%
(Schäfer 2007). Suicidal ideation is seen in 5-6% of patients, while suicide attempts
have been reported in single cases (Janssen 1994, Sockalingam 2010). Mania has
been reported as a sporadically appearing side effect. Contrary to assumptions,
patients with pre-existing psychiatric disturbances do not appear to have a greater
risk for development of depression or attempting suicide (Schaefer 2007, Schaefer
2003, Pariante 2002). However, patients with intravenous drug abuse not stabilized
in a substitution treatment program (e.g., methadone) seem more likely to
discontinue treatment in the first three months compared to controls (Schaefer 2003,
Mauss 2004, Schaefer 2007).
Antidepressants frequently used in trials are selective serotonin re-uptake
inhibitors (SSRIs) such as citalopram, escitalopram, paroxetine or sertraline. The
introduction of SSRIs and other current antidepressants has markedly improved the
adverse event profile of antidepressants. Therefore, depending on the major
symptoms, current sedating or activating antidepressants, especially SSRIs, are
treatment of choice for interferon-induced depressive mood disorders (Table 2). In
patients with predominantly agitation and aggression, other strategies, e.g., modern
antipsychotics, may be added.
The efficacy of antidepressants for the treatment of interferon α-induced
depression has been shown in several open uncontrolled cohorts (Farah 2002,
Gleason 2002, Kraus 2001, Schramm 2000, Hauser 2002, Gleason 2005). In a first
prospective randomized controlled trial an improvement of depressive symptoms
after treatment of IFN-associated depression was shown with citalopram compared
to placebo (Kraus 2008). In particular because of the favourable adverse event
profile, SSRIs seem to be most appropriate for treatment of IFN α-associated
depressive symptoms. However, antidepressants with different receptor profiles
(i.e., mirtazapine) and classic antidepressants (i.e., nortriptyline) are also effective
(Kraus 2001, Valentine 1995). Nevertheless, tricyclic antidepressants should be
used as second choice because of pharmacological interactions, anticholinergic side
effects, a higher risk for development of delirium, and liver or myocardial toxicity.
To reduce early occurring adverse events of SSRIs (headache, nausea, agitation),
treatment with antidepressants should be started at a low dose with subsequent dose
increase depending on the effect and tolerability. In general, a therapeutically
relevant antidepressive effect cannot be expected before day 8-14 of treatment. In
case of non-response, the dose can be escalated. Treatment adherence should be
assessed by monitoring serum levels before patients are switched to a different
antidepressant.
Benzodiazepines can be given for a short period in case of severe sleep
disturbances, irritability or depression. However, benzodiazepines should be
avoided in patients with a history of IV drug or alcohol over-use because of their
potential to induce addiction.
In the case of psychotic symptoms, antipsychotics (e.g., risperidone, olanzapine)
can be used at low doses, but patients should be monitored carefully by a

266 Hepatology 2012
psychiatrist. One important risk factor for the development of psychotic symptoms
is a history of drug use.
Although history of major depression or suicide attempts is considered a
contraindication for interferon-based therapy, treatment of patients with pre-existing
psychiatric disorders can be initiated in close collaboration with an experienced
psychiatrist in a well-controlled setting (Schaefer 2004, Schaefer 2007).
It must be mentioned that so far there is no systematic experience with
combination of the recently approved HCV protease inhibitors and antidepressants.
While the new antiviral drugs telaprevir or boceprevir do not induce specific
psychiatric side effects, drug-drug interactions may complicate the use of
antidepressants and sleep medications.

Preemptive therapy with antidepressants
One double-blind randomised study with patients with malignant melanoma
demonstrated that 14 days of pre-treatment with 20 mg paroxetine per day reduced
the incidence of depression during interferon therapy significantly (Musselmann
2001). Pre-treatment with paroxetine also had a positive effect on the development
of fears, cognitive impairments and pain during interferon treatment, but not on
symptoms such as fatigue, sleep disturbances, anhedonia and irritability (Capuron
2002). A recent prospective controlled trial with HCV-infected patients
demonstrated that pre-treatment with citalopram significantly reduced depression
during the first 6 months of antiviral therapy in patients with psychiatric illness
compared to controls (Schaefer 2005). Furthermore, prophylactic treatment with
SSRIs was shown to reduce the severity of depressive symptoms in patients who
had suffered from severe depression during previous treatment of hepatitis C with
interferon α (Kraus 2005). A recent trial confirmed a protective effect of preemptive
initiation of treatment with antidepressants before starting interferon-based therapy
in cases of elevated depression scores (Raison 2007). However, three other trials
could not show significant effects on reduction of depressive symptoms nor overall
incidence of major depression, although these trials were either small in size or had
short observation times (Morasco 2007, Morasco 2010, Diez-Quevedo 2010). In
summary, current data support the view that all patients with pre-existing depressive
symptoms should receive a prophylactic treatment with antidepressants. However,
larger prospective controlled studies are needed in order to answer the question if
antidepressants should be given before antiviral plus interferon-based therapy,
independent of pre-existing psychiatric disorders.

Sleep disturbances
Patients who have difficulties in falling asleep can be treated with zopiclone or
trimipramine. Zolpidem may be used for patients with interrupted or shortened sleep
patterns. Although the risk of addiction is markedly reduced compared with other
benzodiazepines, only small amounts of zoplicon or zolpidem should be prescribed
at a time and therapy should be limited to the period of interferon-based therapy. As
sleeping disorders can be an early symptom of depression, it is also important to
assess the possible presence of other depressive symptoms when considering the use
of sleeping aids.

Management of Adverse Drug Reactions 267

Hematological and immunologic effects
Interferon-based therapy is accompanied by a marked drop in white blood cells in
general, neutrophils and absolute, although not relative, CD4+ cell count. This
change of the cellular immune system does not result in an increased number of
serious infections even in HIV-coinfected patients (Fried 2002, Manns 2001,
Torriani 2004). In general, the incidence of serious infections is low (<5%) in
patients on interferon-based therapy.
G-CSF increases neutrophils in patients treated with interferon-based therapies.
However G-CSF has not been proven to have a clinical benefit in clinical trials for
this purpose and its use is off-label. Hemolytic anemia induced by ribavirin is
further aggravated by the myelosuppressive effect of interferon inhibiting
compensatory reticulocytosis (De Franceschi 2000). As a consequence, anemia (<10
g/dl) is reported in up to 20% of patients (Hadziyannis 2004). In severe cases of
anemia dose reduction of ribavirin is required. In rare cases red blood cell
transfusion may be necessary. Erythropoietin can be successfully used to correct the
ribavirin-induced anemia at least partially and to avoid ribavirin dose reduction or
red blood cell transfusions. In addition erythropoietin use was associated with an
improved quality of life. However, prospective controlled trials have not shown a
positive effect on the efficacy of hepatits C therapy in patients who take
erythropoietin (Afdahl 2004, Pockros 2004, Shiffman 2007). At present,
erythropoietin is not approved for correction of ribavirin-induced anemia in
hepatitis C therapy.
Mild to moderate thrombocytopenia is frequently seen in patients with advanced
liver fibrosis and may complicate interferon-based therapy. Reduction of interferon
dosing may be indicated to reverse severe thrombocytopenia. In studies
eltrombopag has been used successfully to increase platelet count in patients with
hepatitis C associated thrombocytopenia (McHutchison 2007). In recent trials
eltrombopag even increased efficacy of hepatitis C treatment in cirrhotic patients,
although the occurrence of portal vein thrombosis was observed in a number of
patients cautioning its widespread use (Afdhal 2011).

Skin disorders and hair loss
Some skin disorders such as lichen ruber planus, necrotising vasculitis or porphyrea
cutanea tarda are associated with hepatitis C infection. The effects of hepatitis C
therapy are often not well-studied and based only on information gathered through
cohorts (Berk 2007).
Interferon and ribavirin therapy may have an effect on the skin itself including dry
skin, itching, eczema and new or exacerbated psoriasis. Ointments with rehydrating
components, urea or steroids can be used depending on the nature of the skin
disorders. In severe cases a dermatologist should be involved. In particular, eczema
and psoriasis may last substantially longer than the treatment period with interferonbased therapy.
Local skin reactions to the injection of pegylated interferon are common and
usually present as red indurations lasting days to weeks. Repeated injections at the
same site may cause ulcers and should be avoided. Hypersensitivity reactions to
pegylated interferons are reported anecdotally.
Hair loss is frequent, usually appearing after the first months of therapy and
continuing for some weeks after the cessation of therapy. Alopecia is very rare and

268 Hepatology 2012
hair loss is usually fully reversible, although the structure of the hair may be
different after therapy.

Adverse events with telaprevir and boceprevir
Triple combination therapy of pegylated interferon, ribavirin plus one of the newer
HCV protease inhibitors telaprevir or boceprevir is standard of care for the
treatment of most genotype 1 patients. This treatment provides better efficacy, but
also new challenges for adherence and management of adverse events. In general all
adverse events caused by interferon and ribavirin remain, however some may be
accentuated or new adverse events may occur.
In addition, boceprevir and telaprevir are simultaneous inducers and inhibitors of
multiple enzymes of the cytochrome P450 system. For this reason, drug-drug
interactions are not easy to predict and involve frequently used drugs such as
sedatives, antidepressants, antibiotics, immunosuppressants, oral corticosteroids,
statins and calcium channel blockers. As this is an evolving area, for updated
information, the website www.hep-druginteractions.org should be checked
regularly.
Boceprevir and telaprevir have to be taken three times a day with food or a fatcontaining meal, respectively. Pill burden is high with 12 pills for boceprevir and 6
for telaprevir. Dosing and taking the medication not fasting are crucial for efficacy.
Boceprevir or telaprevir doses should never be reduced in case of toxicities, but
rather discontinued or kept at the standard dose. Reducing the dose of these HCV
protease inhibitors will result in treatment failure due to lower drug exposure.
Frequent adverse events seen with telaprevir are itching and rash, with the first
occurring in the majority of patients. Itching can be orally treated with
antihistamines, e.g., cetirizine, but efficacy seems limited. Rash is usually mild to
moderate while serious skin reactions seem to be rare. Discontinuation is rarely
necessary. Use of corticosteroid-based ointments, e.g., betamethasone 0.1% together
with rehydrating and/or urea-containing creams are the treatments of choice for
rash. For a serious case of psoriasis a consultation of an experienced dermatologist
is advisable. Anal symptoms ranging from discomfort to pain and bleeding are also
common. Depending on the severity, local therapy with a zinc paste or
corticosteroid ointments are used.
A more frequent and more pronounced anemia than what is seen with interferon
plus ribavirin may require dose adjustment of ribavirin or red blood cell transfusion.
The use of erythropoietin for mitigation of anemia is not approved, but can be tried
where reimbursement is possible.
Nausea and diarrhea are frequently seen in patients on telaprevir and may require
symptomatic therapy (Hézode 2009, McHutchison 2009, McHutchison 2010,
Marcellin 2010).
With boceprevir, anemia is the most important adverse event requiring dose
adjustment of ribavirin or red blood cell transfusion in a considerable number of
patients. Dysgeusia is another frequent complaint that resolves upon discontinuation
(Bacon 2011, Poordad 2011).

Management of Adverse Drug Reactions 269

Adherence
Adherence data from retrospective analyses suggest that at least 80% of the
cumulative doses of ribavirin and interferon should be taken by patients as a
prerequisite for treatment success. Cumulative doses of less than 80% were
associated with a steep drop in sustained virologic response (Camma 2005).
Another surrogate of adherence is the premature treatment discontinuation rate,
which usually ranges from 10–15% with pegylated interferon and ribavirin (Fried
2002, Manns 2001).
The added mandatory intake of food as a complication of HCV protease inhibitors
has heightened the adherence concerns.
Despite these increased complications, discontinuation rates in the triple-therapy
arms were only slightly higher in the registration trials, leading to the approval of
boceprevir and telaprevir, indicating a better management of drug-related toxicities
(Bacon 2011, Marcellin 2010, McHutchinson 2010, Poordad 2011).

Conclusion
In summary, the toxicity of interferon-based therapy plus ribavirin is considerable
and requires active management and profound knowledge, particularly about the
management of psychiatric adverse events.
The first generation of HCV protease and polymerase inhibitors will improve the
efficacy of therapy, in particular in HCV genotype 1 patients, but at the cost of
increased toxicities during therapy. Early assessment and robust management of
adverse events may prevent premature treatment discontinuations and improve the
efficacy of hepatitis C therapy.

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272 Hepatology 2012

16. Extrahepatic Manifestations of Chronic
HCV
Karl-Philipp Puchner, Albrecht Böhlig and Thomas Berg

Introduction
Patients with chronic hepatitis C virus (HCV) infection are at risk of a great number
of extrahepatic manifestations (EHMs) (Table 1) – up to 40-76% of patients
infected with HCV develop at least one EHM during the course of the disease
(Cacoub 2000, Cacoub 1999). EHMs may often be the first and only clinical sign of
chronic hepatitis C infection. Evidence of HCV infection should always be sought
out in cases of non-specific chronic fatigue and/or rheumatic, hematological,
endocrine or dermatological disorders. The pathogenesis of EHM is still not fully
understood, although most studies suggest that the presence of mixed
cryoglobulinemia, particular lymphotropism of the virus, molecular mimicry and
non-cryoglobulinemic autoimmune phenomena constitute the major pathogenic
factors (Ferri 2007). Nevertheless, pathogenesis and epidemiology of many EHMs
requires further investigation (Figure 1). Our aim is to give a brief insight into the
epidemiology, pathogenesis, clinical relevance and therapeutic management of
HCV-associated EHM (Zignego 2007a).

Mixed cryoglobulinemia
Cryoglobulinemia refers to the presence of abnormal immunoglobulins in the
serum, which have the unusual property of precipitating at temperatures below 37°C
and redissolving at higher temperatures. The phenomenon of cryoprecipitation was
first described in 1933 (Wintrobe 1933). Cryoglobulins (CGs) are nowadays
classified, on the basis of their clonality, into three types (Table 2). Type II CG and
type III CG, consisting of monoclonal and/or polyclonal immunoglobulins, are
prevalent in patients with a chronic HCV infection, while type I CGs, consisting
exclusively of monoclonal components, are mostly found in patients with
lymphoproliferative disorders (multiple myeloma, B cell lymphoma, Waldenström
macroglobulinaemia). Type II or type III mixed cryoglobulinemia is found in 19%50% of patients with chronic HCV, but leads to clinical manifestations, through

Extrahepatic Manifestations of Chronic HCV 273
vascular precipitation of immunocomplexes, in only 30% of them (Lunel 1994;
Wong 1996). Asymptomatic mixed cryoglobulinemia, during the course of chronic
HCV infection, may evolve into symptomatic disease. Patients with symptomatic
mixed cryoglobulinemia exhibit higher cryoglobulin concentrations (cryocrit >3%)
(Weiner 1998) and lower concentrations of complement factors C3 and C4. Thus
CG-triggered complement activation may constitute a key incidence in
cryoglobulinemia-derived pathogenesis. Factors that seem to favour the
development of MC are female sex, age, alcohol intake (>50g/d), advanced liver
fibrosis and steatosis (Lunel 1994, Wong 1996, Saadoun 2006).
Table 1. Extrahepatic manifestations of chronic hepatitis C infection.
Organ/System involved

Manifestation

Endocrine

• Autoimmune thyroidopathies
(in particular, Hashimoto thyroiditis)
• Insulin resistance/diabetes mellitus*
• GH-insufficiency

Rheumatic disorders

• Mixed cryoglobulinemia*
• Cryoglobulinemic vasculitis*
• Peripheral neuropathy*
• Membrano-proliferative glomerulonephritis (GN)*
• Membranous GN*
• Rheumatoid arthralgias/oligopolyarthritis
• Rheumatoid factor positivity*
• Sicca syndrome

Hematologic disorders

• Lymphoproliferative disorders/Non-Hodgkin Lymphomas*
• Immune thrombocytopenic purpura (ITP)
• Monoclonal gammopathies*
• Autoimmune hemolytic anemia

Dermatologic disorders

• Palpable purpura
• Porphyria cutanea tarda (PCT)
• Lichen planus
• Pruritus

Miscellaneous

• Chronic fatigue*, subclinical cognitive impairment,
psychomotoric deceleration, symptoms of depression*
• Myopathy
• Cardiomyopathy/Myocarditis
• Idiopathic pulmonal fibrosis

•

* Associations that rest upon strong epidemiological prevalence and/or clear pathogenetic
mechanisms.

274 Hepatology 2012
Table 2. Types of cryoglobulinemia.
Type

Clonality

Type I

Monoclonal immunoglobulins (IgG or IgM)

Type II

Polyclonal immunoglobulins (mainly IgG) and monoclonal IgM with
rheumatoid factor activity (RF)

Type III

Polyclonal IgG and IgM

Autoimmune haemolytic anaemia

D

GHInsuffiency
Myocarditis

Myopathy

Non-cryogloblinaemic
neuropathies

Idiopatic pumlmonary fibrosis

C

Thrombocytopenia
Autoimmune
thyroiditis
Diabetes
mellitus II

Rheumatoid
arthritis

Non-cryoglobulinaemic GN
Sicca syndrom

B

Porphyria cutanea
tarda

Lichen
planus

Lymphoproliferative
disorders

A
MC-related disorders

Figure 1. Schematic representation of EHM categories (modified after Zignego 2007a). A)
Associations with strong epidemiological evidence and clear pathogenetic mechanisms; B)
Associations with high prevalence, but unclear pathogenetic mechanisms; C) Associations for
which the high prevalence in HCV collectives could be due to HCV infection and/or confounding
factors; D) Anecdotal observations.

Diagnosis
Detection of CG is carried out by keeping patient serum at 4°C for up to 7 days.
After cryoprecipitate is visible, CG can be purified and characterized using
immunfixation electrophoresis. In case of evidence of mixed cryoglobulinemia in
HCV-positive patients, the presence of cryoglobulinemic syndrome must be sought.
Vigilant monitoring is required, as asymptomatic mixed cryoglobulinemia patients
may develop MC-related disorders in the course of the disease. The diagnosis of the
MC syndrome is based on serologic, pathologic and clinical criteria (Table 3).

Extrahepatic Manifestations of Chronic HCV 275
Table 3. Diagnostic criteria of cryoglobulinemic syndrome.
Serologic

Histopathologic

Clinical

• C4 reduction
• Positive rheumatoid factor
(RF)
• CGs type II or III
• HCV antibodies

• Leukocytoclastic vasculitis
• Infiltrates of monoclonal Bcells

• Purpura
• Fatigue
• Arthralgia
• Membranoproliferative GN
• Peripheral neuropathy

In the presence of mixed CG, low C4 counts, leucocytoclastic vasculitis and
purpura, a definite symptomatic MC can be diagnosed. Rheumatoid factor (RF)
determination constitutes a reliable surrogate parameter for detection of CG.
Finally, presence of CG may impair HCV RNA determination as viral RNA can
accumulate in precipitated cryocrit (Colantoni 1997).

Clinical presentation
HCV-related MC proceeds mostly asymptomatically and has no significant
influence on the course of chronic liver inflammation. On the other hand,
symptomatic mixed cryoglobulinemia is associated with higher mortality (Ferri
2004).
Systemic vasculitis
HCV-related vasculitis relies on a deposition of immunocomplexes containing CGs,
complement and large amounts of HCV antigens in the small- and medium-sized
blood vessels. HCV accumulates in the CG-immunoglobulins. Pathohistological
findings reveal a leucocytoclastic vasculitis (Agnello 1997). The most common
symptoms of mixed cryoglobulinemic vasculitis are weakness, arthralgia and
purpura (the Meltzer and Franklin triad). Mixed cryoglobulinemic vasculitis may
also lead to Raynaud’s Syndrome and Sicca Syndrome, glomerulonephritis and
peripheral neuropathy.
Renal impairment
The predominant renal impairment associated with mixed cryoglobulinemia is the
membranous proliferative glomerulonephritis (MPGN), characterized in most cases
by proteinuria, mild hematuria and mild renal insufficiency. The presence of kidney
impairment is considered to be a negative prognostic factor in the course of the
disease (Ferri 2004). In 15% of patients, MC-related nephropathy may progress
towards terminal chronic renal failure requiring dialysis (Tarantino 1995).
Peripheral neuropathy
Peripheral neuropathy, on the basis of endoneural microangiopathy, constitutes a
further typical complication of mixed cryoglobulinemia. MC-related neuropathy,
presenting clinically as mononeuropathy or polyneuropathy, is mostly sensory and
is characterized by numbness, burning skin crawling and pruritus, predominantly in
the hands and feet (Tembl 1999, Lidove 2001). Epidemiological data from Italy
suggests that peripheral neuropathy is the second most common symptom after the
Meltzer and Franklin triad in patients with symptomatic HCV-associated mixed
cryoglobulinemia (Ferri 2004).

276 Hepatology 2012
Cirrhosis
The causal association between CG and progression of liver fibrosis suggested by
numerous authors has not been confirmed in a recently published 10-year
prospective study. The 10-year rates of progression to cirrhosis were similar in
cryoglobulinemic and non-cryoglobulinemic HCV-infected patients (Vigano 2007).
With respect to this data, it is unlikely that mixed cryoglobulinemia constitutes an
independent risk factor for the progression of liver fibrosis.

Malignant lymphoproliferative disorders/NHL
The association between infectious agents and potentially reversible “antigen
driven” lymphoproliferative disorders, such as Helicobacter pylori-related gastric
marginal zone B cell lymphoma has been known for many decades. Recent data
suggest a causative association between HCV and Non-Hodgkin Lymphoma (NHL)
(Mele 2003, Duberg 2005, Giordano 2007). HCV infection leads per se to a twofold
higher risk of developing NHL (Mele 2003, Duberg 2005). The most prevalent
HCV-associated lymphoproliferative disorders according to the REAL/WHO
classification are: follicular lymphoma, B cell chronic lymphocytic leukemia/small
lymphocyte lymphoma, diffuse large B cell lymphoma and marginal zone
lymphoma, including the mucosa-associated lymphoid tissue lymphoma. Overall,
marginal zone lymphoma appears to be the most frequently encountered low grade
B cell lymphoma in HCV patients.
HCV-associated lymphoproliferative disorders (LPDs) are observed over the
course of MC. 8-10% of mixed cryoglobulinemia type II evolve into B cell NHL
after long-lasting infection. However, a remarkably high prevalence of B cell NHL
was also found in HCV patients without mixed cryoglobulinemia (Silvestri 1997).
Genetic predisposition and other factors seem to have a major impact on the
development of LPDs in HCV-positive patients (Matsuo 2004).

Etiology and pathogenesis of LPDs in patients with HCV
infection
In the development of LPDs direct and indirect pathogenic HCV-associated factors
(Figure 2) are seen. Sustained B cell activation and proliferation, noticed during
chronic HCV infection, is an indirect pathogenic mechanism.
Direct pathogenic mechanisms are based on lymphotropic properties of HCV,
hence on the very invasion of HCV into the B cells. HCV RNA sequences were first
detected in mononuclear peripheral blood cells (Zignego 1992). Especially CD19+
cells seem to be permissive for certain HCV quasispecies (Roque Afonso 1999).
Active replication of the HCV genome in B cells is associated with activation of
anti-apoptotic gene bcl-2 and inhibition of p53 or c-Myc-induced apoptosis
(Sakamuro 1995, Ray 1996). In this light, direct involvement of HCV in the
immortalisation of B cells can be envisioned (Zignego 2000, Machida 2004).

Extrahepatic Manifestations of Chronic HCV 277

Fig 2. Pathomechanisms involved in the development of malignant lymphoproliferative
disorders in patients with chronic HCV infection. Indirect pathomechanism: Sustained
antigen stimulation, like binding of viral envelope protein to CD81 receptor, leads to excessive
B cell proliferation, which in turn favors development of mixed cryoglobulinemia and/or genetic
aberrations, such as t(14;18) translocation. Direct pathomechanism: Viral infection of B cells, as
viral replication in them may result in activation of proto-oncogenes (i.e., Bcl-2) and/or inhibition
of apoptotic factors (i.e., p53, c-myc). One of the factors favoring this polyclonal B cell activation
and proliferation is probably the HCV E2 protein, which binds specifically to CD81, a potent B
cell activator (Cormier 2004).

Treatment of lymphoproliferative disorders
Because of the close correlation between the level of viral suppression and
improvement of HCV-associated extrahepatic symptoms, the most effective
antiviral strategy should be considered when dealing with HCV-related extrahepatic
diseases. The protease inhibitors boceprevir and telaprevir have been shown to
improve significantly sustained virologic response rate in HCV type 1-infected
patients when given in combination with peg-interferon plus ribavirin as compared
to peg-interferon and ribavirin alone, and can be therefore regarded as the treatment
of choice in HCV type 1-infected patients with extrahepatic manifestations.
However, certain protease-inhibitor-associated contraindications, especially drugdrug interaction due to their metabolism via the CYP3A isoenzymes, have to be
taken into account and all concomitant medications need to be assessed and
adjusted. For further information, see the other HCV chapters.

Mixed cryoglobulinemia
While asymptomatic MC per se does not constitute an indication for treatment,
symptomatic mixed cryoglobulinemia should always be treated. Because
asymptomatic cryoglobulinemia may evolve into symptomatic in the course of

278 Hepatology 2012
disease, vigilant monitoring is required and introduction of antiviral therapy in
terms of prophylaxis should be considered.
Because a causal correlation between HCV infection and mixed cryoglobulinemia
has been established, the therapeutic approach of symptomatic mixed
cryoglobulinemia should primarily concentrate on the eradication of the virus.
Indeed, clinical improvement of MC is reported in 50 to 70% of patients receiving
antiviral therapy with IFN α and RBV and mostly correlates with a drastic reduction
of HCV RNA concentrations (Calleja 1999). However, cryoglobulinemic vasculitis
following successful antiviral treatment persists in a small collective (Levine 2005).
IFN α has been shown to be a promising therapeutic tool irrespective of virologic
response. Due to its antiproliferative properties on IgM-RF producing B cells and
stimulation of macrophage-mediated clearance of immunocomplexes, IFN α may
lead to clinical amelioration even in virological nonresponders. Therefore,
therapeutic success should be primarily evaluated on the basis of clinical response
irrespective of virologic response. In case of treatment failure of antiviral therapy
and/or fulminant manifestations, contraindications or severe side effects, alternative
therapeutic strategies such as cytostatic immunosuppresive therapy and/or
plasmapheresis should be considered (Craxi 2008) (Figure 3, Table 4). Recent data
show rituximab as an effective and safe treatment option for MC even in advanced
liver disease. Moreover, B-cell depletion has been shown to improve cirrhotic
syndrome by mechanisms that remain to be further studied (Petrarca 2010).

Systemic vasculitis
In cases of severe systemic vasculitis, initial therapy with rituximab, a monoclonal
chimeric antibody against CD20 B cell specific antigen, is suggested. Its efficacy
and safety have also been demonstrated in patients with symptomatic MC resistant
to IFN α therapy, even though HCV RNA increased approximately twice the
baseline levels in responders (Sansonno 2003). In light of this, combined application
of rituximab with PEG-IFN α plus ribavirin in cases of severe mixed
cryoglobulinemia-related vasculitis resistant to antiviral therapy seems to be the
optimal therapeutic strategy, achieving amelioration of MC-related symptoms and a
complete eradication of HCV in responders (Saadoun 2008). In severe rituximabrefractory mixed cryoglobulinaemia-related vasculitis or acute manifestations,
cycles of plasma exchange plus corticosteroids and eventually cyclophosphamide
are indicated. Further studies showed that low dose interleukin-2 can lead to clinical
improvement of vasculitis and has immunologic effects such as recovery of
regulatory T cells (Saadoun 2011).

Peripheral neuropathy
Effectiveness of antiviral therapy on cryoglobulinemic-induced peripheral
neuropathy is still being debated. While HCV-related peripheral neuropathy
responsive to antiviral therapy with IFN α and ribavirin in 4 patients with chronic
HCV has been reported (Koskinas 2007), several authors report on an aggravation
of cryoglobulinemic neuropathy or even de novo occurance of demyelinating
polyneuropathy during IFN α and PEG-IFN α treatment (Boonyapist 2002, Khiani
2008). Therefore, application of IFN α in presence of HCV-related neuropathy
requires a cautious risk-benefit assessment.

Extrahepatic Manifestations of Chronic HCV 279

Figure 3. Therapy algorithm for symptomatic HCV-related mixed cryoglobulinemia
(modified from Craxi 2008). Antiviral therapy, on the basis of PEG-IFN α and ribavirin
plus/minus protease inhibitors, is regarded as first line therapy in cases of mild/moderate
manifestations. In case of contraindications, patients should be treated primarily with
corticosteroids. Non-response to antiviral therapy or drug-induced aggravation makes
application of corticosteroids essential. Long-term therapy with corticosteroids may result in
elevation of viral load and progression of hepatic disease. In light of this, rituximab represents
an attractive alternative, because in this case, drug-induced viral load elevation is minor. In
patients with severe manifestations, treatment should focus on immunosupression (±
plasmapheresis). Due to its excellent immunosuppressive properties and relatively mild side
effect profile, use of rituximab should be favored. In case of good clinical response, consecutive
antiviral treatment with PEG-IFN α and ribavirin may serve as maintenance therapy. Therapy
refractory cases require individual treatment according to the particular center’s experience.
Supplementation of therapeutic strategy with antiviral therapy should be considered.

As eradication of Helicobacter pylori may lead to complete remission of MALT
lymphoma, antiviral therapy can lead to regression of low-grade NHL in patients
with HCV-related malignant lymphoproliferative disorders. PEG-IFN α plus
ribavirin (± protease inhibitors) should be regarded in such cases as first-line
therapy (Giannelli 2003, Vallisa 2005). Thus, remission of the hematologic
disorders is closely associated with virologic response or rather achievement of
sustained virologic response. Effectiveness of IFN α in this context should be
ascribed primarily to the drug’s antiviral and less to its anti-proliferative properties.

280 Hepatology 2012
Table 4. Treatment of cryoglobulinemia-related disorders in patients with chronic HCV
infection.

Author

Patients

Treatment

Result

Zuckerman

N=9
symptomatic-MC
non-responders to
IFN α monotherapy

IFN α 3x/wk
+ ribavirin 15 mg/kg/d

CGs undetectable within
6 weeks in 7/9 patients;
clinical improvement in
9/9 within 10 weeks

Sansonno

N=20
Rituximab 375 mg/m /
4x/wk
MC vasculitis and
peripheral neuropathy
resistant to IFN α
montherapy

Saadoun

N=16
MC vasculitis in
relapsers or nonresponders to IFN
α/PEG-IFN α + RBV

Bruchfeld

2

16 patients complete
clinical response;12
sustained response
throughout follow-up.
Viremia increase in
responders.

Rituximab 375 mg/m /
4x/wk;
PEG-INF α 1.5 ug/kg/wk
+ RBV (600-1200 mg/d)
for 12 months

2

10/16 complete clinical
response; CGs and RNA
HCV undetectable in
responders

N=7
HCV-related renal
manifestations
(2/7 MC-related)

IFN α + low-dose ribavirin
(200-600 mg)
or PEG-INF α + low-dose
ribavirin

Improvement of GRF and
proteinuria in 4/7 patients
and sustained viral
response in 5/7.

Roccatello

N=6
MC systematic
manifestations
predominantly renal
(5/6)

Rituximab 375
2
mg/m /4x/wk
2
+ rituximab 375 mg/m
1 month and 2 months
later

Decrease of cryocrit and
proteinuria at months 2,
6, 12.

Koskinas

N=4
MC patients with
severe sensory-motor
polyneuropathy

INF α-2b 1.5ug/kg/wk +
ribavirin 10.6 mg/kg/d for
48 weeks

Significant improvement
of neurological
parameters in 4/4;
undetectable HCV RNA
and lower CG levels in
3/4 at the end of therapy.

Treatment of HCV-infected patients with high-grade NHL should be based on
cytostatic chemotherapy. HCV infection does not constitute a contraindication for
cytostatic chemotherapy. Unlike HBV infection, antiviral prophylaxis before
chemotherapy introduction is not obligatory. Chemotherapy may lead to a
substantial increase in viremia. Consecutive exacerbation of the infection, making
discontinuation of chemotherapy mandatory, is unlikely to occur. However,
treatment-related liver toxicity is more frequent in HCV-positive NHL and is often
associated with severe hepatic manifestations (Besson 2006, Arcaini 2009). Current
data suggest that antiviral treatment may serve as maintenance therapy for achieving
sustained remission of NHL after chemotherapy completion (Gianelli 2003).

Extrahepatic Manifestations of Chronic HCV 281

Further hematological manifestations
HCV-associated thrombocytopenia
Thrombocytopenic conditions (platelet counts below 150 x 103/uL) are often
observed in patients with chronic hepatitis C and result mainly from advanced liver
fibrosis and manifest cirrhosis (Wang 2004). Lack of hepatic-derived
thrombopoietin can inter alia be recognized as an important causal factor (Afdhal
2008). As HCV RNA can be abundant in platelets (Takehara 1994) and
megakaryocytes of thrombocytopenic patients, direct cytopathic involvement of
HCV can be hypothesized (Bordin 1995, De Almeida 2004). Furthermore, it has
been suggested that exposure to HCV may be a causative factor for the production
of platelet-associated immunoglobulins, inducing thrombocytopenia through a
similar immunological mechanism to that operating in immune thrombocytopenic
purpura (ITP) (Aref 2009). There is a high HCV prevalence in patients with ITP
(García-Suaréz 2000), and these patients exhibit diverse characteristics to HCVnegative patients with ITP, which supports the hypothesis of direct viral
involvement in the development of thrombocytopenia (Rajan 2005).
There is no consensus regarding the optimum treatment of HCV-related ITP.
Along with classical therapeutic approaches such as corticosteroids, intravenous
immunoglobulins and splenectomy, antiviral therapy constitutes another option. A
substantial increase of platelets after application of antiviral therapy is registered in
a significant percentage of patients with HCV-related ITP (Iga 2005), although
evidence from further studies is required to confirm this hypothesis. However,
caution is recommended in thrombocytopenic patients treated with PEG-IFN α plus
ribavirin, as significant aggravation of HCV-related ITP may occur on this regimen
(Fattovich 1996). On the other hand, long-term use of steroids or
immunosuppressive drugs respectively is limited by an increased risk of fibrosis
progression or a substantial elevation of virus. A new orally active thrombopoietin
receptor agonist, eltrombopag, may be used in thrombocytopenic HCV patients in
the future. Its efficacy was recently documented in patients with HCV-related ITP
(Bussel 2007) as well as in HCV-positive patients suffering from thrombocytopenia
due to cirrhosis (McHutchison 2007). However in a recent study treating patients
with eltrombopag in combination with PEG-IFN α and ribavirin, portal vein
thrombosis was observed in a number of patients as an unexpected complication
(Afdhal 2011). In case of refractory disease or aggravation during the course of
antiviral therapy, rituximab should be considered (Weitz 2005).

HCV-related autoimmune hemolytic anemia
Interpretation of autoimmune hemolytic anemia (AHA) as a possible EHM is based
mainly on a few well-documented case reports (Chao 2001, Fernandéz 2006,
Srinivasan 2001). AHA has been frequently observed in HCV patients treated with
IFN α with and without ribavirin and consequently recognized as a possible side
effect of antiviral treatment (De la Serna-Higuera 1999, Nomura 2004). Recently, a
large-scale epidemiological study confirmed a high incidence of AHA in HCV
patients undergoing antiviral treatment. However, the incidence rate of AHA in
treatment-naïve HCV patients was statistically insignificant (Chiao 2009). In this
light, there is, for the time being, little evidence for regarding AHA as a possible
EHM of chronic HCV infection.

282 Hepatology 2012

HCV-related glomerulonephritis
Glomerulonephritis (GN) constitutes a rare extrahepatic complication of chronic
HCV. Predominant manifestations are cryoglobulinemic or non-cryoglobulinemic
membranous proliferative GN and mesangioproliferative GN. Far less common is
membranous nephropathy (Arase 1998). Other forms of GN do not correlate
significantly with HCV infection (Daghestani 1999). Microhematuria and
proteinuria are among the most frequent medical findings in patients with
membranous proliferative GN. Approximately 50% of patients exhibit a mild renal
insufficiency. 20-25% may present an acute nephritic syndrome (hematuria,
hypertension and proteinuria), as in 25% of patients nephrotic syndrome represents
the initial manifestation. In contrast, >80% of patients with HCV-related
membranous nephropathy suffer primarily a nephrotic syndrome (Doutrelepont
1993, Rollino 1991). The mesangioproliferative form proceeds mostly
asymptomatically, with typical findings such as hematuria and proteinuria often
missing (McGuire 2006).
The pathomechanism of renal impairment is yet not fully understood. It can be
hypothesized that glomerular injury is primarily caused by a deposition of
circulating immunocomplexes containing anti-HCV antibodies, HCV antigens and
complement factors. Formation and deposition of such immunocomplexes occurs
also in the absence of CGs. HCV-proteins in glomerular and tubulointerstitial
structures are immunohistologically detectable in approximately 70% of patients
with chronic HCV (Sansonno 1997). Further possible pathomechanisms of
glomerular injury encompass formation of glomerular autoantibodies, glomerular
impairment due to chronic hepatic injury, or IgM overproduction with consecutive
glomerular IgM deposition as result of HCV-triggered cryoglobulinemia type II. GN
prevalence in HCV patients is estimated at 1.4% and is comparably high due to its
prevalence among blood donors (Paydas 1996).
HCV-induced GN has mostly a benign prognosis (Daghestani 1999). 10-15% of
patients with nephritic syndrome experience spontaneous complete or partial
remission. Frequently persisting mild proteinuria exhibits no tendency to
progression. It is estimated that only approximately 15% of the patients with HCVrelated GN develop terminal renal failure requiring dialysis (Tarantino 1995).
Nevertheless, presence of kidney impairment is considered to be a negative
prognostic factor for long-term survival (Ferri 2004).
Patients with HCV-related GN should be primarily treated with antivirals. In
cases of mild renal impairment, sustained viral response normally leads to
amelioration of proteinuria or even full remission of GN. With high baseline
viremia and advanced renal insufficiency, antiviral therapy is subject to certain
limitations (Sabry 2002). Despite amelioration of proteinuria achieved after antiviral
therapy, significant improvement of renal function is often lacking (Alric 2004).
PEG-IFN and ribavirin dosage must be cautiously adjusted to glomerular filtration
rate (GFR), in order to mainly prevent ribavirin accumulation with consecutive
hemolytic anemia (Fabrizi 2008). Even in advanced renal failure, use of ribavirin is
recommended due to the superior efficacy of combination therapy vs. IFN
monotherapy (Bruchfeld 2003, Baid-Agrawal 2008). In patients with GFR <30
ml/min ribavirin dosage should not exceed 600 mg/week. Careful dosage
augmentation may be undertaken in the absence of side effects. Ribavirin dosages

Extrahepatic Manifestations of Chronic HCV 283
up to 100-400 mg/day was done under vigilant blood level monitoring in dialysis
patients. Ribavirin-induced hemolytic anemia was efficiently treated by
administration of erythropoietin and erythrocyte concentrates (van Leusen 2008).
As determination of ribavirin blood levels is not an established laboratory
procedure, implementation of such a therapeutic approach in clinical routine
remains arduous. No dose reduction is required with respect to renal impairment for
the two licensed protease inhibitors boceprevir and telaprevir (see also Chapters 14
and 15).
Fulminant manifestations with impending acute renal failure make administration
of corticosteroids, immunosuppressive drugs such as cyclophosphamide and
eventually plasmapheresis necessary (Garini 2007, Margin 1994). In cases of
simultaneous bone marrow B cell infiltration and/or resistance to conventional
therapy, application of rituximab is indicated (Roccatello 2004). Rituximab may be
used as an alternative first line therapy in severe renal manifestations (Roccatello
2008). Antiviral and immunosuppressive therapy should always be supplemented
with ACE inhibitors or AT1 receptor antagonists (Kamar 2006).

Endocrine manifestations
Thyroid disease is found more commonly in patients with chronic HCV infection
than in the general population. About 13% of HCV-infected patients have
hypothyroidism and up to 25% have thyroid antibodies (Antonelli 2004). There is
also evidence that IFN α may induce thyroid disease or unmask preexisting silent
thyroidopathies (Graves disease, Hashimoto thyroiditis) (Prummel 2003). In
addition, some studies suggest that thyroid autoimmune disorders were significantly
present in patients with chronic hepatitis C during but not before IFN α therapy
(Marazuela 1996, Vezali 2009). Therefore, the role of chronic hepatitis C infection
per se in the development of thyroid disorders remains to be determined. The
presence of autoantibodies against thyroid with/or without clinical manifestations
increases the risk of developing an overt thyroiditis significantly during antiviral
therapy. Therefore, monitoring of the thyroid function should be performed during
treatment.
Association between chronic HCV infection and development of insulin
resistance and diabetes mellitus has been discussed in the past (Knobler 2000;
Mason 1999, Hui 2003, Mehta 2003). In the meantime, a causal association is
backed up by studies demonstrating that antiviral therapy with consecutive
sustained viral response correlates with improved diabetic metabolic status and
resolution of insulin resistance (Kawaguchi 2007). A recently published metaanalysis of retrospective and prospective studies confirms a high risk for the
development of diabetes mellitus type II in patients with chronic HCV infection
(OR=1.68, 95%, CI 1.15-2.20) (White 2008). Viral induction of insulin resistance
seems to be HCV-specific, as prevalence of diabetes mellitus in HBV-infected
patients is significantly lower (White 2008, Imazeki 2008). The pathomechanism of
HCV-induced insulin resistance is yet not fully understood. It has been suggested
that the appearance of insulin resistance could correlate with certain genotypes of
HCV. Furthermore, HCV-dependent upregulation of cytokine suppressor SOC-3
may be responsible for the induction of cell desensitisation towards insulin. Insulin

284 Hepatology 2012
resistance in turn represents an independent risk factor for progression of liver
fibrosis in patients with chronic HCV infection (Moucari 2008, Kawaguchi 2004).
Finally, a link between HCV, growth hormone (GH) insufficiency and low
insulin-like growth factor (IGF-1) has been hypothesized. Reduced GH secretion
could be the result of a direct inhibitory effect of HCV infection at the level of the
pituitary or hypothalamus (Plöckinger 2007).

Dermatologic and miscellaneous manifestations
A multitude of cutaneous disorders has been sporadically associated with chronic
HCV infection (Hadziyannis 1998). Epidemiologic studies have confirmed the
existence of a strong correlation between the sporadic form of porphyria cutanea
tarda (PTC) and HCV, though the presence of HCV in PTC patients seems to be
subject to strong regional factors. Indeed, HCV prevalence in PTC patients is higher
than 50% in Italy, while only 8% in Germany (Fargion 1992, Stölzel 1995).
Strong evidence of a close association between HCV and lichen planus was
provided by studies performed in Japan and southern Europe (Nagao 1995,
Carrozzo 1996), yet these observations do not apply to all geographic regions
(Ingafou 1998). HLA-DR6 has been recognized as a major predisposing factor for
development of lichen planus in HCV-positive patients. One hypothesis suggests
that geographical fluctuation of HLA-DR6 is responsible for the diverse prevalence
among HCV patients (Gandolfo 2002).
Idiopathic pulmonary fibrosis (IPF) represents potentially an EHM, as prevalence
of anti-HCV in patients with this disease is notably high (Ueda 1992). Interestingly,
alveolar lavage in therapy-naïve HCV patients yielded frequent findings consistent
with a chronic alveolitis. Alveolar lavage in the same patients after completion of
antiviral therapy showed a remission of inflammatory activity (Yamaguchi 1997).
Involvement of CGs in the genesis of IPF is also probable (Ferri 1997).
Numerous central nervous manifestations have been described in association with
HCV infection. Cryoglobulinemic or non-cryoglobulinemic vasculitis of cerebral
blood vessels may be responsible for the relatively high prevalence of both ischemic
and hemorrhagic strokes in young HCV-positive patients (Cacoub 1998).
Transverse myelopathies leading to symmetrical paraparesis and sensory deficiency
have been recently observed (Aktipi 2007).
Furthermore, chronic HCV infection is associated with significant impairment of
quality of life. 35-68% of HCV patients suffer from chronic fatigue, subclinical
cognitive impairment and psychomotor deceleration. Symptoms of depression are
evident in 2-30% of HCV patients examined (Perry 2008, Forton 2003, Carta 2007).
Psychometric as functional magnetic resonance spectroscopy studies suggest altered
neurotransmission in HCV-positive groups (Weissenborn 2006, Forton 2001). In
addition, significant tryptophan deficiency is detectable in patients with chronic
HCV infection. Resulting deficiency of the tryptophan-derived serotonin is likely to
favor an occurrence of depressive disorders. There is evidence to suggest that
antiviral therapy can lead to elevation of tryptophan blood levels and thus contribute
to amelioration of depressive symptoms in HCV patients (Zignego 2007c).
Occasionally, chronic HCV infection has been seen in association with cardiac
pathologies such as chronic myocarditis and dilatative/hypertrophic

Extrahepatic Manifestations of Chronic HCV 285
cardiomyopathy. Pathogenesis seems to rely on genetic predisposition and is
assumed to be immunologically triggered (Matsumori 2000).

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Management of HBV/HIV coinfection 291

17. Management of HBV/HIV coinfection
Stefan Mauss and Jürgen Kurt Rockstroh

Introduction
The prevalence and transmission routes of HBV coinfection in the HIV+ population
vary substantially by geographic region (Alter 2006, Konopnicki 2005). In the
United States and Europe the majority of HIV+ homosexual men have evidence of
past HBV infection, and 5-10% show persistence of HBs antigen with or without
replicative hepatitis B as defined by the presence of HBV DNA (Konopnicki 2005).
Overall, rates of HBV/HIV coinfection are slightly lower among intravenous drug
users compared to homosexual men and much lower among people infected through
heterosexual contact (Núñez 2005).
In endemic regions of Africa and Asia, the majority of HBV infections are
transmitted vertically at birth or before the age of 5 through close contact within
households, medical procedures and traditional scarification (Modi 2007). The
prevalence among youth in some Asian countries has substantially decreased since
the introduction of vaccination on nationwide scales (Shepard 2006). In Europe
vaccination of children and members of risk groups is reimbursed by health care
systems in most countries.
The natural history of hepatitis B is altered by simultaneous infection with HIV.
Immune control of HBV is negatively affected leading to a reduction of HBs
antigen seroconversion. If HBV persists, the HBV DNA levels are generally higher
in untreated patients (Bodsworth 1989, Bodsworth 1991, Hadler 1991). In addition,
with progression of cellular immune deficiency, reactivation of HBV replication
despite previous HBs antigen seroconversion may occur (Soriano 2005). In the
untreated HIV-positive population, faster progression to liver cirrhosis is reported
for HBV/HIV-coinfected patients (Puoti 2006). Moreover, hepatocellular carcinoma
may develop at an earlier age and is more aggressive in this population (Puoti 2004,
Brau 2007).
Being HBV-coinfected results in increased mortality for HIV-positive individuals,
even after the introduction of highly active antiretroviral combination therapy
(HAART), as demonstrated by an analysis of the EuroSIDA Study, which shows a
3.6-fold higher risk of liver-related deaths among HBsAg-positive patients
compared to HBsAg-negative individuals (Konopnicki 2005, Nikolopoulos 2009)
(Figure 1). In the Multicentre AIDS Cohort Study (MACS), an 8-fold increased risk
of liver-related mortality was seen among HBV/HIV-coinfected compared to HIV-

292 Hepatology 2012
monoinfected individuals, particularly among subjects with low nadir CD4-postive
cell counts (Thio 2002). An independent observation from a large cohort confirming
this association is the reduction in mortality for HBV/HIV-coinfected patients
treated with lamivudine compared to untreated patients (Puoti 2007). This result is
even more remarkable because lamivudine is one of the least effective HBV
polymerase inhibitors due to a rather rapid development of resistance. In general,
due to its limited long-term efficacy, lamivudine monotherapy for HBV cannot be
considered as appropriate therapy (Matthews 2011).

Figure 1. Association of HBV/HIV coinfection and mortality (Konopnicki 2005). More than
one cause of death allowed per patient; p-values from chi-squared tests.

These two large cohort studies (EuroSIDA and MACS) plus data from HBV
monoinfection studies showing a reduction in morbidity and mortality justify
treatment of hepatitis B in HBV/HIV-coinfected patients. HBV is often treated
simultaneously with HIV, as some nucleoside and nucleotide reverse transcriptase
inhibitors are active as HBV polymerase inhibitors as well. Therefore, antiretroviral
therapy should be adjusted according to HBV status wherever possible to avoid
higher pill burden and additional toxicities. A less frequent but more challenging
situation is the initiation of HBV therapy in HIV-coinfected individuals who are not
on antiretroviral therapy. Treatment with interferon is one possible therapeutic
option in this situation. The main limitation of some HBV polymerase inhibitors
may be induction of HIV resistance by the anti-HBV agents as they act
simultaneously as HIV reverse transcriptase inhibitors.

Management of HBV/HIV coinfection 293

Figure 2. Treatment algorithm for therapy of HBV in HIV-coinfected patients (EACS 2011).
a) Cirrhotic patients should be referred for variceal assessment, have regular HCC monitoring
and be referred early for transplant assessment.
b) See Figure 5 for assessment of HBV Rx indication. Some experts strongly think that any
HBV-infected patient requiring HAART should receive TDF + 3TC or FTC unless history of TDF
intolerance, particularly in HIV/HBV coinfected patients with advanced liver fibrosis (F3/F4).
c) If patient is unwilling to go on early HAART, adefovir and telbivudine may be used as an
alternative to control HBV alone. Recently a case report suggested anti-HIV activity of
telbivudine. In vitro data using an assay able to demonstrate anti-HIV activity of entecavir failed
to detect an influence of telbivudine on the replicative capacity of HIV-1. Treatment duration: in
patients not requiring HAART and on treatment with telbivudine +/– adefovir, or those on
HAART where nucleoside backbone needs changing, anti-HBV therapy may be stopped
cautiously in HBeAg+ patients who have achieved HBe seroconversion or HBs seroconversion
for at least six months or, after HBs seroconversion; for at least six months in those who are
HBeAg-.
d) Treatment length: 48 weeks for PEG-INF; on-treatment quantification of HBsAg in patients
with HBeAg-negative chronic hepatitis B treated with PEG-INF may help identify those likely to
reach HBs-antigen seroconversion with this therapy and optimize treatment strategies.
e) In some cases of tenofovir intolerance (i.e., renal disease), entecavir or tenofovir in doses
adjusted to renal clearance in combination with effective HAART may be advisable. NRTI
substitution should only be performed if feasible and appropriate from the perspective of
maintaining HIV suppression. Caution is warranted in switching from a tenofovir-based regimen
to drugs with a lower genetic barrier, e.g., FTC/3TC, in particular in lamivudine-pretreated
cirrhotic patients, as viral breakthrough due to archived YMDD mutations has been observed.
This has also been described in individuals with previous 3TC HBV resistance who have been
switched from tenofovir to entecavir.

HBV therapy in HBV/HIV-coinfected patients
without HIV therapy
The recommendations of the updated European AIDS Clinical Society (EACS) for
the treatment of chronic hepatitis B in HIV-coinfected patients without antiretroviral
therapy are shown in Figure 2 (EACS 2011). Starting hepatitis B therapy depends
on the degree of liver fibrosis and the HBV DNA level. Using the level of HBV

294 Hepatology 2012
replication as the basis for treatment decisions is an important change of paradigm
in HBV therapy. This decision is based on the results of the REVEAL study (Iloeje
2006). REVEAL followed the natural course of chronic hepatitis B without liver
cirrhosis in about 3700 Taiwanese patients for more than 10 years. In these HBVmonoinfected patients an HBV DNA of >10,000 copies/ml (i.e., 2000 IU/ml) had a
markedly increased risk of developing liver cirrhosis and hepatocellular carcinoma
(Figure 3). This association was even observed in patients with normal ALT levels
(Chen 2006) (Figure 4).

Figure 3. REVEAL Study: Association of HBV DNA levels and liver cirrhosis (Iloeje 2006).

Figure 4. REVEAL Study: Association of HBV DNA with the development of
hepatocellular carcinoma (Chen 2006).

Management of HBV/HIV coinfection 295

It should be mentioned that this cohort consisted of Asian patients without HIV
coinfection predominantly infected at birth or in early childhood. However, the
results were considered too important not to form part of the management of HIVcoinfected patients.
Usually patients with an HBV DNA of less than 2000 IU/ml have no substantial
necroinflammatory activity in the liver and therefore a benign course of fibrosis
progression and a low risk for the development of hepatocellular carcinoma.
However, especially in patients harbouring HBV precore mutants, fluctuations in
HBV DNA and ALT are not rare. Monitoring of the activity of the HBV DNA and
ALT accompanied by an abdominal ultrasound every 6-12 months is recommended.
In the case of HBV DNA <2000 IU/ml and elevated transaminases and/or signs of
advanced liver fibrosis, alternative causes of hepatitis and liver toxicity should be
excluded.
For patients with HBV DNA >2000 IU/ml the ALT level is the next decision
criterion. Patients with normal ALT should be assessed for liver fibrosis by liver
biopsy or elastometry. In case of lack of substantial liver fibrosis (METAVIR stage
F0/1) monitoring of the activity of the HBV DNA and ALT accompanied by an
ultrasound every 3-6 months is recommended. In the presence of liver fibrosis of
METAVIR F2 or higher, hepatitis B treatment should be initiated.
For patients with HBV DNA >2000 IU/ml and increased ALT, treatment for HBV
is an option particularly in the presence of relevant liver fibrosis.
In patients not taking antiretroviral therapy, pegylated interferon α-2a or -2b
seems a suitable option. However, data in the literature for HIV-coinfected patients
on interferon therapy for HBV infection are limited and not very encouraging
(Núñez 2003). For pegylated interferons no data from larger cohorts exist and one
study combining pegylated interferon with adefovir did not show encouraging
results (Ingiliz 2008). Favourable factors for treatment success with interferon are
low HBV DNA, increased ALT, HBV genotype A or infection with HBV wild type.
Alternatively patients can be treated with polymerase inhibitors. However, due to
their antiretroviral activity tenofovir, emtricitabine and lamivudine are
contraindicated in the absence of effective HIV therapy. In contrast to in vitro data
reported by the manufacturer, antiretroviral activity and induction of the HIV
reverse transcriptase mutation M184V was reported for entecavir (MacMahon
2007). Currently only telbivudine and adefovir are considered reasonably safe
treatment options. There is limited in vivo data for adefovir to support this
recommendation (Delaugerre 2002; Sheldon 2005). For telbivudine in vitro data are
available showing a specific inhibitory activity on the HBV polymerase and no
effect on HIV (Avilla 2009). However, in contrast with this, two case reports have
suggested antiretroviral activity of telbivudine (Low 2009, Milazzo 2009).
Because of its greater antiviral efficacy, telbivudine is preferred by most experts
to adefovir (Chan 2007). Alternatively an add-on strategy of telbivudine to adefovir
in the case of not fully suppressive antiviral therapy or primary combination therapy
of both drugs can be considered although clinical data are not yet available for this
strategy.
As both drugs have limitations in the setting of HBV-monoinfected patients due
to considerable development of resistance to telbivudine and the limited antiviral
efficacy of adefovir, the initiation of antiretroviral therapy using tenofovir plus

296 Hepatology 2012
lamivudine or emtricitabine should be considered, particularly in HIV-coinfected
patients with advanced liver fibrosis.
The treatment duration is determined by HBe antigen or HBs antigen
seroconversion, like with HBV-monoinfected patients. In case of infection with a
precore mutant HBs antigen seroconversion is the biological endpoint.

Treatment of chronic hepatitis B in HBV/HIVcoinfected patients
For patients on antiretroviral therapy a wider choice of polymerase inhibitors is
available. In principle, the treatment algorithm of Figure 5 is based on the same
principles as outlined above (EACS 2011).
Initiating antiretroviral therapy with tenofovir resulted in higher rates of HBe
antigen loss and seroconversion as expected from HBV-monoinfected patients
(Schmutz 2006, Piroth 2010). This may be due to the additional effect of immune
reconstitution in HIV-coinfected patients adding another aspect to the
immunological control of HBV replication.
For patients with HBV DNA <2000 IU/ml and no relevant liver fibrosis no
specific antiretroviral regimen is recommended. However when choosing an HBV
polymerase inhibitor, the complete suppression of HBV DNA is important to avoid
the development of HBV resistance mutations. The activity of the HBV infection in
these patients should be assessed at least every six months as part of routine
monitoring of the HIV infection including an ultrasound due to the slightly
increased risk of hepatocellular carcinoma.

Figure 5. Treatment algorithm for HBV therapy in patients with antiretroviral therapy
(EACS 2011).

When HBV DNA is above 2000 IU/ml in naïve patients a combination of
tenofovir plus lamivudine/emtricitabine to treat both infections is recommended.
Even for patients who harbour lamivudine-resistant HBV due to previous therapies

Management of HBV/HIV coinfection 297
this strategy stands. The recommendation to continue lamivudine/emtricitabine is
based on the delay of resistance to adefovir seen when doing so (Lampertico 2007).
For patients with liver cirrhosis a maximally active continuous HBV polymerase
inhibitor therapy is important to avoid hepatic decompensation and reduce the risk
of developing hepatocellular carcinoma. Tenofovir plus lamivudine/emtricitabine is
the treatment of choice. If the results are not fully suppressive, adding entecavir
should be considered (Ratcliffe 2011). At least every six months, assessement of the
liver by ultrasound for early detection of hepatocellular carcinoma is necessary. In
patients with advanced cirrhosis gastroscopy should be performed as screening for
esophageal varices.
For patients with hepatic decompensation and full treatment options for HBV and
stable HIV infection, liver transplantation should be considered, as life expectancy
seems to be the same as for HBV-monoinfected patients (Coffin 2007, Tateo 2009).
Patients with hepatocellular carcinoma may be considered liver transplant
candidates as well, although according to preliminary observations from small
cohorts, the outcome may be worse than for HBV-monoinfected patients with
hepatocellular carcinoma (Vibert 2008).
In general, tenofovir can be considererd the standard of care for HBV in HIVcoinfected patients, because of its efficacy and its strong HBV polymerase activity.
Tenofovir has been a long-acting and effective therapy in the vast majority of
treated HBV/HIV-coinfected patients (van Bömmel 2004, Mathews 2009, MartinCarbonero 2011, Thibaut 2011). No conclusive pattern of resistance mutations has
been identified in studies or cohorts (Snow-Lampart 2011). But resistance is likely
to occur in patients with long-term therapy as with any other antiviral. In
prospective controlled studies tenofovir was clearly superior to adefovir for
treatment of HBe antigen-positive and HBe antigen-negative patients (Marcellin
2008).
The acquisition of adefovir resistance mutations and multiple lamivudine
resistance mutations may impair the activity of tenofovir (Fung 2005, Lada 2008,
van Bömmel 2010), although even in these situations tenofovir retains activity
against HBV (Berg 2008, Petersen 2009).
In lamivudine-resistant HBV the antiviral efficacy of entecavir in HIV-coinfected
patients is reduced, as it is in HBV monoinfection (Shermann 2008). Because of this
and the property of tenofovir as an approved antiretroviral, tenofovir is the preferred
choice in treatment-naïve HIV-coinfected patients who have an antiretroviral
treatment indication. The use of entecavir, telbivudine
or adefovir as an add-on to tenofovir or other drugs in the case of not fully
suppressive antiviral therapy has not been studied in HIV-coinfected patients so far.
The decision to do so is made on a case-by-case basis.
It was a general belief originating from the history of antiretroviral therapy that
combination therapy of tenofovir plus lamivudine/emtricitabine would be superior
to tenofovir monotherapy, in particular in patients with highly replicative HBV
infection. However, to date no conclusive studies supporting this are available
(Schmutz 2006, Mathews 2008, Mathews 2009).
In the case of development of HIV resistance to tenofovir it is important to
remember its HBV activity before switching to another regimen without antiviral
activity against HBV. Discontinuation of the HBV polymerase inhibitor without

298 Hepatology 2012
maintaining the antiviral pressure on HBV can lead to necroinflammatory flares that
can result in acute liver decompensation in serious cases.
A matter of concern is the potentially nephrotoxic effect of tenofovir. In patients
treated with tenofovir monotherapy nephrotoxicity is rarely observed (Heathcote
2011, Mauss 2011). However in HIV-infected patients treated with tenofovir as part
of an antiretroviral combination therapy renal impairment has been frequently
reported and may be associated in particular with the combined use of tenofovir and
HIV protease inhibitors (Mauss 2005, Fux 2007, Goicoecha 2008). Regular
monitoring of renal function in HBV/HIV-coinfected patients including estimated
glomerular filtration rate and assessment of proteinuria is necessary.

Management of resistance to HBV polymerase
inhibitors
Issues concerning the avoidance and management of resistance to HBV polymerase
inhibitors are discussed in detail in Chapter 10.

Conclusion
The number of available HBV polymerase inhibitors for chronic hepatitis B has
increased substantially over the last few years. In general though, the choice is
confined to two mostly non-cross-resistant classes, the nucleotide and nucleoside
compounds. In HIV-coinfected patients where antiretroviral therapy is not indicated
the choice is more limited with only adefovir and telbivudine as treatment options.
Alternative options in these patients may be interferon therapy or the initiation of
full antiretroviral therapy, which is currently preferred by most experts, although
both toxicities and costs may increase.
For HBV/HIV-coinfected patients on antiretroviral therapy the treatment of
choice is tenofovir in the majority of treatment-naïve or lamivudine-pretreated
cases. Due to rapid development of resistance in not fully suppressive HBV therapy
lamivudine or emtricitabine monotherapy should not be considered in the vast
majority of cases. A combination of tenofovir plus lamivudine or emtricitabine as a
primary combination therapy has theoretical advantages, but studies supporting this
concept have not been published to date.
In general, treatment of HBV as a viral disease follows the same rules as HIV
therapy, aiming at a full suppression of the replication of the virus to avoid the
development of resistance. Successful viral suppression of hepatitis B results in
inhibition of necroinflammatory activity, reversion of fibrosis and the ultimate goal
of immune control of the infection.

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302 Hepatology 2012

18. Management of HCV/HIV Coinfection
Christoph Boesecke, Stefan Mauss, Jürgen Kurt Rockstroh

Epidemiology of HIV and HCV coinfection
HIV and HCV share transmission pathways, which explains the high rate of
coinfection with both viruses. Of the 33.3 million HIV-infected persons worldwide
in 2009 it is estimated that at least 5 million of them had concomitant hepatitis C
virus infection. While both viruses are transmitted with high efficacy via blood-toblood contact, HCV is less easily transmitted sexually. Thus, the prevalence of
hepatitis C coinfection within different countries, regions and populations is closely
related to the prevalence of blood-borne transmission (mainly intravenous drug use)
of HIV. Among HIV-infected patients in Europe, Australia and the US, at least one
out of four is coinfected with hepatitis C (Rockstroh 2004). Hepatitis C coinfection
rates as high as 70% can be found in Eastern European countries like Belarus and
the Ukraine and in Middle Eastern countries such as Iran where intravenous drug
use (IVDU) is the main route of HIV transmission (SeyedAlinaghi 2011). On the
other hand, in Central European countries such as Belgium, Austria or Germany,
where sexual intercourse dominates as mode of HIV transmission, hepatitis C
coinfection rates are rather low, between 10 and 15% (Rockstroh 2005, CDC 2011).
Similar rates can be found in HIV-positive patients in Australia (Jin 2009) and the
UK (Turner 2009). Interestingly, recent data from the US indicate that 25% to 35%
of patients with HIV are coinfected with HCV (Singal 2009, CDC 2011) reflecting
the contribution of at-risk populations such as prison inmates to the overall
numbers. 65-70% of HIV-positive prisoners in the US are coinfected with hepatitis
C, in contrast to 18-25% of the general US HIV-positive population (Weinbaum
2005, CDC 2011). In Asia, coinfection rates of up to 85% have been observed
among Chinese plasma donors whereas in countries with predominantly
heterosexual HIV transmission like Thailand coinfection rates are around 10%
(Qian 2006). In sub-Saharan Africa, where again the primary route of transmission
of HIV is sexual, HCV coinfection rates so far have been reported to be relatively
low.
Although the traditional route of HCV transmission is blood-borne and includes
IVDU, snorting drugs, sharing toothbrushes/razors, and tattooing (Bollepalli 2007),
recent epidemic outbreaks among HIV-positive men who have sex with men
(MSM) from several major European cities such as London, Paris, Amsterdam, and
Berlin as well as more recent reports from the US, Canada Australia and Taiwan

Management of HCV/HIV Coinfection 303
document that HCV may well be sexually transmitted and should therefore also be
taken into account at regular STD screenings (Gotz 2005, Danta 2007, Vogel 2009,
Vogel 2010, Matthews 2011, Schmidt 2011).
HCV is detected in 4-8% of infants born to HCV-infected mothers (Bevilacqua
2009). Dual HCV/HIV infection increases the risk for transmission of both viruses
and high levels of HCV viremia in the mother increases the risk of perinatal HCV
transmission (Zanetti 1995). However, in HIV/HCV-coinfected mothers receiving
HAART and undergoing cesarean section the risk of HCV transmission is strikingly
reduced to less than 1%.
In summary, the prevalence of hepatitis C within the HIV-infected population is
far higher than in the general population where the global burden of hepatitis C is
estimated to be roughly 2%. This highlights the importance of preventing further
spread of hepatitis C infection as one of the major co-morbidities in HIV-infected
individuals. The average estimated risk of transmission for hepatitis C in HIV is
depicted in Table 1. Although they share common routes of infection, the viruses
are transmitted with varying efficacy depending upon the mode of transmission.
Table 1. Average estimated risk of transmission for HIV, HCV and HCV/HIV
coinfection.
Mode of
transmission

HIV

HCV

HCV / HIV coinfection

Perinatal
Sexual contact*
Needle stick injury

7-50%
1-3%
0.3%

1-7%
<1%
<1%

1-20%
<4%
Unknown

* On sexual contact the sexual risk refers to cumulative exposure

Diagnosis of HCV in HIV coinfection
The presence of HCV can be confirmed serologically by the detection of antibodies
to the virus via ELISA testing. Loss of HCV antibodies observed in rare cases in
very advanced immune deficiency in HIV/HCV coinfection does not necessarily
indicate viral clearance (Cribier 1999). Therefore, a single negative HCV antibody
ELISA does not necessarily exclude HCV infection in HIV-positive patients,
especially in severe immune deficiency. Additionally, a rise of liver transaminases
has been proven to be more sensitive in the detection of acute HCV infection in
HIV-positive patients than repeated testing for the presence of antibodies against
HCV (Thomson 2009). However, in more than 80% of HIV-positive individuals
with positive HCV antibodies, HCV RNA is detected in the blood. Higher
concentrations of HCV RNA are found in HIV-positive individuals than in HIVnegative patients with hepatitis C (Perez-Olmeda 2002). Interestingly, recent data
from a cross-trial comparison showed that HIV-positive patients were less likely to
present with elevated serum ALT and clinical signs or symptoms of hepatitis than
HIV-negative patients (Vogel 2009). In observations from hemophiliac patients,
mean HCV RNA concentrations increased by 1 log10 over the first two years after
HIV seroconversion (Eyster 1994). Levels of HCV viremia increase eight times
faster in HIV-positive individuals than in HIV-negative patients with hepatitis C.
The highest concentrations for HCV viremia have been reported in patients who
subsequently developed liver failure.

304 Hepatology 2012
Interestingly, spontaneous clearance of HCV RNA has been observed in some
HIV/HCV-coinfected patients experiencing significant immune reconstitution
following HAART initiation (Fialaire 1999, Thomson 2009). In contrast, there are
also patients with positive HCV antibodies and negative HCV RNA in which HCV
RNA was noted to re-emerge frequently in combination with a flare of liver
transaminases after initiation of HAART. Therefore, regular monitoring of HCV
RNA levels is warranted in HIV/HCV-coinfected patients.
The distribution of HCV genotypes in HIV-positive patients reflects the route of
transmission. Genotype 1b accounts for 2/3 of post-transfusion HCV infections and
is the predominant genotype in hemophiliacs. In contrast, genotypes 1a and 3a are
more common in intravenous drug users (Pol 1994, Soriano 2008).

Natural course of hepatitis C in HIV coinfection
Various studies have demonstrated that underlying HIV infection weakens the
immune response to hepatitis C, thereby diminishing the chance of spontaneous
viral clearance of HCV infection. Interestingly, data from the European epidemic of
sexually transmitted acute hepatitis C infection in HIV-positive individuals suggest
that despite underlying HIV infection spontaneous resolution of HCV may occur in
up to 20-30% of newly infected patients (Vogel 2010, Thomson 2010). Recently,
genome-wide association studies identified single nucleotide polymorphisms (SNP)
near the IL28B gene encoding for interferon lambda that comprise a crucial part of
the host’s innate immune defence against HCV in HCV monoinfection (Thomas
2009). Individuals with the CC genotype were more than three times likely to clear
HCV RNA and to better respond to standard HCV therapy compared with
individuals with CT and TT genotypes (Rauch 2010, Grebely 2010, Nattermann
2011, Rallón 2011). Similar observations have been made in HIV/HCV coinfected
individuals (Clausen 2010). Interestingly, these SNPs could explain differences in
spontaneous clearance rates between different ethnicities as the frequency of the
protective allele varies across ethnic groups with a much lower frequency in those
of African origin compared to Asian patients with Europeans being in-between
(Thomas 2009).
Numerous large cohort studies have demonstrated that once chronic hepatitis C is
established the presence of HIV leads to a faster HCV clinical progression due to
the lack of critical CD4+ T cell responses against HCV (Danta 2008). In the
American multicenter Hemophiliac Cohort Study liver failure occurred in 9% of
multitransfused HCV/HIV-coinfected adult hemophiliacs without an AIDS-defining
opportunistic infection or malignancy (Eyster 1993). In the same time period, no
case of liver failure was observed in HCV-positive HIV-negative hemophiliacs.
Subsequently, several studies have confirmed the unfavorable course of hepatitis C
in HIV-coinfected hemophiliacs, particularly in the setting of progressive
immunodeficiency and lower CD4 counts (Rockstroh 1996, Puoti 2000).
In addition, the time interval between HCV exposition and development of
cirrhosis was found to be shortened in coinfected subjects. Indeed, within 10-15
years of initial HCV infection, 15-25% of HIV-coinfected patients develop cirrhosis
compared with 2-6% of HIV-negative patients (Soto 1997). Importantly, mortality
due to advanced liver disease occurs ten years earlier in coinfected hemophiliacs
than in HIV-negative hemophiliacs with hepatitis C (Darby 1997). The incidence of

Management of HCV/HIV Coinfection 305
hepatocellular carcinoma seems also to be higher in coinfected patients (Giordano
2004).

Effect of hepatitis C on HIV infection
As clear as HIV’s influence on the accelerated disease progression for HCVassociated liver disease is, HCV’s influence on the course of HIV disease is
conflicting. The Swiss Cohort first revealed a blunted CD4 cell response associated
with a faster progression to AIDS after initiation of HAART in HIV/HCVcoinfected patients (Greub 2000). Interestingly, four-year follow-up data from the
same cohort study did not detect significant differences with regard to CD4 cell
count recovery between HCV-positive and HCV-negative HIV-positive patients
(Kaufmann 2003). Subsequent studies have indeed found that after adjusting for use
of HAART, no difference in CD4 cell count recovery can be observed (Sulkowski
2002). Updated information from an analysis of the EuroSIDA cohort, after taking
into account ongoing chronic (persistent HCV replication) and resolved (positive
HCV antibodies but negative HCV RNA) hepatitis C infection, confirm that no
difference in CD4 cell count recovery is observed in patients with chronic hepatitis
C infection and detectable HCV RNA in comparison to HIV-monoinfected patients
(Rockstroh 2005). In addition, data from the same cohort revealed that CD4-positive
T cell recovery in HIV-positive patients with maximal suppression of HIV
replication is not influenced by HCV serostatus in general or HCV genotype or level
of HCV in particular (Peters 2009).

Effect of HAART on hepatitis C
In HIV/HCV-coinfected patients starting antiretroviral therapy a transient increase
in HCV RNA levels may occur at week 4 but thereafter no significant changes in
concentrations of HCV RNA happen over the first six months of treatment
(Rockstroh 1998). However, a 1 log10 decrease of HCV RNA has been reported in
HIV/HCV-coinfected individuals receiving more than 12 months of HAART and
having significant immune reconstitution. Other investigators, however, have not
observed this decrease in HCV RNA. Moreover, eradication of HCV has been
reported in individual patients receiving HAART following CD4 count recovery.
There is increasing evidence that HAART-induced immune reconstitution might
reverse the unfavorable accelerated course for hepatitis C in patients with severe
HIV-associated immune deficiency (Verma 2006, Vogel 2009). Taking into account
that liver disease progresses especially in those whose CD4 count drops below
200/µl it is appealing to think that CD4 increases on HAART may impact the
further course of liver disease. In an early study of 162 individuals with HIV/HCV
coinfection who underwent liver biopsy, the use of protease inhibitors as part of
their HAART regimen was associated with significantly lower rates of progression
of liver fibrosis that could not be explained by other cofactors (Benhamou 2000).
These findings were then confirmed by several cohort analyses which showed that
HIV/HCV-coinfected individuals on HAART had significantly lower liver-related
mortality than patients receiving either suboptimal (one or two nucleoside reverse
transcriptase inhibitors) or no antiretroviral therapy (Qurishi 2003).

306 Hepatology 2012
One paper also addressed the amount of immune reconstitution achieved on
HAART and the subsequent risk for developing hepatic decompensation in
HIV/HCV-coinfected individuals commencing HAART (Pineda 2007). Those
patients who experienced the highest CD4 cell count gain on HAART were the least
likely to develop further complications of liver disease, again highlighting a
favourable impact of HAART-induced immune reconstitution on the course of liver
disease. As a consequence, the current antiretroviral treatment guidelines of the
European AIDS Clinical Society recommend earlier initiation of antiretroviral
therapy in HIV patients with HCV coinfection (CD4 T cell count between 350500/µl in asymptomatic patients).
Short-term and long-term virologic success rates of HAART in HIV/HCV
coinfection are, however, limited by an increased risk of hepatotoxicity. Various
studies have shown that the presence of HCV is independently associated with an
increased risk of rises in serum aminotransferases highlighting the need for close
monitoring.

Treatment of hepatitis C in HIV coinfection
The most important reason to treat hepatitis C in HIV-coinfected individuals is the
unfavourable course of hepatitis C in the setting of HIV coinfection particularly
with the increased life expectancy gained by successful HAART. An increased risk
of hepatotoxicity after HAART initiation in HIV/HCV-coinfected patients, possibly
limiting the long-term benefit of HAART in this particular group, further underlines
the need for successful treatment of hepatitis C (Sulkowski 2000). Several studies
have been able to demonstrate that successful treatment of hepatitis C dramatically
reduces subsequent complications of preexisting liver disease. This implies that
once viral clearance is achieved with hepatitis C combination therapy the prognosis
of liver disease dramatically improves (even in the presence of already developed
liver cirrhosis) and once HCV infection is eradicated further liver complications are
very unlikely.
The goal of hepatitis C treatment is to achieve persistently negative HCV RNA
levels. This is generally referred to as a sustained virologic response (SVR). It is
defined as negative HCV RNA six months after completion of HCV therapy.
Negative HCV RNA at the end of the treatment period is described as an end-oftreatment response (EOT). Negative HCV RNA after four weeks of HCV treatment
initiation is referred to as rapid treatment response (RVR). Failure to respond to
treatment is referred to as non-response.
The combination of pegylated interferon and ribavirin is still regarded as standard
therapy in coinfected patients. Table 2 summarizes the main results from
randomized clinical trials investigating the efficacy of pegylated interferon and
ribavirin in HIV/HCV-coinfected individuals. Data from the GESIDA study show
similar efficacy and safety for both pegylated IFN α-2b and pegylated IFN α-2a in
the treatment of chronic HCV infection in HIV-infected patients (Berenguer 2009).
Overall, SVR rates of up to 50% can be achieved (Torriani 2004; Nunez 2007).
The difference in rates of SVR in various studies can be explained mainly by
differences in ribavirin dosages used, fibrosis stage and probably variations in the
IL28B genotype. In the initial HCV treatment trials in HIV-coinfected individuals,
due to the fear of interactions between ribavirin and commonly used NRTIs for HIV

Management of HCV/HIV Coinfection 307
treatment, 800 mg daily dose of ribavirin was chosen for most patients independent
of the prevailing genotype. This led to suboptimal SVR rates. However, in the
PRESCO trial, where weight-adjusted daily ribavirin dosages of 1000-1200 mg
were used independent of genotype, SVR rates almost doubled in comparison to
some of the earlier studies such as APRICOT, most likely due to the higher ribavirin
levels. In spite of this, data from the PARADIGM trial, a double-blind, multicenter
study comparing 800 vs 1000/1200 mg of ribavirin plus PEG-IFN in HCV/HIVcoinfected patients showed no significant differences in the rates of SVR
(Rodriguez-Torres 2009).
In the current guidelines, daily administration of ribavirin 1000 mg (<75 kg body
weight) and 1200 mg (>75 kg body weight) BID is recommended for HCV therapy
in HIV coinfection for all genotypes in combination with pegylated interferon.
Table 2. Results from randomized clinical trials investigating the efficacy of
pegylated interferon plus ribavirin in HIV/HCV-coinfected individuals.

Patients (n)
PEG-INF α
IV drug use
Liver cirrhosis
Genotype 1,4
Normal ALT
Mean CD4+
HAART
Discontinuation
rate due to AE*
Discontinuation
rate due to
other reasons
EOT (ITT)**
SVR (ITT)***

ACTG5071

APRICOT

RIBAVIC

Laguno

PRESCO

66
2a
11%
77%
34%
495
85%
12%

289
2a
62%
15%
67%
0%
520
83%
25%

194
2b
80%
39% (F3-F4)
61%
16%
477
83%
17%

52
2b
75%
19%
63%
0%
570
94%
17%

389
2a
90%
28% (F3-F4)
61%
0%
546
74%
9%

-

31%

39%

23%

7%

41%
27%

49%
40%

35%
27%

52%
44%

67%
50%

*adverse events, **end-of-treatment response, intent-to-treat analysis, ***sustained virological
response, intent-to-treat

The standard dosage for PEG-IFN α-2a is 180 µg s.c. once weekly and for PEGIFN α-2b 1.5 µg/kg body weight s.c. once weekly. Duration of therapy is
individualized taking into account factors for HCV treatment response such as
genotype, baseline viral load and virologic response (see Figure 1). Results from the
PRESCO trial indicate that at least some patients may benefit from a longer duration
of HCV combination therapy, of up to 72 weeks (see Figure 1). This mainly refers
to patients infected with HCV genotypes 1 and 4 (Núñez 2007) for whom poorer
response rates have been extensively shown when compared with genotypes 2 and
3.
Based on 4 baseline variables (serum HCV RNA, HCV genotype, liver fibrosis
staging using elastometry, and IL28B genotyping), the Prometheus index has
recently been developed and can optionally be used as a risk calculator for
predicting the likelihood of SVR using PEG-IFN/ribavirin therapy in HIV-HCVcoinfected patients. It is freely available on the web (http://goo.gl/oPBJ9), like the
Framingham score for predicting cardiovascular risk (EACS 2011).

308 Hepatology 2012
With the registration of the first oral direct acting antivirals (DAAs) telaprevir and
boceprevir, treatment recommendations for hepatitis C genotype 1 patients will
change depending on the availability of these new agents. So far, only interim data
is available for both agents (24-week treatment response data) showing significantly
higher rates of undetectable HCV RNA with triple therapy when compared with
standard PEG-IFN plus ribavirin in coinfected patients, similar to the rates seen in
Phase II and III trials in HCV monoinfection (Sherman 2011, Sulkowski 2011). For
patients with HCV genotype 1 infection, telaprevir can be added to PEG-IFN/RBV
standard treatment for 12 weeks at 750 mg every 8 hours. In case of successful
treatment response at week 4 (HCV RNA <1000 IU/mL), telaprevir should be
continued until week 12. If HCV RNA at week 12 is still <1000 IU/mL, dual
therapy with PEG-IFN/ RBV should be continued until week 24. If HCV RNA is
<20 IU/mL at week 24, dual therapy with PEG-IFN/RBV should be continued for
another 24 weeks resulting in a total treatment duration of 48 weeks.

Stop

Figure 1. Algorithm for management of hepatitis C in HIV coinfection. Proposed optimal
duration of hepatitis C virus (HCV) therapy in HIV/HCV-coinfected patients (w: week; G:
genotype) (modified according to Rockstroh 2009).
*In patients with low baseline viral load (<400,000 IU/l) and minimal liver fibrosis.

Due to drug-drug interactions and limited drug-interaction studies, telaprevir can
currently only be safely combined with raltegravir, boosted atazanavir or efavirenz
(with efavirenz, telaprevir doses need to be increased to 1125 mg every 8 hours) in
combination with tenofovir and emtricitabine or abacavir and lamivudine. Recently
published pharmacokinetic (PK) data on the combination of telaprevir with
raltegravir shows no influence on telaprevir PK, but telaprevir increased raltegravir
levels by about 30%. This interaction is not considered clinically significant and
suggests that these drugs can be used together without dose adjustment or concerns
regarding effect on safety or efficacy (van Heeswijk 2011). Treatment with
boceprevir for patients with HCV genotype 1 infection is different, with a
mandatory 4-week lead-in Phase with PEG-IFN/RBV to reduce hepatitis C viral
load and subsequently lower the risk of rapidly developing resistance against
boceprevir. If a >2 log drop in HCV RNA is achieved at week 4 boceprevir is added

Management of HCV/HIV Coinfection 309
to PEG-IFN/RBV. Therapy duration then depends on response rates at subsequent
time-points. So far, no official recommendations exist as clinical trials are ongoing.
While boceprevir seemed to have a lower potential for drug-drug interactions with
ART vs. telaprevir due to its simultaneous metabolisation by the aldo-keto reductase
and the cytochrome P450 pathway (Dore 2011), recent study results have changed
that view. Coadministration of boceprevir reduced mean trough concentrations of
ritonavir-boosted atazanavir, lopinavir and darunavir by 49, 43 and 59 percent,
respectively. Mean reductions of 34 to 44 percent and 25 to 36 percent were
observed in AUC and Cmax of atatzanavir, lopinavir and darunavir.
Coadministration of ritonavir-boosted atazanavir with boceprevir did not alter the
exposure of boceprevir, but coadministration of boceprevir with lopinavir/ritonavir
or ritonavir-boosted darunavir decreased the exposure of boceprevir by 45 and 32
percent, respectively. Due to these interactions the concomitant use of boceprevir
with HIV protease inhibitors is not recommended (Merck 2012).
Unlike HAART, HCV treatment offers the possibility of eradicating HCV within
defined treatment periods and this clearly appears potentially advantageous for the
subsequent management of the patient’s HIV infection. Every patient should be
considered for HCV treatment when the benefits of therapy outweigh the risks.
Benefits of therapy also need to be measured in the context of rapid liver fibrosis
progression in HIV/HCV coinfection and improved HCV treatment outcome under
optimized management in these patients. Information on liver fibrosis staging is
important for making treatment decisions in coinfected patients. However, a liver
biopsy is not mandatory for decisions on treatment of chronic HCV infection.
Recently introduced noninvasive markers such as blood tests or transient
elastography constitute new and exciting means of assessing liver disease in HIV
and hepatitis-coinfected individuals (Rockstroh 2009, Resino 2011). When liver
biopsy or non-invasive tests for assessing hepatic fibrosis (e.g., elastometry by
Fibroscan®, Echosense, France) demonstrate lower grades of liver fibrosis (F0-F1)
regardless of HCV genotype, treatment can be deferred. Assessment of fibrosis
should be repeated frequently to monitor progression. It is especially important to
perform a liver disease stage assessment in patients with a low likelihood of
achieving SVR. In addition, insulin resistance (which can be determined using the
homeostasis model assessment of insulin resistance [HOMA-IR] score) has been
reported as a negative predictor of achieving SVR and therefore may also be
considered during evaluation.
Current therapy is particularly recommended in all patients with a high likelihood
of achieving an SVR, i.e., patients infected with genotype 2 or 3 and those infected
with genotype 1 if the viral load is below 600,000 IU/ml and/or if the IL28B-CC
genotype is present (EACS 2011). If chronic hepatitis C is detected early in the
course of HIV infection (before the initiation of HAART) treatment for chronic
HCV is advised. However, if a coinfected patient has severe immune deficiency
(CD4 count <200 cells/ml), the CD4 count should be improved using HAART
before beginning HCV treatment. Patients with a CD4 relative percentage of >25%
are more likely to achieve SVR than those with lower CD4 percentages (Opravil
2008). If an early virologic response of at least 2 log10 reduction in HCV RNA
compared with baseline is not achieved by week 12, treatment should be
discontinued as an SVR is unlikely. The current European recommendations for
treatment initiation of PEG-INF and ribavirin for HIV/HCV-coinfected patients are

310 Hepatology 2012
shown in Figure 1. The procedures for diagnosis of hepatitis C, assessment of liver
disease stage and control examinations before and during HCV therapy are
summarized in Table 3.
Table 3. Diagnostic procedures for hepatitis C in HIV coinfection (adapted from
Rockstroh 2008).
Diagnosis of hepatitis C
HCV Ab (positive 1-5 months after infection, may rarely be lost with immunosuppression)
HCV RNA levels (while not prognostic for progression, it is for response to treatment)

Status of liver damage
Grading of fibrosis (e.g., Fibroscan, liver biopsy, serum fibromarkers)
Hepatic synthetic function (e.g., coagulation, protein, albumin, CHE)
Ultrasound and AFP every 6 months in cirrhotics (gastroscopy upon diagnosis of cirrhosis
and every 1-2 years thereafter)

Before HCV treatment
HCV genotype and serum HCV RNA

The choice of antiretrovirals while on HCV therapy
The choice of the best-tolerated HIV drugs is crucial for completing the planned
treatment duration of hepatitis C therapy of 24-72 weeks (Vogel 2010). ddI use has
been independently associated with increased adverse event rates including lactic
acidosis and hepatic decompensation in patients who have liver cirrhosis prior to
commencement of PEG-IFN/RBV therapy (Mauss 2006). Apparently, ribavirin
enhances the phosphorylation of ddI and thereby leads to an increased risk of
pancreatitis and mitochondrial toxicity in subjects receiving concomitant ribavirin
and ddI therapy (Moreno 2004). ddI use is therefore contraindicated in combination
with ribavirin, especially in patients who have already developed liver cirrhosis. The
use of HIV antiretrovirals such as AZT and d4T are also discouraged whenever
possible, as increased toxicity can be expected. RBV + AZT is associated with
enhanced anemia (Alvarez 2006) while RBV + d4T is associated with increased
mitochondrial toxicity and weight loss and a high potential to worsen pre-existing
lipoatrophy. Patients on atazanavir-containing HAART may develop jaundice due
to an increase in total serum bilirubin levels following initiation of ribavirin
(Rodriguez-Novoa 2008). As abacavir and ribavirin are both guanosine analogs it is
speculated that there may be interference or competition in the phosphorylation
pathway. Data from cohorts using lower dosages of ribavirin suggest lower SVR
results in patients on abacavir-containing HAART (Bani-Sadr 2007). However, in
the presence of therapeutic ribavirin levels no difference was observed between
abacavir and other nucleosides in achieving SVR in HIV/HCV-coinfected patients
receiving PEG-IFN/ribavirin therapy and concomitant HAART in other cohorts
(Laufer 2008, Amorosa 2010, Berenguer 2011).

Treatment of HCV for relapsers or non-responders
Patients with a history of previous HCV therapy who were either non-responders or
who relapsed while on previous HCV therapy need to be reassessed with regard to
new HVC treatment optimizing the dose and duration (see Table 4) as well as
potentially adding a new HCV protease inhibitor in HCV GT 1 patients. As soon as

Management of HCV/HIV Coinfection 311
sustained virologic response results from currently ongoing pilot trials of the new
HCV protease inhibitors in HIV/HCV coinfection become available, treatment
recommendations for hepatitis C genotype 1 in HIV-positive patients will change.
Recent results from the SLAM-C trial (ACTG 5178) have attenuated hopes that
maintenance therapy with PEG-INF might be beneficial for non-responders.
Table 4. Classification of and interventions for HCV/HIV-coinfected patients who are
non-responders/relapsers to prior IFN-based therapies.
Category
Suboptimal treatment

Optimal treatment with
virologic failure

Subgroup

Recommended intervention

Suboptimal schedule
• Interferon monotherapy
• Low doses of ribavirin
• Short length of therapy

Re-treatment using
combination therapy of
PEG-IFN α plus weight-based
dose of ribavirin

Limiting toxicities &
poor adherence

Optimal support (SSRI,
paracetamol/NSAID*,
adherence
support, use of
hematopoietic
growth factors**)

Relapse (HCV RNA
negative at the end
of treatment)

Re-treatment using
combination
therapy of PEG-IFN
plus weight-based
ribavirin dosing
(consider longer
treatment duration)

Non-response
(no HCV RNA
negativization
during treatment)

Wait until new antivirals
become available either
through clinical trials or
upon licensure

*NSAID, non-steroidal anti-inflammatory drugs; PEG, polyethylene glycol; SSRI, selective
serotonin reuptake inhibitors.
**Data on the use of hematopoietic growth factors in HIV/HCV co-infection so far is limited to an
improvement in quality of life but not antiviral efficacy; treatment with growth factors is currently
mostly off-label in Europe.

Treatment of acute HCV in HIV
As SVR rates following treatment of acute HCV infection are higher than for
treatment of chronic HCV, HCV RNA should be measured at initial presentation
and 4 weeks later in patients with acute HCV infection. Treatment should be offered
in patients without a decrease of 2 log10 of HCV RNA at 4 weeks compared with
initial HCV RNA and to patients with persistent serum HCV RNA 12 weeks after
diagnosis of acute HCV. Duration of treatment should be based on rapid virologic
response (RVR) regardless of genotype. Patients who do not achieve a ≥2 log10
decrease in HCV RNA level at week 12 should discontinue therapy (NEAT 2010).
Uncontrolled pilot studies of treatment of acute HCV infection in HIV-coinfected

312 Hepatology 2012
patients demonstrate SVR rates above 60% mostly with combination therapy of
PEG-IFN/RBV for 24-48 weeks (Boesecke 2011). Furthermore, recently presented
data from a large European cohort for the first time revealed the beneficial influence
of GT 2/3 infection on treatment outcomes in the setting of acute hepatitis C
suggesting different cure rates depending on HCV genotype similar to the genotype
effects seen in chronic HCV therapy. In this cohort, patients with GT 2/3 infection
were almost three times more likely to reach SVR than patients with GT 1/4
infection (Boesecke 2011). Unfortunately, clear guidance on treatment duration or
the role of ribavirin is difficult at this point due to the lack of controlled data.

Liver transplantation in HIV/HCV-coinfected
patients
In general, HIV/HCV-coinfected individuals develop more rapid HCV-related
hepatic injuries such as liver fibrosis and cirrhosis. Additionally, HIV/HCV
coinfection is associated with an increased rate of hepatocellular carcinoma (HCC).
Typically HCC occurs in HIV/HCV-coinfected patients at an earlier age and the
course is more aggressive with a shorter survival compared to HCV-monoinfected
individuals. Therefore, the presence of esophageal varices using uppergastrointestinal endoscopy should be monitored in patients with liver cirrhosis every
1-2 years, and an ultrasound of the liver and a serum α-fetoprotein determination
should be performed at least every 6 months in patients with F3/F4 fibrosis
according to the recommendations of the European Consensus Guidelines (Alberti
2005).
Liver transplantation should be considered in patients with decompensated liver
cirrhosis, as this is a contraindication for HCV treatment. To fulfill the selection
criteria for a liver transplant in HIV/HCV-coinfected individuals the CD4+ count
has to be at least 100 cells/ml. Additionally, the patient has to have either
undetectable HIV viremia (<400 copies/ml) or at least rational treatment options to
control HIV infection successfully after liver transplantation. Further
contraindications for transplantation are opportunistic diseases, ongoing alcohol or
drug abuse, HCC metastasis in other organs, a second malignant disease,
cardiopulmonary disease or older age with an elevated risk of mortality related to
the operation. Recent data from a large US cohort sheds light on survival rates after
liver transplantation (Mindikoglu 2008). The estimated 2-year survival rate was
found to be somewhat lower in HIV-positive patients (70%) compared with HIVnegative patients (81%). This was mostly attributable to HBV or HCV coinfection.
Other studies have shown good outcome results in the setting of HBV/HIV
coinfection when compared to HBV mono-infection (Vogel 2005, Baccarani 2011).
This highlights the major problem in HCV/HIV-coinfected transplant recipients:
HCV re-infection of the transplanted organ. Recurrence of chronic hepatitis C in the
liver graft is frequently observed in HIV-positive patients and a more rapid
progression to graft cirrhosis and liver disease-related mortality compared to HCVmonoinfected patients has been reported. Therefore, combination therapy with
pegylated interferon plus ribavirin seems to be the best management option 1-3
months after liver transplantation and after re-infection with hepatitis C virus is
detected.

Management of HCV/HIV Coinfection 313
In the context of post-transplant immunosuppression, it is important to point out
that there are crucial pharmacokinetic drug-drug interactions at the level of the
cytochrome P450 metabolism and P-glycoprotein induction between the key
immunosuppressive drugs tacrolimus or cyclosporin A and the antiretroviral agents
used for HIV therapy. In addition the HCV protease inhibitors telaprevir and
boceprevir increase the drug levels of tacrolimus and cyclosporin A substantially.
Determinations of the plasma levels of the antiretroviral drugs and tacrolimus or
ciclosporine are necessary. Furthermore, the doses of cyclosporin A or tacrolimus
usually need to be reduced when the patient is treated concomitantly with an HIV
protease inhibitor, especially if boosted with ritonavir (Vogel 2004). By contrast,
NNRTIs can lower the concentrations of immunosuppressive drugs. The increase of
drug levels of tacrolimus and to a lesser extent ciclosporin A by telaprevir may
prevent the concomitant use of these drugs with telaprevir.

Conclusion
HIV has been shown to accelerate the progression of hepatitis C and to result in
higher liver disease-related mortality and morbidity in HIV/HCV-coinfected
patients compared to HCV- or HIV-monoinfected individuals. Enhanced
hepatotoxicity of HAART as well as drug-drug interactions between HAART and
ribavirin clearly underline the need for specific treatment strategies. A number of
important clinical studies have established PEG-IFN plus ribavirin combination
therapy as the current gold standard allowing sustained virologic response rates of
almost 50% in HIV/HCV-coinfected individuals under optimized management
conditions (weight-based ribavirin and individualized treatment duration).
Nevertheless, the proportion of patients not treatable or those who relapse,
especially in patients with genotype 1 infection, remains high. However as soon as
sustained viral response results from currently ongoing pilot trials of the new HCV
protease inhibitors in HIV/HCV coinfection become available treatment
recommendations for hepatitis C genotype 1 in HIV patients will change.

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318 Hepatology 2012

19. HBV/HCV Coinfection
Carolynne Schwarze-Zander and Jürgen Kurt Rockstroh

Epidemiology of HBV/HCV coinfection
Hepatitis B (HBV) and hepatitis C (HCV) viruses are the most common causes of
chronic liver disease worldwide. Due to shared routes of transmission, coinfection
with HBV and HCV is not uncommon among individuals in HBV endemic areas
who also have a high risk of parenteral infections, such as injection drug users
(Pallas 1999), patients on hemodialysis (Reddy 2005), patients undergoing organ
transplantation (Aroldi 2005) and HIV-positive individuals (Zhou 2007). Due to a
lack of large-scale population-based studies the exact number of HBV/HCV
coinfected patients is unknown. Dual infection ranges from 9% to 30%, depending
on the geographic region (Zarski 1998, Liaw 1995). These numbers may
underestimate the true number of people with HBV/HCV coinfection as there is a
well-known entity of occult HBV infection (patients with negative hepatitis B
surface antigen [HBsAg] but detectable serum HBV DNA) in patients with chronic
hepatitis C (Cacciola 1999).

Screening for HBV/HCV coinfection
Persons with a first episode of acute hepatitis should be screened for all viral causes
including HBV and HCV (see Chapter 8 on diagnostic tests in acute and chronic
hepatitis B and Chapter 12 for hepatitis C). Some patients may be inoculated with
both viruses simultaneously and will present with acute hepatitis due to both
viruses. In addition, HBV superinfection in patients with chronic hepatitis C, and
HCV superinfection in patients with chronic hepatitis B have both been reported
(Liaw 2004, Liaw 2000, Liaw 2002). Therefore, episodes of acute hepatitis in
patients with known chronic HBV or HCV infection, especially those with ongoing
risk behaviour for infection with the other virus such as injection drug users, should
prompt screening for superinfection. In addition, in patients with chronic hepatitis
C, ruling out occult HBV infection beyond HBsAg testing, i.e., by polymerase chain
reaction (PCR), should be done when clinically indicated.

HBV/HCV Coinfection 319

Viral interactions between HBV and HCV
Patients with both HBV and HCV infections may show a large spectrum of
virologic profiles. HCV infection can suppress HBV replication and it has been
shown that HBV/HCV-coinfected patients have lower HBV DNA levels, decreased
activity of HBV DNA polymerase, and decreased expression of HBsAg and
hepatitis B core antigen in the liver (Chu 1998). Moreover, patients with chronic
HBV infection who become superinfected with HCV can undergo seroconversion of
HBsAg (Liaw 1994, Liaw 1991). Several authors have reported that HBV can
reciprocally inhibit HCV replication as well (Sato 1994). Specifically, HBV DNA
replication has been shown to correlate with decreased HCV RNA levels in
coinfected patients (Zarski 1998). Furthermore, coinfected patients have been
shown to have lower levels of both HBV DNA and HCV RNA than corresponding
monoinfected controls, indicating that simultaneous suppression of both viruses by
the other can also occur (Jardi 2001). Thus, HBV or HCV can play the dominant
role, HBV and HCV can inhibit each other simultaneously and they can alternate
their dominance (Liaw 1995). Both viruses have the ability to induce
seroconversion of the other. The chronology of infection may have a role in
determining the dominant virus. The overall effect appears to be HCV suppression
of HBV (Liaw 2001). Interestingly, recent in vitro studies found no evidence of
direct interference between the two viruses, making also interindividual differences
in innate and/or adaptive host immune responses responsible for viral interference
observed in coinfected patients (Bellecave 2009, Eyre 2009).

Clinical scenarios of HBV and HCV infection
Different scenarios of infection have been described with HBV/HCV coinfection
including acute hepatitis with HBV and HCV (Alberti 1995), occult HBV
coinfection of chronic hepatitis C (Sagnelli 2001), and superinfection by either virus
in patients with preexisting chronic hepatitis due to the other virus (Figure 1).
Frequently the sequence of infection cannot be defined.

Acute hepatitis by simultaneous infection of HBV and HCV
Simultaneous coinfection with HBV and HCV is rarely seen, but the interaction of
HBV and HCV appears to be similar to chronic infection. In acute infection with
HBV and HCV, patients showed delayed HBsAg appearance and a shorter hepatitis
B surface antigenemia compared to those with acute HBV alone (Mimms 1993).
Biphasic alanine aminotransferase (ALT) elevation was found in some patients,
although rates of viral clearance were similar to those in HBV or HCV monoinfected patients (Alberti 1995).

HCV superinfection
HCV superinfection is frequent in endemic areas of HBV infection, such as Asia,
South America and sub-Saharan Africa (Liaw 2002, Liaw 2004), which can result in
the suppression of HBV replication and termination of HBsAg carriage. However,
long-term follow-up analyses have described a higher rate of liver cirrhosis and
hepatocellular carcinoma (Liaw 2004). Fulminant hepatic failure was significantly
higher among patients with underlying HBV infection than those without (23% vs.
3%) (Chu 1999, Wu 1994, Chu 1994).

320 Hepatology 2012

HBV superinfection
HBV superinfection is less common in HCV-infected patients and very limited data
is available. In one report a patient became seronegative for HCV RNA after HBV
superinfection, indicating that superinfection of HBV may lead to suppression of
HCV (Liaw 2000, Wietzke 1999). Other reports have shown that HBV
superinfection may be associated with acute deterioration of liver function among
patients with chronic HCV infection, and the risk of fulminant hepatitis may be
increased (Sagnelli 2002).

Occult HBV infection in patients with HCV infection
Occult HBV infection, defined as detectable HBV DNA in liver or serum and
undetectable HBsAg (Ozaslan 2009, Torbenson 2002), has been identified in up to
50% of patients with chronic HCV. Importantly, a relation to HCV treatment
outcomes has been described (Zignego 1997, Fukuda 2001, Sagnelli 2001). HCV
infection with occult HBV infection has been associated with higher ALT levels,
greater histological activity index and liver disease more often progressing to liver
cirrhosis (Fukuda 1999, Cacciola1999, Sagnelli 2001).

Chronic hepatitis in HBV/HCV coinfection
Patients with detectable serum HBV DNA and HCV RNA are at highest risk of
progression to cirrhosis and liver decompensation and therefore should be
considered for treatment. Active HCV infection (HCV RNA+) in the setting of
inactive HBsAg (HBsAg+/HBV DNA-) behaves similarly to patients with HCV
monoinfection. Another possibility is active HBV infection in patients with inactive
or prior HCV infection (HBV DNA+/HCV RNA-/anti-HCV+). This immune profile
is less common, and may indicate HBV suppression of HCV. A longitudinal study
of virologic monitoring of 103 HBV/HCV coinfected patients revealed fluctuation
of the virological pattern (Raimondo 2006). Thus, close follow-up of levels of
viremia is needed for correct diagnosis and decision on what would be the most
successful treatment.
Table 1 shows the immune profiles found in patients with chronic HBV/HCV
infection.
Table 1. Immune profiles in patients with chronic HBV/HCV hepatitis.
HBV and HCV
active
HBsAg
HBV DNA
Anti-HCV
HCV RNA

+
+
+
+

Occult HBV in
chronic active HCV

–
+
+
+

HCV active in
HBsAg carrier

+
–
+
+

HBV/HCV Coinfection 321

Figure 1. Clinical scenarios of HBV/HCV coinfection (modified after Crockett & Keeffe
2005).

322 Hepatology 2012

Cirrhosis
Higher rates of cirrhosis have been demonstrated in HBV/HCV-coinfected patients.
In comparison to patients with HBV monoinfection, higher rates of cirrhosis (44%
vs. 21%) and decompensated liver disease (24% vs. 6%) were demonstrated in
coinfected patients (Fong 1991). Compared to HCV monoinfected patients a higher
rate of cirrhosis (95% vs. 49%) and more decompensated liver disease (Child-Pugh
class C 37% vs. 0%) were found in HBV/HCV-coinfected patients (Mohamed Ael
1997).

Hepatocellular carcinoma
In many studies coinfection with HBV and HCV has been shown to be associated
with an increased risk of HCC development (Kaklamani 1991, Mohamed Ael 1997).
In one longitudinal study incidence of HCC was 6.4 per person years in
HCV/HBV-coinfected patients compared to 2.0 in HBV and 3.7 in HCV
monoinfection. The cumulative risk of developing HCC after 10 years was 45% in
HBV/HCV-coinfected patients compared to 16% in HBV- and 28% in HCVmonoinfected patients (Chiaramonte 1999). HBV/HCV-coinfected patients should
undergo a screening routine for HCC with liver ultrasound and α-fetoprotein levels
in serum at least every 6 months.

Treatment of HBV and HCV coinfection
Currently there are no well-established treatment guidelines for HBV/HCVcoinfected patients. Generally, treatment guidelines for monoinfected patients
should be applied to coinfected patients. In patients with HBV/HCV coinfection
treatment should be initiated when inclusion criteria for standard treatment
guidelines of HBV and HCV monoinfection are met (see Chapter 9 on therapy of
HBV and Chapter 13 on treatment of HCV). As with HBV and HCV
monoinfection, treatment of coinfected patients should be started in patients with
active chronic hepatitis or cirrhosis before liver decompensation occurs. Due to the
variety of virological profiles in HBV/HCV coinfection it is important to assess the
dominant virus prior to initiating therapy.
Due to loss of viral suppression from the successfully treated dominant virus,
deterioration of liver disease has been reported (Yalcin 2003), thus caution must be
exercised upon initiation of therapy.
In coinfected patients with dominance of HCV infection, treatment with IFN
(Weltman 1995, Villa 2001, Utili 1999) and IFN plus ribavirin (Chuang 2005, Hung
2005, Liu 2003) has been well-studied and proven effective. However, more recent
studies show that combination therapy with pegylated IFN and ribavirin are even
more efficient in inducing virological results (see Table 2).
HCV RNA response was similar to results seen in HCV monoinfection with up to
83% in HCV genotype 2/3 and 72% in HCV genotype 1 achieving sustained
virological response (Liu 2009). In one small study including 17 HCV/HBVcoinfected patients these successful results were not confirmed (Senturk 2008).
Importantly, HBV replication may become detectable in up to 36% of patients with
undetectable pretreatment HBV DNA levels (Potthoff 2009, Liu 2009). Thus, close
monitoring of both viruses is recommended during and after combination therapy.

HBV/HCV Coinfection 323
Table 2. Peg-IFN plus ribavirin treatment trials in HBV/HCV-coinfected patients.
Patients (n)

HCV SVR (%) HBV DNA
HBsAg loss
negative (%) (%)

Reference
HBV
reactivation #
(%)

19

74

33

0

31

Potthoff, 2008

161

72*, 83**

56

11

35

Liu, 2009

17

6

na

na

na

Senturk, 2008

50

40*, 75**

100

0

24

Yu, 2009

*HCV GT1,** HCV GT2/3, na=not applicable, # HBV DNA negative pretreatment

In patients with dominance of HBV disease IFN +/- HBV polymerase inhibitors
are an option, although until now there is only data for lamivudine in a small cohort
of 8 HBV (HBeAg and HBV DNA-pos) and HCV (HCV RNA-pos) coinfected
patients (Marrone 2004). In this study, clearance of HBeAg was found in 3/8, two
patients showing HBeAg seroconversion, and clearance of HBV DNA was observed
in 3/8 at the end of therapy. HBV DNA became detectable again in 2 patients at the
end of follow-up. HCV clearance was observed in 50%. Based on these
observations nucleos(t)ide analogs such as tenofovir, adefovir, entecavir and
telbivudine showing a higher genetic barrier in combination with PEG-IFN are a
possible treatment option. However, studies are needed to estimate the treatment
value of these newer drugs in this clinical scenario.

Conclusion
Coinfection with HBV and HCV is not uncommon, especially within areas of high
hepatitis B prevalence. HBV/HCV coinfection is a challenge for clinicians due to
the complex interaction of HBV and HCV, and the propensity for developing severe
liver disease. No treatment standard has been established for HBV/HCV-coinfected
patients. Treatment decisions must be made based upon identification of the
dominant virus. Combination therapy of PEG-IFN plus ribavirin has been shown to
be highly effective in inducing virological response of HCV in patients with
HBV/HCV coinfection. The availability of direct acting antivirals against HCV will
open new pathways in treatment, which should be replicated in HBV/HCV
coinfection. However, to date, in coinfection of HBV/HCV no treatment experience
with these new agents has been reported. Finally, caution must be exercised in
treating coinfected patients, as flares of the untreated virus may occur.

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326 Hepatology 2012

20. Assessment of Hepatic Fibrosis in
Chronic Viral Hepatitis
Frank Grünhage and Frank Lammert

Introduction
Non-invasive methods for the assessment of liver fibrosis are increasingly being
used versus invasive liver biopsy thanks to patient acceptance and the low but everpresent morbidity of biopsies. Non-invasive tests should be able to discriminate
between non-significant (stages F0-F1) and significant (stages ≥F2) fibrosis to help
either delay or initiate antiviral treatment. In addition, non-invasive markers should
be able to reliably predict liver cirrhosis in order to initiate further diagnostics to
exlude portal hypertension and to initiate surveillance strategies with progressive
fibrosis. Non-invasive strategies are also warranted for monitoring the disease while
on therapy and ideally document a regression of fibrosis in the long term.
Yet, despite recent advances in the use of surrogate markers and the development
of new technical developments such as elastography, liver histology remains the
gold standard for fibrosis staging (Goodman 2007). Currently an intense debate
regarding non-invasive tests is going on and a number of participants of this
discussion have suggested to not accept the claim of liver histology as the gold
standard and define the role of histology as the best available standard (Bedossa
2009). Nevertheless, until today most experts agree that non-invasive techniques
will not replace liver biopsies completely although they will help reduce the number
of biopsies required (Leroy 2007, Pinzani 2005, Sebastiani 2006). While noninvasive fibrosis tests are suitable for the diagnosis of liver cirrhosis, they have been
questioned for clinical practice as they lack the potential to discriminate the stages
of fibrosis. This specific dogma is now being questioned, and entirely non-invasive
algorithms have been developed that include a differentiation of the stages of
fibrosis (Boursier 2011a, Boursier 2011b).

Assessment of Hepatic Fibrosis in Chronic Viral Hepatitis 327

Mechanisms of liver fibrosis in chronic viral
hepatitis
Liver fibrosis is characterised by the loss of hepatocytes, destruction of hepatic
(micro)architecture, proliferation of hepatic (myo)fibroblasts, and excess deposition
of extracellular matrix components (Friedman 2008). Endstage liver fibrosis
(cirrhosis) may include insufficient detoxification, hepatocellular carcinoma, portal
hypertension, renal and pulmonary failure, and is associated with excess mortality.
In chronic viral hepatitis, fibrosis develops as a consequence of the host
immunological response. This immunological response activates antiviral defence
mechanisms that aim to clear infected hepatocytes. The mechanisms underlying
fibrogenesis in HBV or HCV are complex (Friedman 2007).
A key feature of hepatic fibrosis is the activation and proliferation of hepatic
stellate cells. Quiescent hepatic stellate cells store vitamin A and reside in the
subendothelial space of Disse. Chronic liver injury leads to activation of these cells,
which become contractile, produce extracellular matrix components and secrete proinflammatory cytokines and chemokines like transforming growth factor ß. The
activation of these cells is believed to represent the key event in hepatic fibrogenesis
(Friedman 2008). Hepatic stellate cell activation depends on signalling by Kupffer
cells, endothelial cells, hepatocytes, and platelets. The deposition of the
extracellular matrix is constantly opposed by degradation of these proteins. In
progressive liver fibrosis, this balance is skewed in favour of excess extracellular
matrix deposition. Matrix metalloproteinases and their regulators (tissue inhibitors
of metalloproteinases, TIMPs) control matrix deposition and degradation. In liver
fibrogenesis, TIMP-1 is also produced by activated hepatic stellate cells.
Liver histology, by helping visualise the fibrosis, has been considered the gold
standard for assessment and measurement of progression of fibrosis. However, the
disadvantages of this method have motivated researchers and clinicians to look into
more non-invasive strategies. These strategies are based either on single serum
surrogate markers, compositional scores derived from combinations of different
surrogate markers, or modifications of imaging techniques.

Liver biopsy – the gold standard for staging of
liver fibrosis
In the majority of liver centres worldwide, liver biopsy is performed as a “blind” or
ultrasound-guided puncture, as either an out- or in-patient procedure. Liver
punctures are considered to be relatively safe procedures with complication rates
ranging from 0.75% up to 13.6% (Myers 2008, Piccinino 1986, van der Poorten
2006). The most frequent complications are minor bleeding or pain. After efficient
substitution with clotting factors, percutaneous liver biopsy is also possible in
patients with inherited bleeding disorders with no obvious increase of complication
rates (DiMichele 2003, Schwarz 2008). Procedure-related mortality rates are
reported to range from 0.001 to 0.003% (Piccinino 1986). Of note, excess rates with
severe bleedings and biopsy related deaths have been reported after percutaneous
biopsy in populations with advanced fibrosis, cirrhosis, or hepatic tumors (Terjung
2003). Thus, liver biopsies in these patients should always be performed as in-

328 Hepatology 2012
patient procedures, since >90% of complications are detected within the first 24
hours (Piccinino 1986).
Transjugular puncture of the liver via cannulation of a hepatic vein is an
alternative, which can be performed in patients with severe coagulation deficiencies.
It is resource intensive and carries a risk of intrahepatic hemorrhage or capsule
perforation with intraabdominal bleeding. Complication rates are lower as compared
to percutaneous biopsies and range from 2.5% (Mammen 2008) to 6.5% with a
reported mortality rate of up to 0.09% in high-risk groups (Kalambokis 2007).
However, the quality of specimens from transjugular biopsies may be lower because
of the higher fragmentation of specimens and the lower numbers of portal fields in
transjugular biopsies (Cholongitas 2006).
Laparoscopy and mini-laparoscopy are even more invasive procedures for
obtaining liver biopsies. A recent randomized trial showed a higher detection rate of
liver cirrhosis as compared to percutaneous biopsies with lower complication rates
for laparoscopy (Denzer 2007). No data is available for detection of lesser fibrosis
stages. Thus, we recommend this procedure only in selected cases if the results will
have an impact on the clinical management of the patient (Helmreich-Becker 2003).
The quality and reliability of fibrosis staging via histopathological assessment of
liver biopsy specimens depends largely on the size of the specimen and the number
of portal fields. The biopsy should be 20-25 mm long and more than 11 portal tracts
should be visible (Bedossa 2003, Cholongitas 2006, Rousselet 2005). However, in
daily practice these requirements may not be easy to achieve; and even if a large
enough biopsy is acquired, the specimen only reflects about 1/50,000 of the whole
liver. Thus, liver biopsies are particularly prone to sampling errors and may – like
non-invasive markers – have difficulties in discriminating between adjacent stages
of fibrosis (i.e., F1 vs. F2 or F2 vs. F3). Recent studies report up to one stage
difference between specimens from the right and the left lobe in up to 38% of
biopsies (Regev 2002, Siddique 2003). Discrepancies of more than one stage are
rare (Regev 2002, Siddique 2003, Skripenova 2007). Intra- and inter-observer
variability may be unaffected by specimen sizes but can lead to discrepancies in up
to 20% of cases, even if one stage difference between estimates is accepted
(Gronbaek 2002, Petz 2003). Standardized automatic staging via image analysis
may improve interobserver variability (Hui 2004, Calvaruso 2009).
All staging systems for liver fibrosis are based on the definition of categorical
stages of liver fibrosis that describe the increase of deposition of collagen and the
progressive destruction of liver architecture ranging from no fibrosis to cirrhosis
with a variable number of intermediate stages (Table 1). The use of categories
decreases interobserver variation, but also results in a loss of information that may
be covered by more detailed scoring systems (Standish 2006).
Whereas the METAVIR score is considered best in HCV fibrosis, there is a wide
variability in the use of other staging systems in patients with chronic viral hepatitis.
In Germany, current guidelines recommend the staging system defined by Desmet
& Scheuer (Table 1) (Batts 1995, Desmet 1994, Ishak 1995, Knodell 1981,
Schirmacher 2004, French Cooperative Study Group 1994).

Assessment of Hepatic Fibrosis in Chronic Viral Hepatitis 329
Table 1. Commonly used liver fibrosis staging scores.
Staging System

Fibrosis stages

Remark

METAVIR Score

F0, F1, F2, F3, F4

Best evaluated in
HCV fibrosis

Knodell Score

F0, F1, F3, F4

Desmet
& Scheuer

Analogous to
METAVIR

Batts & Ludwig

Similar to METAVIR

No intermediate
stage
Recommended by (Desmet 1994, Schirmacher
German
2004)
guidelines for the
assessment of
liver fibrosis
(Batts 1995)

Ishak Score

F0, F1, F2, F3, F4,
F5, F6

(The French METAVIR
Cooperative Study Group
1994)
(Knodell 1981)

(Ishak 1995)

Surrogate markers of liver fibrosis in chronic viral
hepatitis
Liver fibrosis develops as a continuous process rather than in a stepwise manner.
Thus, so-called surrogate markers, which are also continuous variables, may provide
more precise information. Surrogate makers can be subdivided into direct and
indirect markers. Direct markers reflect changes in the content of extracellular
matrix proteins (such as collagen) in the liver. In contrast, indirect markers reflect
alterations in hepatic function, increase in portal hypertension with subsequent
splenic enlargement, and/or grade of hepatic inflammation that may correlate with
liver fibrosis stage (Table 2) (see http://hepatologytextbook.com/link.php?id=7).
Direct and indirect markers may be used alone or - more commonly - in
combination (“composite scores”). The calculation of such scores can be simple or
based on complicated formulas (e.g., Fibrotest/Fibrosure) (Table 2). Most studies of
non-invasive markers were performed in HCV patients, while studies in HBV or
coinfected cohorts are sparse (Pinzani 2008). Primary endpoints of the studies that
evaluated surrogate markers vary from discrimination of no fibrosis and cirrhosis to
the determination of fibrosis stages. However, for the clinical management of
patients with chronic viral hepatitis both are needed. Whereas the former is needed
to identify patients in need of urgent treatment, the latter may separate those patients
with an indication for antiviral treatment due to significant fibrosis from those with
no or minor fibrosis in whom treatment may be postponed.
From the whole range of surrogate markers only a few are in clinical use. The
simple APRI score has been widely studied in HCV and HBV as well as in
coinfected patients (Cacoub 2008, Lebensztejn 2005, Vallet-Pichard 2008, Wai
2006). A recent comprehensive meta-analysis of the performance of the APRI test
showed that its major strength is the exclusion of significant fibrosis, defined as F2F4, or cirrhosis with cut-offs of 0.5 and 1.0, respectively. However, the authors
conclude that using this marker alone, only about one third of all biopsies can be
avoided. Importantly, the test performance varied with the quantity of advanced
fibrosis in the different patient groups (Shaheen 2007, Shaheen 2008). Fibrotest has
also achieved some clinical significance. However, this test may not be available for
all patients. Recent meta-analyses of the predictive performance of Fibrotest

330 Hepatology 2012
summarize that the reliability for the detection of advanced fibrosis or cirrhosis is
adequate for clinical practice, and a cut-off of 0.6 has been suggested (Poynard
2007, Shaheen 2008, Shaheen 2007). Of note, the reliability for the detection of
earlier fibrosis stages appears to be relatively low (Poynard 2007, Shaheen 2008). In
summary, surrogate markers may support the clinical decision making process, but a
single surrogate marker or score cannot replace the liver biopsy. On the other hand,
attempts have been made to combine different surrogate markers and biopsy in
clinical decision algorithms that aim to reduce the need for liver punctures (Table
2). Increasingly efforts are made to combine surrogate markers with transient
elastography, and it seems that some progress can be expected in precise prediction
of different fibrosis stages, which may eventually replace liver biopsy for fibrosis
staging.

Transient elastography
Transient elastography (TE) is a non-invasive technique to assess liver fibrosis
(Sandrin 1999). TE allows the assessment of liver fibrosis by calculating the
velocity of a low-frequency transient shear wave produced by a mechanical probe
that is placed directly on the skin of the patient. The velocity of the wave that
penetrates the liver tissue depends on the actual stiffness of the liver, which in turn
correlates with the extent of liver fibrosis. In practice, a probe is placed in an
intercostal space at a position that is comparable to the position for standard liver
biopsy. Ten successful measurements are usually necessary for the assessment of
liver stiffness. This can be done in less than 5 minutes. At present TE machines are
exclusively available from Echosense (FibroScan®). Liver stiffness is expressed in
kilo Pascal (kPa). The method is easy to learn, quick, results are available
immediately, and a technical assistant can perform the procedure. TE displays
robust intra- and inter-observer variability (Fraquelli 2007) and may be applied in
children and adults (de Ledinghen 2007). Recently a special S probe for testing
children and patients with small intercostals spaces was introduced, and normal
reference values for different ages were defined (Engelmann 2011).
Evaluation of liver stiffness in subjects without apparent liver disease shows that
liver stiffness is influenced by gender and body mass index (BMI). In general, liver
stiffness is higher in men than in women (5.81±1.54 vs. 5.23±1.59 kPa) (Roulot
2008). Interestingly, TE may be used as a screening tool for the general population
to identify patients with unrecognized liver disease (Roulot 2011).
It is important to note that the applicability of TE is limited to relatively lean
patients (BMI <28 kg/m2), patients without ascites, and “cooperative” patients. A
special probe for obese patients has recently broadened the applicability of TE and
is recommended for patients with a skin-caspular distance of >2.5 cm but below 3.5
cm (Myers 2011). In addition, TE is hampered in those with acute liver injury such
as acute viral or alcoholic hepatitis, or chronic viral hepatitis flares, which may lead
to an overestimation of liver fibrosis (Arena 2008, Coco 2007, Sagir 2008). Unlike
liver histology, no published data is available on the variability (“sampling error”)
of TE results. TE correlates well with other surrogate markers of liver fibrosis such
as APRI and FIB-4 (Vidovic 2010). In patients with chronic liver disease eligible
for TE, liver stiffness values correlate well with the stage of fibrosis, irrespective of
the underlying disease etiology. TE has been evaluated in patients with chronic viral

Assessment of Hepatic Fibrosis in Chronic Viral Hepatitis 331
hepatitis, PBC, PSC, and NASH. Due to high acceptance by patients, it can easily
be used to monitor progression or regression of fibrosis in patients under
observation or on therapy (Wilson 2006, Wong 2011). TE has been evaluated for
the detection of liver fibrosis in patients with acute and chronic viral hepatitis and
has also been positively evaluated for HIV/HCV-coinfected patients and in patients
with HCV reccurrence post-transplantation (Carrion 2006, de Ledinghen 2006,
Maida 2007). In chronic viral hepatitis, it is unknown whether there is a difference
in TE results between patients with chronic HBV, HCV, and/or HIV/HCVcoinfected patients.
In some clinical situations, e.g., older patients or patients with risk factors for
therapy, a positive decision for treatment of chronic hepatitis B and C is guided by
the diagnosis of significant fibrosis. The presence of F2 fibrosis indicates significant
liver fibrosis, which justifies treatment according to treatment guidelines for chronic
hepatitis B, C and coinfected patients (Sarrazin 2010).
Recent studies comparing TE with liver biopsy demonstrate both high sensitivity
and specificity for the detection of advanced fibrosis and cirrhosis. However, TE
performance is less reliable for the detection of fibrosis stages ≥2 as compared to
more advanced stages of liver fibrosis (sensitivity 56-67%), resulting in moderate
negative predictive values. Thus, assessment of liver fibrosis by TE alone may
result in the underestimation of liver fibrosis in some patients. Vice versa, if TE
predicts significant fibrosis, a biopsy will not be necessary. One drawback in
clinical practice is that the different TE studies suggest slightly different cut-off
values (Table 3). A recent meta-analysis that evaluated the predictive performance
of TE in patients with chronic liver disease suggested that the optimal cut-off value
for the diagnosis of significant fibrosis is 7.65 kPa and 13.01 kPa for cirrhosis
(Friedrich-Rust 2008). This cut-off proved to be robust, especially in patients with
chronic HCV infection.
In addition to the assessment of liver fibrosis stages, TE may also be used to
predict the presence of portal hypertension and thus the need to evaluate the patient
for the presence of esophageal varices (Rockey 2008). Whether TE is reliable
enough to predict the stage of cirrhosis is still debatable and needs further studies
(Foucher 2006).
Increasing knowledge from studies on transient elastography also revealed a
number of conditions that may produce false positive results the user should be
aware of. These conditions include acute and chronic cardiac failure, valsalva
maneuver, hepatitis flair, pulmonary hypertension, amyloidosis, cholestasis,
pregnancy and steatosis, with the latter being more relevant for HCV than for HBVinfected patients (Fraquelli 2007, Arena 2008).
Another relevant artifact is the examination of a patient within 2 hours after a
meal, which may increase resistance by up to 2 kPa (Mederacke 2009).
The spectrum of interpretation of elevated TE results has been broadened
recently. For instance, a cut-off value of >25 kPa has been associated with >45-fold
increased risk to develop HCC in viral hepatitis. However, the risk seems to
increase in a linear fashion starting from a cut-off of 10 kPa (Fung 2011, Masuzaki
2009). Furthermore, TE values >21.1 are associated with portal hypertension as well
as the risk of portal hypertension-related complications and suggest endoscopy to
confirm or exclude esophageal varices and to initiate or decline the need for primary
prophylaxis with propranolol (Castera 2011, Robic 2011).

332 Hepatology 2012
Table 3. Cut-off values for transient elastography in different study populations.
Study

Population

Castera

HCV
N=183

Ziol

HCV
N=327

Cut off (kPa)
F=0
n.d.

F≥1
n.d.

F≥2
7.1
Se: 0.67
Sp: 0.95
PPV: 0.95
NPV: 0.48

F≥3
9.5
Se: 0.73
Sp: 0.91
PPV: 0.87
NPV: 0.81

F=4
12.5
Se: 0.87
Sp: 0.91
PPV: 0.77
NPV: 0.97

n.d.

n.d.

8.8

9.6

14.6

n.d.

n.d.
Se: 0.56
Sp: 0.91
PPV: 0.88
NPV: 0.56

Se: 0.86
Sp: 0.85
PPV: 0.71
NPV: 0.93

Se: 0.86
Sp: 0.96
PPV: 0.78
NPV: 0.97

Foucher

HCV / HBV
N=711

n.d.

n.d.

7.2
Se: 0.64
Sp: 0.85
PPV: 0.90
NPV: 0.52

12.5
Se: 0.65
Sp: 0.95
PPV: 0.90
NPV: 0.80

17.6
Se: 0.77
Sp: 0.97
PPV: 0.91
NPV: 0.92

Ogawa

HCV / HBV
N=229

3.5

6.4

9.5
Se: 0.67
Sp: 0.95
PPV: .95
NPV: .48

11.4
Se: 0.67
Sp: 0.95
PPV: .95
NPV: .48

15.4
Se: 0.67
Sp: 0.95
PPV: .95
NPV: .48

6.3

6.7

9.1
Se: 0.67
Sp: 0.95
PPV: 0.95
NPV: 0.48

13.7
Se: 0.67
Sp: 0.95
PPV: 0.95
NPV: 0.48

26.4
Se: 0.67
Sp: 0.95
PPV: 0.95
NPV: 0.48

7.8
Se: 0.83
Sp: 0.82
PPV: 0.83
NPV: 0.79
4.5
Se: 0.93
Sp: 0.18
PPV: n.d.
NPV: n.d.

10.8
Se: 0.91
Sp: 0.94
PPV: 0.89
NPV: 0.95
n.d.

14.8
Se: 0.94
Sp: 0.92
PPV: 0.73
NPV: 0.98
11.8
Se: 1.0
Sp: 0.93
PPV: n.d.
NPV: n.d.

Arena

HCV
N=150

De
Ledinghen

HIV/HCV
N=72

n.d.

n.d.

Assessment of Hepatic Fibrosis in Chronic Viral Hepatitis 333

Other imaging techniques for the
assessment of liver fibrosis
A number of different imaging techniques such as conventional ultrasound, realtime elastography, acoustic radiation force imaging (ARFI), portal venous transit
time, NMR imaging and CT have been used for the assessment of liver fibrosis.
None of these methods has yet achieved an overall clinical acceptance regarding the
detection of early liver fibrosis, either due to low sensitivity and/or specificity, or
high costs. The most promising candidate for everyday usability may be the ARFI
technique that has been integrated in high-end ultrasound machines. This technique
allows the measurement of liver fibrosis in an area of interest rather than a global
assessment as with the transient elastography method. This may be an advantage as
different regions of the liver may be studied separately but may – like histology –
also be a source of “sampling bias” and low reproducability. Compared to transient
elastography the available data from large populations is sparse. A recent metaanalysis managed to combine data from 518 individuals. Nevertheless, in this
analysis the overall accuracy for the prediction of fibrosis stage ≥F2, ≥F3 and
cirrhosis as determined by ROC analysis were 0.87, 0.91. 0.93, respectively
(Friedrich-Rust 2012).

Clinical decision algorithms
Until now, no non-invasive marker for the staging of liver fibrosis has been able to
replace the liver histology as the gold standard. This is largely due to the fact that
outcome studies with clear endpoints like mortality have not been performed and
that a clear differentiation of fibrosis stage by non-invasive strategies has been
unreliable. But the advantages of these non-invasive tests in comparison to liver
biopsy are striking. In order to overcome test limitations and to benefit from their
specific advantages, a frequent strategy is to combine different noninvasive tests,
thereby using liver biopsy only in case of doubt. However, former algorithms vary
greatly in performance and acceptance. Whereas some authors have calculated a
reduction in liver biopsies of 30%, others have estimated reductions of up to 80%
(Leroy 2007, Sebastiani 2004, Sebastiani 2006, Sebastiani 2007). New strategies
with sophisticated algorithms may overcome all these limitations and combination
of TE with FibroMeter achieved results that may give detailed and reliable
information on liver fibrosis stage without any need for histology. However, only
one study from France has described this method, which needs to be cross-validated
by independent groups (Boursier 2011a, Boursier 2011b).

334 Hepatology 2012

Figure 1. Potential clinical decision algorithm for safer liver biopsies in patients
with chronic viral hepatitis.

Summary
Non-invasive tests have not replaced liver biopsies today, but smart combinations of
non-invasive tools can save many patients from the more invasive procedure.
Whatever the current standard of care, the patient should be informed about the noninvasive tests, their applicability and their limitations. The decision to biopsy should
ultimately be made together with the informed patient.

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338 Hepatology 2012

21. Diagnosis, Prognosis & Therapy of
Hepatocellular Carcinoma
Ulrich Spengler

Classification of HCC
Tumors are classified in order to stratify patients with respect to their survival
prognosis, to select and offer optimised therapeutic options at any tumor stage. In
HCC the Barcelona Clinic Liver Cancer (BCLC) Classification has been adopted as
the international standard, which is recommended by both the American Association
for the Study of Liver Diseases (AASLD) and the European Association for the
Study of the Liver (EASL) (Table 1). The BCLC classification takes into account
several aspects of the disease: the patient’s general state of health, the severity of the
liver disease as well as the extent of tumor spread (Llovet 1999). Patients in stages
BCLC 0 and A have a considerably better prognosis than patients in advanced
stages of liver cancer (Mazzaferro 1996). Nevertheless approximately only 25% of
patients with liver cancer are diagnosed at an early stage. Both EASL and AASLD
guidelines also provide recommendations regarding which therapy is best suited to
treat patients at each stage of the BCLC classification. Unlike classification schemes
in other types of malignancies, the BCLC classification is particularly helpful
because it is entirely based on clinical parameters - molecular characteristics are not
yet able to reliably assess individual prognosis of patients with HCC.
Table 1. Barcelona Clinic Liver Cancer (BCLC) Classification.
Tumor stage

General state of health

Tumor characteristics

Child stage

0 Very early

Good

Single nodule <2 cm

A&B

A Early

Good

Single nodule <5 cm,
3 nodules <3 cm

A&B

B Intermediate

Good

Large, multiple nodules

A&B

C Advanced

Reduced

Vascular invasion,
extrahepatic secondaries

A&B

D Terminal

Severely reduced

Any form

C

Diagnosis, Prognosis & Therapy of Hepatocellular Carcinoma 339

Epidemiology
HCC constitutes the sixth most frequent form of cancer worldwide, and it holds
third place concerning malignancy-related mortality (Parkin 2005). Incidence rates
of HCC are steadily rising both in Europe and the US.
Chronic hepatitis B is the major risk factor for developing HCC in Africa and
Asia, while in the US, Europe and Japan chronic hepatitis C is the leading cause of
HCC. Eighty percent of liver cancers are found in cirrhotic livers, which themselves
carry a high risk for HCC. Chronic carriers of hepatitis B virus (HBV) have a 100fold increased risk as compared to a non-infected healthy reference population.
Recent reports from Taiwan indicate a direct link between HBV viral loads and the
risk of developing liver cancer within 10 years (Chen 2006, Iloeje 2006). The risk of
HCC is significantly increased once HBV viral loads exceed 2000 IU/ml
irrespective of the degree of hepatic inflammation. In developing countries exposure
to aflatoxins further increases this risk of HCC.
Approximately 130-170 million people are infected with the hepatitis C virus
worldwide, 20 to 30% of whom will develop liver cirrhosis, which carries a 3-5%
annual risk of ultimately progressing to liver cancer. In practical terms this means
that approximately one third of cirrhotic patients with hepatitis C will go on to
develop HCC. Unlike hepatitis B a close relationship between HCV viral loads and
the risk of developing HCC apparently does not exist (Bralet 2000). As a general
rule patients will not develop liver cancer in chronic hepatitis C before their disease
has progressed to the stage of cirrhosis. Consumption of alcohol or tobacco
enhances the risk of HCC (Donato 2002, Gelatti 2005). Beyond that, obesity (Calle
2003) and diabetes mellitus (Davila 2005) must be considered neglected but
nevertheless pivotal factors that can multiply the risk of liver cancer in western
countries resulting in 4 to 40-fold increased HCC rates among patients with chronic
viral hepatitis. Finally a genetic polymorphism in the adiponutrin gene seems to
predispose patients with alcoholic and non-alcoholic fatty liver disease to develop
cirrhosis and HCC (Fallet 2011, Nischalke 2011)

Surveillance of patients at high risk and early HCC
diagnosis
Surveillance is cost effective if the expected HCC risk exceeds 1.5% per year in
hepatitis C and 0.2% per year in hepatitis B. Surveillance has to be based on
ultrasound examination at 6-month intervals. When 3- versus 6-month surveillance
intervals were compared in a randomized study involving 1200 patients, there was
no evidence that the shorter interval improved rates of early diagnosis and
therapeutic outcomes. However, if patients with cirrhosis harbour nodular lesions,
the 3-month control interval is preferred due to the high potential of malignancy and
growth characteristics of such lesions (Yao 2006). Alpha-fetoprotein (AFP) has
insufficient sensitivity and specificity, and thus is no longer recommended for HCC
surveillance. The consistent use of ultrasound in patients with a high risk for HCC
enables us to diagnose early carcinoma in 30% of patients who then have a
reasonable chance of curative therapy.

340 Hepatology 2012

Diagnosis
The diagnosis of HCC can either be made by detecting malignantly transformed
hepatocytes in a liver biopsy or by dynamic contrast-enhanced radiological imaging
techniques demonstrating intense arterial uptake followed by wash-out of contrast in
the delayed venous phases reflecting arterialised perfusion of the tumor. Contrastenhanced ultrasound may falsely suggest HCC in some patients with
cholangiocarcinoma, and it should not be used as the only diagnostic tool for HCC
(Vilana 2010). Nevertheless, novel diagnostic algorithms enable the diagnosis of
HCC in a cirrhotic liver without histopathology or reference to elevated tumor
markers.
The distinction between a dysplastic nodule and early HCC poses a particularly
challenging task for the pathologist. Staining for glypican 3, heat shock protein 70,
and glutamine synthetase is advised in this situation, and positivity for any two of
these three markers confirms the presence of HCC (International Working Party
2009).
Radiological diagnosis of HCC uses detection of hyper-vascularized nodular
lesions. Contrast-enhanced computed tomography (CT) or nuclear magnetic spin
resonance tomography (MRT) are considered to be equivalent diagnostic tools, and
international consensus guidelines accept a diagnosis of HCC without
histopathology if the patient with a nodular lesion in a cirrhotic liver exhibits the
following sequence of events: in the arterial phase, HCC enhances more intensely
than the surrounding liver, because arterial blood in the liver is diluted by venous
blood from the portal venous circulation, whereas HCC contains only arterial blood.
In the venous phase, HCC enhances less than the liver, reflecting the fact that HCC
does not have a portal venous blood supply and that the arterial blood flowing into
the lesion no longer contains contrast. This phenomenon is termed “washout”. In the
delayed phase “washout” persists, and occasionally HCC can only be detected in
this phase of a dynamic study. Thus, a four-phase dynamic study is needed to
reliably make a diagnosis of HCC (unenhanced, arterial, venous and delayed venous
phases). Contrast enhancement in the early arterial phase, which disappears in the
late venous phase, is highly specific for HCC.
The current recommendations for the diagnosis of HCC are summarized in Figure
1. For lesions smaller than 1 cm detailed investigation is not recommended because
most lesions will represent regenerative nodules rather than HCC. However, close
follow-up in 3-month intervals should be offered using the same imaging technique
that detected the lesion in the first place.
For lesions larger than 1 cm, either dynamic MRI or multidetector CT scans
should be performed. If findings are characteristic for HCC as described above, a
firm diagnosis of HCC can be made and no further studies are necessary. If the
findings are not typical for HCC, then the alternative dynamic imaging technique
should be applied. If this supplementary radiological investigation yields typical
features, the diagnosis of HCC is confirmed. Otherwise, a guided biopsy of the
lesion should be performed. Contrast-enhanced CT and MRI exhibit excellent
diagnostic sensitivity and specificity if the rules regarding early hypervascularity
and washout are strictly applied. The presence of arterial hypervascularisation alone
is not sufficient for a diagnosis of HCC, which requires the presence of venous
washout as an essential second diagnostic component. In equivocal situations the

Diagnosis, Prognosis & Therapy of Hepatocellular Carcinoma 341
diagnosis must be clarified by biopsies, which may have to be repeated within a
short period of time.

Figure 1. Diagnostic algorithm for the diagnosis of hepatocellular carcinoma depending
on tumor size.

Stage-adapted therapy for liver cancer
Patients with early HCC have excellent chances for curative cancer treatment. They
can achieve 5-year survival rates of 50-70% by surgical resection, liver
transplantation or percutaneous ablative procedures. With more advanced HCC,
local transarterial embolisation and multikinase inhibitor therapy can still prolong
life. Figure 2 gives a summary and concise overview of stage-adapted therapy for
hepatocellular carcinoma.

Potentially curative therapy in BCLC stages 0-A
Surgical resection constitutes the backbone of curative treatment in patients with
early HCC. It is the treatment of choice in patients with localised tumor spread and
small-sized cancers and tumors in a non-cirrhotic liver (evidence grade IIIA).
Prognosis after surgical resection is excellent, if the tumor is not larger than 2 cm in
diameter (5-year survival rates 70-90% with rates of tumor recurrence below 10%).
Excluding patients with poor liver function keeps perioperative mortality below 5%.
Favourable criteria for surgical resection comprise single nodules less than 5 cm in
size or a maximum of 3 nodules in a single liver lobe. Patients should be carefully
selected to diminish the risk of postoperative liver failure. Patients should have only
moderately impaired liver function (cirrhosis stage Child’s A), should not have

342 Hepatology 2012
portal hypertension (hepatic-portal-vein pressure gradient >10 mm Hg, presence of
esophageal varices or splenomegaly together with reduced platelet counts
<100,000/µl) and should have a serum bilirubin in the normal range. Right
hepatectomy in cirrhotic patients has a higher risk of inducing hepatic
decompensation than left hepatectomy.
Liver transplantation is an alternative therapeutic option, if the liver cancer
cannot be cured by local resection due to anatomical reasons, if residual liver
function after resection is anticipated to be poor, or if there is multi-nodular tumor
spread into both liver lobes (evidence grade IIIA). Commonly patients with HCC
are selected for liver transplant according to the so-called Milan criteria, i.e., the
patient has a single nodule of less than 5 cm in diameter or at most 3 nodules, none
of which exceeds 3 cm in diameter (Mazzaferro 1996). Milan criteria patients
usually achieve survival rates of 80% and 70% one and five years after liver
transplantation. However, it has been demonstrated that selected patients with more
extensive stages of liver cancer can be transplanted with reasonable long-term
outcomes (Yao 2001). Selection of patients according to the so-called San Francisco
criteria comprises solitary large nodules up to 6.5 cm as well as multi-nodular HCC
with a maximum of 3 nodules, each of which must be smaller than 4.5 cm with a
total sum of all nodule diameters below 8 cm. Patients who remain within these
extended selection criteria can still reach 70-80% five-year survival rates after liver
transplantation. However, there is very limited data to support extending selection
criteria for liver transplantation (Pomfret 2010).
A central issue in liver transplantation is the process of fair organ allocation.
Shortage of donor organs is particularly critical in patients with liver cancer,
because the tumor will continue to expand while the patient is on the waiting list,
and can ultimately reach a stage that makes liver transplantation a futile option. It
has been estimated that after one year on the waiting list approximately 40% of
patients can no longer be cured by liver transplant (Poon 2007). In the
Eurotransplant registry donor livers are allocated to patients according to their
MELD scores. To circumvent the problem that patients with early HCC who are
eligible for liver transplantation have rather low MELD scores, Eurotransplant
accepts the diagnosis of HCC within the Milan criteria as a so-called standard
exemption, allocating additional points on top of the patient’s lab-MELD score in an
incremental time-dependent fashion.
Most transplant centres treat liver cancers locally while the patient is on the
waiting list, mostly using transarterial chemoembolisation. This strategy probably
also facilitates patient selection for liver transplantation, because those with stable
disease after chemoembolisation achieve a greater than 90% five-year survival rate
after liver transplantation, while only 35% of patients in the group with progressive
tumor expansion survive five years post-liver transplantation (Otto 2006).
Radiofrequency ablation can also cure HCC that is limited to a distinct region of
the liver and is especially applied in older patients or patients with significant comorbidity. A cohort study on percutaneous radiofrequency ablation demonstrated
that complete ablation of lesions smaller than 2 cm is possible in more than 90% of
patients with local recurrence in less than 1% (Livraghi 2008). In larger tumors fiveyear survival rates are somewhat lower, at 70-80% for nodules less than 3 cm in
diameter, and 50% for tumors between 3 and 5 cm (Lopez 2006). A cumulative

Diagnosis, Prognosis & Therapy of Hepatocellular Carcinoma 343
meta-analysis has suggested that survival is better after radio frequency ablation
than after ethanol injection (Cho 2009).
Adjuvant therapy, in the context of resection, liver transplantation or localablative procedures, does seem to offer additional benefits. Thus far, antiviral
treatment of hepatitis B with nucleos(t)ide analogs remains the single approved
treatment after removal or local destruction of HCC.
Tumor recurrence is frequent after putatively curative treatment of HCC. The
best predictors of HCC recurrence are microvascular invasion and/or additional
tumor sites besides the primary lesion. There is no effective adjuvant therapy to
reduce recurrence rates. However, it is noteworthy that even the most modern CT
and MRT scanner still underestimate the extent of vascular invasion in 30% of
patients with early HCC. Treatment of recurrence is a poorly studied area. Solitary
nodules might be amenable to repeat resection, but HCC recurrence is frequently
multifocal owing to intrahepatic dissemination of the tumor. Some patients with
HCC recurrence after primary resection might benefit from salvage transplantation.

Palliative therapy in BCLC stages B and C
Palliative treatment remains the only therapeutic option for patients with advanced
stages of liver cancer that cannot be controlled by local therapy.
Arterial chemoembolisation is the most frequent palliative intervention offered
to patients with HCC and is considered for patients with non-surgical HCC that are
also not suited for percutaneous ablation and do not have extrahepatic tumor spread.
HCC exhibits intense neoangiogenic activity, so that even well-differentiated HCCs
become highly dependent on arterial blood supply. Thus, hepatic arterial obstruction
is performed either by angiographic transarterial embolisation or transarterial
chemoembolisation. Usually lipiodol combined with an embolising agent such as
gelatin or microspheres is mixed with cytostatic drugs and applied to the liver via an
intra-arterial catheter. Suitable cytotoxic agents are doxorubicin, mitomycin and cisplatinum, but the optimal combination of drugs and treatment schedules has not
been established. In randomised studies demonstrating a benefit of
chemoembolisation, doxorubicin or cis-platinum were administered in 3-4
angiographic sessions per year. Chemoembolisation carries the risk of ischemic
damage to the liver, potentially leading to fulminant liver failure. To minimize this
risk chemoembolisation should be offered only to patients with good residual
hepatic function, who have asymptomatic multi-nodular liver cancer without
vascular invasion or extrahepatic tumor spread. Vice versa patients with
decompensated liver disease (liver cirrhosis, Child’s B or C) or imminent hepatic
failure should not undergo chemoembolisation. The side effects of interarterial
chemoembolisation are the same as for systemic chemotherapy and comprise
nausea, vomiting, bone marrow depression, alopecia and renal damage. As a
complication of hepatic ischemia, more than 50% of patients also develop a socalled post-embolisation syndrome with fever, abdominal pain and a moderate
degree of ileus. Fasting and fluid replacement is mandatory, but the postembolisation syndrome is usually self-limited and patients can be discharged safely
after 2 days.
Objective response rates vary between 16% and 60%, but less than 2% of patients
achieve complete remission. Residual tumor cells recover their blood supply and the
tumors continue to grow. Thus, repeated therapy is needed.

344 Hepatology 2012
Chemoembolisation is currently the only palliative treatment demonstrated to
significantly improve patient survival in controlled studies (Llovet 2002). However,
it cannot be offered to patients with portal vein thrombosis or HCC-induced portal
vein occlusion.
Radiotherapy applying Yttrium-90 microspheres has been developed as a
novel alternative palliative treatment of liver cancer with unexpectedly impressive
anti-tumoral activity in selected individual cases (Sangro 2006, Jacobs 2007, Salem
2006, Liu 2004). Of note, unlike chemoembolisation, some types of microspheres
do not occlude the blood vessels and can also be applied in the presence of portal
vein thrombosis. Radioembolisation has been shown to induce tumor necrosis.
However, there are no data comparing its efficacy to other palliative treatment
modalities.
Molecular-targeted therapeutic strategies offer new hope for effective
palliative therapy in liver cancer. Sorafenib (Nexavar®) is an orally available multikinase inhibitor acting on several distinct tyrosine kinases (VEGFR2, PDGFR, c-kit
receptor) as well on serine/threonine kinases (b-Raf and p38). Thus, by inhibiting
angiogenesis and cellular proliferation, sorafenib can block two of the major
signalling pathways of HCC expansion. In a Phase III study (the SHARP trial)
involving 602 patients, sorafenib 400 mg BID was well tolerated and associated
with improved survival in 44% of patients resulting in 3 months extended survival
in treated patients (10.7 months in the sorafenib arm versus 7.9 months in the
control arm). Diarrhea, weight loss, hand-foot syndrome and hypophosphatemia
were the most important side effects in patients on sorafenib. The efficacy of
sorafenib has been confirmed in a second randomized placebo-controlled trial,
mostly involving patients with HBV-associated HCC (Cheng 2009). Sorafenib has
established itself as first option in patients with HCC who can no longer be treated
with potentially more effective local therapies. The SHARP trial largely included
patients with preserved liver function. Although the pharmacologic profile is
favourable, data in Child-Pugh class B patients are scarce (Abou Alfa 2011). It has
been demonstrated that sorafenib can be safely combined with chemoembolisation
therapy (Pawlik 2011), although it remains unclear if this strategy actually offers
any clinical benefit to the patients. Finally, further antagonists targeting VEGFR,
EGFR, ERBB2, Akt/mTor or Wnt/β-catenin signal transmission pathways are
currently under evaluation in Phase II studies.
Systemic chemotherapy has been proven repeatedly not to offer survival
benefits, whether given as a single agent or as part of combination chemotherapy
(Llovet 2003). Likewise, anti-hormonal therapy with tamoxifen or octreotide has
not provided improved patient survival when studied under controlled conditions
(Gallo 2006, Yuen 2002).

Diagnosis, Prognosis & Therapy of Hepatocellular Carcinoma 345

Figure 2. Overview of stage-adapted therapy of liver cancer relative to the BLCL criteria.

Prophylaxis of liver cancer
Despite conspicuous progress concerning the diagnosis and therapy of HCC, its
prognosis has not improved very much over time. Thus, prophylactic measures are
of pivotal importance. Vaccination against HBV, as is now recommended by many
national vaccination councils, has been proven in Taiwan to markedly reduce HBV
infection rates along with the incidence of HCC as a complication of chronic
hepatitis B in later life (Lok 2004).
Patients with chronic HBV and patients with chronic hepatitis C should be offered
antiviral therapy as effective secondary prophylaxis of HCC. Although HBeantigen-positive (van Zonneveld 2004) and HBe-antigen-negative patients with
chronic hepatitis B showed reduced incidence rates of HCC when successfully
treated with interferon (Papatheoridis 2001, Brunetto 2002, Lampertico 2003),
antiviral therapy with nucleos(t)side analogs seems to reduce the risk of HCC less
convincingly (Papatheoridis 2010, Papatheoridis 2011). Also several meta-analyses
suggest that successful interferon therapy will reduce the risk of HCC in chronic
hepatitis C (Camma 2001, Paptheoridis 2001a, Veldt 2004). However, patients who
have cirrhosis prior to starting antiviral therapy should be maintained on close HCC
surveillance programs, since the risk of developing liver cancer remains high in
these patients even after sustained virologic elimination is achieved (Yu 2006).

346 Hepatology 2012
Therapeutic management of additional risk factors such as obesity and poorly
controlled diabetes mellitus provide additional chances for prophylactic measures to
reduce the risk of HCC development. Finally, consumption of two or more cups of
coffee per day seems to reduce the risk of liver cancer by 40-50% in patients with
chronic viral hepatitis (Gelatti 2005, Bravi 2007, Larsson 2007, Wakai 2007).

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Update in Transplant Hepatology 349

22. Update in Transplant Hepatology
S. Beckebaum, G. Gerken, V. R. Cicinnati

Introduction
The first attempt at heterotopic grafting of a liver in a dog was reported more than
50 years ago (Welch 1955). The first known experimental orthotopic liver
transplantation (LT) was reported in 1956 at the University of California (Cannon
1956). In the early sixties, a human-to-human LT was performed in a 3-year-old
child with congenital biliary atresia who died intraoperatively (Starzl 1963). The
next two transplant recipients lived for 22 days and 1 week, respectively (Starzl
1963). Starzl finally transplanted several patients with success in 1967 (Starzl
1968).
With the advances in immunosuppression, surgical techniques, organ preservation
and improvements in patient management, LT has become the gold standard in the
treatment of advanced chronic liver disease and fulminant hepatic failure. This
chapter focuses on important issues in the field of transplant hepatology and may
provide helpful information to physicians involved in the care of adult LT
recipients. It includes indications for LT, current organ allocation policy,
pretransplant evaluation, management while on the waiting list, living donor liver
transplantation (LDLT), and management of early and long-term complications
post-LT.

Timing and indications for liver transplantation
Appropriate selection of candidates and timing of LT is crucial in reducing
mortality and improving outcomes in LT recipients. A patient is considered too
healthy to undergo LT if the expected survival is greater without LT. Therefore,
criteria are needed in order to select patients who can most benefit from
transplantation. In 2002, the Organ Procurement and Transplantation Network,
along with the United Network of Organ Sharing (UNOS), developed a new system
based on the model for end-stage liver disease (MELD) (Table 1) to prioritize
patients on the waiting list. In the Eurotransplant countries, the Child-Pugh Turcotte
score was replaced by the MELD score in December 2006.

350 Hepatology 2012
The lab MELD score is a numerical scale using the three laboratory parameters
depicted in Table 1 and ranging from 6 (less ill) to 40 (severely ill).
In a large study (Merion 2005) investigating the survival benefit of LT candidates,
those transplanted with a MELD score <15 had a significantly higher mortality risk
as compared to those remaining on the waiting list, while candidates with a MELD
score of 18 or higher had a significant transplant benefit.
Table 1. Calculation of the MELD* Score.
MELD Score = 0.957 x log (creatinine mg/dL)
0.378 x log (bilirubin mg/dL)
1.120 x log (INR**)
+ 0.643
*Model of End-stage Liver Disease
**International Normalized Ratio

However, the MELD score does not accurately predict mortality in approximately
15-20% of patients. Therefore MELD-based allocation allows exceptions for
patients whose score may not reflect the severity of their liver disease. These
exceptions
include
hepatocellular
carcinoma
(HCC),
non-metastatic
hepatoblastoma, adult polycystic liver degeneration, primary hyperoxaluria type 1,
small for size syndrome, cystic fibrosis, familial amyloid polyneuropathy,
hepatopulmonary syndrome, portopulmonary hypertension, urea cycle disorders,
hereditary
hemorrhagic
telangiectasia
(Osler-Weber-Rendu
disease),
hemangioendothelioma of the liver, biliary sepsis, primary sclerosing cholangitis
(PSC) and cholangiocarcinoma. Patients with standard exceptions will be assigned a
higher MELD score (match MELD) than that assigned by the patient’s laboratory
test results (lab MELD). This results in an increasing proportion of patients
transplanted for HCC and other exceptions over time (Massie 2011).
MELD has proved to be accurate as a predictor of waiting list mortality, but has
shown to be less accurate to predict post-transplant outcome. For instance, MELD
allocation resulted in decreased waiting list mortality; whereas post-transplant
morbidity has increased due to transplantation of a higher proportion of sicker
recipients with MELD scores >30 (Dutkowski 2011). Moreover, since the
introduction of MELD, the quality of donor organs has been impaired and the
threshold for organ allocation has increased from a match MELD of 25 to 34
(Schlitt 2011).
A potential modification of the MELD allocation system currently under
investigation is to allocate organs by not only taking into account pretransplant
mortality but also donor-related factors for estimation of the donor risk index (DRI)
(Feng 2006) and post-transplant mortality.
Furthermore, standardization of laboratory assays and variants of MELD
including incorporation of parameters such as sodium or cholinesterase have been
proposed to overcome the limitations of the current scoring system (Choi 2009,
Weissmüller 2008).
Candidates for LT must have irreversible acute or chronic end-stage liver disease.
Hepatitis C virus (HCV)- or alcohol-induced liver disease account for the most
common disease indications in adults with liver cirrhosis (http://www.eltr.org)
(Figure 1). Other indications include cholestatic liver disorders (primary biliary

Update in Transplant Hepatology 351
cirrhosis [PBC], PSC), hepatitis B virus (HBV) infection, autoimmune hepatitis
(AIH), inherited metabolic diseases (Wilson’s Disease, hemochromatosis, α-1antitrypsin deficiency), nonalcoholic steatohepatitis, HCC, and acute or acute-onchronic hepatic failure. In children, biliary atresia and metabolic liver diseases are
the most common indications. Contraindications for LT include active alcohol and
drug abuse, extrahepatic malignancies, sepsis, uncontrolled pulmonary
hypertension, and coexistent medical disorders such as severe cardiopulmonary
condition, technical or anatomical barriers such as thrombosis of the entire portal
and superior mesenteric venous system. Previous malignancy history must be
carefully considered and likelihood of recurrence estimated.

Figure 1. Indications for liver transplantation (LT). Primary diseases leading to LT in Europe
1988 - 2010 (Data kindly provided from European Liver Transplant Registry,
http://www.eltr.org).

Patient evaluation
Evaluation of a potential transplant candidate is a complex and time-consuming
process that requires a multidisciplinary approach. Requirements for evaluation may
differ slightly between transplant centers. The evaluation process must identify
extrahepatic diseases that may exclude the patient from transplantation or require
treatment before surgical intervention. The protocol we use for evaluation of
potential transplant candidates is shown in Table 2.

Pretransplant management issues
In cases of recurrent variceal hemorrhage despite prior interventional endoscopic
therapy (and non-selective beta-blockade) or refractory ascites, transjugular
intrahepatic portosystemic shunts (TIPS) have been used as an approach to lower
portal pressure and as a bridging therapy for transplant candidates. The
identification of predisposing factors and the application of lactulose, nonabsorbed
antibiotics and protein-restricted diets remain essential for prophylaxis and
management of hepatic encephalopathy (HE).

352 Hepatology 2012
Table 2. Evaluation protocol for potential transplant candidates.
 Physical examination
 Diagnostic tests (baseline laboratory testing; serologic, tumor/virologic, and
microbiological screening; autoantibodies; thyroid function tests)
 Ultrasonography with Doppler
 Abdominal MRI or CT scan
 Chest X-rays
 Electrocardiogram (ECG), stress ECG, 2-dimensional echocardiography (if
abnormal or risk factors are present: further cardiological screening)
 Upper and lower endoscopy
 Pulmonary function testing
 Mammography (females >35 years)
 Physician consultations (anesthesiologist, gynecologist, urologist, cardiologist,
neurologist, dentist, ENT physician)
 A meticulous psychosocial case review (medical specialist in psychosomatic
medicine, psychiatry or psychology)

Hepatorenal syndrome (HRS) represents a complication of end-stage liver disease
and is a risk factor for acute kidney injury (AKI) in the early postoperative phase
(Saner 2011). It is classified into type 1 HRS characterized by a rapid impairment of
renal function with a poor prognosis; type 2 HRS is a moderate steady renal
impairment. Vasoconstrictors including commonly used terlipressin in combination
with volume expansion, have been shown to be effective for restoration of arterial
blood flow and serve as a bridging therapy to LT. Extracorporeal liver support
systems based on exchange or detoxification of albumin have been successfully
employed in indicated cases. After wait-listing, laboratory values must be updated
according to the recertification schedule shown in Table 3.
Table 3. Recertification schedule of MELD data.
Score

Recertification

Lab values

≥25
24-19
18-11
≤10

every 7 days
every 30 days
every 90 days
every year

≤48 hours old
≤7 days old
≤14 days old
≤30 days old

Special attention regarding specific, disease-related therapy prior to surgery
should be given to transplant candidates undergoing LT for HCC or virally-related
liver diseases.

Waiting list monitoring of hepatitis B liver transplant
candidates
The goal of antiviral therapy in HBV patients on the waiting list is to achieve viral
suppression to undetectable HBV DNA levels using sensitive tests (Figure 2)
(Cornberg 2011). Several studies have demonstrated clinical benefits under viral
suppression in patients with decompensated cirrhosis as reflected by a decrease in
CPT score, improvement of liver values and resolution of clinical complications
(Kapoor 2000, Schiff 2007, Nikolaidis 2005).

Update in Transplant Hepatology 353

Figure 2. Management of HBV patients prior to liver transplantation (LT). In all viremic
patients awaiting LT for HBV-related liver damage, efficient antiviral therapy is required.
Suppression of HBV DNA may lead to clinical stabilisation resulting in removal from the waiting
list or in a delay in the need for LT. Neg., negative, pos., positive.

A major concern of long-term lamivudine (LAM) therapy is the emergence of
mutations in the YMDD motif of the DNA polymerase which could result in clinical
decompensation in patients with liver cirrhosis (Beckebaum 2008, Beckebaum
2009). Therefore potent nucleos(t)ide analogs (entecavir [ETV] or tenofovir [TDF])
with a high resistance barrier are preferred.

Waiting list monitoring and treatment of hepatitis C liver
transplant candidates
The number of studies investigating the tolerability and efficacy of antiviral therapy
in HCV patients before LT is limited (Crippin 2002, Iacobellis 2007, Everson 2005,
Triantos 2005). Wait-listed patients who have a viral response on antiviral therapy
have a lower reinfection rate and better outcome after LT (Thomas 2003, Picciotto
2007). Thus, there is an indication for therapy with pegylated interferon (PEG-IFN)
plus ribavirin (RBV) in patients with compensated HCV cirrhosis on the waiting
list. Results from antiviral clinical studies show sustained viral response (SVR) rates
between 20% and 40% (Melero 2009). Adverse effects are frequent including
cytopenias, bacterial infections and hepatic decompensation requiring dose
reduction or treatment withdrawal. Hematopoietic growth factors have shown to
increase patient compliance and to avoid dose reductions, but it remains
questionable whether they result in higher SVR rates. Antiviral therapy in
decompensated cirrhosis and with MELD score ≥18 should be restricted to selected
cases and monitored by a transplant center.

Adjunctive treatment and staging of HCC transplant
candidates
Under MELD allocation, patients must meet the Milan criteria (one tumor ≤5 cm in
diameter or up to three tumors, all ≤3 cm) to qualify for exceptional HCC waiting
list consideration. Diagnosis of HCC is confirmed if the following criteria are met
according to the German
Guidelines for
Organ
Transplantation
(Bundesärztekammer 2008): (1) liver biopsy-proven or (2) AFP >400 ng/mL and
hypervascular liver lesion detectable in one imaging technique (magnetic resonance

354 Hepatology 2012
imaging [MRI], spiral computed tomography [CT], angiography) or (3)
hypervascular liver lesion detectable in 2 different imaging techniques. Patients are
registered at a MELD score equivalent to a 15% probability of pretransplant death
within 3 months. Patients will receive additional MELD points equivalent to a 10%
increase in pretransplant mortality to be assigned every 3 months until these patients
receive a transplant or become unsuitable for LT due to progression of their HCC.
The listing center must enter an updated MELD score exception application in order
to receive additional MELD points. The US National Conference on Liver
Allocation in Patients with HCC recommended the introduction of a calculated
continuous HCC priority score, that incorporates the MELD score, AFP level and
rate of tumor growth, for identifying patients with a good vs. a poor outcome
(Pomfret 2010). Further investigations are necessary to determine the survival
benefit of HCC patients considering these features.
Pre-listing, the patient should undergo a thorough assessment to rule out
extrahepatic spread and/or vascular invasion. The assessment should include CT
scan or MRI of the abdomen, pelvis and chest. We perform trimonthly routine
follow-up examinations (MRI or CT scan) of wait-listed HCC patients for early
detection of disease progression. It has been shown that waiting list drop-out rates
can be reduced by the application of bridging therapies such as transarterial
chemoembolisation or radiofrequency ablation (Roayie 2007). Recently,
transarterial radionuclide therapies such as Yttrium-90 microsphere transarterial
radioembolisation (TARE) have been tested for bridging therapy in selected cases
(Toso 2010, Khalaf 2010). Bridging therapy should be considered in particular in
patients outside of the Milan criteria, with a likely waiting time of longer than 6
months and those within the Milan criteria with high-risk characteristics of HCC.
Sorafenib has been administered in a few studies before LT to investigate the safety
and efficacy of this oral multikinase inhibitor in the neoadjuvant setting (Fijiki
2011, Di Benedetto 2011).
Accurate discrimination of HCC patients with good and poor prognosis by
specific criteria (genomic or molecular strategies) is highly warranted to select
appropriate treatment options (Tournoux-Facon 2011, Marsh 2003, Finkelstein
2003). In patients with alcohol-related liver disease and HCC, a multidisciplinary
approach and thorough work-up of both the alcoholic and oncologic problem is
mandatory (Sotiropoulos 2008a).

Living donor liver transplantation: indications,
donor evaluation, and outcome
LDLT was introduced in 1989 with a successful series of pediatric patients
(Broelsch 1991). Adult-to-adult LDLT (ALDLT) was first performed in Asian
countries where cadaveric organ donation is rarely practiced (Sugawara 1999,
Kawasaki 1998). LDLT peaked in the US in 2001 (Qiu 2005) but therafter the
numbers declined by 30% over the following years (Vagefi 2011). A decline over
time was also observed in Europe, although LDLT activity increased in Asia (Moon
2011).
The evaluation of donors is a cost-effective although time-consuming process.
Clinical examinations, imaging studies, special examinations, biochemical
parameters, and psychosocial evaluation prior to donation varies from center to

Update in Transplant Hepatology 355
center and has been described elsewhere (Valentin-Gamazo 2004). Using Germany
as an example, the expenses for evaluation, hospital admission, surgical procedure,
and follow-up examinations of donors are paid by the recipient’s insurance. Due to
the increasing number of potential candidates and more stringent selection criteria,
rejection of potential donors has been reported in about 69-86% of cases (ValentinGamazo 2004, Pascher 2002). The advantages of LDLT include the feasibilty of
performing the operation when medically indicated and the short duration of cold
ischemia time.
The surgical procedures for LDLT are more technically challenging than those for
cadaver LT. In the recipient operation, bile duct reconstruction has proven to be the
most challenging part of the procedure with biliary complications ranging from 15%
to 60% (Sugawara 2005).
Regarding donor outcome, morbidity rates vary considerably in the literature
(Patel 2007, Beavers 2002). Possible complications include wound infection,
pulmonary problems, vascular thrombosis with biliary leaks, strictures, and
incisional hernia. Biliary complications are the most common postoperative
complication in LDLT and occur in up to 7% of donors (Perkins 2008, Sugawara
2005). Liver regeneration can be documented with imaging studies and confirmed
by normalization of bilirubin, liver enzymes, and synthesis parameters. LDLT
should be performed only by established transplant centres with appropriate medical
expertise.

Perioperative complications
Cardiac decompensation, respiratory failure following reperfusion, and kidney
failure in the perioperative LT setting constitutes a major challenge for the intensive
care unit. Early dialysis has been shown to be beneficial in patients with severe
acute kidney injury (AKI) (stage III according to the classification of the Acute
Kidney Injury Network) (Bellomo 2004), whereas treatment with dopamine or loop
diuretics have shown to be associated with worse outcome. Preventative strategies
of AKI include avoidance of volume depletion and maintenance of a mean arterial
pressure >65 mmHg (Saner 2011).
Despite advances in organ preservation and technical procedures, postoperative
complications due to preservation/reperfusion injury have not markedly decreased
over the past several years. Typical histological features of preservation and
reperfusion injury include centrilobular pallor and ballooning degeneration of
hepatocytes. Bile duct cells are more sensitive to reperfusion injury than
hepatocytes (Washington 2005) resulting in increased levels of bilirubin, gammaglutamyl transpeptidase (GGT) and alkaline phosphatase (AP). Vascular
complications such as hepatic artery thrombosis (HAT) occur in 1.6-4% of patients.
Thus, Doppler exams of the hepatic artery and portal vein are frequently performed
in the early postoperative setting. HAT in the early postoperative period can be
managed with thrombectomy. Late HAT with complication of bile duct strictures is
managed by interventional endoscopic retrograde cholangiography (ERC) but
requires retransplantation in the majority of patients. Early portal vein thrombosis is
rare (<1%) but may lead to graft loss if not revascularized.
Primary non-functioning graft (PNFG) may be clinically obvious immediately
after revascularization of the allograft. Early signs of liver dysfunction include
prolonged coagulation times, elevated liver enzymes (transaminases, cholestasis

356 Hepatology 2012
parameter) without a downward trend, rising lactate, and hypoglycemic episodes.
PNFG is a critical situation and requires immediate retransplantation.
Death within the first year after LT is often associated with bacterial infections.
Management of infections due to multidrug-resistant gram-positive pathogens
represent a major therapeutic challenge in the transplant setting (Radunz 2011).
Overall incidence of fungal infections in LT recipients has declined due to early
identification and treatment of high-risk patients. However, overall mortality rate
for invasive candidiasis and aspergillosis remains high (Liu 2011).
The clinical symptoms of acute cellular rejection are non-specific, may not be
apparent or may manifest as fever, right upper quadrant pain, and malaise. A liver
biopsy is indispensable for confirming the diagnosis of acute rejection. High dose
corticosteroids (3 days of 500-1000 mg methylprednisolone) are the first-line
treatment for acute rejection.

Long-term complications after liver
transplantation
Management issues for the long term include opportunistic infections, chronic
ductopenic rejection, side effects due to immunosuppression including
cardiovascular complications and renal dysfunction, de novo malignancies, biliary
complications, osteoporosis and disease recurrence.

Opportunistic infections
Opportunistic infections in the medium and long term after LT are primarily viral
and fungal in origin. Opportunistic bacterial infections are uncommon after 6
months in patients receiving stable and reduced maintenance doses of
immunosuppression with good graft function.
Cytomegalovirus (CMV) infection plays an important role in the LT setting
(Andrews 2011) (Figure 3). Current guidelines recommend antiviral prophylaxis
over pre-emptive therapy in preventing CMV disease in high-risk LT recipients
(CMV-seronegative recipients of organs from CMV-seropositive donors [D+/R-]).
Delayed-onset CMV disease occurs in 15-38% of CMV D+/R- LT patients with
prophylactic treatment over 3 months (Eid 2010). A controlled clinical trial
demonstrated that valganciclovir, an oral prodrug of ganciclovir, is as effective and
safe as intravenouos (IV) ganciclovir for the prophylaxis of CMV disease in solid
organ (including liver) transplant recipients (Paya 2004). In the kidney transplant
setting, the Impact study demonstrated a marked reduction in the incidence of CMV
disease extending valganciclovir prophylaxis from 100 to 200 days (Humar 2010).
However, side effects and financial burden of this prolonged approach need to be
considered. In cases of ganciclovir-resistant CMV disease, alternative therapeutic
options include CMV hyperimmune globulins, or in rare cases, antiviral medication
(foscarnet, cidofovir or leflunomide) (Eid 2010).
Occurrence of post-transplant lymphoproliferative disease (PTLD) in the first
year after solid-organ transplantation is typically related to Epstein-Barr virus
(EBV) infection. EBV-seronegativity of the recipient before infection, high EB viral
load, intensity of immunosuppression and young age have been reported as risk
factors for PTLD (Smets 2002). Outcomes have improved since rituximab has been
incorporated into treatment regimens (Kamdar 2011). Therapeutic management

Update in Transplant Hepatology 357
options include reduction of immunosuppression, rituximab, combination
chemotherapy, and adoptive immunotherapy.
Oral reactivation of human herpes simplex virus-1 (HSV-1) after LT is common.
Development of varicella-zoster virus (HHV-3) after LT is typically related to
intense immunosuppressive therapy and its therapy does not differ from the nontransplant setting. There is a potential role of human herpes virus (HHV)-6 and
HHV-7 as copathogens in the direct and indirect illnesses caused by CMV. To what
extent HHV-6 and HHV-7 may be directly causing symptomatic disease is not clear
(Razonable 2009).

Figure 3. Cytomegalovirus (CMV) infection of the upper gastrointestinal tract. A. Livertransplanted patient complaining of dysphagia and epigastric discomfort with multiple
longitudinal esophageal ulcers seen at upper endoscopy. B. Endoscopic findings of deep
esophageal ulcerations with fibrinoid necrosis in another immunocompromised patient. In both
cases, lesions were caused by CMV infection. Diagnosis depends on a positive mucosal
biopsy, which should include specimens from the ulcer margins and ulcer base. Hematoxylin
and eosin staining typically reveals "owl's-eye" cytoplasmic and intranuclear inclusion bodies.

Chronic ductopenic rejection
Advances in immunosuppressive regimens have greatly reduced the incidence of
chronic ductopenic rejection and allograft failure. Chronic rejection begins within
weeks to months or years after LT and affects about 4% to 8% of patients
(Neuberger 1999). The most widely recognized manifestation of chronic rejection is
obliterative arteriopathy and damage or loss of small ducts (Demetris 1997).
Chronic rejection may appear indolently and might only become apparent as liver
test injury abnomalities (GGT, AP, bilirubin, transaminases). The diagnosis needs to
be confirmed by histopathologic examination. Switching the baseline
immunosuppression from cyclosporine A (CSA) to tacrolimus (TAC) and initiating
mycophenolate mofetil (MMF) rescue therapy represents a treatment option in these
patients (Daly 2002).

358 Hepatology 2012

CNI-induced nephrotoxicity and alternative
immunosuppressive protocols
Despite the introduction of new immunosuppressive agents (Table 4), calcineurin
inhibitors (CNI) remain the key drugs of most immunosuppressive regimens. Both
CSA and TAC inhibit the calcineurin-calmodulin complex and therefore IL-2
production. Renal failure, mainly due to CNI nephrotoxicity, is the most common
complication following orthotopic LT. The incidence of chronic renal dysfunction
has been reported in up to 70% of patients in the long term after LT (Afonso 2008,
Ziolkowski 2003). End stage renal disease has been described to occur in 18% of
patients during a post-transplant follow-up of 13 years (Gonwa 2001).
In LT patients with CNI-induced nephrotoxicity, a complete replacement of CNI
with conversion to MMF has shown conflicting results with respect to occurence of
rejection ranging between 0% and 60% (Creput 2007, Moreno 2003, Schmeding
2011, Moreno 2004). MMF inhibits inosine monophosphate dehydrogenase, a
critical enzyme in the de novo pathway of purine synthesis. Results from previous
studies with immunosuppressive regimens including MMF and minimal CNI
treatment suggest a significant improvement in renal function in this patient group
(Beckebaum 2011, Cicinnati 2007a, Beckebaum 2004a, Cantarovich 2003, Garcia
2003, Raimondo 2003).
De novo immunosuppression with MMF combined with induction therapy and
delayed CNI introduction is another approach to reduce CNI-related nephrotoxicity
especially in patients with higher MELD score or significant renal dysfunction. In a
randomized clinical trial, a daclizumab/MMF/delayed low-dose TAC-based
regimen was compared with a standard TAC/MMF regimen (Yoshida 2005). In
both study arms, corticosteroids were tapered over time. Statistically significant
higher median GFR were found in the delayed CNI group, although acute rejection
episodes were not statistically significant different in either group. Similar results
have been found in two retrospective studies in LT patients receiving thymoglobulin
induction therapy and delayed initiation of CNI (Bajjoka 2008, Soliman 2007). The
group from Regensburg initiated a single arm pilot study to determine the safety and
efficacy of a CNI-free combination therapy (basiliximab induction/MPA and
delayed [10 days post-transplant] sirolimus [SRL]) in patients with impaired renal
function (GFR <50 ml/min and/or serum creatinine >1.5 mg/dL) at LT
(Schnitzbauer 2010a). The study design stipulates that if at least 8 of the 9 patients
do not present with steroid-resistant acute rejection within 30 days after LT, an
additional 20 patients will be included. Results from this “bottom-up”
immunosuppressive strategy need to be awaited to determine if this strategy works
to prevent or avoid further renal dysfunction during follow-up after LT. Moreover, a
flaw of combined MMF and mTOR inhibitors therapy are agonistic side effects
such as bone marrow suppression, which may limit their combined use in a
substantial proportion of patients.
Another approach to maintain renal preservation is replacement of CNI by mTOR
inhibitors such as SRL or everolimus (EVL) (Sanchez 2005, Harper 2011, Saliba
2011, Kawahara 2011). Reported side effects of mTOR inhibitors include increased
incidence of wound infection and dehiscence, hepatic artery thrombosis,
hyperlipidemia, thrombocytopenia, leucopenia, and anemia. The antifibrotic effect
of mTOR inhibitors may provide an explanation for impaired wound healing
(Watson 1999).

Update in Transplant Hepatology 359
In a recently published randomized controlled study, patients were treated with
CSA for the first 10 days, then randomized to receive EVL plus CSA up to day 30,
and then either continued on EVL monotherapy (EVL group) or maintained on CSA
with or without MMF (control group) (Masetti 2010). One-year results showed that
MDRD was significantly better in the EVL monotherapy group as compared to the
control group. Results from two multicenter, randomized, Phase III studies
(ClinicalTrials.gov Identifiers: NCT00378014 & NCT00622869) comparing EVLbased regimen versus CNI-based regimen in de novo LT recipients will add further
information with respect to the role of mTOR inhibitors for renal preservation after
LT.
Table 4. Clinically used immunosuppressive agents in liver transplantation.
Immunosuppressant Class

Immunosuppressive Agent

Corticosteroids

Prednisone, prednisolone, methylprednisolone

Calcineurin inhibitors

Cyclosporin A, tacrolimus

Antimetabolites

Mycophenolate mofetil, azathioprine

mTOR Inhibitors

Sirolimus, everolimus

Polyclonal antibodies

Antithymocyte globulin (ATG)

Monoclonal anti-CD3 antibodies

Muromonab-CD3 (OKT3)

Chimeric monoclonal antibodies

Anti-IL-2 inhibitors (basiliximab)

Monoclonal anti-CD52 antibodies

Alemtuzumab (campath-1H)

Other side effects of CNI
Beside potential nephrotoxicity, CNI therapy is associated with side effects that
include cardiovascular complications, tremor, headache, electrolyte abnormalities,
hyperuricemia, hepatotoxicity, and gastrointestinal symptoms. Neurotoxicity,
including tremor, paresthesia, muscle weakness, and seizures, more often occurs in
TAC-treated patients; whereas gingival hyperplasia, as a rare event, and hirsutism
are associated with CSA treatment.
Cardiovascular side effects due to CNI and steroids include hyperlipidemia,
arterial hypertension, and diabetes (Beckebaum 2004b).
The prevalence of new-onset diabetes mellitus after LT has been reported to occur
in 9-21% of patients (John 2002, Konrad 2000). The prevalence of post-transplant
diabetes is even higher if cofactors such as hepatitis C are present. In various
studies, the diabetogenic potential has been reported to be higher in patients
receiving TAC than in those receiving CSA. In contrast, CSA has a more
pronounced effect on lipid levels. CSA can act by modulating the activity of the
LDL receptor or by inhibiting the bile acid 26-hydroxylase that induces bile acid
synthesis from cholesterol.

Corticosteroid minimization/avoidance protocols
There is ongoing discussion of steroid avoidance due to dyslipidemia, osteoporosis,
development of cataracts, weight gain, hypertension, and a deleterious impact on
glucose control. A recently published literature review (Lerut 2009) analysed the
actual status of corticosteroid minimization protocols in LT based on a detailed
analysis of 51 peer- and 6 non-peer-reviewed studies. Results from the majority of

360 Hepatology 2012
studies showed that these protocols have clearly metabolic benefits and are safe
with respect to graft and patient survival. Other research groups have reported
encouraging findings with steroid-free protocols including basiliximab induction
therapy (Filipponi 2004, Llado 2008, Becker 2008). A steroid-free alemtuzumab
induction regimen resulted in less hypertension and rejection but with more
infectious complications. So far, the overall benefit of alemtuzumab induction in LT
recipients remains questionnable (Levitsky 2011).

Figure 4. Non-melanoma skin scancers and liver transplantation (LT). Organ transplant
recipients have an increased risk of development of non-melanoma skin cancers as compared
to the non-transplant setting. Premalignant lesions such as actinic keratoses [A] are
predominantly located on sun-exposed areas. Squamous cell carcinoma [B,C] is the most
frequent skin cancer after LT followed by basal cell carcinoma [D] (Photographs kindly provided
by PD Dr. Hillen, Transplant Dermatology Outpatient Unit, Department of Dermatology,
University Hospital Essen, Germany).

De novo malignancies
Incidence of malignancies is higher in transplant patients and depends on the length
of follow-up, characteristics of the transplant population, choice of
immunosuppression and era in which the LT was performed (Buell 2005, Fung
2001). A cumulative risk has been reported of 10%, 24%, 32% and 42% at 5, 10, 15
and 20 years, respectively, for development of de novo cancers after LT
(Finkenstedt 2009). The highest risks in the transplant setting are nonmelanoma skin
cancers, mainly squamous cell carcinoma and basal cell carcinoma (Figure 4).
Premaligant lesions such as actinic keratoses are mostly located on sun-exposed
areas. Squamous cell carcinoma and basal cell carcinoma are increased by factors of
~65-200 and ~10, respectively, in organ transplant recipients as compared to the
immunocompetent population (Ulrich 2008). An annual routine dermatological

Update in Transplant Hepatology 361
follow-up exam, limitation of sun exposure and protective measures including
sunscreens are highly recommended for transplant patients.
A higher incidence of colon cancer in patients with inflammatory bowel disease
who are transplanted for PSC has been reported in the literature (Hanouneh 2011). It
is therefore necessary to maintain an adequate colonoscopic surveillance in these
patients even in the absence of bowel symptoms or active disease at regular
intervals (Fevery 2011). A trend has been recently reported toward an increased
incidence of advanced colon polyps and colon carcinoma in patients transplanted for
other diseases than PSC after LT. However, larger studies are needed to determine
whether post-transplant colon cancer surveillance should be performed more
frequently than in the non-transplant setting (Rudraraju 2008).
Recent studies reported a significantly higher incidence of aerodigestive cancer
including lung cancer among patients who underwent LT for alcohol-related liver
disease (Vallejo 2005, Jimenez 2005). A Spanish transplant group recommended
annual screening for oropharyngeal tumors in patients with a history of alcohol
overconsumption (Benlloch 2004). SRL exerts antiangiogenic activities that are
linked to a decrease in production of vascular endothelial growth factor (VEGF) and
to a markedly inhibited response of vascular endothelial cells to stimulation by
vascular endothelial growth factor (VEGF) (Guba 2002). Furthermore, the ability of
SRL to increase the expression of E-cadherin suggests a mechanism for blocking
regional tumor growth and for inhibiting metastatic progression. Therefore, we give
special consideration for mTOR inhibitor-based immunosuppressive regimens not
only patients transplanted for HCC but also those with de novo malignancies after
LT.

Biliary complications
Biliary leaks generally occur as an early post-transplant complication. In patients
with biliary stones, endoscopic sphincterotomy and stone extraction are the
treatment of choice.
Biliary strictures are one of the most common complications after LT, with a
reported incidence of 5.8-34% (Graziadei 2006). Early anastomotic strictures (AS)
usually have a technical origin; while strictures appearing later have a multifactorial
origin. Nonanastomotic strictures (NAS) without underlying hepatic artery
thrombosis are commonly referred to as ischemic-type biliary lesions (ITBL).
Risk factors for ITBL include preservation-induced injury, prolonged cold and
warm ischemia times, altered bile composition, ABO blood incompatibility and
immunological injury (Verdonk 2007, Buis 2009).
Endoscopic retrograde cholangiography (ERC) or percutaneous transhepatic
cholangiography (PTC) have typically been used as the primary approach, leaving
surgical intervention for those who are nonresponsive to endoscopic interventions or
who have diffuse intrahepatic bile duct damage. Novel radiological methods such as
magnetic resonance cholangiopancreaticography (MRCP) have been introduced as
an additional diagnostic tool for biliary complications.
The long-term efficacy and safety of endoscopic techniques have been evaluated
in various transplant centers (Qin 2006, Zoepf 2006, Pascher 2005).
Nonanastomotic strictures are commonly associated with a less favourable response
to interventional endoscopic therapy in comparison to anastomosis stenosis (Figure
5). An Austrian group found anastomotic strictures in 12.6% of patients transplanted

362 Hepatology 2012
between October 1992 and December 2003 and nonanastomic strictures in 3.7%
during a mean follow-up of 53.7 months after LT (Graziadei 2006). Interventional
endoscopic procedures were effective in 77% of patients with anastomosis stenosis;
whereas treatment of nonanastomotic strictures showed long-term effectiveness in
63% of patients. A surgical approach was required in 7.4% of transplant recipients.

Figure 5. Biliary tract complications after liver transplantation. A. Endoscopic retrograde
cholangiography (ERC) showing post-transplant short filiform anastomotic biliary stricture in a
46-year-old patient transplanted for HCV and alcohol-related cirrhosis 6 months earlier.
Therapy sessions include dilatation and an increasing number of bile duct endoprostheses at
short intervals of every 2-3 months. Prior to endoscopic therapy an endoscopic spinkterotomy is
performed. B. ERC of a 41-year-old patient transplanted for HCV diagnosed with ischemic-type
biliary lesions (type 3) with long nonanastomotic stricture extending proximally from the site of
the anastomosis and strictures throughout the entire liver.

Zoepf et al. (2006) retrospectively analyzed results from 75 transplanted patients
undergoing ERC for suspected anastomic strictures. Balloon dilatation alone and
combined dilatation and endoprotheses placement was efficacious in 89% and 87%
of cases respectively, but recurrence occurred in 62% and 31% of cases
respectively. We therefore use dilatation plus stenting with endoscopic reassessment
in anstomotic strictures. Repeated ERC sessions are performed with increasing
endoprothesis diameter in trimonthly time intervals and double or triple parallel
stenting in selected cases. Up to 75% of patients are stent-free after 18 months of
endoscopic intervention (Tung 1999).
Medical treatment for bile duct strictures consists of UDCA and additional
antibiotic treatment in stricture-induced cholangitis. Complications related to
bilioenteric anastomosis require PTC or surgical intervention.

Metabolic bone disease
Liver cirrhosis and therapy with corticosteroids are risk factors for the development
of osteoporosis. Screening with bone densitometry should therefore begin prior to
LT (Wibaux 2011). A further increase in bone turnover has been described after LT
and may be associated with resolution of cholestasis, increased parathormone
secretion and/or CNI administration (Moreira Kulak 2010). Metabolic bone disease

Update in Transplant Hepatology 363
is therefore a common cause of morbidity after LT. Factors such as vitamin D
deficiency, hypogonadism, secondary hyperparathyroidism and adverse lifestyle
factors should be addressed and corrected. There are no specific therapies for posttransplant osteoporosis other than for nontransplanted patients. General
interventions to reduce fracture risk include adequate intake of calcium and vitamin
D. Bisphosphonates are currently the most effective agents for treatment of posttransplant osteoporosis (Moreira Kulak 2010) (www.dv-osteologie.org). A metaanalysis and systematic review of randomised controlled trials demonstrated that
bisphosphonate therapy within the first 12 months after LT is associated with
reduced accelerated bone loss and improved bone mineral density at the lumbar
spine (Kasturi 2010).

Recurrent diseases after liver transplantation
Disease recurrence may occur in patients transplanted for viral hepatitis, tumor
disease, autoimmune or cholestatic or alcohol-related liver diseases. With universal
recurrence of HCV in all replicative patients, hepatitis C continues to pose one of
the greatest challenges for preventing disease progression in the allograft.

Recurrence of hepatitis B in the allograft
Combined use of hepatitis B immunoglobulin (HBIG) and nucleos(t)ide analogs has
emerged as treatment of choice in transplant HBV recipients (Figure 6) (Yan 2006,
Marzano 2005, Cai 2011) and its efficacy has been investigated extensively. HBV
recurrence using combined prophylactic regimens is less than 5%. However,
recurrence rates differ among various studies as most of them are small, with
varying proportions of patients with active viral replication at LT and varying
follow-up periods after LT. Furthermore there is a high variability (dose, duration
and method of HBIG administration) in the prophylactic protocols. According to the
German guidelines (Cornberg 2011) patients receive 10,000 IU HBIG intravenously
(IV) in the anhepatic phase followed by 2000 IU during the first postransplant week.
For long-term HBIG prophylaxis, trough anti-HBs levels at or above 100 IU/L
should be maintained. Subcutaneous (SC) HBIG application has various advantages
over intramuscular (IM) and IV administration (Beckebaum 2008b, Yahyazadeh
2011) and can be administered in stable and compliant patients after the early posttransplant period. It is well tolerated and patients can perform injections in a home
setting, thus reducing physician consultation time. Nucleos(t)ide analogs given prior
to LT should be continued indefinitely post-transplant.
Economic issues have led to a controversial discussion of whether indefinite
passive immunisation is necessary and if nucleos(t)ide analog therapy is sufficient
for antiviral prophylaxis (Naoumov 2001, Buti 2007, Gane 2007, Angus 2007, Neff
2007, Lo 2005, Wong 2007, Nath 2006, Yoshida 2007, Weber 2010, Karlas 2011).
Post-transplant studies have described unacceptable 2-4 year rejection rates of
approximately 25-50% with LAM monotherapy and with no initial phase of HBIG
therapy (Table 5) (see http://hepatologytextbook.com/link.php?id=9) (Marzano
2001, Jiao 2007, Zheng 2006).
A prospective, open-label, multicenter study was conducted on the safety and
efficacy of combined LAM/ADV therapy in HBsAg-positive LT recipients (Gane
2007) (Table 5) (see http://hepatologytextbook.com/link.php?id=9). Patients with

364 Hepatology 2012
clinical and virologic LAM resistance were excluded. Combined nucleos(t)ide
analog treatment started upon wait-listing. The median duration of antiviral therapy
prior to LT was 3.6 months. HBIG (800 IU IM) was administered for only one week
post-transplant. During the study, 19 patients were transplanted, and of those, none
had recurrent HBV during a median follow-up of 11.7 months.

Figure 6. Prophylaxis of HBV recurrence after liver transplantation (LT). Combined use of
nucleos(t)ide analog(s) and hepatitis B immunoglobulin (HBIG) is the current gold standard for
prophylaxis of HBV reinfection after LT. HBIG therapy can be withdrawn in the long term after
LT in selected low-risk (HbsAg-negative) cases. Those who are anti-hepatitis B core (anti-HBc)positive and without detectable anti-hepatitis B surface (anti-HBs) titers or anti-HBs titers <100
IU/L should be vaccinated according to the German Guidelines (Cornberg 2011). In case of no
or little response (anti-HBs <100 IU/L) to vaccination, lamivudine (LAM) monotherapy can be
initiated. In patients who have protective anti-HBs titers of >100 IU/L, antiviral therapy is not
necessary but long-term monitoring of HBV serology including anti-HBs titers is required. Neg.,
negative; pos., positive.

The same group conducted a study comparing patients who were switched from
HBIG/LAM to LAM/ADV versus those who maintained on HBIG/LAM therapy
(Table 5) (see http://hepatologytextbook.com/link.php?id=9) (Angus 2007). One
patient in the switch group became HBsAg-positive, but remained HBV DNAnegative after 5 months; all others remained HBsAg- and HBV DNA-negative at a
median of 17.2 months from randomization.
In another study, sixteen patients with LAM resistance who had treatment at LT
with LAM plus ADV therapy were investigated (Lo 2005). Half of the patients were
administered HBIG for a median of 24 months. None of them had detectable HBV
DNA, 13 were HBsAg-negative, and 2 without combined HBIG therapy maintained
HBsAg-positive after a follow-up period of 7.7 and 9.5 months, respectively.
A small cohort of non-HBV replicating patients who were switched from
HBIG/LAM (150 mg/d) to ADV (5 mg/d)/LAM (150 mg/d) therapy after a mean
post-LT period of 6.5 months was retrospectively investigated (Neff 2007) (Table 5)

Update in Transplant Hepatology 365
(see http://hepatologytextbook.com/link.php?id=9). The mean length of follow-up
from therapy switch was 21 months. They found that none of the patients showed an
increase in transaminases while on dual nucleos(t)ide analog therapy. Although the
authors mentioned that HBV serologic testing was performed, no results were given
post-therapy switch.
In a recently published study from Hong Kong, HBIG-free monoprophylaxis with
ETV was evaluated. Only 26% of patients had undetectable HBV DNA at the time
of LT. HBsAg loss occurred in 91% within 2 years post-transplant but 13% had
reappearance of HBsAg and 22.5% were HBsAg-positive at the time of their last
follow-up visit (Fung 2011).
The efficacy of a switch after at least 12 months of HBIG/LAM to combination
therapy with an oral nucleoside and nucleotide analog was investigated (Saab 2011).
Estimated HBV reinfection rate was 1.7% at 1 year after HBIG withdrawal.
HBV prophylactic postransplant studies to date are limited, small and with short
follow-up periods (Table 5) (see http://hepatologytextbook.com/link.php?id=9).
Larger prospective studies are needed to show if nucleos(t)ide analogs can be safely
applied in the majority of HBV transplant patients against recurrent hepatitis B
infection. Presently, withdrawal of HBIG prophylaxis and maintenanance with
nuclesos(t)ide analog therapy can be considered in stable HBsAg-negative patients
in the long term post-LT.
There is no rationale for continuing HBIG therapy in case of viral breakthrough
with detectable HBV DNA. The choice of antiviral therapy in patients with HBV
recurrence depends on the current antiviral medication, the viral load, and the
resistance profile. Antiviral drug resistance can easily be established by genotypic
assays that identify specific mutations known to be associated with decreased
susceptibility to particular drugs.

Recurrence of hepatitis C in the allograft
The influence of HCV infection on allograft histology is highly variable. The liver
injury can vary from absent or mild disease despite high viral burden to cirrhosis in
the allograft (approximately 20-30% of recipients within 5-10 years of follow-up)
(Rubin 2011). Patient and graft survival in HCV-infected transplant recipients is
worse compared to those with other indications (Berenguer 2007, Forman 2002,
Testa 2000). After a diagnosis of cirrhosis, the decompensation risk appears to be
accelerated (17% and 42% at 6 and 12 months, respectively) (Berenguer 2000) and
patient survival is significantly decreased (66% and 30% at 1 and 5 years,
respectively) (Saab 2005). Female gender has been reported as a risk factor for
advanced recurrent HCV disease and graft loss after LT (Lai 2011). Several other
factors have been suggested that may accelerate HCV reinfection of the allograft
(Belli 2007, Berenguer 2003, Iacob 2007, Saab 2005) (Table 6).
There are insufficient and somewhat controversial data regarding the relationship
between immunosuppressive agents and clinical expression of HCV recurrence
(Table 7) (Berenguer 2011). TAC and CSA do not seem to be significantly different
(Berenguer 2006a, Lake 2003, Martin 2004, Berenguer 2011) with respect to their
impact on the course of hepatitis C recurrence. Results from the multicenter ReViSTC cohort study, conducted in 14 Spanish liver centres, revealed that CSA-based
immunosuppression regimens may be advantageous against viral relapse after

366 Hepatology 2012
antiviral therapy as compared to TAC-based immunosuppression (ReViS-TC Study
Group 2011).
Table 6. Factors that may accelerate histological progression in HCV patients after
liver transplantation.
Donor factors

Recipient factors

Age
Liver graft steatosis

Surgical factors (cold/warm ischemia time)
Age
Gender
Non-caucasian race
High viral load pre-transplant/early post-transplant
Genotype 1b
Muromonab-CD3 (OKT3®)
Bolus corticosteroids
Rapid tapering of corticosteroids

Various studies have demonstrated that slowly tapering corticosteroids over time
may prevent progression to severe forms of recurrent disease (Brillanti 2002,
McCaughan 2003).
Induction with MMF has been associated with more severe recurrence of HCV
(Berenguer 2003). Other investigators have found that MMF has no impact on
patient survival, rejection, or rate of viral recurrence in HCV-infected transplant
recipients based on biochemical changes and histological findings (Jain 2002). In a
recent systematic review, recurrent HCV was less severe in 5/9 studies with AZA
compared with 2/17 with MMF (Germani 2009). Significantly better patient
survival and graft survival was shown for HCV-infected patients treated with MMF,
TAC, and steroids than for patients treated only with TAC and steroids, with 4-year
patient survival rates of 79.5% vs. 73.8% and 4-year graft survival rates of 74.9%
vs. 69.5% (Wiesner 2005). MMF in combination with a CNI taper for 24 months
had a positive effect on fibrosis progression, graft inflammation, and alanine
aminotransferase levels (Bahra 2005). This may be due to the antifibrotic effects of
MMF through an antiproliferative effect on myofibroblast-like cells.
Interleukin-2 receptor inhibition can be safely used in HCV transplanted patients
(Togashi 2011, Klintmalm 2007, Calmus 2002) while OKT3 has shown to increase
severity of HCV recurrence resulting in impaired patient and allograft survival
(Rosen 1997).
Sufficient data from randomised controlled studies in HCV patients are lacking
with respect to the role of mTOR inhibitors in patients and graft outcome
(Samonakis 2005, Schacherer 2007, Asthana 2011). Results of one study suggest
that de novo SRL-based immunosuppression does not significantly affect the
severity of HCV recurrence (Asthana 2011). A lower fibrosis extent and rate of
progression was found among HCV transplant recipients with SRL as primary
immunosuppression as compared to controls with an SRL-free immunsosuppressive
regimen (McKenna 2011).

Update in Transplant Hepatology 367
Table 7. Factors that may accelerate histological progression in HCV patients after
liver transplantation.
Immunosuppressive agent

Severity of HCV recurrance

Calcineurin inhibitors

No difference between cyclosporin A and tacrolimus

Bolus corticosteroids

Higher risk of fibrosis progression, increase in viral load

Azathioprine

Controversial debate

Mycophenolate mofetil

Controversial debate

Monoclonal CD3-antibodies
(OKT3)

Increased risk of graft failure

Interleukin-2 receptor antibodies

No disadvantage on graft survival

mTOR inhibitors

Controversial (reduction of vs. no impact on fibrosis
progression)

Regular histological evaluation of post-transplant chronic hepatitis C in 1-year (or
maximum 2-year) intervals is recommendable to determine the grade of
inflammation and stage of fibrosis. In particular, the biopsy result is important for
therapy decision, to exclude signs of rejection prior to antiviral therapy and to
determine the efficacy of antiviral therapy. In addition, there are some published as
well as ongoing studies evaluating the role of non-invasive measurement of fibrosis
in HCV and non-HCV transplant recipients (Cross 2011, Beckebaum 2010).
IFN α and RBV therapy may prevent the development of HCV graft cirrhosis
(Hashemi 2011, Gordon 2009, Cicinnati 2007b). This treatment is however
associated with more side effects and is far less effective than in the non-transplant
setting. The most applicable treatment strategy is treatment of established HCV
recurrence with PEG-IFN α and ribavirin, which results in an SVR of 20-30%
(Gordon 2009). Preemptive antiviral therapy (Shergill 2005, Sugawara 2004,
Chalasani 2005, Bzowej 2011) has not shown superior effects as compared to
established HCV therapy (Berenguer 2008, Chalasani 2005, Bizollon 2005, Castells
2005, Toniutto 2005) and should only be considered in cases of rapid progression of
HCV infection in the early post-transplant period. Most published studies in the
transplant setting are not controlled, monocentric and/or comprise a small patient
cohort (Shergill 2005, Sugawara 2004, Gane 1998, Kizilisik 1997, Ghalib 2000).
Recently published results indicate that RBV pre-treatment increased the
tolerability of the antiviral treatment, and improved its efficacy in LT patients
(Merli 2011).
The NS3/4 protease inhibitors telaprevir and boceprevir are metabolized primarily
by the cytochrome P450 CYP3A4. Coadministration with CNI metabolised
primarily by CYP3A4 has shown to result in increased dose-normalized CSA and
TAC exposure by ~4.6-fold and ~70-fold (Garg 2011).
Optimal onset, dose and duration of therapy are not known yet. Positive predictive
factors for SVR include use of erythropoietin, patient compliance, treatment with
PEG-IFN (versus standard IFN) and an early histological response (Berenguer
2006b).
The proportion of patients who need a dose reduction of their antiviral therapy
due to anemia or leucopenia may be reduced by the use of erythropoietin or
granulocyte-macrophage colony-stimulating factor (not approved for this
indication). Reported risk of rejection is low if antiviral therapy is closely monitored

368 Hepatology 2012
(Gane 1998, Kizilisik 1997). Therapy needs to be withdrawn in case of significant
histologically-proven rejection.

Recurrence of cholestatic liver diseases and autoimmune
hepatitis
Data about the frequency of recurrent cholestatic and AIH-related liver disease vary
in the literature depending on the follow-up period and criteria chosen for definition
of disease recurrence.
The post-transplant prognosis for PBC patients is excellent, with an
approximately 80% 5-year survival reported by most large centres (Carbone 2011,
Silveira 2010). It has been reported that HLA-A, -B, and -DR mismatches between
the donor and the recipient decrease the risk of disease recurrence in PBC patients
(Morioka 2007a, Hashimoto 2001). A recently published study reported recurrent
PBC in one-third of patients at 11-13 years post-transplant (Charatcharoenwitthaya
2007). Various other studies reporting recurrent PBC are depicted in Table 8 (Jakob
2006, Liermann-Garcia 2001, Montano-Loza 2010, Hytiroglou 2008).
Table 8. Recurrence rates in patients transplanted for autoimmune-related or
cholestatic liver disease.
Reference

Patients, n

Follow-up after liver
transplantation

Recurrence
rate

AIH

Duclos-Vallée 2003

17

>120 months

41%

AIH

Prados 1998

27

mean 44 months

33%

AIH

Molmenti 2002

55

median 29 months

20%

AIH

Campsen 2008

66

median 81 months

36%

AIH

Vogel 2004

28

mean 100 months

32%

PBC

Charatcharoenwitthaya 2007 154

mean 130 months

34%

PBC

Jakob 2006

100

up to 17 years

16%

PBC

Liermann-Garcia 2001

400

mean 56 months

17%

PBC

Montano-Loza 2010

108

mean 88 months

26%

PBC

Hytiroglou 2008

100

mean 44 months

16%

PSC

Cholongitas 2008

69

median 110 months

13%

PSC

Alabraba 2009

230

median 55 months

24%

PSC

Vera 2002

152

median 36 months

37%

PSC

Graziadei 1999

150

mean 54 months

20%

PSC

Goss 1997

127

mean 36 months

9%

Diagnosis of PBC in the transplanted liver is usually more challenging than
diagnosis in the native liver. Immunoglobulin M and anti-mitochondrial antibodies
(AMA) often persist, and elevated cholestatic enzymes may be due to other causes
of bile duct damage such as ischemic cholangiopathy or chronic ductopenic
rejection. Recurrent PBC is a histological diagnosis, typically appearing as
granulomatous cholangitis or duct lesions. The frequency of recurrence will be
considerably underestimated if a liver biopsy is carried out only when clinical
features are apparent.
Some investigators have found that CSA-based immunosuppressive therapy is
associated with lower PBC recurrence rates as compared to TAC-based

Update in Transplant Hepatology 369
immunosuppression (Wong 1993, Montano-Loza 2010). However, long-term
survival has been shown to be not significantly different between CSA-and TACtreated patients (Silveira 2010). The impact of UDCA on the natural history of
recurrent disease remains unknown. In the Mayo Clinic transplant cohort, 50% of
recurrent PBC patients receiving UDCA showed normalization of serum alkaline
phosphatase and alanine aminotransferase levels over a 36-month period compared
with 22% of untreated patients (Charatcharoenwitthaya 2007). Although no
significant differences in the rate of histological progression could be detected
between the treated and untreated subgroups, the proportion of individuals with
histological progression was significantly lower in those that showed improvement
of biochemical parameters regardless of treatment.
The reported recurrence rate for PSC after LT ranges between 9% and 37%
(Cholongitas 2008, Alabraba 2009, Vera 2002, Graziadei 1999, Goss 1997). A
British liver transplant group found significantly better recurrence-free survival
rates in patients who underwent colectomy before or during LT and in those with
with non-extended donor criteria allografts (Alabraba 2009).
Recurrent PSC is diagnosed by histology and/or imaging of the biliary tree and
exclusion of other causes of nonanastomotic biliary strictures. Histopathological
findings in PSC include fibrous cholangitis, fibro-obliterative lesions, ductopenia,
and biliary fibrosis. In a study conducted by the Mayo clinic, recurrence of PSC was
defined by strict cholangiographic and histological criteria in patients with PSC, in
whom other causes of bile duct strictures were absent (Graziadei 2002). However,
due to the lack of a histological gold standard, the diagnosis of PSC recurrence is
based primarily on cholangiographic features. Due to its responsiveness to steroid
therapy, IgG4-associated cholangitis instead of suspected or recurrent PSC should
be considered in patients with atypical features including history of pancreatitis.
Interestingly, despite immunosuppression, a significantly higher corticosteroid
requirement was reported in the transplant as compared to the nontransplant setting,
with 20% of PSC patients becoming corticosteroid dependent after LT (Ho 2005). A
recent study reported that maintenance steroids (>3 months) for ulcerative colitis
post-LT were a risk factor for recurrent PSC (Cholongitas 2008).
AIH recurrence has been reported in about one-third of patients within a posttransplant follow-up period of ≥5 years (Mendes 2011, Tripathi 2009, DuclosVallee 2003, Campsen 2008, Vogel 2004). Incidence increases over time as
immunosuppression is reduced (Prados 1998). A long-term follow-up study (>10
years) by a French group found AIH recurrence in 41% of the patients. The authors
recommended regular liver biopsies, because histological signs precede abnormal
biochemical liver values in about one-fourth of patients (Duclos-Vallee 2003). The
diagnosis of recurrent AIH may include histological features, the presence of
autoantibodies, and increased gamma globulins. The majority of published studies
did not confirm a post-transplant prognostic role of antibodies in patients
undergoing LT for AIH. Conflicting data exist regarding the presence of specific
HLA antigens that predispose patients to AIH recurrence after LT (Gonzalez-Koch
2001, Molmenti 2002). Histological signs of recurrence include interface hepatitis,
lymphoplasmacytic infiltration, and/or lobular involvement. In an analysis of data
from 28 patients with AIH, 5-year survival rate was not significantly different from
controls with genetic liver diseases (Vogel 2004). Patients had more episodes of
acute rejection though, in comparison to the control group.

370 Hepatology 2012
Patients with AIH typically receive low-dose steroid therapy after LT. The
transplant centre in Colorado that found that recurrence was not strongly influenced
by steroid withdrawal in their cohort attempts to minimise or stop steroid therapy in
AIH transplant patients (Campsen 2008).

Outcome in patients transplanted for hepatic malignancies
The results of early studies of LT for HCC were disappointing. More than 60% of
patients developed tumor recurrence within the first two transplant years (Ringe
1989). Currently, there are recurrence rates of 10-15% in patients fulfilling the
Milan criteria (Zavaglia 2005). In an analysis of predictors of survival and tumorfree survival in a cohort of 155 HCC LT recipients, histological grade of
differentiation and macroscopic vascular invasion were identified as independent
predictors of survival and tumor recurrence (Zavaglia 2005). Others identified
MELD score >22, AFP >400 ng/mL and age >60 years as negative predictors for
survival in HCC (Sotiropoulos 2008b, Jelic 2010). For patients having an indication
for LT despite exceeding the Milan criteria, the use of marginal grafts or
performance of LDLT has been considered as a reasonable option.
Expansion beyond the Milan criteria to University of California San Francisco
(UCSF) criteria (single tumour <6.5 cm; two to three tumours, none >4.5 cm or total
diameter <8 cm, no vascular invasion) or even more liberal criteria (no portal
invasion, no extrahepatic disease) have been discussed widely (Sotiropoulos 2007,
Silva 2011, Jelic 2010). Centers such as the San Francisco Transplant Group as well
as the UCLA Transplant Group have demonstrated 5-year survival rates of 50-80%
after LT for tumours beyond the Milan criteria but within UCSF criteria (Duffy
2007, Yao 2007).
Recently, the 'up to seven' criteria (with 7 being the sum of the size and number of
tumors for any given HCC) were suggested as an approach to include additional
HCC patients as transplant candidates. However, acceptance of a more liberal organ
allocation policy would result in a further increase of HCC patients on the waiting
list and in denying the use of these organs to other non-HCC patients.
Expansion of criteria in the LDLT setting is even more challenging due to the
donor risk and the risk of selection of tumours with unfavorable biology following
the concept of fast-tracking (Hiatt 2005). Novel molecular biology techniques, such
as genotyping for HCC, may become relevant for determining recurrence-free
survival and improving patient selection but these biomarkers can not yet been used
for clinical decision making.
Recently, a satisfactory outcome and potential survival benefit were reported in
studies and a meta-analysis of controlled clinical trials with SRL-based
immunosuppression in patients transplanted for HCC (Kneteman 2004, Zimmerman
2008, Toso 2007, Liang 2011). These results are in line with a retrospective analysis
based on the Scientific Registry of US Transplant Recipients, which included 2491
HCC LT recipients and 12,167 recipients with non-HCC diagnoses. Moreover, the
SILVER Study, a large prospective randomized controlled trial, comparing SRLcontaining versus SRL-free immunosuppression will provide further results and
details with respect to the impact of SRL on HCC tumour recurrence (Schnitzbauer
2010b).
Neoadjuvant chemoradiation and subsequent LT has shown promising results for
patients with localized, unresectable hilar cholangiocellular carcinoma (CCC) (Rea

Update in Transplant Hepatology 371
2005, Masuoka 2011). In a recently published US study, outcome of 38 patients
who underwent LT was compared to that of 19 patients who underwent combined
radical bile duct resection with partial hepatectomy (Hong 2011). Tumor was
located in intrahepatic bile duct in 37 patients and in hilar bile duct in 20 patients.
Results demonstrated that LT combined with neoadjuvant and adjuvant therapies is
superior to partial hepatectomy with adjuvant therapy. Challenges of LT attributable
to neoadjuvant therapy include tissue injury from radiation therapy and vascular
complications that include hepatic artery thrombosis. Predictors of response to the
neoadjuvant protocol prior to LT need to be determined (Heimbach 2008).
Increasing age, high pretransplant tumor marker, residual tumour size in the explant
>2 cm, tumour grade, previous cholecystectomy and perineural invasion were
identified as predictors of recurrence following LT (Knight 2007).
Metastatic lesions originating from neuroendocrine tumours (NET) may be
hormone-producing (peptide hormones or amines) or may present as nonfunctional
tumours (Frilling 2006, Lehnert 1998). They are characterized by slow growth and
frequent metastasis to the liver, and their spread may be limited to the liver for
protracted periods of time. Most studies in patients transplanted for NET are limited
and usually restricted to small numbers of patients. A recently published analysis
based on the UNOS database including patients transplanted for NET between
October 1988 and January 2008, showed that long-term survival of NET patients
was similar to that of patients with HCC. Excellent results can be obtained in highly
selected patients and a waiting time for LT longer than 2 months (Gedaly 2011).
Long-term results from prospective studies are needed to further define selection
criteria for patients with NET for LT, to identify predictors for disease recurrence,
and to determine the influence of the primary tumour site on patient post-transplant
survival.

Recurrent alcohol abuse after liver transplantation for
alcoholic liver disease
Alcoholic liver disease has become a leading indication of LT in Europe and the
United States. Patient and graft survival is excellent in those maintaining alcohol
abstinence after LT. Severe chronic alcohol consumption after LT significantly
decreases the medium- and long-term survival (Pfitzmann 2007). A recent study has
shown that urine ethyl glucuronide is a reliable marker for detection of alcohol
relapse after LT (Staufer 2011). Studies evaluating recurrent alcohol use have
reported a mean incidence of relapse in one-third of patients ranging from 10% to
50% in up to 5 years of follow-up (Burra 2005).
According to results from the European Liver Transplant Registry (ELTR),
mortality and graft failure were more often related to de novo tumors,
cardiovascular and social factors in alcoholic LT patients as compared to patients
transplanted for other etiologies (Burra 2010). The role of the length of pretransplant abstinence as a predictor of post-transplant abstinence has been widely
discussed. Many studies have assessed possible risk factors for alcoholic relapse
after LT. The following factors have been identified as risks for recurrent alcohol
abuse: a shorter length of abstinence before LT, more than one pretransplant alcohol
withdrawal, alcohol abuse in first relatives, younger age, and alcohol dependence
(Perney 2005). Accordingly, the results from the Pittsburgh Transplant Center
revealed that the prognosis regarding continued abstinence post-transplant is much

372 Hepatology 2012
more favourable for individuals with a diagnosis of overconsumption (abuse) than
for those who meet criteria for alcohol dependence (DiMartini 2008).
A recently published study reported that poorer social support, family alcohol
history, and pretransplant abstinence of ≤6 months showed significant associations
with relapse (Dew 2008). In addition, an Australian study identified the presence of
psychiatric comorbidities, or a score higher than 3 on the High-Risk Alcoholism
Relapse (HRAR) scale as factors predictive of relapse into harmful drinking (Haber
2007).

Experiences with liver transplantation in inherited
metabolic liver diseases in adult patients
LT is regarded as an effective treatment strategy for patients with Wilson’s Disease
which presents as deterioration of cirrhosis not responsive to treatment, as acute on
chronic disease or fulminant hepatic failure (Moini 2010). LT reverses the
abnormalities of copper metabolism by converting the copper kinetics from a
homozygous to a heterozygous phenotype, thus providing an adequate increase of
ceruloplasmin levels and a decrease of urinary copper excretion post-transplant. The
King's College Hospital reported excellent long-term results after LT in patients
who have undergone LT for Wilson’s Disease since 1994 with 5-year patient and
graft survival rates of 87.5% (Sutcliffe 2003). There are several reports in the
literature indicating a reversal of neurological symptoms after LT (Martin 2008).
However, the course of neurological symptoms remains unpredictable and it is still
a matter of debate if LT should be considered in patients with severe neurological
impairment (Pabón 2008).
Αlpha-1-antitrypsin (AAT) deficiency is a common genetic reason for pediatric
LT, but a rare indication in adults. The Z allele is most commonly responsible for
severe deficiency and disease. LT corrects the liver disease and provides complete
replacement of serum AAT activity. 567 AAT recipients who underwent LT
between 1995 and 2004 were retrospectively investigated (Kemmer 2008). Results
based on UNOS data revealed 1-, 3-, and 5-year patient survival rates of 89%, 85%,
and 83%, respectively.
In hemochromatosis, iron depletion therapy prior to LT may be associated with a
better outcome after LT and is therefore strongly recommended (Weiss 2007). It has
been reported that the survival of patients who undergo LT for hereditary
hemochromatosis is markedly lower in comparison to other indications (Dar 2009,
Brandhagen 2001). Reduced post-transplant survival in patients with
hemochromatosis has been attributed to cardiac problems and increased infectious
complications. Findings derived from the UNOS database revealed 1-year and 5year survival rates of 75% and 64% in patients with iron overload, as compared to
83% and 70% in those without iron overload (Brandhagen 2001). More recent
results from patients with hemochromatosis (n=217) transplanted between 19972006 revealed excellent 1-year (86.1%), 3-year (80.8%), and 5-year (77.3%) patient
survival rates, which were not different from those transplanted for other liver
diseases (Yu 2007).

Update in Transplant Hepatology 373

Outcome after liver transplantation for acute
hepatic failure
Acute hepatic failure (AHF) accounts for 5-12% of LT activity worldwide. Druginduced liver injury due to acetaminophen overdose is the most common cause of
LT for acute liver failure in developed countries (Craig 2010, Au 2011). Other
etiologies comprise idiosyncratic drugs (such as isoniazid/rifampicin, cumarins,
acetaminophen, ectasy, tricyclic antidepressants), Budd-Chiari syndrome, Wilson’s
Disease, hepatitis A, B and E infection or autoimmune disease.
Patients with acute fulminant liver disease should be transferred to an ICU at a
medical centre experienced in managing AHF, with LT capabilities. Bioartificial
hepatic devices may serve as bridging therapy to native liver recovery or to LT.
Early postoperative complications in patients transplanted for AHF include sepsis,
multisystem organ failure, and primary graft failure. Serum creatinine
concentrations above 200 µmol/L pretransplant, non-white race of the recipient,
donor body mass index >35 kg/m2 and recipient age >50 years have been suggested
as risk factors for post-transplant mortality (Wigg 2005). Others reported that
extended donor criteria rates and severe cerebral edema were associated with worse
outcome (Chan 2009). The Edinburgh LT centre investigated the impact of
perioperative renal dysfunction on post-transplant renal outcomes in AHF patients.
They found that older age, female gender, hypertension, CSA and nonacetaminophen-induced AHF but not the severity of perioperative renal injury were
predictive for the development of chronic kidney injury (Leithead 2011).
The results in patients transplanted for AHF have improved within the last decade
due to the establishment of prognostic models, improved intensive care management
and the option for LDLT which has a limited role in the US and Europe but plays a
major role in Asia (Lo 2008). AHF was the indication for LDLT in more than 10%
of the cohort reported by two Asian groups (Morioka 2007b, Lo 2004).
Available data document that survival in patients with AHF is inferior to that of
recipients with nonacute indications for LT within the first year but comparable in
the long-term (Chan 2009, Wigg 2005).

Conclusion
LT is challenging due to a shortage of organs and a prolonged waiting-list time. The
large disparity between the number of available cadaver donor organs and recipients
awaiting LT has created an ongoing debate regarding the appropriate selection
criteria. The rationale of allocation systems utilizing the MELD score is to prioritize
patients with severe liver dysfunction ("the sickest first"). This results in decreased
waiting list mortality from 20 to 10% in the Eurotransplant region but also in a
reduction of 1-year post-transplant survival by approximately 10%. A potential
modification of the MELD allocation system or rather development of an improved
prognostic scoring system incorporating donor-related factors, pretransplant
mortality and post-transplant outcome is urgently warranted to optimize organ
allocation in the future.
Due to the availability of antiviral drugs, the survival of patients undergoing LT
for HBV infection has dramatically improved and has become comparable to or
even better than the survival of patients with non-virus-related liver diseases. HBIG-

374 Hepatology 2012
free therapeutic regimens with new promising nucleos(t)ide analog combinations
are currently being investigated for their efficacy and safety as first-line therapy in
clinical studies.
HCV has become a leading indication for LT in Europe and the United States.
There is ongoing research aiming to define host or viral factors that predict
recurrence, the impact of immunosuppressive regimens, and the appropriate
timepoint and dosing for combined PEG-IFN and RBV therapy. The overall risk
and benefit of new antiviral treatment strategies including protease inhibitors remain
to be evaluated.
Data about the frequency of disease recurrence in cholestatic and autoimmune
liver diseases vary in the literature. Diagnosis of disease relapse in cholestatic and
autoimmune liver disease is more challenging than in the non-transplant setting.
Most studies report excellent medium-term and long-term results despite limited
therapeutic options for disease recurrence.
LT in HCC patients provides excellent outcomes and low recurrence rates
following the Milan criteria. Expansion of transplantation criteria beyond the Milan
criteria has been discussed at length. The acceptance of a more liberal organ
allocation policy may result in a further increase of the proportion of patients
transplanted for HCC and denying the use of these organs to other patients for
whom better results may be achieved. Recent developments in genomic and
proteomic approaches may allow the identification of new biomarkers for prediction
of HCC recurrence.
Non-use of alcohol of ≥6 months pretransplant is widely considered the
prerequisite time for listing for LT. There are few reliable predictors of relapse in
alcoholic patients after LT. Survival rates in patients with alcohol-related liver
disease are similar or even better when compared to the outcomes of patients who
undergo transplant for other types of chronic liver disease. In contrast, survival is
worse in patients with heavy alcohol consumption after LT.
The management of cardiovascular, renal, coagulopathic, cerebral and infectious
complications in patients with AHF is clinically challenging. Prognostic models are
helpful but not entirely accurate in predicting those who will require LT. Due to
advances in intensive care medicine and surgical techniques, outcomes for patients
with AHF have progressively improved during the last 2 decades.
Much attention has been directed to reducing CNI-associated long-term
complications. Cardiovascular comorbidities due to metabolic complications such as
diabetes mellitus, dyslipidemia, obesity, and arterial hypertension account for 3070% of long-term morbidity. Current trends of immunosuppressive strategies
include CNI-sparing/-free protocols including MMF- and/or mTOR-based
immunosuppressive regimens and corticosteroid-avoidance protocols. CNI delay
with induction therapy for bridging the early postoperative phase should be
considered especially in patients with high MELD scores. Finally, "individually
tailored immunosuppressive" protocols may optimize drug efficacy, minimise drug
toxicity and improve transplant outcome.

Update in Transplant Hepatology 375

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386 Hepatology 2012

23. End-stage Liver Disease, HIV Infection
and Liver Transplantation
José M. Miró, Fernando Agüero, Montserrat Laguno, Christian Manzardo,
Montserrat Tuset, Carlos Cervera, Neus Freixa, Asuncion Moreno, JuanCarlos García-Valdecasas, Antonio Rimola, and the Hospital Clinic OLT in
HIV Working Group

Introduction
Liver disease due to chronic hepatitis B and C is currently one of the leading causes
of morbidity and mortality among HIV-positive patients in the developed world and
its burden is increasing (Joshi 2011). Non-coinfected patients with chronic hepatitis
C tend to progress to end-stage liver disease (ESLD) in 20-30 years, whereas
coinfected patients have higher rates of progression (Mohsen 2003; Poynard 2003).
Furthemore, a higher overall adjusted relative risk (RR) of histological cirrhosis or
decompensated liver disease has been observed in patients coinfected with HIV and
the hepatitis C virus (HCV) when compared to HCV-monoinfected patients
(Graham 2001). End-stage liver disease (ESLD) in coinfected patients has become a
leading clinical state in many of these patients. Medical management is essential in
this scenario and should be considered as a bridge to organ liver transplantation
(OLT), which can play an important role as a therapeutic tool in this subset of
patients. We summarize recent developments in the management of ESLD.
Nevertheless this is an evolving field and many issues remain unclear (Miró 2011).

Epidemiology
Of the approximately 40 million persons infected with HIV globally, 2 to 4 million
are chronically infected with the hepatitis B virus (HBV) and 4 to 5 million have
chronic HCV (Alter 2006).
The prevalence of HCV and/or HBV coinfection is high in developed countries.
Studies performed in European HIV-positive patients showed rates of 33% and 9%,
respectively (Rockstroh 2005, Konopnicki 2005), while in the US figures are very
similar, 28% and 9% (Fung 2004). Other authors have addressed the significance of
HCV as a cause of non–AIDS-related death (Palella 2006, Crum 2006, Lewden
2005). One single-centre study (Martínez 2007) in Spain analysed the cause of 235

End-stage Liver Disease, HIV Infection and Liver Transplantation 387
deaths in 4471 patients (5%) on combination antiretroviral therapy (cART) from
1997 until 2004. The number of patients who died from ESLD increased from 8% in
1997 to 41% in 2004, and in recent years this condition has become the leading
cause of death in HIV-positive patients. In comparison with the general population
of a similar age, deaths due to liver disease were 11 times more frequent in HIVpositive patients. Another prospective multicenter study (Rosenthal 2007) in France
determined mortality due to ESLD in a nationwide population of HIV-positive
patients. The authors followed a total of 21,000 HIV-positive patients, 4000 (19.9%)
of whom were coinfected, and showed that, in 2003, mortality due to ESLD
represented 23.7% of non–AIDS-related deaths. In this population, ESLD was fatal
in 1.5% of patients in 1995, 6.6% in 1997, 14.3% in 2001, and 12.6% in 2003, and
92.6% of patients who died from ESLD had chronic HCV infection. Another
prospective study of 11 cohorts carried out in Europe, the United States and
Australia (D:A:D Study 2006) included 23,500 HIV-1–infected patients (22.5%
were HCV-positive) and followed them from December 1999 until February 2004.
This study showed that, of the 1250 deaths recorded, those related to AIDS were the
most frequent (31.1%), while liver disease was the most frequent non–AIDS-related
cause (14.5%). HCV infection was shown to be an independent predictor of liverrelated death (D:A:D Study 2006). As for hepatocellular carcinoma (HCC), one
study comparing liver-related deaths in HIV-positive patients (Salmon-Ceron 2009)
described an increase in mortality due to HCC from 15% in 2000 to 25% in 2005
(p=0.04).

Clinical features of coinfected patients with ESLD
154 patients with a new diagnosis of Child-Turcotte-Pugh class A compensated
cirrhosis were followed and 36 of them (23.4%) developed a first hepatic
decompensation during follow-up (mean 36 months) (Pineda 2009). The probability
of developing decompensated cirrhosis at 3 and 5 years was 26% and 33%,
respectively. Factors predicting the emergence of an episode of hepatic
decompensation at 5 years were Child-Turcotte-Pugh stage (HR, 3.33 [95% CI,
1.39-7.69]), lack of anti-HCV therapy (HR, 3.38 [95% CI, 1.14-5.04]), and baseline
CD4 cell count below 300 cells/mm3 (HR, 2.40 [95% CI, 1.09-10.53]).
In a retrospective study, the same authors (Pineda 2005) described the frequency
of specific events, such as first decompensation and cause of death in HIV-negative
and HIV/HCV-coinfected subjects. Ascites and jaundice were more frequent among
HIV-positive patients, while upper gastrointestinal bleeding and HCC were more
frequent in monoinfected patients. Hepatic encephalopathy (HE) as both first
decompensation and cause of death was higher in coinfected patients.
The clinical characteristics and outcome of spontaneous bacterial peritonitis
(SBP) were evaluated in an HIV-positive population with cirrhosis (Shaw 2006).
Thirty-five HIV-positive patients with cirrhosis were compared with 70 HIVnegative patients with cirrhosis. An aetiologic diagnosis was made in almost 80% of
the HIV-positive cases and bacteraemia was present in more than 50%, with both
rates being higher than those observed in HIV-negative patients. An important
bacteriological finding in this study was the high incidence of Streptococcus
pneumoniae as the etiologic agent of SBP among HIV-positive patients, second
only to Escherichia coli.

388 Hepatology 2012
HCC has a faster and worse outcome in HIV/HCV-coinfected patients than in
HCV-monoinfected patients (Puoti 2004, Bruno 2006, Berretta 2011). Of note, this
disorder is expected to rise significantly in patients with HIV infection and chronic
VHC/VHB hepatitis.
The findings of HCC in 41 HIV-positive and 2384 HIV-negative patients were
compared in an Italian study (Puoti 2004). The authors found a more aggressive
course of HCC in HIV-positive patients, with an independent association between
HIV infection and a more advanced stage of HCC at clinical presentation, in
addition to a higher rate of infiltrating neoplasm and extrahepatic-extranodal
metastasis. Furthermore, portal vein thrombosis was more frequent among HIVpositive patients with HCC.
A retrospective study from Canada and the US compared 63 HIV-positive
patients with HCC and 226 HIV-negative patients with HCC (Bräu 2007) and
revealed that HIV+ patients were younger and more frequently symptomatic. In this
cohort, median survival was similar between HIV-positive (7 months) and HIVnegative patients (7.5 months, p=0.44, log-rank), as was tumour stage.
Recently, these findings were confirmed grouping a comparison of 104 HIVinfected patients who had HCC with 484 HIV-uninfected patients with HCC
(Berretta 2011). The group of patients with HIV infection was younger, had better
BCLC stage at diagnosis and the median survival time was significantly shorter.
One of the factors independently associated with survival was the HCC diagnosis
via a screening program. However, there are no data available on the costeffectiveness of screening for HCC in cirrhotic patients with HIV infection (Joshi
2011).

Prognosis after decompensation
The survival rate of HIV-positive patients with decompensated cirrhosis is much
lower than that of HIV-negative patients – approximately 50% at 1 year (Pineda
2005, Merchante 2006, Murillas 2009). In a multicentre case-control study (Pineda
2005), the outcome of cirrhosis after the first decompensation in coinfected patients
was much worse than in the monoinfected population. Survival at 1, 2, and 5 years
for the coinfected/monoinfected population was 54%/74%, 40%/61%, and
25%/44%, respectively. In another study (Merchante 2006) severity of liver disease
(Child-Turcotte-Pugh score or HE as the first hepatic decompensation) and the level
of cellular immunosuppression (<100 CD4 cells) were identified as independent
predictors of poor outcome in coinfected patients. On the other hand, HAART was
associated with a reduced mortality rate.
104 HIV-positive patients with HCC or cirrhosis after their first hepatic
decompensation were analysed (Murillas 2009). The median survival time of this
cohort was 14 months, similar to that observed by Merchante (13 months). This
study included HCV and non–HCV-infected patients, and it did not find significant
differences in survival based on the aetiology of cirrhosis, suggesting that HIVpositive patients have an overall poor outcome regardless of the nature of their liver
disease. Furthermore, the MELD score and the inability to reach an undetectable
plasma HIV-1 viral load at any time during follow-up were the only variables
independently associated with the risk of death (p<0.001). This is particularly
relevant because the MELD score has been increasingly used to establish the

End-stage Liver Disease, HIV Infection and Liver Transplantation 389
prognosis of patients with cirrhosis and, consequently, to indicate liver
transplantation.
Recently, a Spanish study (Lopez-Diéguez 2011) showed that the mortality rate
for patients with decompensated cirrhosis was 27.1 deaths/100 person years and 4.0
deaths/100 person years for patients with compensated cirrhosis. The risk of first
hepatic decompensation was relatively low but the time from the first liver
decompensation to the next decompensation was critically reduced. HIV-positive
patients with cirrhosis have a poor prognosis after the development of SBP (Shaw
2006). HIV infection was associated with a more than 6-fold increase in the
probability of dying within a month of the first episode of SBP. Impaired renal
function at diagnosis and severity of liver disease were identified as predictors of
death. HIV-positive patients also had a dramatically shorter survival time than HIVnegative patients: only 50% of patients were still alive 3 months after the first
episode of SBP and only 23% were alive after 1 year. Death was mostly related to
complications of advanced liver disease rather than to AIDS-related conditions.
High mortality rates among coinfected patients with ESLD waiting for liver
transplantation have also been reported in observational studies (Maida 2005, Prieto
2008, Murillas 2009). One study (Prieto 2008) analysed 18 patients who were on the
waiting list for OLT. Eight (44%) received a transplant, 8 (44%) died while on the
waiting list, and 2 (12%) were still on the waiting list at the end of the study (Prieto
2008). 10 (67%) out of 15 patients on the transplant waiting list died after a median
follow-up of 5 months, and 5 (33%) underwent liver transplantation (Murillas
2009).
Two case-control studies have analysed mortality rates among coinfected patients
with ESLD waiting for liver transplantation. In the first (Ragni 2005), mortality
rates during the pre-transplant evaluation in HIV-positive (N=58) and HIV-negative
(N=1359) patients were 36% and 15%, respectively (p<0.001), although these data
were not confirmed by the second (Subramanian 2009). Waiting list mortality was
14.4% in patients with HIV infection (N=167) and 11.1% in the control group
(N=792) (p=0.30). In a multivariate analysis, a MELD score higher than 25 was the
only variable related to death on the waiting list (Subramanian 2009). Recently, it
was observed that the survival rate at 1 and 3 years after listing were 81% and 55%
in HIV infected vs. 91% and 82% in patients without HIV infection, respectively
(p=0.005) (Vibert 2011).
For these reasons, physicians attending HIV-positive patients with cirrhosis
should follow patients prospectively and evaluate them early for OLT after the first
clinical decompensation of liver disease. Similarly, patients whose cirrhosis is
associated with HCC should also be evaluated (Llovet 2004). Both prevention and
effective treatment of these complications may improve the likelihood of survival
until OLT, and this should be performed also with the HIV-negative patients
(Agüero 2007, Spengler 2011).

Management of cirrhosis complications
Management of the complications of cirrhosis (portal hypertension, ascites,
gastrointestinal bleeding, encephalopathy, SBP, HCC, and hepatorenal syndrome)
must be planned, just as in the HIV-negative population (Arroyo 2008, EASL 2011,
Spengler 2011, Bruix 2011). Medical management also includes prevention of

390 Hepatology 2012
infection. In view of the short survival associated with the development of SBP,
primary antibiotic prophylaxis with quinolones or trimethoprim-sulfamethoxazole
should be considered (Fernández 2007).
It has been observed that transient elastometry could be used to select HIV/HCVcoinfected patients undergoing screening with upper gastrointestinal endoscopy for
oesophageal varices (Pineda 2009). This study found that HIV/HCV-coinfected
patients with cirrhosis who harbour oesophageal varices requiring preventive
therapy for bleeding had liver stiffness values higher than those who did not require
treatment. Liver stiffness values lower than 21 kPa were highly predictive of varices
not at risk for bleeding.
As far as HCC is concerned, patients may benefit from more frequent imaging,
i.e., every 3 months (Bräu 2007). Treatment of HCC may not be successful,
depending on the stage. Other issues that may delay the progression of liver disease,
such as avoidance of hepatotoxic drugs (e.g., didanosine) and vaccination for
hepatitis A and B, should be kept in mind.

Substance abuse
Smoking has been linked to HCC (Kuper 2000) and increased hepatic fibrosis
(Pessione 2001). It may also increase histological activity in chronic HCV patients
irrespective of alcohol consumption (Hezode 2003).
According to one study (Rosenthal 2007), alcohol consumption was more
frequent among coinfected patients who died from ESLD (92%), and another study
suggested that excess alcohol consumption increases HCV RNA levels (Cooper
2005).
In addition, daily cannabis smoking was significantly associated with the presence
of moderate to severe fibrosis in patients with chronic HCV infection and those with
hepatitis C cirrhosis should abstain from or reduce cannabis use (Ishida 2008).

HCV/HBV management
Specific treatment for infection with HBV or HCV is possible, although more
difficult, in patients with advanced cirrhosis, especially for HCV infection
(Rockstroh 2008, GESIDA 2010).
One of the objectives when treating HCV-monoinfected patients with advanced
liver cirrhosis using pegylated-interferon plus ribavirin is to obtain undetectable
plasma HCV RNA levels at the time of OLT in order to reduce the risk of HCV
recurrence posttransplant. One study (Everson 2005) using a low accelerating
dosage regimen (LADR) of anti-HCV therapy in monoinfected patients on the OLT
waiting list showed that 30 (24%) of 124 patients achieved a sustained virologic
response (SVR) and 12 (80%) of 15 patients who were HCV RNA-negative before
OLT remained HCV RNA-negative 6 months or more after transplantation. This
approach has not yet been addressed in the HIV infection setting. Safety data is also
available from the APRICOT substudy (Mauss 2004). Hepatic decompensation was
observed only in HIV/HCV-coinfected patients with markers of advanced cirrhosis,
and its incidence was 10.4% (14/134). However, 6 (43%) of the 14 patients died as
a result of hepatic decompensation. Antiretroviral treatment with didanosine was an
associated risk factor. In contrast, no hepatic decompensation was noted in
HIV/HCV-coinfected patients without cirrhosis. Therefore, anti-HCV treatment

End-stage Liver Disease, HIV Infection and Liver Transplantation 391
during the pretransplant evaluation or while patients are on the waiting list should
be individualized (e.g., patients with Child-Turcotte-Pugh class A and HCC or
genotypes 2/3) and patients must be monitored closely because of their high risk of
hepatic decompensation and death. In this sense, the AADSL Practice Guidelines
for HCV infection (Ghany 2009), state that HIV-infected patients with
decompensated liver disease (CTP class B or C) should not be treated with peginterferon + ribavirin, and should be considered candidates for liver transplantation
(Grading IIa, C).
Promising results are available related to the new HCV protease inhibitors
(telaprevir and boceprevir) in HCV-monoinfected patients. However, data from
larger trials regarding the HIV/HCV-coinfected patient is lacking. Their role in this
subset of patients is unknown.
Since HBV replication is a contraindication for OLT and only patients without
HBV viraemia are accepted for OLT, treatment of this infection should be a priority.
HIV-positive patients who require antiretroviral therapy and have chronic HBV
infection can be treated with lamivudine (or emtricitabine) and tenofovir as part of
their triple antiretroviral therapy (Rockstroh 2008, Soriano 2008, GESIDA 2010).
Adefovir and tenofovir have proven useful against HBV and could be used in cases
of resistance to lamivudine (Rockstroh 2008, Soriano 2008, GESIDA 2010).
Recently, one study (Heathcote 2011) showed that tenofovir disoproxil fumarate
(TDF) is safe and effective in the long-term management of patients with chronic
hepatitis B.

Combination antiretroviral therapy (HAART)
The role of HAART in the progression of liver disease and in overall mortality in
HCV/HIV-coinfected patients remains controversial (Tedaldi 2003, Qurishi 2003).
Nevertheless, permanent discontinuation of HAART was independently associated
with risk of first hepatic decompensation and a poorer survival rate (Lopez-Diéguez
2011) and to increased risk of fibrosis progression (Thorpe 2011). Antiretroviral
drug regimens should be carefully planned in persons with HIV and ESLD. These
patients should follow general recommendations (GESIDA 2011, DHHS 2011) and
their liver function must be closely monitored for signs of hepatotoxicity. Careful
consideration of drug prescriptions and possible interactions is essential.
Furthermore, some antiretroviral drugs may be contraindicated in cirrhotic patients
(e.g., didanosine, nevirapine, full-dose ritonavir) and their dosing should be adjusted
according to the degree of hepatic impairment (Wyles 2005, Back 2011, Tuset
2011).
Therapeutic drug monitoring (TDM) may be useful for efavirenz and protease
inhibitors. Indinavir and atazanavir can increase unconjugated bilirubin levels by
inhibiting UDP-glucuronosyltransferase. As total bilirubin is a component of both
the Child-Turcotte-Pugh and MELD scores, results in patients taking these drugs
should be interpreted with caution.
It is noteworthy that the newer antiretroviral drug raltegravir, which is not a
substrate of CYP450, can be used in HIV-1 OLT recipients. One study (Tricot
2009) enrolled 13 patients with HIV-1 infection who underwent solid organ
transplantation (8 liver and 5 kidney) and received raltegravir. The authors found a
lack of significant interaction between raltegravir and calcineurin inhibitors that

392 Hepatology 2012
allowed simplified management of immunosuppressive treatment, excellent
tolerability, and no events related to outcome (acute rejection) or HIV infection.
Therefore, the combination of two nucleos(t)ide reverse transcriptase inhibitors
(tenofovir + emtricitabine or abacavir + lamivudine) + raltegravir is the
antiretroviral regimen of choice in HIV-infected liver transplant recipients.
Finally, given the speed with which new antiretrovirals appear and thus new
interactions, physicians should consult updated databases on drug interactions (Back
2011, Tuset 2011).

Orthotopic liver transplant (OLT)
OLT is the only therapeutic option for patients with ESLD. HIV infection is not a
contraindication for liver transplantation (Miró 2007, Stock 2007, Samuel 2008).
There are 3 different classes of criteria for including HIV-positive patients on the
liver transplant waiting list: liver disease, HIV infection, and other criteria.

Liver disease criteria
These are the same as for the non–HIV-infected population; the main indication for
OLT in HIV-positive patients is ESLD caused by HCV coinfection. Less frequent
indications are HBV coinfection (either acute or ESLD) and liver cancer.
In the UK guidelines (O’Grady 2005), indications for liver transplantation include
acute liver failure, decompensated liver disease - with ascites, encephalopathy (it is
important to exclude HIV-related dementia), variceal bleeding that is difficult to
manage with standard therapy, and poor liver function (albumin <30 g/l, INR >1.5,
and elevated serum bilirubin >450 mmol/l) - and HCC detected during regular
tumour surveillance. In the Eurotransplant region these criteria have been replaced
by the MELD score. The criteria for liver transplantation in patients with HCC are
as follows: no more than 3 tumour nodules, no nodule greater than 5 cm in diameter,
absence of macroscopic portal vein invasion, and absence of recognizable
extrahepatic disease.
A new indication for liver transplant in HIV+ patients has been described in a
recent study (Tateo 2009) in which 3 patients underwent liver transplantation and
the cause of ESLD was nodular regenerative hyperplasia (NRH). OLT is the only
therapeutic option in cases of severe portal hypertension such as that observed in
these patients.

HIV infection criteria
Most liver transplant groups from Europe and North America use similar HIV
criteria. These are summarized in Table 1 (O’Grady 2005, Grossi 2005, Miró 2007,
Anonymous 2004).

Clinical criteria
Some authors are in favour of withdrawing exclusion criteria for some opportunistic
infections that can be effectively treated and prevented, such as tuberculosis,
candidiasis, and Pneumocystis jiroveci pneumonia (Roland 2003, Neff 2004,
Radecke 2005). In fact, the NIH-sponsored study has updated the inclusion criteria
for opportunistic complications and only untreatable diseases continue to be an
exclusion criteria for liver transplantation (e.g., progressive multifocal

End-stage Liver Disease, HIV Infection and Liver Transplantation 393
leukoencephalopathy, chronic cryptosporidiosis, multidrug-resistant systemic fungal
infections, primary CNS lymphoma, and visceral Kaposi’s sarcoma) (Roland 2006).
Table 1. HIV criteria for OLT in some European countries and the US.

Previous C events
Opportunistic
infections
Neoplasms
CD4 cell
3
count/mm

Spain
(Miró 2005)

Italy
(Grossi 2005)

UK
US
(O’Grady 2005) (Anon 2004)

Some*

None in the
previous year.
No

Some**

>200 or >100 if
decompensated
cirrhosis

None after ARTinduced
immunological
reconstitution.
>200 or >100 if
portal
hypertension

Yes

Yes

Yes

No
>100***

Plasma HIV-1 RNA
viral load BLD on
Yes
HAART****

No
>100***

* In Spain, patients with previous tuberculosis, Pneumocystis jiroveci pneumonia (PCP) or
esophageal candidiasis can be evaluated for OLT; ** In the US, PCP and esophageal
candidiasis were not exclusion criteria; *** Patients with previous OIs should have >200 CD4
3
cells/mm ; **** If PVL was detectable, post-OLT supression with HAART should be predicted in
all patients. BLD, Below the level of detection

Immunological criteria
All groups agree that the CD4+ lymphocyte count should be above 100 cells/mm3
for OLT (Roland 2003, Neff 2004). This figure is lower than that for kidney
transplantation (CD4 >200 cells/mm3), because patients with cirrhosis often have
lymphopenia due to hypersplenism, which leads to a lower absolute CD4+ count,
despite high CD4 percentages and good virologic control of HIV. In Spain and the
US, the CD4+ count must be greater than 200 cells/mm3 in patients with previous
opportunistic infections (Miró 2005, Anonymous 2004).
In Italy (Grossi 2005) and the UK (O’Grady 2005) the CD4+ cut-off is 200 cells/
mm3, unless patients have decompensated cirrhosis or portal hypertension. In these
scenarios, they use the same CD4+ cell threshold as in Spain and the US (100
cells/mm3).

Virologic criteria
The essential criterion for OLT is that the patient must be able to have effective,
safe and long-lasting HAART during the post-transplant period (Neff 2004, Fung
2003). The ideal situation is one in which the patient tolerates HAART before
transplant and is ready for the transplant with undetectable HIV viral load by ultrasensitive techniques (<50 copies/ml). Some patients do not have an indication for
HAART, as they are long-term non-progressors with no immunological criteria
(CD4+ lymphocyte count above 350 cells/mm3) or clinical criteria to start HAART
and a detectable plasma viral load. In this setting, it is unknown whether and when
(pre-transplant or post-transplant) it would be beneficial to initiate HAART in order
to reach an undetectable plasma viral load.

394 Hepatology 2012

Other criteria
To be included on the OLT waiting list, an HIV-infected patient must have a
favourable psychiatric evaluation. One observational prospective study found that
HIV-1-infected patients with ESLD improved on all the items of a psychometric
score (MADRS) at the follow-up evaluation (Barbanti 2009). In this study, the score
variation was 10.20 at baseline and 4.09 at follow-up (p<0.001).
Patients who actively consume drugs should not be placed on the waiting list. In
Spain, patients must undergo a 2-year consumption-free period for heroin and
cocaine (Miró 2005), and 6 months with no consumption of other drugs (e.g.,
alcohol). Patients who are on stable methadone maintenance programmes can be
included and can continue on the maintenance programme after the procedure (Liu
2003). Finally, as is the case with any transplant candidate, HIV-positive patients
must show an appropriate degree of social stability in order to ensure adequate care
in the post-transplant period.

Outcome of OLT in HIV-positive patients
Overall short-term survival rates of HIV-positive patients who undergo OLT have
been reported to be similar to those of HIV-negative patients when there is no HCV
coinfection (Ragni 2003, Neff 2003, Fung 2004, Norris 2004, De Vera 2006,
Schreibman 2007, Vennarecci 2007, Duclos-Vallee 2008, Tateo 2009, Sioutis 2010,
Coffin 2010, Antonini 2011, Cherian 2011) (Table 2).
HIV+ patients have not been shown to have an increased risk of post-operative
complications or a higher incidence of opportunistic infections or tumours than
HIV-negative patients (Samuel 2008). However, some concerns have lately arisen.
Findings from a case-control study (81 HIV/HCV-coinfected liver transplant
recipients vs. 213 control patients) found that coinfected individuals were about
twice as likely to have treated acute rejection than HCV-monoinfected patients
(35% vs 18%, p=0.001) (Terrault, 2009). Moreover, a recent retrospective study
(Cherian 2011) has observed a 12% (3/24) incidence of post-transplant hepatic
artery thrombosis (HAT) while in people without HIV infection that number is
around 4.4 % (Bekker 2009). The prothrombotic state associated with HIV and liver
disease could be the underlying factor involved.
Regarding infections, it has been observed that bacterial infections are common in
liver (43%) and kidney recipients (35%), whereas HCV infection was the only
factor associated with an increased risk of bacterial infection (liver recipients only)
(Blumberg 2008). A high rate of severe (43%) and opportunistic (11%) infections in
a cohort of 84 HIV/HCV-coinfected patients who underwent liver transplantation
has also been noted (Moreno 2011). Bacterial infections occurred in 38 patients
(45%), CMV infection in 21 (25%), uncomplicated herpes virus infection in 13
(15%), and fungal infections in 16 patients (19%, 7 invasive cases). A pretransplant
MELD score >15, history of category C AIDS-defining event and non-tacrolimus
based immunosupression regimes were factors independently associated with severe
infections. Further studies are needed in order to obtain more robust conclusions.

End-stage Liver Disease, HIV Infection and Liver Transplantation 395
Table 2. Liver transplantation in HIV-infected patients: main cohorts of cases (≥10)
in the late HAART era (2003-2011).
Author

Year Country

Virus
Nº
cases

Follow-up Survival
(months)

Ragni
Neff
Fung
Norris

2003
2003
2004
2004

International
US
US
UK

24
16
29
14

De Vera

2006

US

27

HCV-62%; HBV-29%
HCV or HVB
HCV-90%
HCV-50%; HBV/OH50%
HCV-100%

17
12
18
12
19
27

18 (75%)
14 (87%)
20 (69%)
2 (29%)
7 (100%)
13 (48%)

Schreibman
Vennarecci
DuclosVallée
Roland
Tateo

2007
2007
2008

US
Italy
France

15
12
35

HCV-40%;HBV-33%
HCV-91%
HCV-100%

74
26
44

10 (67%)
6 (50%)
22 (63%)

2008
2009

US
France

11
13

HCV-55%; VHB-45%
HBV-100%

36
32

Sioutis
Coffin
Antonini

2010
2010
2011

Germany
US
France

26
22
59

HCV-58%; HBV-34%
HBV-100%
HCV-100%

Di
Benedetto
Baccarani
Cherian
Spanish
study

2011

Italy

23

HCV-87%

30
42
Not
reported
24

7 (64%)
13
(100%)
17 (65%)
19 (86%)
34 (58%)

2011
2011
2011

Italy
UK
Spain

27
24
248

HCV-78%
HCV-50%; HBV-33%
HCV-96%

26
88
50

13 (57%)
23 (85%)
15 (71%)
193
(72%)

HIV/HCV coinfection
Mid-term survival is affected by recurrent hepatitis C (de Vera 2006). After OLT,
recurrence of HCV infection is universal, regardless of whether the patient is
infected by HIV or not. In fact, it is currently the number one cause of death. Some
studies have suggested that recurrence of HCV in coinfected patients tends to be
more severe and occurs earlier (de Vera 2006, Castells 2006, Antonini 2011). The
outcomes of 27 coinfected patients were compared to 54 HCV-monoinfected
patients who underwent OLT (de Vera 2006). The researchers found that HIVpositive patients had a higher likelihood of developing cirrhosis or dying of an
HCV-related complication than HIV-negative patients (RR=2.6; 95%CI, 1.06-6.35).
Cumulative 1-, 3- and 5-year survival for coinfected and monoinfected patients was
67% vs. 76%, 56% vs. 72%, and 33% vs. 72%, respectively (p=0.07).
In a retrospective study (Mindikoglu 2008) in the US that enrolled 138 HIVpositive patients who underwent liver transplant during the HAART era (19962006), the rate of survival at years 2 and 3 was significantly lower in HIV-positive
patients (70% and 60%) than in the general population (n=30,520) (81% and 77%),
although this difference was observed only in the HCV/HIV- and HBV/HIVcoinfected groups. None of the 24 HIV-monoinfected recipients died. Therefore,
liver transplant in HIV-positive patients does not have higher short-term mortality
(1-2 years). Nevertheless, the management and outcome of HCV reinfection could
affect survival in the medium term (3-5 years) and long term (5-7 years).

396 Hepatology 2012
In France, data from 35 HIV/HCV-coinfected patients were analysed and
compared with those of 44 HCV-monoinfected patients. Survival rates at 2 and 5
years were 81%/91% and 51%/73% in HIV/HCV-coinfected patients/HCVmonoinfected patients, respectively (p=0.004) (Duclos-Vallée 2008).
In Spain, data from a multicentre case-control study show that survival of
HIV/HCV-coinfected patients (N=84) at 1 year was similar to that of HCVmonoinfected patients (N=252) - 88% vs. 89% (NS) - but it was significantly lower
at 3 and 5 years: 62% vs. 77% and 48% vs. 75%, respectively (p<0.01). The
variables independently associated with mortality were HCV genotype 1 infection,
non-traumatic donor death, number of units of blood transfused during surgery, and
development of invasive fungal infection after transplant (Miró 2009). However, a
recent Italian case-control study (Baccarani 2011) that included 27 HIV-positive
and 27 HIV-negative recipients found that patient survival at 1, 2, and 5 years were
88%, 83%, and 83% for HIV-positive patients vs. 100%, 73%, and 73% for HIVnegative patients (p=0.95). The estimated graft survival rate at 1, 3 and 5 years was
92%, 87%, and 87% for HIV-positive patients vs. 95%, 88%, and 82% for HIVnegative patients (p=0.59), respectively. The median follow-up was only 26 months
(range 1-64) and 27 months (range 1-48) for HIV+ recipients and HIV-negative
recipients (p=0.85), respectively, and the aetiology of ESLD was HCV in most
cases (78% vs. 67%, respectively).
In order to definitively answer the survival question, additional cohort studies
analysing donor and recipient characteristics, issues related to the activity of both
viruses and the efficacy and safety of antiviral therapies are necessary for
determining the long-term prognosis of this procedure.
Rapid progression of HCV-related liver disease in HIV-positive recipients would
represent a major drawback and would shorten life expectancy in this group of
patients. In fact, it is currently the primary cause of death. A French study observed
that progression to fibrosis (≥F2) was significantly higher in HIV-positive patients
(p<0.0001) (Duclos-Vallée 2008) and MELD was the only significant predictor of
mortality, although donor age was of borderline significance (p=0.06). 11 (19%) out
of 59 patients who underwent OLT developed fibrosing cholestatic hepatitis (FCH)
(Antonini 2011). Nine of them (82%) died of liver failure after developing FCH.
Survival rate was significantly lower in the FCH group when compared to non-FCH
patients: 26 vs 76 months (p=0.004)
There is insufficient experience on the efficacy and safety of therapy with
pegylated-interferon and ribavirin in coinfected transplant patients. One study (Miró
2007) summarized the reports evaluating the effectiveness of treatment of HCV
reinfection in OLT with pegylated-interferon + ribavirin (Fung 2004, de Vera 2006,
Vennarecci 2007, Castells 2007, Duclos-Vallée 2008, Di Benedetto 2011, Miró
2011). These patients were treated when they had histological criteria. Only 29
(21.3%) out of 136 HCV/HIV-coinfected patients achieved an SVR. SVRassociated factors were investigated in 23 HIV/HCV-coinfected liver recipients and
found that donor age <60 years (p=0.02), genotype other than 1 (p=0.001), and use
of cyclosporin A (p=0.002) were independently associated with SVR (Krishnan
2008). New strategies are necessary to improve the outcome of HCV recurrence in
this setting. In this sense, a German study showed that SVR was obtained in 6 out of
7 patients treated within the first 3 months after OLT (Emmelkamp 2007). The low
accelerating dosage regimen (LADR) approach has recently been addressed in 10

End-stage Liver Disease, HIV Infection and Liver Transplantation 397
coinfected patients who developed fibrosing cholestatic hepatitis after OLT
(Antonini 2011). None of these patients displayed a sustained virological response
while adverse effects were reported in all patients. Anemia was present in the vast
majority of cases and 4 died in the context of the anti-HCV virus. Finally, 2 cases of
spontaneous clearance of HCV RNA after OLT have been described. This
phenomenon is very infrequent and its mechanism is not known (Bhagat 2008).
Table 3. Summary of studies evaluating the effectiveness of the treatment of HCV
reinfection in OLT with pegylated interferon + ribavirin.
Author + Year of
Publication

HIV/HCV coinfected patients

Non-HIV HCV-monoinfected
patients (Control Group)

No. of cases

No. of cases

Fung 2004
b
De Vera 2006
d
Vennarecci 2007
e
Castells 2007
Duclos-Vallée 2008
Di Benedetto 2011
Spanish study 2011

12
15
9
5
19
9
67

SVR
No. (%)
2 (17)
4 (27)
1 (11)
1 (20)
3 (16)
4 (44)
14 (21)

Total

136

29 (21)

27
9
20

SVR
No. (%)
c
7 (28)
1 (11)
15 (75)

-

-

-

-

b,Most cases were genotype 1. Three patients were treated with classical interferon plus
ribavirin; c,Rate of sustained virological response was not specified. Data show the rate of
virologic response (clearance of HCV RNA from serum); d,The authors did not specify the type
of interferon used; e,These patients were included in the Spanish study and were not taken into
account for the overall response rate; f,3/27 (11%) genotypes1-4 and 6/13 (46%) genotypes 23. SVR: sustained virological response (modified from Miró 2007).

On the other hand, in two recent genome-wide association studies (Ge 2009,
Thomas 2009), a single nucleotide polymorphism (rs12979860) 3 kilobases
upstream of the IL28B gene, which encodes the type III interferon-l, was shown to
be associated with natural clearance of HCV among HIV-negative individuals of
both European and African ancestry and with more than a twofold difference in
response to anti-HCV drug treatment with pegylated-interferon and ribavirin in
HCV-monoinfected patients. In a Swiss study (Rauch 2010), this antiviral effect
was stronger in patients with HCV genotypes 1 or 4. Similar results have been
recently communicated in HCV/HIV-coinfected patients (Rallon 2011). Moreover,
IL28B polymorphisms predicted treatment response in HIV/HCV-coinfected
patients with prior relapse while only predicted response in prior nonresponders
carrying HCV genotypes 1 or 4 (Labarga 2011). The role of IL28B polymorphysms
of the donor and their impact on the natural history of HCV recurrence and response
to antiviral therapy in liver transplant recipients is not yet known.

HIV/HVB coinfection
Cohorts of HIV/HBV-coinfected patients are not as large as those of HIV/HCVinfected patients. Nevertheless, the outcome of HBV infection after OLT is much
better (Tateo 2009, Coffin 2010). The survival rate in the short and medium term in
HBV/HIV-coinfected patients is high and similar to that observed in HBVmonoinfected patients, probably due to the low incidence of HBV reinfection. A
French study (Tateo 2009) that included 13 HIV/HBV-coinfected patients (3 out of

398 Hepatology 2012
6 patients with positive anti-HCV serology had HCV RNA detectable before OLT),
revealed 100% graft and patient survival after a mean follow-up of 32 months. They
compared outcomes of 22 HBV/HIV patients and 20 HBV monoinfected patients
who underwent OLT (Coffin 2010). Cumulative patient and graft survival at one
and three years was 85% in the coinfected patients and 100% in the monoinfected
group (p=0.08).

Hepatocellular carcinoma
Preliminary Italian data show good results in 7 HIV-1–infected patients with HCC
who underwent OLT. They observed an 86% overall patient and graft survival rate
after a mean follow-up of 8 months, and recommend OLT in HIV-infected patients
with early stage HCC (Di Benedetto 2006, Di Benedetto 2008). The same authors
found no recurrence in 7 HIV-1 OLT recipients after a median follow-up of 13
months (Di Benedetto 2008). Recently, data have published from a case (VIH+)
control (VIH-) study of patients with HCC who underwent OLT (Vibert 2011).
They observed that the proportion of patients who exceeded the Milan criteria were
similar in both groups (4/21 (19%) vs. 17/65 (26%), respectively, p=0.5) while no
differences were seen in number of nodules, maximum diameter and microvascular
invasion grade. Time on waiting list was similar (6.4 vs. 4.1 months, respectively
p=0.2). 16 out of 21 HIV-infected patients underwent OLT vs. 58 out of 65 in the
control group. The survival rate at 1 and 3 years was 81% and 55% vs. 91% y 82%
respectively (p=0.005). These authors found a high rate of HCC recurrence in the
case group 5/16 (30%) vs. 9/58 (15%) in the control group. Further studies with
more patients and longer follow-up are needed to precisely define the recurrence
rate in this setting.

Liver retransplantation
The experience of liver retransplantation (re-OLT) in the HIV-infected population is
scarce and most of the cases are mentioned in articles published with patients from a
single center (de Vera 2006, Vogel 2005, Polard 2005). The incidence and outcome
of liver retransplantation in patients with HIV infection is unknown. Currently, in
people without HIV infection, re-OLT accounts for approximately 10% of all liver
transplants (Pfitzmann 2007, Reese 2009) and overall post-retransplant patient
survival rate is between 15 and 20 percentage points lower than the primary OLT
survival rate (Carrión 2010), which is a concern due to the significant disparity
between the number of patients waiting for their first OLT and the shortage of
available organs. A recent report (Gastaca 2011) observed a 6.7% of re-OLT in a
cohort of 227 patients with HIV infection. Overall survival (95% confidence
interval) at 30 days, 1 year, and 3 years after re-LT for HIV-positive and HIVnegative patients was 93% (59%-99%) vs. 85% (78%-90%), 40% (13%-66%) vs.
72% (64%-78%), and 40% (13%-66%) vs. 64% (55%-71%), respectively
(p=0.231). More patients at risk are needed to provide more conclusive results.

End-stage Liver Disease, HIV Infection and Liver Transplantation 399

Conclusions
ESLD is an increasingly frequent clinical scenario in the setting of HIV/HCV-HBV
coinfection, and its burden is expected to continue to increase.
Early diagnosis of ESLD complications is particularly important and should be
actively monitored and treated. In general terms, the management of ESLD in HIVpositive patients should be the same as in those without HIV infection.
Physicians taking care of ESLD patients should follow them prospectively and
evaluate them for OLT after the first clinical decompensation of liver disease.
OLT is a life-saving procedure in this population, and is safe and effective in
patients with HBV infection. However, recurrence of HCV infection in coinfected
patients can affect both graft and patient survival in the medium- and long-term.
Prospective and larger studies with a longer follow-up must be carried out in order
to determine the benefit of OLT in this setting.
The members of the Hospital Clinic OLT in HIV Working Group are: JM Miró, A. Moreno, C.
Manzardo, M. Laguno, JL Blanco, J Mallolas, C. Cervera, M. Tuset, M. Monras, N. Freixa, J.
Blanch, I.Perez, D. Paredes, J. Fuster, C. Fontdevila, JC García-Valdecasas, JM Gatell, A.
Rimola, (Hospital Clinic – IDIBAPS. University of Barcelona, Barcelona); C. Tural and A. Jou
(Hospital Germans Trías i Pujol, Badalona, Barcelona, Spain); and J. Murillas and M.
Peñaranda (Hospital Son Dureta, Palma de Mallorca, Spain).

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Metabolic Liver Diseases: Hemochromatosis 405

24. Metabolic Liver Diseases:
Hemochromatosis
Claus Niederau

Definition and classification of iron overload
diseases
Hereditary hemochromatosis is classified into 4 subtypes (Table 1). Type 1 is the
well known form of iron overload due to an autosomal-recessive genetic metabolic
malfunction; the homozygous C282Y mutation of the HFE gene on chromosome 6
accounts for more than 90% of clinical phenotypes in populations of Caucasian
origin (Feder 1996). The mutation leads to an inadequately high intestinal iron
absorption that after decades may cause iron overload and damage to various organs
(Figure 1). Types 2a and 2b of genetic hemochromatosis are juvenile forms of iron
overload that lead to a severe outcome prior to age 30, with cardiomyopathy and
hypogonadism. The corresponding mutations are located in the hemojuveline and
hepcidin genes, respectively (Roetto 1999). Type 3 has mainly been described in
Italian families and refers to a mutation in the transferrin receptor 2 gene (Girelli
2002). Clinical consequences of type 3 hemochromatosis are similar to type 1.
Types 2 and 3 are autosomal-recessive traits. The mutations of the autosomaldominant type 4 hemochromatosis are located in the gene coding for the basolateral
iron transporter ferroportin 1 (Njajou 2001). In contrast to the other types, iron is
accumulated in type 4 mainly in macrophages; ferritin values are markedly elevated
although transferrin saturation is only slightly higher.
Secondary hemochromatosis is usually caused by multiple blood transfusions in
hemolytic anemias such as thalassemia, sickle cell anemia and myelodysplasia
syndrome. Iron first accumulates in RES macrophages and is later transferred to
parenchymal cells. With frequent blood transfusions, iron may accumulate faster
than with genetic hemochromatosis; iron overload often leads to severe
cardiomyopathy and liver cirrhosis, limiting effective prognosis. Therapy consists of
iron chelators because phlebotomies cannot be done due to the underlying anemia.
This review will focus on type 1 HFE hemochromatosis, the most prevalent genetic
form in Germany. Most consequences of iron overload are similar, whatever the

406 Hepatology 2012
cause. Thus, the pathophysiology of tissue and organ damage by iron excess is
discussed in detail only for HFE hemochromatosis.

Figure 1. Scheme of natural history of type 1 genetic hemochromatosis.

Table 1. Classification of hemochromatosis.
I) Genetic hemochromatosis
Types

Gene defect on

Affected gene

Inheritance

High
prevalence

Type 2a

Chromosome 1

Hemojuveline

Autosomalrecessive

Juvenile form

Type 2b

Chromosome 19

Hepcidin

Autosomalrecessive

Juvenile form

Type 3

Chromosome 7

Transferrin
receptor 2

Autosomalrecessive

Italy

Type 4

Chromosome 2

Ferroportin 1

Autosomaldominant

Italy

Neonatal

Unknown

Unknown

Unknown

Very rare

Others

Unknown

Unknown

Unknown

Of nonCaucasian origin

II) Secondary hemochromatosis
a) Chronic anemias (thalassemia, sickle cell disease, MDS, other rare hemolytic
anemias)
b) Multiple blood transfusions in general
c) Long-term oral intake of high amounts of iron (diet-related or IV)
III) Non-classified, ill-defined iron overload syndromes
a) iron overload in Bantu Africans
b) iron overload in aceruloplasminemia

Type 1 HFE hemochromatosis
History
The association between liver cirrhosis, pigment deposits in the liver, and diabetes
mellitus was recognized over a century ago (Trosseau 1865, Troisier 1871, Hanot

Metabolic Liver Diseases: Hemochromatosis 407
and Schachmann 1886). The term hemochromatosis was first introduced in the 19th
century (Recklinghausen 1889), but was not generally accepted until used as the
title of a classic monograph (Sheldon 1935). The controversy over whether
hemochromatosis was merely a form of alcoholic liver cirrhosis (MacDonald 1960)
or a genetic error of iron metabolism (Sheldon 1935, Crosby 1966) lasted almost a
century until the association between special HLA haplotypes and hemochromatosis
which recognized the genetic nature of the disease was described (Simon 1975). The
mode of inheritance was identified as an autosomal recessive disorder (Simon
1977). Finally, the major mutation on the HFE gene associated with clinical
manifestations was identified (Feder 1996).

Epidemiology
Type 1 hemochromatosis is probably the most prevalent genetic metabolic error in
Caucasian populations (Adams 2005). The prevalence of C282Y homozygotes is
approximately 0.5% in central Europe and in the Caucasian population of North
America; the prevalence of C282Y and H63D heterozygotes approaches 40% in
similar populations (Adams 2005). Phenotypic expression also depends on several
non-genetic factors such the amount of dietary iron and blood loss (Figure 2). For
example, females develop clinical consequences of iron overload 5-8 times less
frequently and 10-20 years later than males due to menses. It is now widely
accepted that not all C282Y homozygous men will develop the full clinical
manifestation of hemochromatosis. It is unknown, however, whether 5% or 50%
will show clinical disease during their lifetime and what factors determine that
phenotype.
As mentioned previously, the homozygous C282Y mutation accounts for more
than 90% of the clinical phenotype in Caucasian populations (Feder 1996, Adams
2005) (Table 2). A point mutation at H63D is also frequently identified in the HFE
gene as well as other less frequent mutations. None of these gene alterations or
polymorphisms, found in up to 40% of Caucasians, correlates with the phenotype. A
subject with a C282Y variation on one allele and a H63D variation on the other is
called a "compound heterozygote" (Table 2). Only a small percentage of such
compound heterozygotes are at risk for clinical consequences of iron overload.
C282Y and H63D heterozygotes are at no risk of iron overload (Table 2). In nonCaucasian populations other genes may be involved in causing iron overload.

Etiology and pathogenesis
Intestinal iron absorption and iron losses are finely balanced under physiological
conditions. Approximately 10% of the total daily intake (10-20 mg) is absorbed by
the small intestine (1-2 mg). However, subjects with the homozygous C282Y
mutation may absorb up to 20% of iron intake; i.e., up to 2-4 mg/day. Thus,
homozygotes have an excessive iron intake of approximately 1 mg/day. It may
therefore take several decades until iron stores approach 10 g above which organ
damage is considered to be induced. Many patients at the clinical end stage of
hemochromatosis, including liver cirrhosis and diabetes mellitus, have total body
iron stores of 20-30 g. Their intestinal iron absorption is downregulated when iron
stores increase, as it is in patients with genetic hemochromatosis. This
downregulation, however, occurs on an increased level when compared to subjects
without the HFE gene mutation. Correspondingly, intestinal iron absorption is

408 Hepatology 2012
massively increased in patients with hemochromatosis when iron stores have been
depleted by phlebotomy. Phlebotomies should be continued after iron depletion in
order to prevent reaccumulation. These regulatory processes however do not explain
how HFE gene mutations cause the increase in intestinal iron absorption since the
HFE gene product is neither an iron transporter nor an iron reductase or oxidase.
Only recently have carriers and regulators of cellular iron uptake and release been
identified (Pietrangelo 2002, Fleming 2002, Townsend 2002, Fletcher 2002).
It has also become increasingly evident that some of them interact with the HFE
gene product in the regulation of intestinal iron absorption (Pietrangelo 2002,
Fleming 2002, Townsend 2002, Fletcher 2002). Recent studies have shown that the
Nramp2 protein is the luminal iron carrier. Shortly thereafter, the luminal iron
reductase was identified as the Dcytb protein (duodenal cytochrome B) (Pietrangelo
2002, Fleming 2002, Townsend 2002, Fletcher 2002). At the same time, the
basolateral iron transporter ferroportin 1 (also named Ireg1 or MTP1) was identified
(Donovan 2000, Abboud 2000) as well as the basolateral iron oxidase hephestin
(Vulpe 1999). Mutations in some of these proteins are responsible for the rarer types
2-4 of genetic hemochromatosis, although none of these genes is altered in type 1
hemochromatosis. Recently, two other proteins have been shown to act as important
iron regulating proteins, transferrin receptor 2 and hepcidin (Pietrangelo 2002;
Fletcher 2002; Fleming 2005). Mutations in the transferrin receptor 2 gene may lead
to the rare type 3 hemochromatosis, and mutations in the ferroportin 1 gene to type
4 hemochromatosis. More recent studies also indicate that hepcidin may be the most
important regulator of iron metabolism, involved in iron deficiency and overload.
Hepcidin has been shown to down regulate the basolateral iron carrier ferroportin. It
has also been demonstrated that hepcidin itself is up regulated by HFE. Thus, an
HFE mutation may reduce the upregulation of hepcidin that then does not down
regulate ferroportin; the corresponding increase in ferroportin expression finally
causes the increase in intestinal iron uptake (DeDomenico 2007). There may be
further interactions between HFE, transferrin receptor 2, Nramp2, Dcytb,
ferroportin, hephestin and hepcidin, all of which are currently being studied.

Figure 2. Non-genetic factors that may influence iron absorption.

Metabolic Liver Diseases: Hemochromatosis 409
Table 2. Genotype/phenotype correlation in hemochromatosis.
Mutations/

Prevalence in

Risk of advanced

polymorphisms

Caucasian populations

clinical phenotype

C282Y/C282Y
H63D/C282Y
C282Y/wild type
H63D/wild type
Others

85-95%
3-8%
1%

low if ferritin is <1000 ng/ml
very low
none
none
unknown

Diagnosis
Laboratory tests. Any increase in serum iron should start with the exclusion of
hemochromatosis so as not to overlook early disease. Normal serum iron, however,
does not exclude hemochromatosis and increased serum iron often occurs in the
absence of hemochromatosis. Serum iron values are highly variable and should not
be used either for diagnosis or for screening of hemochromatosis. The determination
of transferrin saturation is a better indicator of iron overload than serum iron. The
increase in transferrin saturation usually precedes the ferritin increase (Figure 1).
Transferrin saturation is more sensitive and specific for detection of
hemochromatosis when compared to serum ferritin. For screening, a threshold of
50% for transferrin saturation may be optimal under fasting conditions. Ferritin on
the other hand is a good indicator of largely increased iron stores and reliably
indicates iron deficiency. It has less value for early detection of hemochromatosis.
In hemochromatosis a slightly increased serum ferritin (300-500 ng/ml) is usually
accompanied by transferrin saturations exceeding 80-90%. Unfortunately, serum
ferritin is also increased, often in the presence of infections and malignancies, and
thus has a low specificity for indicating hemochromatosis (Niederau 1998). Ferritin
increases not due to genetic hemochromatosis are usually associated with normal or
only slightly elevated transferrin saturation. Therefore, transferrin saturation should
be measured in order to correctly interpret ferritin increases.
Liver biopsy and determination of liver iron concentration. Although
simultaneous increases of both serum ferritin and transferrin saturation strongly
indicate a risk for hemochromatosis, diagnosis needs to be confirmed by genetic
testing or by liver biopsy with a determination of iron content in the liver. Hepatic
iron concentration also increases with time in subjects with an HFE gene mutation.
It is recommended to divide the liver iron concentrations by the patient’s age in
order to obtain the “hepatic iron index” (Summers 1990). The semi-quantitative
estimation of liver iron stores by the Berlin blue colour is less sensitive and specific
than the chemical quantification of liver iron concentration. In case of a
homozygous C282Y gene test, liver biopsy is not required for the diagnosis of
genetic hemochromatosis (Table 2).
There may, however, be other reasons to perform a liver biopsy in iron overload:
(1) subjects with biochemical or clinical evidence of iron overload in the absence of
the homozygous C282Y mutation should have a liver biopsy to substantiate iron
overload; (2) in C282Y homozygotes the risk for liver fibrosis and cirrhosis
increases at ferritin values >1000 ng/ml (Loreal 1992); in those patients liver biopsy

410 Hepatology 2012
is recommended because the presence of liver cirrhosis markedly increases later
HCC risk and thus warrants HCC screening.
Deferoxamine testing and ferrokinetic measurements. Determinations of
urinary excretion of iron after administration of deferoxamine allows some
estimation of total body iron stores. The deferoxamine test, however, often only
shows pathological results when serum ferritin and transferrin saturation are
markedly increased and does not allow diagnosis of early disease. Ferrokinetic
measurements today are only done for scientific research or in difficult diagnostic
situations.
Computed tomography (CT), magnetic resonance tomography (MRT) and
biomagnetometry. CT density measurements of the liver allow a semi-quantitative
estimation of iron concentration in the liver. This method however is associated
with radiation and therefore not allowed in many countries where alternative
methods are available. MRT, on the other hand, allows a reliable measurement of
liver iron content, provided that special software is used and the equipment is
calibrated for such measurement. In clinical practice most MRT do not fulfil these
criteria. Biomagnetometry allows the most accurate non-invasive measurement of
liver iron concentration. However, this equipment is expensive and only allows
measurement of iron concentration. Consequently, biomagnetometry is done only at
a few centres worldwide and is primarily used for scientific studies and not in daily
clinical practice. With the availability of reliable and inexpensive genetic testing,
CT, MRT, and biomagnetometry do not need to be done for most patients.

Figure 3. Diagnosis and treatment algorithm for type 1 hemochromatosis.

Genetic tests. As outlined previously, in Caucasian populations the homozygous
C282Y mutation accounts for more than 90% of patients with the clinical phenotype
of type 1 hemochromatosis (Adams 2005, Erhardt 1999). Approximately 5% of
patients with the clinical phenotype are C282Y/H63D compound heterozygotes; the
prevalence of C282Y or H63D heterozygosity in patients with the clinical

Metabolic Liver Diseases: Hemochromatosis 411
phenotype of hemochromatosis is considerably lower than in the general population.
Thus, a subject who is heterozygous for C282Y or H63D per se has no risk of iron
overload. In subjects homozygous for C282Y, both serum ferritin and transferrin
saturation are frequently increased; however, only male subjects have an increased
risk for liver disease when compared to subjects without HFE gene alterations in a
recent large screening study. It is unknown how many C282Y homozygotes will
later develop clinical signs and symptoms due to iron overload. It is increasingly
evident that only a minority of C282Y homozygotes progress to end stage iron
overload with liver cirrhosis and diabetes mellitus. In subjects who are not C282Y
homozygotes but have laboratory, histological or clinical evidence of iron overload,
further genes may be analysed for mutations such as hemojuveline, transferrin
receptor 2, ferroportin 1 and hepcidin.

Early diagnosis and screening
The prevalence of C282Y homozygotes is 0.5 % in Caucasian populations (Adams
2005, Erhardt 1999). Clinical manifestations however are variable and depend on
non-genetic factors such as dietary iron intake and blood loss. Until 1980 most
patients with hemochromatosis were detected with late irreversible complications
such as liver cirrhosis and diabetes mellitus. With a better understanding of the
disease, the broad use of ferritin and transferrin saturation measurements and the
availability of a reliable genetic test, diagnostic efforts have concentrated on the
detection of early disease before liver cirrhosis and diabetes mellitus. Several
studies have shown that iron removal by phlebotomy is associated with normal life
expectancy in patients diagnosed early (Niederau 1985, Niederau 1996, Fargion
1992) (Figure 3). Several other studies have focused on screening procedures in
order to diagnose more subjects with early disease (Edwards 1988). These studies
include populations with special risks, family members, as well as the general
population (Table 3) (see Niederau 2002). It has been shown that an increasing
number of patients are now diagnosed early and that this trend increases survival
(Figure 4).
A large number of studies have shown that screening is useful for detection of
asymptomatic C282Y homozygotes by using transferrin saturation and serum
ferritin as well a genetic test for the C282Y mutation (Edwards 1988, Phatak 1998,
Niederau 1998). A broad screening of the general population however is as yet not
recommended by WHO and CDC mainly because its is unknown how many of the
asymptomatic C282Y homozygotes will later develop clinical disease (see US
Preventive Services Task Force 2007). The largest screening study analyzed HFE
gene mutations in almost 100,000 subjects in North America. In Caucasians, C282Y
homozygosity was found in 0.44%, a value similar to many previous studies in other
populations with a similar background. Asian or Black people in contrast almost
never have an HFE gene mutation (Adams 2005). Among the Caucasian C282Y
homozygotes only males had a significant increase in liver disease when compared
to subjects without an HFE gene variation (Adams 2005). Only further prospective
follow-up studies will determine how many asymptomatic C282Y homozygotes will
develop clinical consequences of iron overload.

412 Hepatology 2012

Figure 4. Survival of 251 patients with genetic hemochromatosis (with and without
cirrhosis) in comparison with matched general population. Modified from Niederau 1996.

Table 3. Methods for early diagnosis of hemochromatosis.

It is also unknown at which ferritin values phlebotomy treatment should be
initiated in asymptomatic C282Y homozygotes (Table 4). The values recommended
by the AASLD (American Association for the Study of Liver Diseases) are based
more on the judgment of experts than on solid data. The only solid data shows that
the risk for liver fibrosis and cirrhosis increases above the threshold of 1000 ng/ml

Metabolic Liver Diseases: Hemochromatosis 413
for serum ferritin (Loreal 1996). The value of screening family members is obvious
when a first-degree relative has clinical hemochromatosis. Such family screening is
easy to do with the genetic test. Heterozygous family members are not at risk for
hemochromatosis unless they have other risk factors.
The clinical phenotype of hemochromatosis is seen in 1-2% of patients with
newly diagnosed diabetes mellitus and in 3-15% of patients with liver cirrhosis
(Niederau 1999). These latter patients should be screened for iron overload although
such screening obviously does not aim at a very early diagnosis. Nevertheless,
cirrhotic and diabetic patients with hemochromatosis can benefit significantly from
phlebotomy therapy. Little is known about the prevalence of hemochromatosis in
patients with arthropathy or cardiomyopathy of unclear etiology. Several smaller
studies indicate that arthropathy may be a rather early clinical sign of iron overload,
whereas cardiomyopathy usually occurs in severe iron overload.

Figure 5. Cumulative survival in 251 patients with genetic hemochromatosis according
to the time of diagnosis. Modified from Niederau 1996.

414 Hepatology 2012
Table 4. Iron overload therapy.
1. Phlebotomy
a) In symptomatic genetic hemochromatosis
•
Aims: complete iron depletion in 12-24 months;
•
Treatment: 1-2 phlebotomies of 500 ml each week until serum ferritin is in the range
of 20-50 ng/ml;
long-term therapy with 4-8 phlebotomies per year to keep ferritin between 20-50
ng/ml and thus prevent reaccumulation of iron
b) In asymptomatic C828Y homozygotes therapy should be initiated above these
ferritin values:
•
Subjects <18 years
>200 ng/ml
•
Men
>300 ng/ml
•
Women (not pregnant)
>200 ng/ml
•
Women (pregnant)
>500 ng/ml
2. Therapy with iron chelators in secondary hemochromatosis and anemia
•
Aims: removal of iron overload by increase of iron excretion in feces and urine
•
In case of further blood transfusions at high frequency at stabilisation of iron
balance and reduction of further iron accumulation
•
Treatment: until recently, 25-50 mg deferoxamine/kg as SC infusion for 10-12 h
daily; today, deferoxamine is largely replaced by the oral chelator deferasirox - 20
mg/kg deferasirox once daily to prevent iron accumulation up to 800 ml erythrocytes
concentrates/month
•
Long-term treatment necessary
•
Normalisation of ferritin and liver iron concentration is often not possible
3. Diet
•
Recommended: avoidance of food with very high iron content (e.g., liver) and ironsupplemented food;
•
A further strict iron-depleted diet is very difficult to adhere to and not recommended
•
A single phlebotomy of 500 ml blood is as effective for iron removal as a very rigid
iron-restricted diet for a full year

Figure 6. Signs and symptoms in 185 patients with genetic
hemochromatosis prior to and after iron removal. Modified
from Niederau 1996.

Metabolic Liver Diseases: Hemochromatosis 415

Complications of iron overload
Liver cirrhosis, diabetes mellitus, and increased skin pigmentation are the classical
trio of genetic hemochromatosis. Cardiomyopathy, cardiac arrhythmias, and
impotence are also typical complications of advanced iron overload. Arthropathy in
contrast may be an early sign of hemochromatosis, which may help with diagnosis
in the precirrhotic stage (Niederau 1996).
Liver disease. The liver is the organ that is affected by genetic iron overload most
early and heavily. At early stages excess iron stores are mainly found in periportal
parenchymal cells as ferritin and hemosiderin. When iron excess further increases,
there is development of perilobular fibrosis and iron stores are also found in bile
ducts and Kupffer cells. Septal fibrosis eventually progresses towards complete
cirrhosis. The stage of fibrosis is closely associated with the degree of excess of
iron. In many affected symptomatic patients with type 1 hemochromatosis there are
some signs of liver disease at the time of diagnosis (Niederau 1985, Niederau 1996).
Many nonspecific symptoms such as abdominal discomfort and fatigue may also be
due to liver involvement. In asymptomatic patients diagnosed by a screening
procedure, signs of liver disease are infrequent. Complications due to cirrhosis such
as ascites, jaundice and portal hypertension are seen only rarely and only in cases of
advanced severe iron overload (Niederau 1985, Niederau 1996). The risk for liver
cirrhosis increases at ferritin values >1000 ng/ml (Loreal 1996). Similar to insulindependent diabetes, liver cirrhosis cannot be reversed by removal of iron (Niederau
1996). However, less advanced stages like hepatic fibrosis and abnormalities in liver
enzymes and function respond well to iron removal (Niederau 1996) (Figure 5).
Survival is significantly reduced in the presence of liver cirrhosis whereas patients
diagnosed in the precirrhotic stage have a normal life expectancy when treated by
phlebotomy (Niederau 1996) (Figure 3).
Association of hemochromatosis with other liver diseases. Some studies indicate
that C282Y heterozygosity may aggravate the progression of concomitant liver
diseases such as porphyria cutanea tarda, chronic hepatitis C, alcoholic hepatitis and
non-alcoholic steatohepatitis (NASH). In these latter patients one might find slightly
elevated liver iron concentrations and serum ferritin levels when they are C282Y
heterozygotes (for review see Erhardt 2003). Most studies however have shown that
these associations are of only minor importance in the clinical course of the disease.
Phlebotomy as yet has only been proven meaningful in porphyria cutanea tarda
because it can ameliorate the cutaneous manifestations.
Liver carcinoma. Liver carcinoma develops in approximately 30% of patients
with hemochromatosis and cirrhosis independent of iron depletion (Niederau 1996).
The interval between complete iron depletion and reported diagnosis of liver cancer
is approximately 9 years in large cohorts in German patients (Niederau 1985
Niederau 1996). The risk of liver cancer is increased in patients with
hemochromatosis 100-200-fold when compared to the general population (Figure
6). Among liver cancers there are hepatocellular carcinomas (HCC) as well as
cholangiocellular carcinomas. Most liver cancers develop in patients with cirrhosis.
Thus, cancer screening by ultrasound and APF (twice a year) is only recommended
for cirrhotic patients. Patients who develop liver cancer usually have the largest
amount of mobilisable iron among various subgroups (Niederau 1996, Niederau
1999).

416 Hepatology 2012

Figure 7. Relative mortality risk of 251 patients with genetic
hemochromatosis in comparison to the general population.
Modified from Niederau 1996.

Diabetes mellitus. In studies the prevalence of diabetes in hereditary
hemochromatosis ranges from 20-50% (Niederau 1996, Adams 1991). The
prevalence and stage of diabetes is related to the degree of iron deposition in the
pancreas. Patients with diabetes have a twofold higher mobilisable iron content than
non-diabetics (Yaouanq 1995). Investigations into the prevalence of unrecognized
genetic hemochromatosis in diabetic patients show some variation in Europe vs.
elsewhere; i.e., screening revealed a prevalence of 5-8 per 1000 unrecognized cases
in Europe (Singh 1992) and 9.6 per 1000 in Australia (Phelps 1989). Diabetes
mellitus and impaired glucose tolerance are frequent features in several chronic liver
diseases (Creutzfeldt 1970, Blei 1982). This author’s study (Niederau 1984) showed
hyperinsulinemia and hence insulin resistance without impaired glucose tolerance in
noncirrhotic hemochromatosis. The increase in circulating insulin concentrations is
likely to be due to a decrease in diminished hepatic extraction of insulin. With the
progression of iron overload and destruction of beta cells, insulin secretion becomes
impaired (Dymock 1972, Bierens de Haan 1973). In end-stage hemochromatosis,
insulin deficiency is associated with severe reduction in the mass of beta cells
(Rahier 1987). Insulin resistance observed in early iron overload may be partially
reversible after phlebotomy therapy (Niederau 1985, Niederau 1996) whereas
insulin-dependent diabetes is irreversible (Niederau 1996). Survival is significantly
reduced in patients with diabetes mellitus at diagnosis compared to patients without
diabetes (Niederau 1996). Survival of non-diabetic patients is virtually identical to
that of a matched normal population.
Heart disease. Cardiomyopathy and cardiac arrhythmias are specific
complications of hemochromatosis caused by iron deposition in the heart (Buja and
Roberts 1971, Short 1981). Clinical or electrocardiographic signs of heart disease
can be found in 20-35% of patients with HFE hemochromatosis (Niederau 1985).
Arrhythmias usually respond well to iron removal (Short 1981, Niederau 1996). In
type 1 hemochromatosis cardiomyopathy is rare and usually associated with
advanced iron overload and an older patient population. However, particularly in
young patients who present with cardiac disease due to hemochromatosis,
cardiomyopathy is a frequent cause of death (Finch 1966, Short 1981). Only

Metabolic Liver Diseases: Hemochromatosis 417
recently has it become clear that young patients with severe cardiomyopathy may be
affected by juvenile type 2 hemochromatosis; these patients may show severe iron
overload, hypogonadism, cardiomyopathy, liver cirrhosis, and amennorrhea by ages
15-24. The type 2-associated cardiomyopathy is often irreversible despite initiation
of phlebotomy or chelation therapy and may require an immediate transplant of the
heart and potentially of the liver as well (von Herbay 1996, Jensen 1993).
Arthropathy. Joint changes in genetic hemochromatosis may occur in two
different ways (Schuhmacher 1964, Dymock 1970, Niederau 1985, Niederau 1996).
The most prevalent changes are seen in the metacarpophalangeal joints II and III, in
the form of cystic and sclerotic changes, cartilage damage and a narrowing of the
intraarticular space. Sometimes other joints of the hands and the feet are affected.
Large joints, i.e., of the knees and hips, may be affected in the form of
chondrocalcinosis. The pathogenesis of joint changes in hemochromatosis remains
unclear. Arthropathy is one of the few complications not associated with the degree
of iron overload. It has been speculated that iron may inhibit pyrophosphatase and
may thereby lead to a crystallisation of calcium pyrophosphates. Alternatively, iron
may have direct toxic effects on the joints. Arthropathy may be an early sign of
hemochromatosis and may help to make the diagnosis at a precirrhotic stage
(Niederau 1996). Hemochromatosis should therefore been considered in all patients
with an arthropathy of unknown etiology.
Endocrine abnormalities. In contrast to the early onset of arthropathic changes,
endocrine abnormalities are a late consequence of iron overload. Sexual impotence
and loss of libido may occur in up to 40% of male patients (Niederau 1985). The
endocrine abnormalities in hemochromatosis are mainly, if not exclusively, due to
pituitary failure. This is in contrast to alcoholic cirrhosis where testicular failure is
predominant (Kley 1985a, Kley 1985b). In contrast to alcoholic cirrhosis, where
estrogen levels are usually increased, estrogen levels were found decreased in
hemochromatosis (Kley 1985a). Most endocrine changes are late and irreversible
complications of genetic hemochromatosis and do not respond well to phlebotomy
treatment (Niederau 1996). Iron overload only infrequently affects other endocrine
organs such as the thyroid and adrenal glands. Severe hypogonadism with
amennorrhea in young women and impotence in young men is today thought to be
due to type 2 hemochromatosis.
Skin. Increased skin pigmentation is mainly seen in areas exposed to sunlight. A
large part of the darkening of pigmentation is thought to be due to an increase in
melanin and not due to iron excess itself. The increase in skin pigmentation is
reversible on iron removal (i.e., phlebotomy).
Other potential complications. Iron overload has been speculated to aggravate
atherosclerosis; however, the evidence for that is rather weak (for review see
Niederau 2000). There have also been reports that extrahepatic malignancies may be
increased in HFE hemochromatosis (Amman 1980, Fracanzani 2001) while other
studies have not found extrahepatic associations (Bain 1984, Niederau 1996,
Elmberg 2003). It is not clear whether HFE gene mutations are involved in the
pathogenesis of porphyria cutanea tarda since the prevalence of both risk factors
vary greatly in different parts of the world; associations between HFE gene
mutations and porphyria have often been described in southern Europe but not in
northern Europe (Toll 2006).

418 Hepatology 2012

Therapy
Phlebotomy treatment. Phlebotomy treatment is the standard of care to remove iron
in genetic hemochromatosis. One phlebotomy session removes approximately 250
mg iron from the body. Since patients with the classical clinical phenotype may
have an excess of 10-30 g iron, it may take 12-24 months to remove the iron
overload when phlebotomies of 500 ml blood are done weekly (Table 4).
Phlebotomy treatment is generally well tolerated and hemoglobin usually does not
drop below 12 g/dl. Several studies have shown that liver iron is completely
removed at such low ferritin values; thus the effect of therapy can be checked by
ferritin measurements and a control liver biopsy is not necessary. After complete
removal of excess iron the intervals of phlebotomies may be increased to once every
2-3 months; serum ferritin should be kept in the lower normal range, between 20-50
ng/ml. Phlebotomy should not be interrupted for longer intervals; there is a risk of
reaccumulation of iron due to the genetic autosomal recessive metabolic
malfunction.
Iron removal by chelators. Deferoxamine therapy for genetic hemochromatosis is
not recommended because phlebotomy is more effective with less side effects and
lower cost. Recently, a Phase II study has started, looking for safety and
effectiveness of the new oral iron chelator deferasirox in genetic hemochromatosis.
As yet, deferasirox is only approved for secondary hemochromatosis.
Diet. An iron-low diet is not recommended for patients with genetic
hemochromatosis. One phlebotomy of 500 ml blood removes approximately 250 mg
iron. A difficult to follow rigid iron-restricted diet for a complete year would have
the effect of a single phlebotomy. It is thus recommended that patients simply do
not eat excessive amounts of food with very high iron content (such as liver) and
that they do not eat food to which iron has been added (Table 4).
Liver transplantation. Advanced liver cirrhosis and carcinoma may be indications
for a liver transplant in hemochromatosis (Kowdley 1995, Brandhagen 2000). The
prognosis of patients who have a liver transplant for hemochromatosis is markedly
worse than that for patients with other liver diseases; a considerable number of
patients with hemochromatosis die after transplant from infectious complications or
heart failure (Brandhagen 2000). Liver transplantation does not heal the original
genetic defect.

Prognosis
Untreated hemochromatosis often has a bad prognosis in the presence of liver
cirrhosis and diabetes mellitus. The prognosis is markedly worse in patients with
cirrhosis than in those without cirrhosis at diagnosis (Figure 3); the same is true for
diabetes mellitus. It is generally accepted that phlebotomy therapy improves the
prognosis. Patients diagnosed and treated in the early non-cirrhotic stage have a
normal life expectancy (Figure 3) (Niederau 1985, Niederau 1996). Thus, early
diagnosis markedly improves the prognosis (Figure 4). Iron removal by phlebotomy
also improves the outcome in patients with liver cirrhosis. The prognosis of liver
cirrhosis due to hemochromatosis is markedly better than those with other types of
cirrhosis (Powell 1971). Hepatomegaly and elevation of aminotransferases often
regress after iron removal (Niederau 1985, Niederau 1996) (Figure 5). Insulindependent diabetes mellitus and hypogondism are irreversible complications despite
complete iron removal (Niederau 1996) (Figure 5). Earlier changes in glucose and

Metabolic Liver Diseases: Hemochromatosis 419
insulin metabolism, however, may be ameliorated after iron removal. For unknown
reasons arthropathy does not respond well to phlebotomy treatment although it may
be an early sign of iron overload (Figure 5). The AASLD consensus guidelines
recommend to start phlebotomy treatment at ferritin values >300 ng/ml in men and
>200 ng/ml in women. The risk for liver fibrosis and cirrhosis is increased only at
ferritin levels >1000 ng/ml. Further studies need to determine whether
asymptomatic C282Y homozygotes with ferritin values between 300 and 1000
ng/ml need to be treated or whether one might wait and monitor ferritin at that
stage.

Juvenile hereditary hemochromatosis
Two genes have been shown to be associated with juvenile hemochromatosis: 90%
of cases are associated with mutations in hemojuveline (HJV) (locus name HFE2A,
which encodes HJV), while 10% of cases are associated with HAMP (locus name
HFE2B, which encodes hepcidin). Despite the nomenclature of HFE2A and
HFE2B, juvenile hemochromatosis is not associated with HFE mutations. In order
to avoid confusion most physicians use the terms type 2A (hemojuvelin mutations)
and type 2B (HAMP mutations). Mutations in hemojuvelin are associated with low
levels of hepcidin in urine suggesting that hemojuvelin regulates hepcidin. Hepcidin
is the key regulator of intestinal iron absorption and iron release from macrophages.
Hepcidin facilitates ferroportin internalisation and degradation. Hepcidin mutations
may thereby lead to an increase in ferroportin and thus iron uptake from the
intestine. Juvenile hemochromatosis is very rare. A clustering of HJV mutations is
seen in Italy and Greece although few families account for this phenomenon.
Mutations in HJV represent the majority of worldwide cases of juvenile
hemochromatosis.
Only a small number of patients have been identified with HAMP-related juvenile
hemochromatosis. Juvenile hemochromatosis is characterized by an onset of severe
iron overload in the first to third decades of life. Clinical features include
hypogonadism, cardiomyopathy, and liver cirrhosis (Diamond 1989, Vaiopoulos
2003). The main cause of death is cardiomyopathy (De Gobbi 2002, Filali 2004). In
contrast to HFE type 1 hemochromatosis, both sexes are equally affected. Mortality
can be reduced in juvenile hemochromatosis when it is diagnosed early and treated
properly. Phlebotomy is the standard therapy in juvenile hemochromatosis as well
and is treated similarly to HFE hemochromatosis (Tavill 2001). In patients with
juvenile hemochromatosis and anemia or severe cardiac failure, administration of
chelators such as deferoxamine have been tried to reduce mortality; some case
reports suggest that this might improve left ventricular ejection fraction (Kelly
1998).

Transferrin receptor 2 (TFR2)-related type 3
hemochromatosis
TFR2-related hemochromatosis is defined as type 3 and is also known as HFE3;
however, the term HFE3 should not be used because the HFE gene is not affected in
type 3 hemochromatosis. TFR2-related hemochromatosis is inherited in an
autosomal recessive manner. TFR2 is a type II 801-amino acid transmembrane
glycoprotein expressed in hepatocytes and at lower levels in Kupffer cells (Zhang
2004). A finely regulated interaction between TFR2, TFR1 and HFE is now thought

420 Hepatology 2012
to affect the hepcidin pathway, and, consequently, iron homeostasis (Fleming 2005).
Patients with homozygous TFR2 mutations have increased intestinal iron absorption
that leads to iron overload. Hepcidin concentrations in urine are low in TFR2
hemochromatosis (Nemeth 2005). TFR2-related hemochromatosis is very rare with
only about 20 patients reported worldwide (Mattman 2002). Age of onset in TFR2related type 3 hemochromatosis is earlier than in HFE-associated type 1 (Piperno
2004, Girelli 2002, Hattori 2003). Progression is, however, slower than in juvenile
type 2 (De Gobbi 2002, Roetto 2001, Girelli 2002). The phenotype is similar to type
1. Many patients present with fatigue, arthralgia, abdominal pain, decreased libido,
or with biochemical signs of iron overload (Roetto 2001, Girelli 2002, Hattori
2003). Complications of type 3 hemochromatosis include cirrhosis, hypogonadism,
and arthropathy. Cardiomyopathy and diabetes mellitus appear to be rather rare.
Hepatocellular carcinoma has not been observed in the small number of cases
diagnosed. Most individuals with type 3 hemochromatosis have an Italian or
Japanese genetic background. Some of the Japanese males have had liver cirrhosis
at diagnosis (Hattori 2003). Similar to type 1 hemochromatosis, the penetration of
type 3 hemochromatosis is also considerably less than 100% (Roetto 2001).
Standard therapy is iron removal by weekly phlebotomy similar to the management
of type 1 disease. Individuals with increased ferritin should be treated similar to
those with HFE hemochromatosis.

Type 4 hemochromatosis – Ferroportin Disease
Ferroportin-associated iron overload (also called Ferroportin Disease) was first
recognised by Pietrangelo (1999) who described an Italian family with an autosomal
dominant non-HFE hemochromatosis. Many family members had iron overload
resulting in liver fibrosis, diabetes, impotence, and cardiac arrhythmias. In addition
to autosomal dominant inheritance, features distinguishing this from HFE
hemochromatosis included early iron accumulation in reticuloendothelial cells and a
marked increase in ferritin earlier than what is seen in transferrin saturation
(Pietrangelo 1999, Rivard 2003, Montosi 2001, Wallace 2004, Fleming 2001).
Several patients showed a reduced tolerance to phlebotomy and became anemic
despite elevated ferritin (Pietrangelo 1999, Jouanolle 2003).
In 2001 this form of non-HFE hemochromatosis was linked to mutations of
ferroportin (Montosi 2001) that had just been identified as the basolateral iron
transporter (Abboud 2000, Donovan 2000). Since that time, numerous mutations in
the gene have been implicated in patients from diverse ethnic origins with
previously unexplained hemochromatosis. Iron overload disease due to ferroportin
mutations has been defined as type 4 hemochromatosis or Ferroportin Disease (for
review see Pietrangelo 2004). The iron export is tightly regulated because both iron
deficiency and iron excess are harmful. The main regulator of this mechanism is the
peptide hepcidin which binds to ferroportin, induces its internalization and
degradation, thereby reducing iron efflux (Nemeth 2004). Increase in iron
absorption may be caused either by hepcidin deficiency or its ineffective interaction
with ferroportin. All recent studies have shown that hepcidin deficiency appears to
be the common characteristic of most types of genetic hemochromatosis (mutations
in HFE, transferrin receptor 2, hemojuvelin, or hepcidin itself). The remaining cases
of genetic iron overload are due to heterozygous mutations in the hepcidin target,
ferroportin. Because of the mild clinical penetrance of the genetic defect there were

Metabolic Liver Diseases: Hemochromatosis 421
doubts about the rationale for iron removal therapy. However, a recent study shows
that there may be clinically relevant iron overload with organ damage and liver
cancer in patients carrying the A77D mutation of ferroportin (Corradini 2007).
Treatment schemes are similar to those described for other types of genetic
hemochromatosis.

Secondary hemochromatosis
Pathophysiology
Most forms of secondary hemochromatosis are due to hemolytic anemia associated
with polytransfusions such as thalassemia, sickle cell disease, and MDS. Most of
these patients need blood transfusions on a regular basis for survival. However, in
the long run, multiple blood transfusions often lead to iron overload if patients are
not treated with iron chelators. In general, iron overload due to blood transfusions is
similar to genetic hemochromatosis; however, secondary iron overload develops
much faster than the genetic forms (McLaren 1983), sometimes as soon as after 1012 blood transfusions (Porter 2001). Subsequently secondary iron overload can
result in more rapid organ damage when compared with genetic hemochromatosis.
Secondary iron overload can obviously not be treated by phlebotomy because a
marked anemia is the clinical marker of the disease. Secondary iron overload often
limits the prognosis of patients with thalassemia; life expectancy deteriorates with
increasing iron concentrations in the liver (Telfer 2000). Therapy with iron chelator
may reduce the transfusional iron burden if the frequency of transfusion is not too
high. The development of HFE versus secondary hemochromatosis not only differs
in terms of the speed of iron accumulation but also in the type of organ damage; in
secondary hemochromatosis cardiomyopathy is often the complication that limits
the prognosis (Liu 1994). It is interesting that heart disease is also very frequent in
juvenile genetic hemochromatosis where there is also rapid iron accumulation. In
general, serum ferritin values closely reflect liver iron concentration and may be
used as an indication for timing of therapy as well as to check the effects of iron
chelation.
Until recently deferoxamine was the only iron chelator available in most
countries; in some countries the drug deferiprone is approved for patients who do
not tolerate deferoxamine (Hoffbrandt 2003). The clinical use of deferiprone was
limited due to side effects such as agranulocytosis and neutropenia (Refaie 1995).
Long-term data prove that deferoxamine can reduce iron overload and its organ
complications (Olivieri 1994, Cohen 1981). Deferoxamine, however, needs to be
given daily subcutaneously or by IV infusion for several hours. Thus, patients with
thalassemia often consider the deferoxamine treatment worse than thalassemia itself
(Goldbeck 2000). There are minor compliance problems that often limit the
beneficial effects of this iron chelator (Cohen 1989).
Without iron chelation, children with thalassemia often develop a severe
cardiomyopathy prior to age 15 (Cohen 1987). After that age, liver cirrhosis is also
a significant complication in secondary iron overload due to thalassemia (Zurlo
1992). Iron chelation should start early to prevent complications of iron overload.
By the ages of 3-5, liver iron concentration may reach values associated with a
significant risk for liver fibrosis in severe thalassemia (Angelucci 1995). Children
younger than 5 should therefore be cautiously treated with chelators if they have

422 Hepatology 2012
received transfusions for more than a year (Olivieri 1997). Deferoxamine can
reduce the incidence and ameliorate the course of iron-associated cardiomyopathy
(Olivieri 1994, Brittenham 1994, Miskin 2003).
Deferasirox is an oral iron chelator with high selectivity for iron III (Nick 2003).
Deferasirox binds iron in a 2:1 proportion with a high affinity and increases the
biliary iron excretion (Nick 2003). This chelator is able to reduce iron overload in
hepatocytes and cardiomyocytes (Nick 2003, Hershko 2001). Due to its half-life of
11-18 hours it needs to be taken only once daily (Nisbet-Brown 2003). Deferasirox
exerted a similar iron chelation when compared with deferoxamine in patients with
thalassemia; the effect of 40 mg/kg deferoxamine was similar to that of 20 mg/kg
deferasirox (Piga 2006). Both in adults and children 20-30 mg/kg/day deferasirox
significantly reduced liver iron concentration and serum ferritin (Cappellini 2006).
Magnetic resonance imaging showed that 10-30 mg/day deferasirox may also
reduce iron concentration in the heart within one year of maintenance therapy.
Deferasirox may cause minor increases in serum creatinine as well as
gastrointestinal discomfort and skin exanthema which are usually self-limiting.
Considering the compliance problems with deferoxamine, deferasirox has a better
cost-effectiveness ratio (Vichinsky 2005). Deferasirox is defined as standard
therapy both in the guidelines of the National Comprehensive Cancer Network
(NCCN) (USA) and in the international guidelines on MDS (Greenberg 2006,
Gattermann 2005).

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NAFLD and NASH 427

25. NAFLD and NASH
Claus Niederau

Introduction
Both non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis
(NASH) are often associated with obesity, diabetes mellitus and asymptomatic
elevations of serum ALT and gamma GT. Ultrasound monitoring can suggest the
presence of a fatty infiltration of the liver; differentiation between NAFLD and
NASH, however, requires a liver biopsy. Such differentiation may be important
because NASH is associated with a much higher risk of liver fibrosis and cirrhosis
than NAFLD. Moderate weight loss due to dietary and life-style modifications is the
only therapy proven to be effective in NASH. Complete alcohol abstinence and
good control of diabetes mellitus are probably also important to reduce the risk of
severe liver disease in NASH.

Prevalence
NAFLD is present in the general population in industrialized countries in 20 to 40%
and is the most prevalent chronic liver disease (Browning 2004, Chitturi 2004,
McCullough 2005). It is more prevalent in obese and diabetic subjects (Bellentani
1994, Wanless 1990, Clark 2002, Chitturi 2004). Among all subjects with NAFLD,
features of non-alcoholic steatohepatitis (NASH) can be seen in 10-20%. The
prevalence of NASH in western countries is approximately 2-6%. In the US, NASH
is estimated to affect 5-6% of the general population (McCullough 2005). It has
been suggested that NASH accounts for more than 50% of cryptogenic cirrhosis
(Ratziu 2002). NAFLD may progress to NASH with fibrosis, cirrhosis, and
hepatocellular carcinoma (Marchesini 2003, Caldwell 2004). The term NASH was
introduced in a description of 20 Mayo Clinic patients with a hitherto unnamed
disease associated with hepatomegaly, abnormal ALT, a fatty liver histology,
lobular hepatitis, and fibrosis mimicking alcoholic hepatitis in the absence of
alcohol intake (Ludwig 1980); most patients had obesity and diabetes mellitus.

428 Hepatology 2012

Demographics and risk factors
In the US, NAFLD is 3-5 times more prevalent in men than in women; such
differences in gender might partly be explained by the fact that men have a higher
BMI and that some male patients with NAFLD drink more alcohol than they report
drinking (Schwimmer 2005, Bahcecioglu 2006, Loguercio 2001). The NAFLD
prevalence in the US is particularly high in people of Hispanic (28%) or Asian
origin (20-30%) (Schwimmer 2005, Weston 2005). Due to the dramatic increase in
obesity in the US and many other industrialized countries, there is also a dramatic
increase in the prevalence of NAFLD and NASH. In the US almost 50% of obese
boys have NAFLD (Schwimmer 2005). In many countries more than 80% of
NAFLD patients have an increased BMI and 30-40% are obese; approximately 50%
show signs of insulin resistance, 20-30% have type 2 diabetes, 80% show
hyperlipidemia, and 30-60% have arterial hypertension. Correspondingly there is a
strong association between NAFLD and NASH and the metabolic syndrome
throughout the world (Marchesini 1999, Bedogni 2005). In comparison with
NAFLD patients, NASH patients are older, more obese and more often have high
serum liver enzymes, diabetes mellitus and metabolic syndrome (Ratziu 2002,
Adams 2005, Hamaguchi 2005, Fassio 2004).

Pathogenesis
The degree of fatty infiltration in NAFLD is graded according to the percentage of
hepatocytes with fat deposits: mild NAFLD involves less than 30% hepatocytes,
moderate NAFLD up to 60%, and severe NAFLD above 60% (Ploeg 1993).
NAFLD may regress if the cause is eliminated. NASH is associated with insulin
resistance, increased circulating levels of leptin, adiponectin, tumour necrosis factor
and some interleukins (Friedman 1998, Marra 2004). It is thought that there is an
increased flow of free fatty acids from visceral fat to the liver contributing to
abnormalities in intracellular lipid metabolism (Hashimoto 1999, Vendemiale
2001). Insulin resistance and increased free fatty acids may both affect
mitochondrial oxidation of fatty acids causing free radical generation in hepatocytes
(Grattagliano 2003). Thus, NASH is caused by two mechanisms or toxic “hits”; the
first mechanism is the hepatic accumulation of triglycerides (NAFLD) due to insulin
resistance and the second is thought to be the generation of free radicals with
subsequent release of mediators and cytokines (McCullough 2006).
Insulin resistance has been closely linked to non-alcoholic fatty liver disease in
both clinical trials and laboratory-based studies (McCullough 2006, Marchesini
2001, Sanyal 2001). The actual process by which NAFLD turns into NASH
however remains ill defined despite this double-hit theory. Likely, genetic factors
(similar to those responsible for the metabolic syndrome) as well as exogenic
factors (like drugs, moderate amounts of alcohol, and other toxins) may contribute
to the evolution of NAFLD into NASH. The role of hepatic iron in the progression
of NASH remains controversial, but in some patients, iron may have a role in the
pathogenesis of NASH by promoting oxidative stress. Iron overload has been shown
to cause lipid peroxidation and to activate hepatic stellate cells (Lee 1995). In some
reports, an increased prevalence of the Cys282Tyr HFE gene mutation in patients
with NASH has been reported (George 1998). The presence of the Cys282Tyr

NAFLD and NASH 429
mutation was associated with increased hepatic iron concentration that in turn is
associated with the severity of the fibrosis. Other studies have shown that other
heterozygote HFE gene mutations are more prevalent in NASH patients when
compared with controls (Bonkowsky 1999). In another clinical cohort, there was no
association between hepatic iron and histological or clinical outcome (Younoussi
1999).

Natural history
The natural history of NAFLD in the general population is not well-defined since
most data come from selected patients and tertiary centres (Dam-Larsen 1996, Lee
1989, Teli 1995). Correspondingly, published mortality and morbidity in
hospitalized NAFLD are approximately 5 times higher than what is seen in the
general population (Matteoni 1999). In the general population the risk for liverrelated death in NAFLD appears to be associated mainly with age, insulin
resistance, and histological evidence of hepatic inflammation and fibrosis (Adams
2005). Probably around 10% of NAFLD patients will progress to NASH over a
period of 10 years (Figure 1). Cirrhosis later develops in 5-25% of patients with
NASH and 30-50% of these patients die from liver-related causes over a 10-year
period (McCollough 2005, Matteoni 1999). Cirrhosis in patients with NASH can
also decompensate into subacute liver failure, progress to hepatocellular cancer
(HCC), and recur after liver transplantation (McCollough 2005). Steatosis alone is
reported to have a more benign clinical course, with cirrhosis developing in only 13% of patients (Day 2004, Day 2005, McCollough 2005, Matteoni 1999). Patients
with NASH and fibrosis also have a significant risk for hepatocellular carcinoma
(El-Serag 2004) (Figure 1).

Figure 1. Natural history of NASH.

430 Hepatology 2012
Table 1. Non-invasive predictors of NASH.
HAIR index (hypertension; ALT >40 U/l; insulin resistance)
≥2 are 80% sensitive, 89% specific for NASH (Dixon 2001)
BAAT index (BMI >28; Age >50 years; ALT >2x UNL; increased trig’s)
≤1 has 100% negative predictive value for NASH (Ratziu 2000)

Diagnosis
NAFLD and NASH require valid reporting about alcohol consumption. Since only
approximately 10% of western populations are completely abstinent from alcohol,
one needs to set a threshold above which one assumes that alcohol at least
contributes to the pathogenic process of NAFLD and NASH. Most authors use a
daily alcohol ingestion of 20 g as such a threshold (Figure 2); others use lower
values such as 10 g/day or as high as 40 g/day for men.

Figure 2. Differentiation of alcoholic liver disease (ASH) and NASH.

The workup of NAFLD and NASH also includes checking into drug abuse, HBV
and HCV infections, hemochromatosis, autoimmune liver disease and, in younger
patients, Wilson’s Disease. In special groups of patients NASH may be
accompanied by drug- and alcohol-induced liver disease and by HCV and HBV
infections. The combination of NAFLD/NASH and HCV infection plays a
particularly important clinical role because in this situation the rate of liver fibrosis
is increased and the success of antiviral therapy is diminished (Ramesh 2004).
NASH can be induced by various drugs and toxins including corticosteroids,
amiodarone, methotrexate, tetracycline, tamoxifen, and valproate (Pessayre 2002)
(Table 4). Thus, one needs to carefully assess the full clinical history of patients. In
practice NAFLD is often diagnosed by combining elevated levels of ALT and
gamma GT with the sonographic appearance of an increase in the echodensity of the
liver. However, a considerable number of patients with NAFLD and even with
NASH and fibrosis have normal serum liver enzymes (Abrams 2004). Usually ALT
is higher than AST unless there is already severe fibrosis or cirrhosis. Fasting serum
glucose should be checked in all patients with NAFLD and NASH; one will also
often find elevated serum insulin, insulin resistance, and/or diabetes (Table 2).

NAFLD and NASH 431
Table 2. Treatment options for NASH.
Moderate weight loss
Drugs for treatment of obesity (e.g., orlistat or sibutramine)
Complete abstinence from alcohol
Good control of diabetes mellitus
Insulin sensitizers (e.g., glitazones)
Surgery for massive obesity (e.g., gastric bypass surgery)
Liver transplant (LTX)

Many authors also recommend to routinely look for metabolic syndrome, which is
diagnosed when three of the following features are seen (Greenland 2003):
− waist circumference ≥102 cm for men and ≥88 cm for women,
− fasting glucose level ≥6.1 mmol/L,
− triglyceridemia ≥1.7 mmol/L,
− increase in high-density lipoprotein cholesterol (>1.3 mmol/L in women; >1.03
mmol/L in men)
− hypertension ≥135/80 mmHg.
Ultrasound of the liver has a high sensitivity and specificity (both approaching
90%) for detection of fatty infiltration but does not allow assessment of the presence
or degree of inflammation and fibrosis (Davies 1991). Therefore, diagnosis of fat in
the liver is easily made by ultrasound but diagnosis of NAFLD or NASH cannot be
made without a liver histology. In addition, liver biopsy is still the only way to
reliably differentiate NASH from NAFLD (Harrison 2003). Today most
pathologists use the Brunt description to score the histological degree of NASH
(Brunt 1999) (Table 3). Since NAFLD is a very frequent but also relatively benign
disease, one aims to identify some risks factors for NASH in order to avoid doing
liver biopsies in all NAFLD patients. Risk factors for NASH include older age,
excessive obesity, diabetes mellitus, other hepatotoxins, and clinical, laboratory or
sonographic signs suggesting severe liver disease; two non-invasive scores have
been used to predict NASH and might be used to identify patients who should have
a liver biopsy (Table 3) (Dixon 2001, Ratziu 2000). Combinations of various serum
markers of liver fibrosis and the results from liver stiffness measured by the
fibroscan have been suggested to predict the presence of NASH and fibrosis
(Rosenberg 2004, Suzuki 2005). These tests have not yet replaced the liver biopsy.

Diet and lifestyle recommendations
Today, the only effective treatment for NAFLD and NASH is a slow and moderate
weight loss, usually associated with other lifestyle modifications. Several studies
have shown that rapid weight loss (very low calorie diet or starving) increases the
risk of progression of liver disease and even liver failure (Grattagliano 2000, James
1998, Neuschwander-Tetri 2003). Patients should therefore be educated not to
induce rapid weight loss, but to aim at a weight loss of less than 10% of their body
weight over 6-12 months (Okita 2001). It is unclear whether special diets are
helpful; probably it is more important that the patients simply eat healthy foods like
vegetables and fruits, rich in fibre and complex carbohydrates with a low glycemic
index; they should avoid meat, saturated fat and products with less complex

432 Hepatology 2012
carbohydrates. Lifestyle modifications should include an increase in physical
activity and sports as well as abstinence from alcohol. With the results of recent
studies, coffee consumption does not need to be limited.
Table 3. Histological Brunt score (Brunt 1999).

Pharmacological treatment
There is no drug proven to be beneficial in NAFLD and NASH; therefore no drug
has been approved by FDA or EMA. Drugs that might reverse insulin resistance
such as metformin and thiazolidinediones (rosiglitazone, pioglitazone) seemed to be
most promising, but have so far not been proven to be benificial (Angelico 2007). In
addition marketing of rosiglitazone was discontinued due to safety concerns. The
distribution of pioglitazone was stopped in some countries due to similar reasons.
In general all drugs that induce weight loss might be beneficial in NAFLD and
NASH, in particular when diet and life-style modification do not work (Table 5).
Both sibutramine and orlistat have shown to improve some characteristics of
NAFLD and NASH such as the sonographic degree of liver steatosis as well as the
histological degree of steatosis and fibrosis (Sabuncu 2003, Derosa 2004, Hussein
2007, Harrison 2007).
Antioxidants and cytoprotective substances have also been proposed to treat
NAFLD and NASH including vitamin E, vitamin C, glutathione, betaine,
acetylcysteine, S-adenosyl-L-methionine and ursodesoxycholic acid. After a recent
Cochrane analysis, none of these substances has shown significant benefit in
validated randomized studies (Lirussi 2007).

Surgery for obesity
Gastric bypass has also recently been shown to improve NASH (Liu 2007, de
Almeida 2006, Furuya 2007); however, surgery is usually restricted to patients with
massive obesity.

NAFLD and NASH 433

Liver transplantation (LTX) for NASH
LTX is the final option for patients with end-stage liver disease due to cirrhosis and
complications of portal hypertension with NASH. Due to the increase in the
prevalence of NASH, there is also an increase in LTX done for end-stage liver
disease caused by NASH (Burke 2004). However, NASH can recur after LTX,
particularly if patients have previously undergone jejunoileal bypass surgery (Kim
1996, Requart 1995, Weston 1998, Contos 2001, Burke 2004). LTX does not cure
the metabolic defect that causes NASH.

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Wilson’s Disease 437

26. Wilson’s Disease
Claus Niederau

Introduction
In 1912, Kinnear Wilson was the first to describe an inherited lethal disease
associated with progressive lenticular degeneration, chronic liver disease and
cirrhosis (Wilson 1912). In the same year, Kayser and Fleischer detected that
patients with Wilson’s Disease (WD) often have brownish corneal copper deposits
now called Kayser-Fleischer rings (Fleischer 1912).
WD is an autosomal recessive error of the metabolism. Its gene ATP7B encodes a
copper-transporting ATPase (Bull 1993, Tanzi 1993, Petrukhin 1993, Yamaguchi
1993). The genetic defect of the ATP7B protein reduces biliary copper excretion
leading to copper accumulation in the cornea and various organs including the liver,
brain and kidney. The alteration of the ATP7B protein also reduces the
incorporation of copper into ceruloplasmin. The corresponding presence of
apoceruloplasmin (ceruloplasmin with no copper incorporation) leads to a decrease
in circulating levels of ceruloplasmin due to the reduced half-life of the apoprotein.
Thus, despite copper accumulation in many organs, circulating levels of copper and
ceruloplasmin are decreased in most WD patients.
The prevalence of WD is rare, estimated at 3 per 100,000 population (Frysman
1990). The clinical presentation may vary. Some WD patients are diagnosed with
liver problems while others present with neurologic or psychiatric symptoms; many
patients show both hepatic and neurological disease (Figure 1). Episodes of
hemolysis and renal abnormalities may also occur. WD typically affects children
and younger adults, and is rarely seen in adults older than 40. WD is fatal unless
appropriately treated. Drugs for treatment of WD are copper chelators such as
penicillamine, and trientine (Walshe 1956). More recently, zinc has been used to
reduce intestinal copper absorption and to detoxify free circulating copper. Patients
with fulminant liver failure or decompensated cirrhosis may have to undergo liver
transplantation (LTX), which cures WD.

Clinical presentation
Screening for WD is useful only in families with an affected member. In all other
circumstances diagnostic procedures are only done when symptoms and findings
suggest WD. These include liver disease, neurological symptoms, renal

438 Hepatology 2012
abnormalities and episodes of hemolysis. WD is diagnosed in the vast majority of
patients between the ages of 5 and 35. There are rare reports of patients diagnosed at
ages 3-5 (Kalach 1993, Wilson 2000) and at ages of up to about 60 years (Gow
2000). Late-onset WD is a frequently overlooked condition (Ferenci 2007).
Diagnostic workup does not rely on a single test but includes identification of
corneal Kayser-Fleischer rings, reduced serum ceruloplasmin and copper as well as
a quantitative determination of liver copper concentration (Scheinberg 1952,
Walshe 1956, Saito 1987, Stremmel 1991, Roberts 2003) (Figure 2).

Figure 1. Clinical course of WD in 53 patients (modified from
Stremmel 1991).

Figure 2. Diagnostic workup for WD.

Genetic tests are usually only done in relatives of a confirmed WD patient. It is
easy to diagnose WD in subjects who present with liver cirrhosis, typical neurologic
manifestations and Kayser-Fleischer rings; many of these patients present at ages 5
to 35 and have decreased serum copper and ceruloplasmin (Sternlieb 1990).
However, a considerable number of WD patients present only with liver disease and
may not have Kayser-Fleischer rings or decreased serum levels of ceruloplasmin
(Steindl 1997). Under these circumstances diagnosis may be difficult; measurement
of 24 hour urinary copper excretion often helps to support the suspicion of WD.

Wilson’s Disease 439
Liver biopsy with measurement of quantitative copper concentration should be done
to corroborate the diagnosis (Stremmel 1991, Roberts 2003).
In general, WD patients diagnosed primarily with liver disease are children and
adolescents and are younger than those diagnosed due to neurological symptoms
(Merle 2007). Many patients who present only with CNS symptoms are 20-40 years
old. Patients with WD may present with a wide spectrum of liver disease ranging
from asymptomatic elevation of serum aminotransferases to fulminant liver failure.
Serum aminotransferases are elevated in most WD patients irrespective of age
(Schilsky 1991). Other WD patients may present with findings and symptoms of
autoimmune hepatitis including autoimmune antibodies and elevated IgG (Scott
1978, Milkiewicz 2000). The clinical picture might also resemble acute or chronic
viral hepatitis, without the viral serum markers. Even liver histology is not
predictive or typical for WD unless copper concentration is measured. Histological
findings may range from fatty liver changes to severe necro-inflammatory and
fibrotic disease and complete cirrhosis. In particular, children and adolescents with
chronic active hepatitis of unknown etiology or autoimmune hepatitis and adult
patients with a suspicion of autoimmune hepatitis or non-response to
immunosuppressants should be evaluated for WD (Roberts 2003).
WD has to be excluded in patients with fulminant liver failure of unknown
etiology, especially at ages under 35 years; WD patients with such presentation
usually have some sort of liver disease (Rector 1984, Ferlan-Maroult 1999, Roberts
2003) associated with Coombs-negative hemolytic anemia and severely increased
prothrombine time non-responsive to vitamin K and progressive renal failure (Sallie
1992). Some patients have bilirubin levels of more than 40 mg/dl while serum
alkaline phosphatase is normal or just slightly elevated (Berman 1991). In contrast
to many types of toxic liver failure, liver failure in WD usually does not start with
high increases in aminotransferases. In many WD patients AST levels exceed ALT
levels (Emre 2001, Berman 1991). In most cohorts, for unexplained reasons, the
ratio of females to males is approximately 2:1 (Roberts 2003). Serum ceruloplasmin
may be decreased while serum copper and 24-hour urinary excretion of copper is
usually elevated. It is extremely helpful when one can identify Kayser-Fleischer
rings in this situation; these patients need to be studied with a slit lamp by an
experienced ophthalmologist. Patients with acute liver failure need a diagnostic
workup as rapidly as possible; if there is a strong suspicion or diagnosis of WD, the
patient should be transferred to a transplant centre the same day.
Neurological symptoms in WD often resemble those seen in Parkinson’s disease
including tremor and rigor. Many patients report that symptoms start with problems
in handwriting and dysarthria. Neurological symptoms may be associated with
slight behavioural alterations, which may later proceed to manifest psychiatric
disease including depression, anxiety and psychosis. With the progression of CNS
involvement WD patients may develop seizures and pseudobulbar palsy associated
with severe dysphagia, aspiration and pneumonia. Although many older WD
patients present with neurological disease, the diagnostic workup often shows
significant liver involvement or even complete liver cirrhosis.
Renal involvement of WD may present with aminoaciduria and nephrolithiasis
(Azizi 1989, Nakada 1994, Cu 1996). There may be various other non-neurological
and non-hepatic complications of WD such as osteoporosis and arthritis,

440 Hepatology 2012
cardiomyopathy, pancreatitis, hypoparathyroidism, and miscarriages (for literature
see Roberts 2003).
Kayser-Fleischer rings are caused by corneal copper deposition (Figure 3).
Sometimes, one can see the rings directly as a band of brown pigment close to the
limbus. In other patients the ring can only be identified using a slit lamp. Very
rarely similar rings may be seen in non-WD patients, e.g., in some patients with
neonatal or chronic cholestasis (Tauber 1993). Kayser-Fleischer rings are detectable
in 50-60% of WD patients in most large cohorts (Tauber 1993, Roberts 2003).
Many young WD patients with liver disease do not have such rings (Giacchino
1997) whereas almost all patients with primarily neurologic symptoms do have
them (Steindl 1997). WD patients may also have other less specific eye changes
including sunflower cataracts (Cairns 1963). Kayser-Fleischer rings usually regress
with chelation therapy or after LTX (Stremmel 1991, Schilsky 1994).

Figure 3. Kayser-Fleischer ring in a patient with WD.

Diagnosis
Serum ceruloplasmin
Ceruloplasmin, the major circulating copper transporter, is synthesized and
secreted mainly by hepatocytes. The 132 kd protein consists of six copper atoms per
molecule of ceruloplasmin (holoceruloplasmin) while the remaining part of the
protein does not carry copper (apoceruloplasmin). Ceruloplasmin acts as an acute
phase reactant and may thus be increased by any inflammatory process; it may also
rise in pregnancy and with the use of estrogens and oral contraceptives. One also
needs to remember that the normal range of serum ceruloplasmin is age-dependent:
it is usually low in infants until the age of 6 months; in older children it may be

Wilson’s Disease 441
somewhat higher than in adults. As explained previously, serum levels of
ceruloplasmin are generally decreased in WD; however, this finding alone is
unreliable because low serum ceruloplasmin may be seen without WD and serum
ceruloplasmin may even be increased in severe WD and liver failure. Non-specific
reductions of ceruloplasmin are usually associated with protein deficiency or any
end-stage liver disease. Long-term parenteral nutrition may also lead to decreased
levels of ceruloplasmin. Low serum ceruloplasmin is also a hallmark of Menkes’
disease, a very rare X-linked inborn error of metabolism that leads to a defect in
copper transport due to mutations in ATP7A (Menkes 1999). Very rarely, one
cannot measure serum ceruloplasmin at all. This aceruloplasminemia is a very rare
genetic disease caused by mutations in the ceruloplasmin gene; however, patients
with aceruloplasminemia develop iron and not copper overload (Harris 1998).
Most patients with WD have a serum ceruloplasmin lower than 20 µg/dl; this
finding is diagnostic for WD however only when there are other findings such as a
Kayser-Fleischer corneal ring. In one prospective screening study, ceruloplasmin
was measured in 2867 patients presenting with liver disease: only 17 of them had
reduced ceruloplasmin levels and only 1 of these subjects had WD (Cauza 1997).
Thus decreased ceruloplasmin had a positive predictive value of only 6% in the
2867 patients tested. In two cohorts, about 20% of WD had normal ceruloplasmin
and no Kayser-Fleischer rings (Steindl 1997, Gow 2000). Most reports, however,
show that more than 90% of WD patients have a reduced serum ceruloplasmin
(Walshe 1989, Lau 1990, Stremmel 1991). Measurement of ceruloplasmin as a
single marker cannot reliably differentiate homozygotes from heterozygotes.

Serum copper
Corresponding to the decrease in serum ceruloplasmin, total serum copper is also
usually decreased in WD. Similar to the diagnostic problems in interpreting
ceruloplasmin data in WD patients with fulminant liver failure, serum copper may
also be normal in this situation – even if serum ceruloplasmin is decreased. In acute
liver failure circulating copper may in fact be elevated because it is massively
released from injured hepatocytes. If ceruloplasmin is reduced, a normal or elevated
serum copper usually suggests that there is an increase in free serum copper (not
bound to ceruloplasmin). The free copper concentration calculated from total copper
and ceruloplasmin values has also been proposed as a diagnostic test and for
monitoring of WD. It is elevated above 25 µg/dL in most untreated patients (normal
values are below 10-15 µg/dL). The amount of copper associated with
ceruloplasmin is 3.15 µg of copper per mg of ceruloplasmin. Thus free copper is the
difference between the total serum copper in µg/dL and 3 times the ceruloplasmin
concentration in mg/dl (Roberts 1998).
Increases in serum free copper, however, are not specific for WD and can be seen
in all kinds of acute liver failure as well as in marked cholestasis (Gross 1985,
Martins 1992). The calculation of the free copper concentration critically depends
on the adequacy of the methods used for measuring total serum copper and
ceruloplasmin; often labs simply state that one of the tests is below a certain value,
which makes it impossible to calculate the amount of free copper.

442 Hepatology 2012

Urinary copper excretion
Most WD patients have an increase in urinary copper excretion above 100 µg/24
hours, which is thought to represent the increase in circulating free copper (not
bound to ceruloplasmin). Some studies suggest that about 20% of WD patients may
have 24-hr urinary copper excretion between 40-100 µg/24 h (Steindl 1997,
Giacchino1997, Gow 2000, Roberts 2003). However, some increase in urinary
copper excretion can be found in severe cholestasis, chronic active hepatitis and
autoimmune hepatitis (Frommer 1981). It has been suggested that urinary copper
excretion stimulated by penicillamine may be more useful than the non-stimulating
test. In children 500 mg of oral penicillamine is usually given at the beginning and
then at 12 hours during the 24-hour urine collection. All WD children looked at had
levels above 1600 µg copper/24 h and all patients with other liver diseases including
autoimmune hepatitis and cholestatic liver disease had lower values. It is not clear
whether this test has a similar discriminative power in adults where it has been used
in various modifications (Tu 1967, Frommer 1981).

Hepatic copper concentration
Hepatic copper content above 250 µg/g dry weight liver is still the gold standard for
diagnosis of WD and is not seen in heterozygotes or other liver diseases with the
exception of Indian childhood cirrhosis (Martins 1992). Biopsies (larger than 1 cm
in length) for measurements of hepatic copper determination should be taken with a
disposable Tru-Cut needle, placed dry in a copper-free container and shipped frozen
(Song 2000, Roberts 2003).

Radiolabelled copper
In WD, incorporation of radiolabelled copper into ceruloplasmin is significantly
reduced. This test is rarely used because of the difficulty in obtaining the isotope
and because of legal restrictions.

Liver biopsy findings
Histological findings in WD range from some steatosis and hepatocellular necrosis
to the picture as seen in severe autoimmune hepatitis with fibrosis and cirrhosis.
Patients diagnosed at a young age usually have extensive liver disease; older
patients who first present with neurological symptoms often have abnormalities in
liver biopsy as well (Stremmel 1991, Steindl 1997, Merle 2007). Detection of
copper in hepatocytes, e.g., by staining with rhodamin using routine histochemistry
does not allow a diagnosis of WD (Geller 2000) (Figure 4).

Wilson’s Disease 443

Figure 4. Liver histology (rhodamine staining for copper) in a WD patient.

Neurology and MRI of the CNS
Neurologic symptoms in WD include Parkinson’s-like abnormalities with rigidity,
tremor and dysarthria. In more severely affected patients there may be muscle
spasms, contractures, dysphonia, and dysphagia. In patients with pronounced
neurological symptoms magnetic resonance imaging (MRI) often identifies
abnormalities in basal ganglia such as hyperintensity on T2 weighted imaging
(Aisen 1995, van Wassanaer 1996). MRI of the CNS is superior to computed
tomography to diagnose WD.

Genetic Studies
The use of mutation analysis in WD is limited by the fact that more than 200
ATP7B
mutations
have
been
described
(see
www.medgen.med.ualberta.ca/database.html). When the mutation is known in a
specific patient, gene analysis may be useful for family screening or prenatal
analysis (Thomas 1995, Shab 1997, Loudianos 1994). Some populations in Eastern
Europe show predominance of the H1069Q mutation (for literature see Roberts
2003).

Treatment
Before 1948, all patients with Wilson’s Disease died shortly after diagnosis. In
1948, intramuscular administration of the copper chelator BAL (dimercaprol) was
introduced as the first treatment of WD (Cumming 1951, Denny-Brown 1951)
followed by the oral chelators penicillamine (1955), trientine (1969) and
tetrathiomolybdate (1984). Other treatment modalities include oral zinc salts (1961)
and liver transplantation (1982). Today, most patients with WD remain on a lifelong

444 Hepatology 2012
pharmacologic therapy usually including a copper chelator and/or a zinc salt (Figure
5). LTX is reserved for fulminant liver failure and irreversible decompensation of
liver cirrhosis. Patients with a successful LTX do not need WD treatment because
LTX heals the biochemical defect. Today, most doctors use oral chelators for initial
treatment of symptomatic patients; many physicians start therapy with penicillamine
while some prefer trientine. Both drugs are probably equally effective, with trientine
having fewer side effects. In patients with advanced neurological disease some
authors recommend tetrathiomolybdate for primary therapy. Combination therapy of
chelators and zinc salts might have additive effects, acting on both urinary copper
excretion and its intestinal absorption. After removal of most accumulated copper
and regression of the most severe clinical problems the chelator dose may be
reduced and later replaced by zinc. Patients presenting without symptoms may be
treated with a rather low dose of a chelator or with a zinc salt from the beginning.
Compliance problems have been shown to regularly cause recurrence of
symptomatic WD and may lead to fulminant liver failure, need for LTX or death.
Table 1. Treatment options in WD.
Penicillamine (600-1800 mg/day)
In case of intolerance to penicillamine:
Trientine (900-2400 mg/day)
For combination of maintenance:
Zinc acetate/sulfate
For neurologic disease − not yet approved:
Tetrathiomolybdate
In acute liver failure/decompensated cirrhosis:
Liver transplantation
Restriction of food with high copper content
(does not substitute for chelators or zinc!)

Penicillamine. Penicillamine was the first oral copper chelator shown to be
effective in WD (Walshe 1955). Total bioavailability of oral penicillamine ranges
between 40 and 70% (Bergstrom 1981). Many studies have shown that
penicillamine reduces copper accumulation and provides clinical benefit in WD
(Walshe 1973, Grand 1975, Sternlieb 1980). Signs of liver disease often regress
during the initial 6 months of treatment. Non-compliance has been shown to cause
progression of liver disease, liver failure, death and LTX (Scheinberg 1987).
However, neurological symptoms may deteriorate at the start of penicillamine
treatment; it remains controversial how often this neurological deterioration occurs
and whether it is reversible; the rate of neurological worsening ranges from 10-50%
in different cohorts (Brewer1987, Walshe 1993). Some authors even recommend
not using penicillamine at all in WD patients with neurological disease (Brewer
2006). Penicillamine is associated with many side effects that lead to its
discontinuation in up to 30% of patients (for literature see Roberts 2003). An early
sensitivity reaction may occur during the first 3 weeks including fever, cutaneous
exanthema, lymphadenopathy, neutropenia, thrombocytopenia, and proteinuria. In
such early sensitivity, penicillamine should be replaced by trientine immediately.
Nephrotoxicity is another frequent side effect of penicillamine, which occurs later

Wilson’s Disease 445
and includes proteinuria and signs of tubular damage. In this case penicillamine
should be immediately discontinued. Penicillamine may also cause a lupus-like
syndrome with hematuria, proteinuria, positive antinuclear antibody, and
Goodpasture’s Syndrome. More rarely the drug can damage the bone marrow
leading to thrombocytopenia or total aplasia. Dermatologic side effects include
elastosis perforans serpiginosa, pemphigoid lesions, lichen planus, and aphthous
stomatitis. There have also been reports of myasthenia gravis, polymyositis, loss of
taste, reduction of IgA, and serous retinitis due to administration of penicillamine.
In order to minimize its side effects pencillamine should be started at 250 mg
daily; the dose may be increased in 250 mg steps every week to a maximal daily
amount of 1000 to 1500 mg given in 2 to 4 divided doses daily (Roberts 2003).
Maintenance doses range from 750 to 1000 mg/d given as 2 divided doses. In
children the dose is 20 mg/kg/d given in 2 or 3 divided doses. Penicillamine should
be given 1 hour before or 2 hours after meals because food may inhibit its
absorption. After starting penicillamine therapy serum ceruloplasmin at first may
decrease. Treatment success is checked by measuring 24-hr urinary copper that
should range between 200-500 µg/day. In the long run ceruloplasmin should
increase and free copper should regress towards normal with penicillamine therapy
(Roberts 2003).
Trientine (triene). The chemical structure of the copper chelator trientine
(triethylene tetramine dihydrochloride, short name triene) differs from
penicillamine. Trientine has usually been used as an alternative or substitute for
penicillamine, in particular when penicillamine’s major side effects are not tolerable
(Walshe 1982). Triene only rarely has side effects. Similar to penicillamine longterm treatment with trientine may cause hepatic iron accumulation in persons with
WD. Trientine is poorly absorbed from the gastrointestinal tract, and only 1%
appears in the urine (Walshe 1982). Doses range from 750 to 1500 mg/d given in 2
or 3 divided doses; 750 or 1000 mg are given for maintenance therapy (Roberts
2003). In children a dose of 20 mg/kg/d is recommended. Similar to penicillamine,
trientine should be given 1 hour before or 2 hours after meals. The effectiveness of
copper chelation by triene is measured as described for penicillamine. Triene
chelates several metals such as copper, zinc, and iron by urinary excretion and it
effectively removes accumulated copper from various organs in persons with WD as
well as in severe liver disease (Walshe 1979, Scheinberg 1987, Santos 1996, Saito
1991). It is still unclear whether penicillamine is a more effective copper chelator
when compared to triene; probably the difference in effectiveness is small (Walshe
1973, Sarkar 1977). Potential deterioration of neurological disease may also be seen
after starting triene therapy; the worsening however is less frequent and less
pronounced than that seen after starting with penicillamine.
Zinc. Most physicians substitute penicillamine or triene with zinc for
maintenance therapy when most copper accumulation has been removed. Zinc can
also be given as initial therapy in asymptomatic patients diagnosed by family
screening. A recent report however shows that WD symptoms may occur despite
zinc prophylaxis in asymptomatic patients (Mishra 2008). In a recent study from
India, 45 WD patients were on both penicillamine and zinc sulfate. The majority of
patients (84%) had neuropsychiatric manifestations. The mean duration of treatment
with penicillamine and zinc, before stopping penicillamine, was 107 months. All
patients had to stop penicillamine due to the financial burden. The patients then only

446 Hepatology 2012
received zinc sulfate for 27 months and 44 of the 45 patients (98%) remained stable.
Only one patient reported worsening in dysarthria (Sinha 2008). Zinc does not act as
an iron chelator but inhibits intestinal copper absorption and has also been
suggested to bind free toxic copper (Brewer 1983, Schilksky 1989, Hill 1987). Zinc
rarely has any side effects. It is still unclear whether zinc as monotherapy is an
effective “decoppering” agent in symptomatic patients. There are some hints that
hepatic copper may accumulate despite zinc therapy including reports about hepatic
deterioration with a fatal outcome (Lang 1993, Walshe 1995). Therefore some
authors use zinc in combination with a chelator. Neurological deterioration is rather
rare under zinc therapy (Brewer 1987, Czlonkowska 1996). The recommended
doses of zinc vary in the literature: according to AASLD practice guidelines dosing
is in milligrams of elemental zinc (Roberts 2003). For larger children and adults,
150 mg/d is administered in 3 divided doses. Compliance with doses given thrice
daily may be problematic; zinc has to be taken at least twice daily to be effective
(Brewer 1998). Other authors recommend using zinc sulfate at 150 mg thrice daily
as a loading dose and 100 mg thrice daily for maintenance. Further
recommendations suggest giving 50 mg as zinc acetate thrice daily in adults. The
type of zinc salt used has been thought to make no difference with respect to
efficacy (Roberts 2003). However, zinc acetate has been suggested to cause the least
gastrointestinal discomfort. When zinc is combined with a chelator the substances
should be given at widely spaced intervals, potentially causing compliance
problems. Effectiveness of the zinc treatment should be checked as described for
penicillamine and zinc (Roberts 2003).
Tetrathiomolybdate. Tetrathiomolybdate is an experimental copper chelator not
approved by FDA or EMA. It has been suggested as the initial treatment of WD
patients with neurological involvement. Early reports say that tetrathiomolybdate
stabilizes the neurological disease and reduces circulating free copper in a matter of
weeks (Brewer 1994, Brewer 1996). A more recent randomized study supports this
view and also suggests that zinc monotherapy is insufficient for treatment of
neurological WD (Brewer 2006).
Vitamin E, other antioxidants and diet. Since serum and hepatic concentrations
of vitamin E levels may be reduced in WD (von Herbay 1994, Sokol 1994) it has
been suggested to complement vitamin E intake. Some authors have also
recommended taking other antioxidants; studies have not proven their effectiveness
as yet.
WD patients should avoid food with high copper content (nuts, chocolate,
shellfish, mushrooms, organ meats, etc). Patients living in older buildings should
also check whether the water runs through copper pipes. Such dietary and lifestyle
restrictions do not replace chelator or zinc therapy (Roberts 2003).
Fulminant hepatic failure and LTX. Most WD patients with fulminant liver
failure need LTX urgently in order to survive (Sokol 1985, Roberts 2003).
However, in a long-term cohort study only two patients died prior to LTX being
available (Stremmel 1991). It is a difficult clinical question whether WD patients
with liver failure can survive without LTX. The prognostic score used to help with
this difficult decision includes bilirubin, AST, and INR (Nazer 1986). In any case,
WD patients with signs of fulminant liver failure need to be transferred immediately
(same day!) to a transplant center.

Wilson’s Disease 447
WD patients with a chronic course of decompensated cirrhosis follow the usual
rules for LTX. LTX cures the metabolic defects and thus copper metabolism returns
to normal afterwards (Groth 1973). Prognosis for WD after LTX is excellent, in
particular when patients survive the first year (Eghtesad 1999). It is still unclear
under which circumstances LTX may be helpful for WD patients with neurological
complications, which do not respond to drug therapy. In some patients CNS
symptoms regress after LTX while other patients do not improve (for literature see
Brewer 2000).
Asymptomatic Patients. All asymptomatic WD subjects - usually identified by
family screening - need to be treated by chelators or zinc in order to prevent lifethreatening complications (Walshe 1988, Brewer 1989, Roberts 2003). It is unclear
whether therapy should begin in children under the age of 3 years.
Maintenance Therapy. After initial removal of excessive copper by chelators,
some centres replace the chelators with zinc for maintenance therapy. It is unclear
when such change is advisable and whether it might be better to reduce the dose of
chelators instead of replacing them with zinc. It is generally accepted that
replacement of chelators with zinc should only be done in patients who are
clinically stable for some years, have normal aminotransferase and liver function, a
normal free copper concentration and a 24-hr urinary copper repeatedly in the range
of 200-500 µg while on chelators (Roberts 2003). Long-term treatment with zinc
may be associated with fewer side effects than chelator treatment. Many patients on
trientine, however, do have significant side effects, and this author believes one
does need to replace trientine with zinc in such patients. In any case, therapy either
with a chelator or with zinc needs to be maintained indefinitely; any interruption
may lead to lethal liver failure (Walshe 1986, Scheinberg 1987).
Pregnancy. Treatment must be maintained during pregnancy because an
interruption has been shown to carry a high risk of fulminant liver failure (Shimono
1991). Maintenance therapy with chelators (penicillamine, trientine) or with zinc
usually results in a good outcome for mother and child, although birth defects have
(rarely) been documented [for literature see Sternlieb 2000). It is recommended that
the doses of both chelators be reduced, if possible by about 50%, in particular
during the last trimester to avoid potential problems in wound healing (Roberts
2003). Zinc does not need to be reduced.

Monitoring of treatment
Monitoring should be done closely during initial treatment in all WD patients to
look for efficacy (Figure 6) and side effects. During the maintenance phase patients
should be checked at least twice a year.
Table 2. Monitoring the treatment efficacy in WD.
Clinical Improvement (Neurologic features, liver disease, hematology)
Regression of Kayser-Fleischer Ring
Circulating free copper <10 µl/dl
24-hr urinary copper excretion (200-500 µg/day on chelators)
Decrease in liver copper content

448 Hepatology 2012
Clinical examinations include neurological, ophthalmologic and psychiatric
consultations (Figure 7). Patients with liver involvement need to be checked
carefully for signs of liver failure.
Laboratory tests include measurements of serum copper and ceruloplasmin,
calculation of free (nonceruloplasmin-bound) copper (see above), and 24-hr urinary
copper excretion (Roberts 2003). While on chelating therapy 24-hr urinary copper
excretion should initially range between 200 and 500 µg; such a value can also
suggest that the patient is adherent to the drug. After removal of copper
accumulation, urinary copper excretion may be lower. Prognosis of WD is
dependent on the initial severity of the disease and then on adherence to the lifelong treatment. Patients treated prior to severe and potentially irreversible
neurological and hepatic complications have a good prognosis approaching a
normal life expectancy (Figure 8). Irreversible liver disease often can be treated
successfully by LTX while some patients with severe neurological disease do not
get better despite optimal therapy.

Figure 5. Findings prior to and after beginning chelating therapy in
53 WD patients (modified from Stremmel 1991).

Figure 6. Cumulative survival in 51 WD patients versus a matched
general population (modified from Stremmel 1991).

Wilson’s Disease 449

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(Abstract)

Autoimmune Liver Diseases: AIH, PBC and PSC 453

27. Autoimmune Liver Diseases: AIH, PBC
and PSC
Christian P. Strassburg

Autoimmune hepatitis (AIH)
Autoimmune hepatitis (AIH) is a chronic inflammatory disease, in which a loss of
tolerance against hepatic tissue is presumed. Autoimmune hepatitis (AIH) was first
described as a form of chronic hepatitis in young women showing jaundice, elevated
gamma globulins and amenorrhea, which eventually led to liver cirrhosis
(Waldenström 1950). He also first described a beneficial effect of steroids in the
patient cohort he reported on and thereby laid the groundwork for the first chronic
liver disease found to be curable by drug therapy. AIH was later recognized in
combination with other extrahepatic autoimmune syndromes, and the presence of
antinuclear antibodies (ANA) led to the term lupoid hepatitis (Mackay 1956).
Systematic evaluations of the cellular and molecular immunopathology, of the
clinical symptoms and of laboratory features has subsequently led to the
establishment of autoimmune hepatitis as a separate clinical entity which is
serologically heterogeneous, treated by a specific therapeutic strategy (Strassburg
2000). An established (Alvarez 1999a) and recently simplified (Hennes 2008b)
revised scoring system allows for a reproducible and standardized approach to
diagnosing AIH in a scientific context. The use and interpretation of
seroimmunological and molecular biological tests permits a precise discrimination
of autoimmune hepatitis from other etiologies of chronic hepatitis, in particular
from chronic viral infection as the most common cause of chronic hepatitis
worldwide (Strassburg 2002). Today, AIH is a treatable chronic liver disease in the
majority of cases. Much of the same initial treatment strategies of
immunosuppression still represent the standard of care. The largest challenge
regarding treatment is the timely establishment of the correct diagnosis.

Definition and diagnosis of autoimmune hepatitis
In 1992, an international panel met in Brighton, UK, to establish diagnostic criteria
for AIH, because it was recognized that several features including histological
changes and clinical presentation are also prevalent in other chronic liver disorders
(Johnson 1993). In this and in a revised report the group noted that there is no single

454 Hepatology 2012
test for the diagnosis of AIH. In contrast, a set of diagnostic criteria was suggested
in the form of a diagnostic scoring system designed to classify patients as having
probable or definite AIH (Table 1). According to this approach the diagnosis relies
on a combination of indicative features of AIH and the exclusion of other causes of
chronic liver diseases. AIH predominantly affects women at any age, and is
characterized by a marked elevation of serum globulins, in particular
gammaglobulins, and circulating autoantibodies. It should be noted that AIH
regularly affects individuals older than 40 but should be considered in all age groups
(Strassburg 2006). The clinical appearance ranges from an absence of symptoms to
a severe or fulminant presentation (Stravitz 2011) and responds to
immunosuppressive treatment in most cases. An association with extrahepatic
autoimmune diseases such as rheumatoid arthritis, autoimmune thyroiditis,
ulcerative colitis and diabetes mellitus and a family history of autoimmune or
allergic disorders has been reported (Strassburg 1995).
Autoantibodies are one of the distinguishing features of AIH. The discovery of
autoantibodies directed against different cellular targets including endoplasmatic
reticulum membrane proteins, nuclear antigens and cytosolic antigens has led to a
suggested subclassification of AIH based upon the presence of 3 specific
autoantibody profiles. According to this approach, AIH type 1 is characterized by
the presence of antinuclear antibodies (ANA) and/or anti-smooth muscle antibodies
(SMA) directed predominantly against smooth muscle actin. AIH type 2 is
characterized by anti liver-kidney microsomal autoantibodies (LKM-1) directed
against cytochrome P450 CYP2D6 (Manns 1989, Manns 1991) (Figure 1) and with
lower frequency against UDP-glucuronosyltransferases (UGT) (Strassburg 1996).
AIH type 3 (Manns 1987, Stechemesser 1993) is characterized by autoantibodies
against a soluble liver antigen (SLA/LP) identified as UGA suppressor serine
tRNA-protein complex (Gelpi 1992, Wies 2000, Volkmann 2001, Volkmann 2010).
However, this serological heterogeneity does not influence the decision of whom to
treat or of what strategy to employ.

Figure 1: Indirect immunofluorescence showing LKM-1 autoantibodies on rat kidney and
liver cryostat sections. Serum of a patient with autoimmune hepatitis type 2. A. Using rat
hepatic cryostat sections a homogeneous cellular immunofluorescence staining is visualized
excluding the hepatocellular nuclei (LKM-1). B. Typical indirect immunofluorescence pattern of
LKM-1 autoantibodies detecting the proximal (cortical) renal tubules but excluding the distal
tubules located in the renal medulla, which corresponds to the tissue expression pattern of the
autoantigen CYP2D6.

Autoimmune Liver Diseases: AIH, PBC and PSC 455
Although the histological appearance of AIH is characteristic, there is no specific
histological feature that can be used to prove the diagnosis (Dienes 1989).
Percutaneous liver biopsy should be performed for grading and staging, as well as
for therapeutic monitoring. Histological features usually include periportal hepatitis
with lymphocytic infiltrates, plasma cells, and piecemeal necrosis. With advancing
disease, bridging necrosis, panlobular and multilobular necrosis may occur and
ultimately lead to cirrhosis. A lobular hepatitis can be present, but is only indicative
of AIH in the absence of copper deposits or biliary inflammation. In addition,
granulomas and iron deposits argue against AIH. A liver biopsy should be obtained
at first diagnosis before therapy for grading, staging and as confirmation of the
diagnosis.
Viral hepatitis should be excluded by the use of reliable, commercially available
tests. The exclusion of ongoing hepatitis A, B and C virus infection is sufficient in
most cases. The exclusion of other hepatotropic viruses such as cytomegalovirus,
Epstein-Barr and herpes group may only be required in cases suspicious of such
infections or if the diagnosis of AIH based on the above-mentioned criteria remains
inconclusive.
The probability of AIH decreases whenever signs of bile duct involvement are
present, such as elevation of alkaline phosphatase, histological signs of
cholangiopathy and detection of AMA. If one or more components of the scoring
system are not evaluated, merely a score pointing to a probable diagnosis can be
compiled (Table 1).

Epidemiology and clinical presentation
Based on limited epidemiological data, the prevalence is estimated to range between
50 and 200 cases per 1 million in Western Europe and North America among the
Caucasian population. The prevalence of AIH is similar to that of systemic lupus
erythematosus, primary biliary cirrhosis and myasthenia gravis, which also have an
autoimmune etiology (Nishioka 1997, Nishioka 1998). Among the North American
and Western European Caucasian population AIH accounts for up to 20% of cases
with chronic hepatitis (Cancado 2000). However, chronic viral hepatitis remains the
major cause of chronic hepatitis in most Western societies. In locales in which viral
hepatitis B and C are endemic, such as in Asia and Africa, the incidence of AIH
appears to be significantly lower. Additional epidemiological analyses are required
to comprehensively elucidate the prevalence and geographical distribution of AIH.
Autoimmune hepatitis is part of the syndrome of chronic hepatitis, which is
characterized by sustained hepatocellular inflammation of at least 6 months duration
and elevation of ALT and AST of 1.5 times the upper normal limit. In about 49% of
AIH patients an acute onset of AIH is observed and rare cases of fulminant AIH
have been reported. In most cases, however, the clinical presentation is not
spectacular and characterized by fatigue, right upper quadrant pain, jaundice and
occasionally also by palmar erythema and spider naevi. In later stages, the
consequences of portal hypertension dominate, including ascites, bleeding
esophageal varices and encephalopathy. A specific feature of AIH is the association
of extrahepatic immune-mediated syndromes including autoimmune thyroiditis,
vitiligo, alopecia, nail dystrophy, ulcerative colitis, rheumatoid arthritis, and also
diabetes mellitus and glomerulonephritis.

456 Hepatology 2012
Table 1. International criteria for the diagnosis of autoimmune hepatitis
(Alvarez 1999).
Parameter
Gender
Female
Male
Serum biochemistry
Ratio of elevation of serum alkaline phosphatase vs aminotransferase
>3.0
1.5-3
<1.5
Total serum globulin, γ-globulin or IgG
(times upper limit of normal)
>2.0
1.5-2.0
1.0-1.5
<1.0
Autoantibodies (titers by immunfluorescence on rodent tissues)
Adults
ANA, SMA or LKM-1
>1:80
1:80
1:40
<1:40
Antimitochondrial antibody
Positive
Negative
Hepatitis viral markers
negative
positive
History of drug use
Yes
No
Alcohol (average consumption)
<25 gm/day
>60 gm/day
Genetic factors: HLA-DR3 or -DR4
Other autoimmune diseases
Response to therapy
complete
relapse
Liver histology
interface hepatitis
predominant lymphoplasmacytic infiltrate
rosetting of liver cells
none of the above
biliary changes
other changes
Seropositivity for other defined autoantibodies

Score
+2
0

-2
0
+2

+3
+2
+1
0

+3
+2
+1
0
-4
0
+3
-3
-4
+1
+2
-2
+1
+2
+2
+3
+3
+1
+1
-5
-3
-3
+2

Interpretation of aggregate scores: definite AIH - greater than 15 before treatment
and greater than 17 after treatment; probable AIH - 10 to 15 before treatment and 12 to
17 after treatment.

Autoimmune Liver Diseases: AIH, PBC and PSC 457

Natural history and prognosis
Data describing the natural history of AIH are scarce. The last placebo-controlled
immunosuppressive treatment trial containing an untreated arm was published in
1980 (Kirk 1980). The value of these studies is limited considering that these
patients were only screened for epidemiological risk factors for viral hepatitis and
were not characterized by standardized diagnostic criteria and available virological
tests. Nevertheless, these studies reveal that untreated AIH had a very poor
prognosis and 5- and 10-year survival rates of 50% and 10% were reported. They
furthermore demonstrated that immunosuppressive treatment significantly improved
survival.
Data reveal that up to 30% of adult patients had histological features of cirrhosis
at diagnosis. In 17% of patients with periportal hepatitis cirrhosis developed within
5 years, but cirrhosis develops in 82% when bridging necrosis or necrosis of
multiple lobules is present. The frequency of remission (86%) and treatment failure
(14%) are comparable in patients with and without cirrhosis at presentation.
Importantly, the presence of cirrhosis does not influence 10-year survival and those
patients require a similarly aggressive treatment strategy (Geall 1968, Soloway
1972).
Almost half of the children with AIH already have cirrhosis at the time of
diagnosis. Long-term follow-up revealed that few children can completely stop all
treatment and about 70% of children receive long-term treatment (Homberg 1987,
Gregorio 1997). Most of these patients relapse when treatment is discontinued, or if
the dose of the immunosuppressive drug is reduced. About 15% of patients develop
chronic liver failure and are transplanted before the age of 18 years.
In elderly patients, a more severe initial histological grade has been reported
(Strassburg 2006). The risk of hepatocellular carcinoma varies considerably
between the different diseases PBC, PSC and AIH. Particular PCS can be
complicated by cholangiocarcinoma, gall bladder carcinoma and hepatocellular
carcinoma. In contrast, occurrence of HCC in patients with AIH is a rare event and
develops only in long-standing cirrhosis.

Who requires treatment?
Autoimmune hepatitis (AIH) is a remarkably treatable chronic liver disease (Manns
2001, Czaja 2010). Untreated, the prognosis of active AIH is dismal with 5- and 10year survival rates between 50 and 10% and a well recognized therapeutic effect
exemplified by the last placebo-controlled treatment trials (Soloway 1972, Kirk
1980). For these reasons the indication for treatment is given in any patient who has
an established diagnosis of AIH, elevations of aminotransferase activities (ALT,
AST), an elevation of serum immunoglobulin G and histological evidence of
interface hepatitis or necroinflammatory activity. This has recently been discussed
in the newest version of the AIH guidelines for the American Association for the
Study of the Liver (AASLD) (Manns 2010a). These points indicate that an initial
liver biopsy specimen is desirable for confirmation of the diagnosis and for grading
and staging. Biopsies are also helpful for observation of aminotransferase activities
in serum reflecting inflammatory activity in the liver, which is not closely correlated
in all cases.

458 Hepatology 2012

Who does not require treatment?
Only very few patients with an established diagnosis of AIH should not be treated.
Rare cases, in which the initiation of standard therapy should be weighed against
potential side effects, are contraindications with steroids or azathioprine, or for
certain other immunosuppressants (see below). In decompensated liver cirrhosis of
patients on the waiting list for liver transplantation and in individuals with complete
cirrhosis and absent inflammatory activity treatment does not appear beneficial
(Manns 2010a).

Standard treatment strategy
Independent of clinically- or immunoserologically-defined type of AIH, standard
treatment is implemented with predniso(lo)ne alone or in combination with
azathioprine. Both strategies are just as effective (Manns 2001, Manns 2010a). The
basic strategy of this treatment is still based upon the findings of studies of almost 3
decades ago that indicated the effectiveness of steroids in AIH. Since that time no
single multicenter randomized treatment trial in AIH patients has been performed.
All advances of alternative treatment strategies are based on small cohorts and on
the need to develop strategies for difficult-to-treat patients. The use of prednisone or
its metabolite prednisolone, which is used more frequently in European countries, is
equally effective since chronic liver disease does not seem to have an effect on the
synthesis of prednisolone from prednisone. Important is the exact differentiation
between viral infection and autoimmune hepatitis. Treatment of replicative viral
hepatitis with corticosteroids must be prevented as well as administration of
interferon in AIH, which can lead to dramatic disease exacerbation.
Standard induction treatment and suggested follow-up examinations are
summarized in Table 2. Please note that there are differences in preferred regimen
in Europe and the US, which are delineated in the AASLD AIH Guideline (Manns
2010a). Therapy is usually administered over the course of 2 years. The decision
between monotherapy and combination therapy is guided principally by side effects.
Long-term steroid therapy leads to cushingoid side effects. Cosmetic side effects
decrease patient compliance considerably (Table 3). Serious complications such as
steroid diabetes, osteopenia, aseptic bone necrosis, psychiatric symptoms,
hypertension and cataract formation also have to be anticipated in long-term
treatment. Side effects are present in 44% of patients after 12 months and in 80% of
patients after 24 months of treatment. However, predniso(lo)ne monotherapy is
possible in pregnant patients. Azathioprine, on the other hand, leads to a decreased
dose of prednisone. It bears a theoretical risk of teratogenicity. In addition,
abdominal discomfort, nausea, cholestatic hepatitis, rashes and leukopenia can be
encountered. These side effects are seen in 10% of patients receiving a dose of 50
mg per day. From a general point of view, a postmenopausal woman with
osteoporosis, hypertension and elevated blood glucose would be a candidate for
combination therapy. In young women, pregnant women or patients with
hematological abnormalities, prednisone monotherapy may be the treatment of
choice.

Autoimmune Liver Diseases: AIH, PBC and PSC 459
Table 2. Treatment regimen and follow-up examinations of autoimmune hepatitis
regardless of autoantibody type.
Monotherapy
Prednis(ol)one

Azathioprine

Examination

Combination therapy

60 mg
30-60 mg
reduction as in monotherapy
reduction by 10 mg/week to
maintenance of 20 mg/wk
reduction by 5 mg to 10 mg
find lowest dose in 2.5 mg
decrements
n.a.
1 mg/kg of body weight (Europe)
(maintenance with azathioprine:
50 mg (US)
monotherapy: 2 mg/kg body weight)
Before
therapy

Physical
Liver biopsy
Blood count
Aminotransferases
Gamma
glutamyltransferase
Gammaglobulin
Bilirubin
Coagulation studies
Autoantibodies
Thyroid function
tests

During
therapy
before
remission
q 4 weeks

Remission
on therapy
q 3-6
months

Cessation
of therapy
q 3 weeks
(x 4)
+

+

+
+

+
+

+
+
+

+
+
+

+
+
+
+

+
+

+
(+/-)
+
+

+

+

+

+
+
+
+

+
+
+
+/-

+
+
+

+

+/-

Table 3. Side effects.
Prednis(ol)one

acne
moon-shaped face
striae rubra
dorsal hump
obesity
weight gain
diabetes mellitus
cataracts
hypertension

Azathioprine

nausea
vomiting
abdominal discomforts
hepatotoxicity
rash
leukocytopenia
teratogenicity (?)
oncogenicity (?)

Remission Evaluation of
post-therapy relapse
q 3-6
months
+
+
+

+
+
+
+

460 Hepatology 2012
One of the most important variables for treatment success is adherence. The
administration of treatment is essential since most cases of relapse are the result of
erratic changes of medication and/or dose. Dose reduction is aimed at finding the
individually appropriate maintenance dose. Since histology lags 3 to 6 months
behind the normalization of serum parameters, therapy has to be continued beyond
the normalization of aminotransferase levels. Usually, maintenance doses of
predniso(lo)ne range between 10 and 2.5 mg. After 12-24 months of therapy
predniso(lo)ne can be tapered over the course of 4-6 weeks to test whether a
sustained remission has been achieved. Tapering regimens aiming at withdrawal
should be attempted with great caution and only after obtaining a liver biopsy that
demonstrates a complete resolution of inflammatory activity. Relapse of AIH and
risk of progression to fibrosis is almost universal when immunosuppression is
tapered in the presence of residual histological inflammation. Withdrawal should be
attempted with caution to prevent recurrence and subsequent fibrosis progression
and should be discussed with the patient and closely monitored.
Outcomes of standard therapy can be classified into four categories: remission,
relapse, treatment failure and stabilization.
Remission is a complete normalization of all inflammatory parameters including
histology. This is ideally the goal of all treatment regimens and ensures the best
prognosis. Remission can be achieved in 65-75% of patients after 24 months of
treatment. Remission can be sustained with azathioprine monotherapy of 2 mg/kg
bodyweight (Johnson 1995). This prevents cushingoid side effects. However, side
effects such as arthralgia (53%), myalgia (14%), lymphopenia (57%) and
myelosuppression (6%) have been observed. Complete remission is not achieved in
about 20% of patients and these patients continue to carry a risk of progressive liver
injury.
Relapse is characterized by an increase in aminotransferase levels and the
reccurrence of clinical symptoms either while on treatment, following tapering of
steroid doses to determine the minimally required dose, or, after a complete
withdrawal of therapy. Relapse can be found in 50% of patients within 6 months of
treatment withdrawal and in 80% after 3 years. Relapse is associated with
progression to cirrhosis in 38% and liver failure in 14%. Relapse requires
reinitiation of standard therapy, consideration of dosing as well as diagnosis, and
perhaps a long-term maintenance dose with predniso(lo)ne or azathioprine
monotherapy.
Treatment failure characterizes a progression of clinical, serological and
histological parameters during standard therapy. This is seen in about 10% of
patients. In these cases the diagnosis of AIH has to be carefully reconsidered to
exclude other etiologies of chronic hepatitis. In these patients experimental
regimens can be administered or ultimately liver transplantation becomes necessary.
Stabilization is the achievement of a partial remission. Since 90% of patients
reach remission within 3 years, the benefit of standard therapy has to be reevaluated
in this subgroup of patients. Ultimately, liver transplantation provides a definitive
treatment option.

Autoimmune Liver Diseases: AIH, PBC and PSC 461

Treatment of elderly patients
The presentation of acute hepatitis, clinical symptoms of jaundice, abdominal pain
and malaise have a high likelihood of attracting medical attention and subsequently
leading to the diagnosis of AIH (Nikias 1994). More subtle courses of AIH may not
lead to clinically relevant signs and may develop unnoticed other than via routine
work-up for other problems or via screening programs. The question of disease
onset in terms of initiation of immune-mediated liver disease versus the clinical
consequences that become noticeable after an unknown period of disease
progression is not easily resolved. Thus, “late onset” AIH may just simply reflect a
less severe course of the disease with a slower progression to cirrhosis. While
LKM-positive patients display a tendency towards an earlier presentation, both
acute and subtle (earlier and late presentation) variants appear to exist in ANApositive AIH. In practice, the diagnostic dilemma is that AIH is still perceived by
many as a disease of younger individuals and that therefore this differential
diagnosis is less frequently considered in elderly patients with “cryptogenic”
hepatitis or cirrhosis. Another relevant question resulting from these considerations
is the issue of treatment. Standard therapy in AIH consists of steroids alone or a
combination with azathioprine. In maintenance therapy azathioprine monotherapy
can also be administered but induction with azathioprine alone is not effective.
From a general standpoint most internists will use caution when administering
steroids to elderly patients, especially in women in whom osteopenia or diabetes
may be present.
Recommendations for the treatment of AIH suggest that the steroid side effects be
weighed against the potential benefit of therapy, and that not all patients with AIH
are good candidates for steroid treatment (Manns 2001). Controversy exists
surrounding the benefit of therapy in this group of elderly patients. One cohort
reported on 12 patients over 65 out of a total of 54 AIH patients. Cirrhosis
developed after follow-up in 26% irrespective of age although the histological grade
of AIH activity was more severe in the elderly group. 42% of the patients over 65
did not receive therapy and yet deaths were reported only in the younger group
(Newton 1997). In another cohort of 20 patients aged >65 years, a longer time to
establishment of the diagnosis (8.5 vs. 3.5 months) was reported, patients presented
mainly with jaundice and acute onset AIH and showed a response rate to
immunosuppression comparable to that of younger patients (Schramm 2001). The
authors also noted that the prevalence of the HLA-A1-B8 allotype was less frequent
in older patients suggesting a role for immunogenetics.
This point was further elaborated by a recent report analyzing 47 patients with
ANA-positive AIH 60 years and older, as well as 31 patients 30 years and younger
in whom DR4+/DR3- prevalence was 47% (older) versus 13% (younger) patients
(Czaja 2006). In the older patients steroid responsiveness was better, which is in line
with previous findings in the same collective (Czaja 1993). Cirrhosis and
extrahepatic immune-mediated syndromes including thyroid and rheumatologic
disease (47% vs. 26%) were more prevalent in older AIH patients. However,
although more treatment failures were observed in the younger patients (24% vs
5%) the rates of remission, sustained remission and relapse were similar.
Interestingly, an assessment of age-stratified prevalence showed an increase after
the age of 40 from 15% to over 20%.

462 Hepatology 2012
From all this data, AIH in elderly patients appears to be characterized by a distinct
clinical feature, a distinct immunogenetic profile, favourable response rates and
higher rates of cirrhosis present at diagnosis, all of which contribute to the
heterogeneity of AIH. In a cohort of 164 patients from the UK including 43
individuals 60 years and older AIH was looked at (Al-Chalabi 2006). The age
groups showed no significant differences regarding serum biochemistry,
autoantibody titers, time to establishment of diagnosis, and mode of presentation.
The authors provided a substratification of patients below and above 40 years of age
and reported that older patients had a higher median histological stage and a
comparable median grade but younger patients had more median relapse episodes
and a higher median stage at follow-up biopsy. The most distinguishing clinical sign
was a higher prevalence of ascites in the older group. However, rates of complete,
partial and failed response were similar, and the median number of relapses was
higher in younger patients, which nevertheless did not lead to differences in liverrelated deaths in either group (12% vs. 15%). In comparison to the study of ANApositive AIH patients from the US (Czaja 2006) the differing findings regarding
HLA association are notewothy. In the UK study there was no differential
distribution of HLA-DR3 and -DR4 and this questions the suggested hypothesis of a
primary influence of immunogenetics on the observed clinical distinctions. The
reasons for the clinical differences of AIH in older and younger patients are unclear.
They may include differences in hepatic blood flow and alterations involving the
regulation of cellular immunity during aging (Talor 1991, Prelog 2006). In
summary, these data suggest that AIH in elderly patients should be considered and
treated (Strassburg 2006).

Alternative Treatments
When standard treatment fails or drug intolerance occurs, alternative therapies such
as cyclosporin, tacrolimus, cyclophosphamide, mycophenolate mofetil, rapamycin,
UDCA, and budesonide can be considered (Table 4). The efficacy of most of these
options has not yet been definitively decided and is only reported in small case
studies.
Table 4. Alternative drugs in autoimmune hepatitis.
Compound

Advantage

Budesonide

Cirrhosis (portosystemic
High first pass effect
shunts) and side effects
Immunosuppressive action
Inactive metabolites
Satisfactory experience
Renal toxicity
Potent immunosuppressant
Transplant immunosuppressant
Potent immunosuppressant
Renal toxicity
Transplant immunosuppressant
Disappointing
Favourable toxicity profile
Transplant immunosuppressant effectiveness
Effective
Continuous therapy
Hematological side effects

Cyclosporine

Tacrolimus
Mycophenolic acid
Cyclophosphamide

Disadvantage

Autoimmune Liver Diseases: AIH, PBC and PSC 463

Budesonide
Budesonide is a synthetic steroid with high first-pass metabolism in the liver, in
principle with limited systemic side effects compared to conventional steroids. In
comparison to prednisone the absolute bioavailability of budesonide is less than 6fold lower (Thalen 1979) but it has an almost 90% first-pass metabolism in the liver,
a higher affinity to the glucocorticoid receptor, acts as an anti-inflammatory and
immunosuppressive drug and leads to inactive metabolites (6-OH-budesonide, 16OH-prednisolone). In a pilot study treating 13 AIH patients with budesonide over a
period of 9 months the drug was well tolerated and aminotransferase levels were
normalized (Danielson 1994). However, in a second study budesonide therapy was
associated with a low frequency of remission and high occurrence of side effects
(Czaja 2000). In that study, 10 patients were treated who had previously been
treated with azathioprine and steroids and had not reached a satisfactory remission.
The conclusion of the authors was that budesonide was not a good treatment option
in those patients. A third study with 12 previously untreated patients was published
(Wiegand 2005). In this study remission was induced with budesonide combination
therapy. The authors performed kinetic analyses and reported that in those with high
inflammatory activity and cirrhosis the area under the curve (AUC) of budesonide
was increased. This finding plausibly demonstrates that in patients with
portosystemic shunts in portal hypertension the effect of high hepatic first-pass
metabolism that would limit typical steroid side effects is reduced.
The main advantage of budesonide for the future treatment of autoimmune
hepatitis would therefore be to replace prednisone in long-term maintenance therapy
and induction therapy to reduce steroid side effects. To this end the first multicenter
placebo-controlled randomised AIH treatment trial in 3 decades was performed with
a total of 207 non-cirrhotic patients from 30 centres in 9 European countries and
Israel (Manns 2010b). In this trial 40 mg prednisone (reduction regimen) and
azathioprine was compared to 3 mg budesonide (TID initially, reduced to BID) in
combination with azathioprine. The data was recently published and shows that
budesonide in combination with azathioprine is efficient in inducing stable
remission, is superior in comparison to a standard prednisone tapering regimen
beginning with 40 mg per day (Manns 2010b) and leads to a substantially superior
profile of steroid-specific side effects. From these data budesonide is emerging as an
alternative first-line treatment strategy for non-cirrhotic patients with AIH (Manns
2010b).

Deflazacort
This alternative corticosteroid has also been studied for immunosuppression in AIH
because of its feature of fewer side effects than conventional glucocorticoids. In a
pilot study 15 patients with AIH type I were treated with deflazacort, who had been
previously treated with prednisone with or without azathioprine until they reached a
biochemical remission. Remission was sustained for 2 years of follow-up. However,
the long-term role of second-generation corticosteroids to sustain remission in AIH
patients with reduced treatment related side effects requires further controlled
studies (Rebollo Bernardez 1999).

464 Hepatology 2012

Cyclosporine A
Cyclosporine A (CyA) is a lipophylic cyclic peptide of 11 residues produced by
Tolypocladium inflatum that acts on calcium-dependent signaling and inhibits T cell
function via the interleukin 2 gene (Strassburg 2008). Out of the alternative AIH
drugs considerable experience has been reported with CyA. In these studies CyA
was successfully used for AIH treatment and was well tolerated (Alvarez 1999b,
Debray 1999). The principal difficulty in advocating widespread use of CyA as
first-line therapy relates to its toxicity profile, particularly with long-term use
(increased risk of hypertension, renal insufficiency, hyperlipidemia, hirsutism,
infection, and malignancy) (Alvarez 1999b, Debray 1999, Fernandez 1999,
Heneghan 2002).

Tacrolimus
Tacrolimus is a macrolide lactone compound with immunosuppressive qualities
exceeding those of CyA. The mechanism of action is similar to that of CyA but it
binds to a different immunophilin (Strassburg 2008). The application of tacrolimus
in 21 patients treated for 1 year led to an improvement of aminotransferase and
bilirubin levels with a minor increase in serum BUN and creatinine levels (Van
Thiel 1995). In a second study with 11 steroid refractory patients improvement of
inflammation was also observed (Aqel 2004). Although tacrolimus represents a
promising immunosuppressive candidate drug, larger randomized trials are required
to assess its role in the therapy of AIH.

Mycophenolic acid
Mycophenolate has attracted attention as a transplant immunosuppressant with an
important role in the steroid-free immunosuppressive therapy of patients
transplanted for chronic hepatitis C infection. Mycophenolate is a noncompetitive
inhibitor of inosine monophosphate dehydrogenase, which blocks the rate-limiting
enzymatic step in de novo purine synthesis. Mycophenolate has a selective action on
lymphocyte activation, with marked reduction of both T and B lymphocyte
proliferation. In a pilot study 7 patients with AIH type 1 who either did not tolerate
azathioprine or did not respond to standard therapy with a complete normalization
of aminotransferase levels were treated with mycophenolate in addition to steroids.
In 5 out of 7 patients normalization of aminotransferase levels was achieved within
3 months. These preliminary data suggested that mycophenolate may represent a
promising treatment strategy of AIH (Richardson 2000). In a recent retrospective
study 37 patients with AIH and azathioprine failure or intolerance were treated with
mycophenolate (Hennes 2008a). There was no statistically significant benefit for
mycophenolate treatment. Less than 50% reached remission and in the azathioprine
non-responders failure was 75%. Although the toxicity profile of mycophenolate
would suggest its use, the retrospective study data does not indicate an effective
second line therapeutic option.

Cyclophosphamide
The induction of remission with 1-1.5 mg per kg per day of cyclophosphamide in
combination with steroids has been reported. However the dependency of continued
application of cyclophosphamide with its potentially severe hematological side
effects renders it a highly experimental treatment option (Kanzler 1996).

Autoimmune Liver Diseases: AIH, PBC and PSC 465

Anti-TNF α antibodies
There is some emerging evidence that anti-TNF antibodies are capable of inducing
remission in AIH patients in whom standard or alternative therapeutic options have
been exhausted (Efe 2010, Umekita 2011). However, the development of AIH has
also been observed under treatment with anti-TNF antibodies (Ramos-Casals 2008).
Future studies will have to define the role of this therapeutic option in difficult-totreat cases of AIH.

Ursodeoxycholic acid
Ursodeoxycholic acid is a hydrophilic bile acid with putative immunomodulatory
capabilities. It is presumed to alter HLA class I antigen expression on cellular
surfaces and to suppress immunoglobulin production. Uncontrolled trials have
shown a reduction in histological abnormalities, clinical and biochemical
improvement but not a reduction of fibrosis in 4 patients with AIH type 1 (Calmus
1990, Nakamura 1998, Czaja 1999). However, its role in AIH therapy or in
combination with immunosuppressive therapy is still unclear.
Other alternative treatment strategies include methotrexate, anti-TNF a antibodies,
and rituximab, but there is currently insufficient data on any of these.

Overlap syndromes and treatment
The term overlap syndrome describes a disease condition that is only incompletely
defined (Strassburg 2006). A valid definition is difficult (Boberg 2011). It is
characterized by the coexistence of clinical, biochemical or serological features of
autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing
cholangitis (PSC), and depending on the definition, also viral hepatitis C (Ben-Ari
1993, Colombato 1994, Duclos-Vallee 1995, Chazouilleres 1998, Angulo 2001,
Rust 2008). In adult patients an overlap of PBC and AIH is most frequently
encountered although it is unclear whether this is true co-existence of both diseases
or an immunoserological overlap characterized by the presence of antinuclear
(ANA) as well as antimitochondrial (AMA) antibodies (Poupon 2006, Gossard
2007, Silveira 2007, Al-Chalabi 2008). In many AMA-negative patients with a
cholestatic liver enzyme profile ANA are present. This has been termed
autoimmune cholangiopathy or AMA-negative PBC (Michieletti 1994).
Apart from coexisting, autoimmune liver diseases can also develop into each
other, i.e., the sequential manifestation of PBC and autoimmune hepatitis. The true
coexistence of AIH and PSC has only been conclusively shown in pediatric patients
(Gregorio 2001). It can be hypothesized whether a general predisposition toward
liver autoimmunity exists which has a cholestatic, a hepatitic and a bile duct facet,
which may be variable depending upon unknown host factors. The diagnosis of an
overlap syndrome relies on the biochemical profile (either cholestatic with elevated
alkaline phosphatase, gamma glutamyltransferase and bilirubin, or hepatitic with
elevated aspartate aminotransferase and alanine aminotransferase levels in addition
to elevated gamma globulins), the histology showing portal inflammation with or
without the involvement of bile ducts, and the autoantibody profile showing AMA
or autoantibodies associated primarily with AIH such as liver-kidney microsomal

466 Hepatology 2012
antibodies (LKM), soluble liver antigen antibodies (SLA) or ANA. In cholestatic
cases cholangiography detects sclerosing cholangitis. In an overlap syndrome the
classical appearance of the individual disease component is mixed with features of
another autoimmune liver disease. Immunoglobulins are usually elevated in all
autoimmune liver diseases.
Regarding a therapeutic strategy the leading disease component is treated. In an
overlap syndrome presenting as hepatitis, immunosuppression with prednisone (or
combination therapy with azathioprine) is initiated. In cholestatic disease
ursodeoxycholic acid is administered. Both treatments can be combined when
biochemistry and histology suggest a relevant additional disease component
(Chazouilleres 1998). Validated therapeutic guidelines for overlap syndromes are
not available. It is important to realize that treatment failure in AIH may be related
to an incorrect diagnosis or an overlap syndrome of autoimmune liver diseases
(Potthoff 2007). Several studies show that treatment of the AIH component of
overlapping syndromes is important to avoid progression to cirrhosis (Chazouilleres
2006, Gossard 2007, Silveira 2007, Al-Chalabi 2008).

Liver transplantation
In approximately 10% of AIH patients liver transplantation remains the only lifesaving option (Strassburg 2004). The indication for liver transplantation in AIH is
similar to that in other chronic liver diseases and includes clinical deterioration,
development of cirrhosis, bleeding esophageal varices and coagulation
abnormalities despite adequate immunosuppressive therapy (Neuberger 1984,
Sanchez-Urdazpal 1991, Ahmed 1997, Prados 1998, Tillmann 1999, Vogel 2004).
There is no single indicator or predictor for the necessity of liver transplantation.
Candidates for liver transplant are usually patients who do not reach remission
within 4 years of continuous therapy. Indicators of a high mortality associated with
liver failure are histological evidence of multilobular necrosis and progressive
hyperbilirubinemia. In Europe, 4% of liver transplants are for AIH (Strassburg
2009). The long-term results of liver transplantation for AIH are excellent. The 5year survival is up to 92% (Sanchez-Urdazpal 1991, Prados 1998, Ratziu 1999) and
well within the range of other indications for liver transplantation. The European
liver transplant database indicates 76% survival in 5 years and 66% survival after 10
years (1647 liver transplantations between 1988 and 2007). When these numbers are
considered it is necessary to realize that patients undergoing liver transplantation
usually fail standard therapy and may therefore have a reduced life expectancy after
liver transplant compared to those who achieve stable complete remission on drug
therapy.

Recurrence and de novo AIH after liver transplantation
The potential of AIH to recur after liver transplantation is beyond serious debate
(Schreuder 2009). The first case of recurrent AIH after liver transplant was reported
in 1984 (Neuberger 1984), and was based upon serum biochemistry, biopsy findings
and steroid reduction. Studies published over the years indicate that the rate of
recurrence of AIH ranges between 10-35%, and that the risk of AIH recurrence is
perhaps as high as 68% after 5 years of follow-up (Wright 1992, Devlin 1995, Götz
1999, Milkiewicz 1999, Manns 2000, Vogel 2004). It is important to consider the
criteria upon which the diagnosis of recurrent AIH is based. When transaminitis is

Autoimmune Liver Diseases: AIH, PBC and PSC 467
chosen as a practical selection parameter many patients with mild histological
evidence of recurrent AIH may be missed. It is therefore suggested that all patients
with suspected recurrence of autoimmune hepatitis receive a liver biopsy,
biochemical analyses of aminotransferases as well as a determination of
immunoglobulins and autoantibody titers (Vogel 2004). Significant risk factors for
the recurrence of AIH have not yet been identified although it appears that the
presence of fulminant hepatic failure before transplantation protects against the
development of recurrent disease. Risk factors under discussion include steroid
withdrawal, tacrolimus versus cyclosporine, HLA mismatch, HLA type, and LKM-1
autoantibodies. An attractive risk factor for the development of recurrent AIH is the
presence of specific HLA antigens that may predispose toward a more severe
immunoreactivity. In two studies recurrence of AIH appeared to occur more
frequently in HLA-DR3-positive patients receiving HLA-DR3-negative grafts.
However, this association was not confirmed in all studies. There have not been
conclusive data to support the hypothesis that a specific immunosuppressive
regimen represents a risk factor for the development of recurrent AIH (Gautam
2006). However, data indicate that patients transplanted for AIH require continued
steroids in 64% versus 17% of patients receiving liver transplants for other
conditions (Milkiewicz 1999).
Based on these results and other studies it would appear that maintenance of
steroid medication in AIH patients is indicated to prevent not only cellular rejection
but also graft-threatening recurrence of AIH (Vogel 2004). Steroid withdrawal
should therefore be performed only with great caution. The recurrence of AIH is an
important factor for the probability of graft loss. Apart from hepatitis C and primary
sclerosing cholangitis a recent report found AIH recurrence to represent the third
most common reason for graft loss (Rowe 2008). Transplanted patients therefore
require a close follow-up and possibly an immunosuppressive regimen including
steroids, although this is controversial and not backed by prospective studies
(Campsen 2008).
In addition to AIH recurrence the development of de novo autoimmune hepatitis
after liver transplantation has been reported (Kerkar 1998, Jones 1999a, Salcedo
2002). The pathophysiology of this is also elusive. From a treatment point of view
de novo autoimmune hepatitis, which appears to occur mostly in patients
transplanted with PBC but may just be the serendipitous occurrence of AIH, is
responsive to steroid treatment (Salcedo 2002).

Primary biliary cirrhosis
Introduction
Primary biliary cirrhosis (PBC) is a chronic inflammatory, cholestatic disease of the
liver with an unknown cause. The clinical observation of a broad array of immunemediated symptoms and phenomena suggests the disease to be of autoimmune
etiology, in the course of which progressive and irreversible destruction of small
interlobular and septal bile ducts progressively and irreversibly ensues (Table 5). As
in other autoimmune diseases PBC affects women in over 80% of cases and is
associated with varying extrahepatic autoimmune syndromes in up to 84%. These
extrahepatic manifestations of immune-mediated disease include the dry gland
syndrome (sicca syndrome with xerophthalmia and xerostomia) but also collagen

468 Hepatology 2012
diseases, autoimmune thyroid disease, glomerulonephritis and ulcerative colitis
(Table 6).
Table 5. Clinical profile of primary biliary cirrhosis (PBC).
Sex
Age

Elevation

Liver biopsy

90% female
40-59 years
pruritus
jaundice
skin pigmentation
alkaline phosphatase (AP), aspartate aminotransferase (AST), bilirubin, IgM
antimitochondrial antibodies (AMA)
associated immune-mediated syndromes
- cellular bile duct infiltration
- granulomas possible
- copper deposits

Table 6. Extrahepatic immune-mediated syndromes in PBC and overlap with
rheumatic diseases.
dry gland “sicca” syndrome
Sjögren’s syndrome
rheumatoid arthritis
autoimmune thyroid disease
renal tubular acidosis
mixed connective tissue disease (MCTD)
polymyositis
polymyalgia rheumatica
pulmonary fibrosis
CREST syndrome
systemic lupus erythematosus (SLE)
pernicious anemia
ulcerative colitis
exogenous pancreatic insufficiency
myasthenia gravis

The striking female predominance (Donaldson 1996, Mackay 1997, Uibo 1999)
and familiar clustering of PBC (Kato 1981, Jones 1999b, Tsuji 1999) suggest that
inheritable genetic factors play a role in this disease. This has focussed attention on
the immunogentics of PBC in order to further define host risk factors (Manns 1994).
Studies have suggested an instability of lymphocytic DNA in PBC patients (Notghi
1990). Immunogentic analyses, however, have only come up with relatively weak
associations with specific human leukocyte antigen haplotypes. An additional
hypothesis is an alteration of bile acid composition and bile fluid composition,
which would indicate a role for transporter proteins in the development of PBC.
Bicarbonate rich bile is believed to be protective for biliary epithelium.

Definition and prevalence of PBC
Primary biliary cirrhosis is an inflammatory, primarily T cell-mediated chronic
destruction of intrahepatic microscopic bile ducts of unknown etiology (Strassburg
2000). It affects women in 80% of cases who exhibit elevated immunoglobulin M,
antimitochondrial antibodies directed against the E2 subunit of pyruvate

Autoimmune Liver Diseases: AIH, PBC and PSC 469
dehydrogenase (PDH-E2), a cholestatic liver enzyme profile with elevated alkaline
phosphatase, gamma glutamyltransferase as well as serum bilirubin levels, and a
variable course of disease leading to cirrhosis over the course of years or decades. A
prominent feature is the presence of extrahepatic immune-mediated disease
associations. In later stages pronounced fatigue, pruritus, marked
hyperbilirubinemia and the consequences of portal hypertension such as ascites,
bleeding esophageal varices, and encephalopathy develop (Strassburg 2004).
The prevalence is estimated at 65 per 100,000 in women and 12 per 100,000 in
men with an incidence of 5 per 100,000 in women and 1 per 100,000 in men. The
prevalence and incidence appear to vary regionally. An increase of PBC incidence
in recent years may be the result of more specific testing of antimitochondrial
antibody reactivity (Strassburg 2004).

Diagnostic principles of PBC
Suspicion of PBC arises when cholestasis and cirrhosis are present in middle-aged
women (Figure 2). Ultrasound is employed to rule out mechanical cholestasis. The
presence of antimitochondrial antibodies (AMA) against PDH-E2 is diagnostic of
PBC. AMA against E2 subunits of members of the inner mitochondrial membraneexpressed oxoacid dehydrogenase complex (PDH, branched chain ketoacid
dehydrogenase [BCKD], and ketoglutarate dehydrogenase [OADC]) are present in
95% of PBC patients. AMA-negative PBC can exhibit antinuclear autoantibodies
with specificity against nuclear dot antigen (SP100), a 210 kDa nuclear membrane
protein (gp210), or nucleoporin p62. In AMA-negative PBC a biopsy is indicated to
contribute to the establishment of the diagnosis; in the presence of AMA against
PDH-E2, histology is used primarily for the staging of cirrhosis and is not necessary
(Strassburg 2004).
Diagnostic role of AMA in PBC
The main aim of AMA determinations is the detection of PBC-specific AMA and
the exclusion of AMA of low diagnostic relevance for the disease. As a screening
test the determination of AMA using indirect immunofluorescence testing on rat
kidney cryostat sections or immobilized Hep-2 cells (Strassburg 1999). The indirect
immunofluorescence on rat kidney sections leads to the staining of the distal and
proximal tubuli (note: proximal staining only is indicative of liver/kidney
microsomal antibodies, LKM). When positive AMA immunofluorescence is
detected, further analysis should include subclassification using molecularly defined
antigen preparations. The detection of PDH-E2, BCKD-E2 can be achieved by
ELISA using recombinant antigen or reference sera. If both are negative, testing
should include OGD-E2. The final step is performed using Western Blot Analyses
to confirm the findings. By Western Blot the indicative 74 kDa (PDH-E2), 52 kDa
(BCKD-E2) and 48 kDa (OGD-E2) bands can be visualized. This multi-step
regimen secures a rational and reliable diagnosis of PBC-specific AMA excluding
those found in drug-induced and infectious diseases.
In the majority of cases the determination of anti-PDH-E2 is sufficient to secure
the diagnosis. Studies will have to evaluate whether the future application of a
single PDH-E2 ELISA as highly specific screening test in suspected PBC represents
an efficient and economic diagnostic approach.

470 Hepatology 2012

Figure 2. Diagnostic algorithm of PBC including clinical presentation, ultrasound and
serology.

Therapeutic principles in PBC
Treatment leading to a cure of PBC is not available (Strassburg 2004).
Ursodeoxycholic acid (UDCA) (15 mg/kg body weight per day) has been shown to
improve serum biochemistry, histology and survival but has no effect on fatigue and
osteoporosis. It has immunomodulatory properties, alters cell signal transduction
and modifies hydrophilicity of the bile. UDCA should not be given in severe
cholestasis and during the first trimester of pregnancy. Immunosuppression in PBC
has shown disappointing results. Symptomatic therapy of the complications of PBC
includes management of pruritus (cholestyramine, induction with rifampicin, opioid
antagonists, serotonin antagonists), ascites (diuretics, beta blockers to control portal
hypertension), osteoporosis (vitamin D and calcium supplementation,
bisphosphonates in some), as well as endoscopic intervention for bleeding
esophageal varices. Fat-soluble vitamin replacement is suggested. When liver
cirrhosis-induced liver failure is progressive liver transplantation remains a
definitive therapeutic option. Ten-year survival rates are 75-80% and recurrence of
PBC after transplant occurs in 10-40%. Recurrence can be expected in 25-30%
(Rowe 2008, Strassburg 2009)
Immunosuppression in PBC
Corticosteroids: Treatment with prednisolone can improve serum
aminotransferase activities, alkaline phosphatase and elevated immunoglobulins. It
does not lead to significant improvement of bilirubin, pruritus, or histology. In a

Autoimmune Liver Diseases: AIH, PBC and PSC 471
placebo-controlled study with 36 asymptomatic patients for over 1 year osteopenia
and cushingoid side effects were noted (Mitchison 1992).
Azathioprin: The classical immunosuppressant azathioprin, which has a
pronounced effect in AIH, did not show significant effects in two different studies
and is not used in PBC (Christensen 1985).
Cyclosporin A: In a large study of 346 patients with a median observation time
of 2.5 years this classical transplant immunosuppressant did not show significant
effects on histological progression (Lombard 1993). Contrasting these findings, in a
small study with 20 patients who were treated for 2 years, histology improved,
which should however be viewed with caution (Wiesner 1990). Because of the
possibility of severe side effects cyclosporin A is not a recommended therapeutic
option.
D-penicillamine: Because PBC is characterized by copper accumulation in the
bile ducts the chelator d-penicillamine was studied. D-penicillamine also has
immunosuppressive and antifibrotic properties. It was tested on a total of 748
patients in 6 studies, without leading to a positive therapeutic effect. However, 30%
of patients had severe side effects (Bodenheimer 1985). D-penicillamine in PBC is
not recommended.
Colchicine: Because of its antifibrotic and anti-inflammatory properties
colchicine was studied in 3 studies in the 1980s. Despite improvement of albumin,
bilirubin, aminotransferases and alkaline phosphatase, an improvement of clinical
symptoms and histology was not observed (Kaplan 1986, Warnes 1987,
Bodenheimer 1988). Severe side effects were not reported but an effect on longterm prognosis was not seen.
Methotrexate: Despite its known hepatotoxicity, methotrexate was used as an
immunosuppressant in PBC. In a placebo-controlled study with 60 patients, lowdose methotrexate (7.5 mg/week) led to an improvement of biochemical parameters
except for bilirubin but no effects were reported regarding necessity of liver
transplantation or survival (Hendrickse 1999). Hepatotoxicity was not observed.
Interstitial pneumonitis, which affects 3-5% of rheumatoid arthritis patients, was
observed in 14% of PBC patients. Methotrexate cannot be recommend outside of
scientific evaluations or studies.
In principle other immunosuppressants (Table 7) such as mycophenolic acid
(mycophenolate mofetil), tacrolimus (FK506) or even monoclonal antibodies
against the interleukin 2 receptor may represent interesting candidate strategies.
However, study data is currently lacking.
Table 7. Effects of immunosuppressants in PBC.

Corticosteroids
Azathioprine
Cyclosporin A
D-penicillamine
Colchicine
Methotrexate

Biochemical
improvement
++
++
++
++

Histological
improvement
++
+

Survival

Side effects/toxicity

+
++
+
-

++
+
++
++
+

472 Hepatology 2012
Ursodeoxycholic acid in PBC (UDCA)
In 1981, a positive effect of UDCA was first observed on elevated liver parameters,
the exact mechanism of which was unclear (Leuschner 1996). On one hand UDCA
leads to a modification of the bile acid pool to a more hydrophilic environment with
lower detergent-like properties, and it leads to increased bile flow. On the other
hand an immunomodulatory activity is suggested regarding HLA antigens expressed
on biliary epithelial cells and altered signal transduction (Paumgartner 2002). The
optimal dose in PBC patients appears to be 13-15 mg/kg. In a meta-analysis of 3
studies that looked at 548 patients with this dose, biochemical improvement and a
slower histological progression to fibrosis was observed (Poupon 1997). These
effects were only evident when follow up extended to 4 years. These data rely
heavily on the positive effects of a single study and it is not surprising that a
subsequent meta-analysis of 8 studies with 1114 patients failed to find positive
associations with UDCA therapy (Goulis 1999).
There are a number of problems with this. Doses varied and protocols included
patients with insufficient dosing, and follow up was under 2 years in some cases. In
a recently published analysis of 367 patients from 4 clinical cohorts an initiation of
UDCA therapy in early stages of PBC (stage I-II) and a treatment duration of 2
years led to a retardation of histological progression, which argues for an early
initiation of UDCA therapy after diagnosis even in the absence of fibrosis or
cirrhosis. UDCA was also shown to improve biochemistry, delay portal
hypertension and varices, and currently has no therapeutic alternative (Poupon
2003). No convincing effect was demonstrable on osteopenia and extrahepatic
manisfestations of PBC. An interesting side effect appears to be the significant
reduction of colonic epithelial proliferation. UDCA therapy is not associated with a
higher prevalence of colonic polyps and appears to delay their reappearance after
polypectomy (Serfaty 2003).
Therapy in non-responders and combination strategies
Non-response is usually defined as a failure to lower cholestatic enzyme activities
or to reach normalisation of these parameters. In patients in whom alkaline
phosphatase and gamma glutamyltransferase activities are not lowered by UDCA
therapy, increased morbidity and progression is likely. Alternative therapeutic
strategies can be considered.
Steroids and UDCA: The combination of immunosuppressants and UDCA was
looked at in smaller studies and included the use of prednisolone (Leuschner 1996),
azathioprine (Wolfhagen 1998) and budesonide (Leuschner 1999, Angulo 2000)
(Table 7). In a randomised, controlled study with 30 patients, who received 10 mg
prednisolone/day an improvement of inflammatory activity was reported (Leuschner
1996). A study with 9 mg budesonide/day showed in 39 patients not only
biochemical but also histological improvement (Leuschner 1999). In an open study
with 22 patients a deterioration of osteopenia was noted (Angulo 2000). The
combination of budesonide and UDCA may have additional beneficial effects
related to the activation of the anion exchanger AE2, which may serve to alter
biliary composition and produce a more protective bicarbonate rich bile.
Sulindac and UDCA: In an open study with 23 patients and incomplete response
to UDCA over 12 months treated with UDCA or UDCA and sulindac a trend

Autoimmune Liver Diseases: AIH, PBC and PSC 473
towards histological improvement and biochemical improvement were reported in
the combination group (Leuschner 2002).
Colchicine and UDCA: In 3 studies the combination of colchicine and UDCA
were studied for 24 months in a total of 118 patients (Raedsch 1992, Ikeda 1996,
Poupon 1996). Although mild biochemical improvement was noted, the effect of
longer treatment remains unclear. Because of the biliary elimination of colchicine
combinations with bile acids, there may be potentially toxic effect.
Methotrexate and UDCA: Several pilot studies and 3 randomized studies have
looked at methotrexate in combination with UDCA. In a recent randomized
placebo-controlled protocol with 60 patients a high rate of side effects without
therapeutic benefit was reported (Van Steenbergen 1996, Bach 2003).

Primary sclerosing cholangitis
Diagnosis of primary sclerosing cholangitis (PSC)
PSC is classically characterized by the progressive destruction of large intra- as well
as extrahepatic bile ducts and – contrasting with AIH and PBC – preferentially
affects male patients with a maximum age of around 25-45 (Strassburg 1996).
About 50-75% of the time, PSC is associated with ulcerative colitis. PSC is
clinically characterized by upper quadrant pain, pruritus, anorexia and fever, but up
to 50% of patients lack any symptoms (Weismüller 2008). The diagnosis is
established by a typical biochemical profile of cholestasis with elevations of
bilirubin, alkaline phosphatase and gamma glutamyl transferase, the characteristic
findings upon cholangiography and a typical biopsy showing ring fibrosis around
the bile ducts, which is not present in all patients. Serology regularly identifies
atypical anti-neutrophil cytoplasmic autoantibodies (xANCA) in up to 80% of
patients (Terjung 2000), although these are not disease specific and can also occur
in patients with ulcerative colitis without PSC. There is a significant association of
PSC with cholangiocarcinoma (10-20%) and colorectal cancer (9% in 10 years). In
a subgroup of patients, small bile duct PSC may be present (Broome 2002), which
lacks typical strictures and pruning of the biliary tree upon cholangiography. In
these cases the diagnosis can be established in the presence of the typical
association with ulcerative colitis in male patients by performing a liver biopsy
(Figure 3).

Differential diagnosis: sclerosing cholangitis
The finding of macroductal sclerosing cholangitis can be brought about by a number
of conditions, which include ischemia, liver transplantation complications, and
drugs. Of note are two additional differential diagnoses that require attention
(Figure 4): secondary sclerosing cholangitis (Gelbmann 2007, Esposito 2008, von
Figura 2009, Al-Benna 2011) and IgG4-associated cholangitis (Webster 2009,
Clendenon 2011, Takuma 2011, Zhang 2011).
Secondary sclerosing cholangitis is an entity with severe infection of the biliary
tree that develops in some patients following systemic infections and sepsis who are
treated with aggressive intensive care unit management. IgG4-associated cholangitis
is an immune-mediated entity often with high plasma levels of IgG4 and IgG4
expression in biliary cells obtained upon brush biopsy. The latter can be treated with

474 Hepatology 2012
immunosuppression and should be diagnosed because of an available medical
therapy.

Figure 3. Diagnostic algorithm of PSC including clinical presentation.

Figure 4a. Examples of different entities of sclerosing cholangitis. A. PSC showing
multiple strictures with narrowing (black arrows) and prestenotic dilatation (white arrows) and
an endoscopic aspect of purulent biliary infection at the biliary papilla.

Autoimmune Liver Diseases: AIH, PBC and PSC 475

Figures 4b, 4c. Examples of different entities of sclerosing cholangitis. B. Secondary
sclerosing cholangitis (SSC) with a similar intrahepatic picture but also biliary casts (dotted
arrows) that can be extracted endoscopically (right panel). C. Cholangiogram of autoimmune
(AIC) IgG4-associated cholangitis mimicking PSC. Black arrows show narrowing, white arrows
show dilatations.

Association of PSC with inflammatory bowel disease
A clinical hallmark of PSC is the high number of patients suffering from
inflammatory bowel disease (IBD). In several studies with 605 PSC patients in the
US (Mayo Clinic), UK (King’s College) and in Sweden. IBD was found in 71%,
73% and 81% of PSC cases (Boberg 1998, Bergquist 2002). In our own experience

476 Hepatology 2012
this is found in 52% of cases (Tischendorf 2007). Ulcerative colitis is more often
associated (UK 71%, Sweden 72%) than Crohn’s disease. IBD is usually diagnosed
before PSC but owing to the symptomatic latency of both IBD and PSC it can also
be diagnosed at the same time or later than PSC. Most commonly ulcerative colitis
is diagnosed more than a year before PSC (67%). In 22% the diagnoses occurred
within 1 year of each other, and only in 11% the was diagnosis of ulcerative colitis
reached more than 1 year after PSC was established. IBD patients with elevated
liver biochemistry are a risk group and require careful hepatological workup for
PSC. About 5% of all patients with ulcerative colitis have PSC.

PSC as a risk factor for cancer
Apart from the risk of developing portal hypertension and cirrhosis, PSC is a severe
risk factor for cancer, which distinguishes this disease from AIH and PBC (Table 8).
The increased risk of cholangiocarcinoma is well described (Bergquist 2001,
Boberg 2002). The numbers reported vary because explanted livers during liver
transplantation, autopsies and in vivo diagnosed cases are taken into account in
different analyses. The diagnosis of cholangiocarcinoma (CC) in PSC patients
continues to represent a difficult task because stenoses upon cholangiography may
be caused by inflammatory activity as well as tumour, and because biochemical
tests and biopsy procedures have a low sensitivity and specificity. Imaging studies
are equally complicated by a lack of sensitivity since tumours frequently grow
intramurally and are diagnosed in late stages precluding curative therapeutic
approaches. Studies from Sweden show that 54% of CC occur within 1 year of the
diagnosis of PSC and 27% are diagnosed at liver transplantation. Overall 12.2% of
Northern European PSC patients develop CC, which is corroborated by our data
from Hannover (Boberg 2002, Tischendorf 2006). These patients suffer from
jaundice, pruritus and abdominal pains and had a longer IBD history. Male gender
and smoking is also a risk factor (Tischendorf 2006, Weismüller 2008). In a Dutch
study there were similar findings of 18 CC out of 174 patients (10%) (Ponsioen
2002). The CC risk of a PSC patient amounts to 1.5% per year and is 161-fold
higher than in healthy controls. It is also important to realize that the risk for
colorectal cancer (CRC) is elevated 10-fold, in addition to a 14-fold risk of
pancreatic cancer (Bergquist 2002). These data point to the need of yearly
colonoscopies and ultrasound studies after diagnosis of PSC to monitor the high
potential for cancer development.
Table 8. Cancer association of PSC.
Cholangiocarcinoma

Colorectal cancer

Pancreatic cancer

10-20% of PSC patients
Yearly risk 1.5%
Frequent within 1 year of diagnosis
Bilirubin, male gender, long-standing ulcerative colitis, abdominal
symptoms, smoking
10-fold risk (PSC and ulcerative colitis)
Yearly colonoscopies in ulcerative colitis
In ulcerative colitis and AP elevation: consider ERC
14-fold risk in PSC patients
Abdominal ultrasound

Autoimmune Liver Diseases: AIH, PBC and PSC 477

Medical therapy of PSC
Present day data and clinical experience does not suggest that PSC represents a
disease curable by medical therapy (Zein 2010, Wiencke 2011). A cure would
include the improvement or normalization of abnormal cholestatic biochemical
features but more importantly the improvement of sclerosing changes to the intraand extrahepatic biliary tree, which ultimately lead to biliary cirrhosis, to episodes
of cholangitis, and, which carry the risk of cholangiocellular carcinoma. The only
available drug that combines a favourable toxicity profile and can lead to a
reduction of cholestatic serum parameters currently is ursodeoxycholic acid
(UDCA). However, controversy surrounds the use of UDCA (Chapman 2010),
which has recently led to guidelines that do not specifically recommend UDCA
treatment in all adult patients (Guidelines 2009, Chapman 2010). In two studies an
improvement was documented using 20 mg/kg body weight, and 25-30 mg/kg body
weight, respectively (Harnois 2001, Mitchell 2001). Both use UDCA doses, which
are considerably higher than those common in the therapy of PBC (15 mg/kg body
weight). From these data a higher dose appeared to be more beneficial in PSC.
However, a study analysing UDCA in bile as a function of oral UDCA dose found
that doses exceeding 25 mg/kg body weight are not likely to be useful since the
maximum transport of UDCA into the bile leveled off at 25 mg/kg with no further
increase (Rost 2004). After these and other initial reports, a meta-analysis was
published in 2002 (Chen 2003), which concluded that UDCA therapy improved
biochemical parameters but that overall beneficial effect in patients with PSC, in
particular survival benefit, was uncertain. A large study appeared to confirm this
view; 219 PSC patients in a placebo-controlled trial (Olsson 2005) received 17-23
mg/kg body weight of UDCA and a trend towards better survival and less need for
transplantation was seen, but did not reach statistical significance. A difference in
the incidence of cholangiocarcinoma was not observed. However, statistical
analyses reported in this study concluded that 346 patients would have been
required to reach statistical significance. Based on the body of literature available, a
positive effect of UDCA at present cannot be excluded, and clearly larger placebocontrolled studies are required. This will only be possible in multicentre trials.
An additional effect of UDCA was reported showing a decrease of the dysplasia
in colon polyps associated with UDCA doses as low as 10-15 mg/kg bodyweight
(Tung 2001, Pardi 2003). Although this requires confirmation in larger studies the
association of PSC with ulcerative colitis in 75% of affected individuals would
make this an interesting ancillary effect of UDCA therapy.
The issue of immunosuppression in PSC is controversial and the majority of
centres and publications do not recommend the routine administration of
corticosteroids and other immunosuppressants (van Hoogstraten 2000, LaRusso
2006). In PSC one of the most feared and unpredictable complicating factors is
bacterial cholangitis and cholangiosepsis (Negm 2011). Immunosuppression would
be expected to aggravate this complication. In rare instances such as overlapping
features of PSC and autoimmune hepatitis (AIH) (Boberg 2011),
immunosuppression may be of benefit but this requires rigorous documentation of
AIH, which includes biopsies, autoimmune serology and suggestive biochemistry
(Boberg 1996, Beuers 2005).

478 Hepatology 2012

Therapy of IBD in PSC
Many PSC patients suffer from a milder course of IBD. Ulcerative colitis is
frequently characterized by pancolitis without severe symptoms, rectal sparing or
backwash ileitis. Nevertheless the risk of dysplasia and CRC remains significantly
higher in PSC patients with ulcerative colitis. Therapeutic intervention is no
different that that for IBD without PSC. In this context UDCA appears to provide a
beneficial effect for dysplasia development. In a study with 59 PSC patients with
ulcerative colitis, UDCA reduced the risk of colonic dysplasia (Tung 2001, Serfaty
2003). UDCA may therefore contribute to the positive modulation of CRC risk in
PSC.
Endoscopic therapy
The most important factor determining the course of PSC is the development of
biliary strictures, which carry and increase the risk of septic cholangitis driving
biliary fibrosis (Figures 4 and 5). Endoscopic dilatation can improve cholestasis, in
some cases biliary stenting (Weismüller 2008), which is not recommended by all
gastroenterologists. The combination of endoscopic intervention and UDCA therapy
appears to lead to a significant prolongation of transplant-free survival. UDCA
alone does not lead to this effect.

Figure 5. Management of PSC by dilatation of a dominant stricture of the common bile
duct (arrows) and subsequent short-term stenting with a plastic stent. In this particular
case it turned out that the biliary biopsy revealed cholangiocarcinoma.

Liver transplantation in PSC (OLT)
In PSC patients survival has been shown to be reduced both in symptomatic and in
asymptomatic patients (Kim 2000, LaRusso 2006), which is in part attributable to
the inherent risk of cholangiocarcinoma affecting 10-20% of these patients, and
renders decision-making for liver transplantation a formidable challenge. In
addition, PSC patients with advanced destructive cholangiopathy frequently exhibit
only mild signs of liver failure based upon coagulation abnormalities,

Autoimmune Liver Diseases: AIH, PBC and PSC 479
hypoalbuminemia, or complications of portal hypertension (Tischendorf 2007,
Strassburg 2009). The course of deterioration to liver failure is often observed after
long periods of clinical stability, and frequently proceeds rapidly following septic
biliary complications. This is not well predicted by the aforementioned PSC scores,
which is also true for the model of end stage liver disease (MELD), the measure for
organ allocation in the US and the Eurotransplant member countries.
Two major problems define the challenges involved in the indication for liver
transplantation in PSC. First, timing is difficult (Wiesner 1992). PSC patients are
young and preemptive liver transplantation carries a higher short-term risk of OLT
itself than the most likely short-term natural course of the disease. On the other
hand, patients that urgently require OLT because of advanced biliary destruction
frequently do not meet priority criteria calculated by the MELD system. Second, the
161-fold increase of cholangiocarcinoma risk (Bergquist 2002) is a risk that may
eliminate the option of liver transplantation altogether when evidence of
cholangiocarcinoma is detected by diagnostic imaging procedures. The diagnosis of
early cholangiocarcinoma is difficult and presently no single diagnostic procedure
with high sensitivity and specificity is available (Tischendorf 2006). Moreover, the
patients at risk cannot be reliably identified.
In terms of practical management the first point can only be addressed by careful
clinical monitoring of PSC patients in experienced hepatological transplant centers,
where the likelihood of early complication diagnosis and management, as well as
the individualized timing of wait-listing for OLT is higher (Tischendorf 2007). The
second point has been addressed in two centres by establishing specific protocols
for the management of hilar cholangiocarcinoma and OLT (Sudan 2002, Rea 2005).
A rigorous algorithm for non-resectable hilar cholangiocarcinoma patients, that
were carefully selected and capable of surviving chemotherapy, radiation therapy
and surgery was reported. A multimodal approach including neoadjuvant chemo/radiation therapy, brachytherapy, chemotherapy, laparotomy and OLT was
employed resulting in a 5-year survival of 82%, which did not differ from results in
PSC patients without cholangiocarcinoma (Rea 2005). However, although
attractive, these interdisciplinary strategies are best limited to studies and
experienced hepatological transplant centers.
Overall the results of liver transplantation in PSC are good, leading to 10-year
survival rates of around 70% (Graziadei 1999). In our centre the median survival of
PSC patients with cholangiocarcinoma was 12.7 months, and all PSC patients,
irrespective of OLT, had a mean survival of 112 months (Tischendorf 2006).
Recurrence after OLT is difficult to diagnose but appears to occur in up to 25% of
patients (Graziadei 1999, Rowe 2008). Liver transplantation continues to represent
the only curative option in PSC. Future developments will have to address the
lacking sensitivity and specificity of early cholangiocarcinoma detection, the
clinical prediction of the disease course and consequently, specific allocation
criteria for patients with PSC.

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488 Hepatology 2012

28. Alcoholic Hepatitis
Claus Niederau

Health and social problems due to alcohol
overconsumption
Mortality due to alcohol overconsumption is high, in particular among young men
(Mokdad 2000). Alcohol overconsumption not only increases the risk for liver
disease but is also responsible for malignancies, accidents, violence, and social
problems (Bellentani 1997, Vaillant 1995). Alcohol consumption in excess of 20-30
g for women and 40-60 g for men per day markedly increases the risk for liver
disease (Becker 1996, Lucey 2008). However, liver cirrhosis is seen only in a
minority of subjects with high alcohol consumption; less than 10% of subjects who
drink more than 120 g of alcohol daily have cirrhosis (Bellentani 1997). In addition
to the level of alcohol consumption, various other factors such as sex, other genetic
characteristics and comorbidities contribute to the risk for liver disease (Nishigushi
1991, Becker 1996, Bellentani 1997, McCollough 1998, de Alwis 2007, Lucey
2009).

Classification and natural history of alcoholic liver
disease
Alcohol overconsumption most often causes fat accumulation of hepatocytes, called
hepatic steatosis (Figure 1). Alcohol-induced steatosis is in general reversible after
alcohol abstinence. Continued alcohol overconsumption in the presence of steatosis
markedly increases the risk for development of hepatitis, fibrosis and cirrhosis (Teli
1995, Cubero 2009). Patients with alcohol-induced cirrhosis have a significantly
increased risk for hepatocellular carcinoma (McCollough 1998). Patients with only
fatty liver in the absence of inflammation and fibrosis have a much lower risk for
development of cirrhosis than those with fatty liver plus presence of inflammation
and fibrosis. The latter group of patients with alcoholic fatty liver, inflammation and
fibrosis is defined as alcoholic steatohepatitis (ASH). The liver histology of patients
with ASH is similar when compared to patients with non-alcoholic steatohepatitis
(NASH) that is often associated with obesity and diabetes (Ludwig 1980, Brunt
1999).

Alcoholic Hepatitis 489
The diagnosis of ASH by liver biopsy thus helps to define the risk for
development of cirrhosis. The histological diagnosis of ASH however should not be
confused with the term “alcoholic hepatitis” that is also called “acute alcoholic
hepatitis” although its course can be a rather chronic one (Lucey 2009). This
overview article concentrates on “alcoholic hepatitis” which is a clinical diagnosis
of a rather acute development of jaundice and liver failure associated with a high
short-term mortality.
It is not exactly known which factor(s) set off the development of severe alcohol
hepatitis. In general, pathogenesis and individual predisposition are governed by
gene-environment interactions in all types of alcoholic liver disease (Figure 1).
Based on the “second hit” or “multiple hits” hypothesis, patients are predisposed to
progressive alcoholic liver disease when a specific combination of gene and
environmental interaction exists (Tsukamoto 2009). A loss or gain of function
genetic model has become a popular experimental approach to test the role of a gene
as a second hit. Significant interactions for progressive development of alcoholic
liver disease have been proven in particular for female gender, obesity, various
drugs, iron overload, and hepatitis B and C viral infections (Mueller 2009, Machado
2009, Cubero 2009). These factors may also interact in the development of
hepatocellular carcinoma (HCC).

Figure 1. Effects of alcohol overconsumption on the liver.

A liver biopsy in someone with “alcoholic hepatitis” is often similar to a
histological feature of ASH. Most patients with histological features of ASH
however will not develop “alcoholic hepatitis”. Alcohol overconsumption leads to a
severe form of hepatitis and liver failure associated with a high short-term mortality
only in some subjects. Such alcoholic hepatitis may be seen with or without
preexisting cirrhosis.

490 Hepatology 2012

Clinical features and diagnosis of alcoholic
hepatitis
Alcoholic hepatitis is a clinical diagnosis characterized by the rapid development of
jaundice and liver failure most often due to long-term alcohol overconsumption
(Naveau 1997, McCollough 1998, Lucey 2009). Further characteristics include
fever, ascites, and in some patients hepatic encephalopathy as well. Usually the liver
is enlarged and tender. Women have a higher risk for alcoholic hepatitis than men
assuming that both genders drink the same amount of alcohol. The type of alcohol is
not associated with the risk. Prevalence was 20% in a cohort of 1604 patients who
had a history of heavy alcohol consumption and underwent a liver biopsy (Naveau
1997).
Laboratory tests show increases in serum aspartate aminotransferase (AST) to
approximately twice the upper limit of normal (ULN), while the increase in alanine
aminotransferase (ALT) is less pronounced. The ratio of AST to ALT is typically
>2 (Cohen 1979, Matloff 1980). Other laboratory abnormalities include increases in
peripheral leukocytes, serum bilirubin, and international normalized ratio (INR)
(Mathurin 2002, Orrego 1979). In the presence of an increase in serum creatinine
there is a high risk for development of an often lethal hepatorenal syndrome
(Multimer 1993).
A liver biopsy usually shows big fat droplets and ballooning of hepatocytes that
may also include alcoholic hyaline (also called Mallory bodies); these changes are
accompanied by neutrophil infiltration and intrasinusoidal fibrosis (Figures 2 & 3)
(MacSween 1986).

Figures 2 & 3. Liver biopsies of alcoholic hepatitis.

The diagnosis of alcoholic steatohepatitis (ASH) requires the presence of fibrosis.
The role of liver biopsy in defining prognosis and treatment of alcoholic hepatitis in
the clinical setting remains unclear. Today, prognosis is usually not based on liver
biopsy but on clinical scoring systems (Lucey 2009).
Ultrasound is routinely done to look for hepatocellular carcinoma, biliary
obstruction, ascites, splenomegaly, portal vein thrombosis, and signs of portal
hypertension. Ascites should be checked for spontaneous bacterial peritonitis
routinely.
Differential diagnosis of alcoholic hepatitis includes severe non-alcoholic
steatohepatitis (NASH), acute or chronic viral hepatitis, drug-induced injury,

Alcoholic Hepatitis 491
autoimmune hepatitis, and Wilson’s disease. NASH shares the histological features
of ASH except for the rapid development of jaundice and liver failure.
After discontinuation of alcohol consumption the majority of patients will recover
from alcoholic hepatitis although jaundice, ascites and encephalopathy may persist
for weeks or months (Alexander 1971). Even so, a considerable percentage of
patients with alcoholic hepatitis still die today despite adequate treatment and
abstinence (Mathurin 2002, Orrego 1979).

Course and severity
Severe alcoholic hepatitis occurs in a small fraction of patients who overconsume
alcohol. The 28-day mortality is high and ranges from 30% to 50% in most cohorts
(Cohen 2009). Various scores have been used to predict the prognosis of alcoholic
hepatitis. Maddrey’s discriminant function (Maddrey 1978) and the Model for EndStage Liver Disease (MELD; http://goo.gl/ksgu4) score may help to identify
patients who can benefit with corticosteriods. Most scores share some important
characteristics such as serum bilirubin and prothrombin time (Srikureja 2005).
Maddrey’s discriminant function is calculated as [4.6x (prothrombin time–control
prothrombin time, in seconds)]+serum bilirubin (mg/dL). A value of >32 indicates
severe alcoholic hepatitis and consequently calls for the use of corticosteroids
(Maddrey 1978). In two retrospective studies, the MELD score predicted short-term
mortality in alcoholic hepatitis as well as or even better than Maddrey’s
discriminant function (Dunn 2005, Srikureja 2005). A MELD score >21 was
associated with a 90-day mortality of 20%. The Lille score is based on pretreatment
data and on the response of serum bilirubin to a 7-day treatment with corticosteroids
and has been used to determine whether corticosteroids should be discontinued after
7 days because of treatment failure (Forrest 2005, Dunn 2005, Louvet 2007).
Patients with Maddrey’s discriminant function of <32 usually have mild disease
with a short-term survival of more than 90% and will not benefit from corticosteroid
treatment.
Investigators reported the results of a stepwise logistic-regression identifying
variables related to survival 1-4 months after hospital admission in patients with
alcoholic hepatitis (Forrest 2005); by using this data the Glasgow alcoholic hepatitis
score was developed (not to be confused with the Glasgow coma score). The score,
which includes age, peripheral leukocytes, urea nitrogen, bilirubin, and prothrombin
time, may help to identify high-risk patients who should receive corticosteroids.
Patients with a Maddrey’s discriminant function >32 and a Glasgow alcoholic
hepatitis score of >9 who were treated with corticosteroids had an 84-day survival
of 59%, while untreated patients had a 38% survival (Forrest 2007). In one study the
Glasgow score indicated which subgroup of patients with a high score of Maddrey’s
discriminant function would benefit from corticosteroid therapy (Forrest 2007).
Child-Pugh (CP) and MELD scores have been widely used to predict survival in
cirrhotic patients. Recent studies have suggested that the addition of serum sodium
to MELD (MELD-Na score) may improve its prognostic accuracy. Another recent
study compared the performance of CP, MELD, and MELD-Na scores in predicting
6-month mortality in patients with alcoholic cirrhosis, and developed a new
prognostic score. In this study two French centres (Boursier 2009) enrolled 520
patients (mean age 56.4±10.2 years) with alcoholic cirrhosis randomly allocated

492 Hepatology 2012
into two groups. MELD, MELD-Na1, and MELD-Na2 were calculated according to
UNOS recommendations. Frequencies of CP classes were: A - 29.6%, B - 25.8%, C
- 44.6%. Of the 520 patients 93 died during the 6-month follow-up. In the whole
population, the values of CP, MELD, MELD-Na1, and MELD-Na2 for prediction of
6-month mortality were similar. Multivariate analysis identified age, bilirubin, urea,
prothrombin time, sodium, and alkaline phosphatase as independent predictors of 6month mortality. The score combining these 6 variables was named the Prognostic
Score for Alcoholic Cirrhosis (PSAC) and compared to the 4 other scores. The
predictive value of PSAC was better than all other scores except for MELD-Na2.
By stepwise multivariate analysis, PSAC was identified as independently associated
with 6-month mortality at the first step, and CP at the second. The new PSAC score
may improve the prognostic accuracy to predict the 6-month outcome (Boursier
2009).
Another recent study analyzed the outcome of 79 patients who were admitted to
an Intensive Care Unit (ICU) because of alcoholic liver disease (Rye 2009). The
value of various scores was analyzed for predicting mortality including the Acute
Physiology, Age and Chronic Health Evaluation (APACHE II), Sequential Organ
Failure Assessment (SOFA), CP, and MELD scores. The major reason for
admission was sepsis (44%). The observed mortality in the ICU was 49% and
hospital mortality 68%. Compared to survivors, non-survivors had a significantly
higher serum bilirubin, creatinine and prothrombin time, and lower GCS and length
of ICU stay. Survival was affected by cardiac arrest pre-admission (mortality 75%)
and number of organs supported (mortality 8% with no organ support, 79% ≥2
organs, 100% ≥3 organs). Renal replacement therapy was associated with 100%
mortality. Mortality due to GI bleeding was only 33%. Thus, cirrhotics admitted to
the ICU with cardiac arrest pre-admission, need for renal replacement therapy, or
multiple organ support have a poor prognosis. The predictive accuracy of SOFA and
MELD scores were superior to APACHE II and Child-Pugh scores in cirrhotic
patients (Rye 2009).
A further study analyzed the mortality of 105 patients presenting with alcoholic
hepatitis (Hussain 2009). Patients were evaluated by the modified discriminant
function (mDF) for alcoholic liver disease, CP score, and Glasgow alcoholic
hepatitis score (GAHS). Mean survival for those alive at the end of the study (n=36)
was 34.6 ± 17.8 months. Mean survival for those who died (n=50) was 13.2 ± 14.4
months. The mDF, CP and GAHS scores were significant predictors of mortality in
this population. Prothrombin time was also a significant predictor of mortality
(Hussain 2009).

Mechanisms of alcohol-related liver injury
Alcoholic liver disease is initiated by different cell types in the liver and a number
of different factors including products derived from alcohol-induced inflammation,
ethanol metabolites, and indirect reactions from those metabolites, as well as genetic
predisposition (Colmenero 2007). Ethanol oxidation results in the production of
metabolites that have been shown to bind and form protein adducts, and to increase
inflammatory, fibrotic and cirrhotic responses. Lipopolysaccharide (LPS) has many
deleterious effects and plays a significant role in a number of disease processes by
increasing inflammatory cytokine release. In alcoholic liver disease, LPS is thought

Alcoholic Hepatitis 493
to be derived from a breakdown in the intestinal wall enabling LPS from resident
gut bacterial cell walls to leak into the blood stream. The ability of adducts and LPS
to independently stimulate various cells of the liver provides for a two-hit
mechanism by which various biological responses are induced and result in liver
injury.
Alcohol (ethanol) can be oxidized by various enzymatic and non-enzymatic
pathways (Figures 2 and 3). In hepatocytes the most important pathway is oxidation
of ethanol via alcohol dehydrogenase (ADH) to acetaldehyde (Figure 4). In
mitochondria, acetaldehyde is converted to acetate and in turn acetate is converted
to acetyl CoA, which leads the two-carbon molecule into the TCA (tricarboxylic
acid cycle).

Figure 4. Oxidation of ethanol to acetaldehyde by enzymatic pathways.

This oxidation generates reducing equivalents, primarily reduced nicotinamide
adenine dinucleotide (NAD), i.e., NADH. The changes in the NADH–NAD+
potential in the liver inhibit both fatty acid oxidation and the TAC and may thereby
increase lipogenesis (You 2004a). Ethanol has also been shown to increase lipid
metabolism by inhibiting peroxisome-proliferator–activated receptor α (PPARα)
and AMP kinase as well as by stimulation of sterol regulatory element-binding
protein (Fischer 2003, You 2004b, Ji 2006). All these mechanisms lead to hepatic
steatosis. Further enzymatic pathways of ethanol oxidation include catalase and the
“Microsomal Ethanol Oxidizing System” (MEOS), a cytochrome P450 component.
Oxidation of ethanol to acetaldehyde may also be due to non-enzymatic free radical
pathways (Figure 5). These include strong oxidizing intermediates such as the
hydroxyl radical which can abstract a hydrogen atom from ethanol, preferentially
producing the 1-hydroxyethyl radical; hypervalent iron complexes may also catalyse
this reaction without involvement of •OH (Reinke 1994, Welch 2002, Qian 1999).
Hydroxyethyl radicals may then react with oxygen to form a peroxy radical
intermediate which can rearrange to release acetaldehyde and superoxide.
Hydroxyethyl radicals can also react with proteins to produce antigenic adducts or
induce mitochondrial permeability transition (Clot 1995, Sakurai 2000).
There are probably various other mechanisms by which ethanol may cause or
contribute to liver disease. Ethanol increases the translocation of lipopolysaccharide

494 Hepatology 2012
(LPS) from the small and large intestines to the portal vein and on to the liver. In
Kupffer cells LPS can bind to CD14, which combines with toll-like receptor 4
(TLR4) thereby activating multiple cytokine genes (Schaffert 2009). In addition,
NADPH oxidase may release reactive oxygen species (ROS) that activate cytokine
genes within Kupffer cells, hepatocytes, and hepatic stellate cells. These cytokines
including TNF-α may cause fever, anorexia, and weight loss. Interleukin-8 and
monocyte chemotactic protein 1 (MCP-1) have been shown to attract neutrophils
and macrophages. Platelet-derived growth factor (PDGF) and transforming growth
factor b (TGF-b) contribute to the activation, migration, and multiplication of
hepatic stellate cells, thereby promoting liver fibrosis.

Figure 5. Oxidation of ethanol to acetaldehyde by non-enzymatic free radical pathways.

In the hepatocyte, ethanol is converted to acetaldehyde by the cytosolic enzyme
alcohol dehydrogenase (ADH) and the microsomal enzyme cytochrome P450 2E1
(CYP2E1). Acetaldehyde is converted to acetate. These reactions produce NADH
and inhibit the oxidation of triglycerides and fatty acids. ROS released by CYP2E1
and mitochondria cause lipid peroxidation. Inhibition of proteosomes due to ethanol
disturbs protein catabolism and may be partly responsible for the formation of
Mallory bodies. Reduction in enzymes that convert homocysteine to methionine
may increase homocysteine, thereby injuring the endoplasmic reticulum. Decrease
in binding of peroxisome proliferator–activated receptor a (PPAR-α) to DNA
reduces the expression of genes involved in fatty acid oxidation.
Glutathione transport from the cytosol into the mitochondria is reduced by
ethanol. Ethanol may also activate Fas and TNF receptor 1 (TNF-R1) thereby
activating caspase 8, causing mitochondrial injury and opening the mitochondrial
transition pore (MTP), releasing cytochrome c, and activating caspases; all these
processes contribute to apoptosis. Activation of TNF-R1 leads to nuclear factor
kappa B (NFkB) activation (Schaffert 2009).
Gut permeability and the circulating LPS endotoxin levels of the outer wall of
gram-negative bacteria are increased in patients with alcoholic liver injury (Uesugi
2002, Bjarnson 1984, Urbaschel 2001). In various animal studies alcohol exposure
promoted the transfer of LPS endotoxins from the intestine into portal blood (West
2005). Oral treatment with antibiotics reduced such increases in LPS endotoxins and
ameliorated alcoholic liver injury in animals (Uesugi 2001, Nanji 1994, Adachi

Alcoholic Hepatitis 495
1995). Activation of Kupffer cells by LPS endotoxins involves CD14, toll-like
receptor 4 (TLR4), and MD2 (Uesugi 2001, Akira 2001, Yin 2001). The
downstream pathways of TLR4 signalling include activation of early growth
response 1 (EGR1), NFkB, and the TLR4 adapter also called toll-interleukin-1
receptor domain-containing adapter inducing interferon-ß (TRIF) (McCuillen 2005,
Zhao 2008). TRIF-dependent signalling may contribute to alcohol-induced liver
damage mediated by TLR4 (Hritz 2008).
Many animal studies have shown that alcohol ingestion increases various markers
of oxidative stress (Meagher 1999, Wu2009). Studies in rats and mice suggest that
activated macrophages (Kupffer cells) and hepatocytes are the main sources of
alcohol-induced free radicals (Bailey 1998, Kamimura 1992). Oxidative stress may
mediate alcohol-induced liver injury, e.g., via cytochrome P450 2E1 (Mansuri 1999,
Lu 2008), leading to mitochondrial damage, activation of endoplasmic reticulum–
dependent apoptosis, and up-regulation of lipid synthesis (Ji 2003, Yin 2001).
Activated Kupffer cells will also release TNF-α; this cytokine plays an important
role in the pathogenesis of alcoholic hepatitis. Circulating TNF-α concentrations are
higher in patients with alcoholic hepatitis than in heavy drinkers with inactive
cirrhosis, heavy drinkers who do not have liver disease and persons who do not
drink alcohol and who do not have liver disease (Adachi 1994, Bird 1990).
Circulating TNF-α concentrations are associated with high mortality (Bird 1990). In
animal studies, knockouts of the TNF receptor 1 and administration of the anti-TNF
agent thalidomide both ameliorated alcohol-induced liver injury (Yin 1999, Imuro
1997, Enomoto 2002). Ethanol was also shown to release mitochondrial cytochrome
c and to induce expression of the Fas ligand which may then cause apoptosis via the
caspase-3 activation pathway (Zhou 2001). Both TNF- and Fas-mediated signals
may increase the vulnerability of hepatocytes (Minagawa 2004).

Treatment
Abstinence from alcohol
After recovery from liver failure all patients with alcoholic hepatitis patients need to
have psychological and social support in order to assure continued abstinence (Saitz
2007).

Supportive therapy
There is still a lack of specific therapy for patients with alcoholic hepatitis although
prednisolone and pentoxifylline may have beneficial effects in severe disease. It is,
however, generally accepted that all complications and risks such as ascites,
encephalopathy, hepatorenal syndrome, and infections should be treated like other
decompensated liver diseases (Kosten 2003, Sanyal 2008, Lim 2008). The daily
protein intake should be at least 1.5 g/kg. Vitamin B1 and other vitamins should be
administered according to recommended references (Barr 2006).

Corticosteroids
Various studies and meta-analyses show controversial results for the use of
corticosteroids in alcoholic hepatitis (Imperiale 1990, Christensen 1999, Imperiale
1999, Rambaldi 2008). In general, corticosteroids have not been shown to increase
survival, in particular during longer follow-up (Rambaldi 2008). However, there is

496 Hepatology 2012
evidence that corticosteroids do reduce mortality in a subgroup of patients with a
Maddrey’s discriminant function >32 or in those presenting with hepatic
encephalopathy (Rambaldi 2008). A meta-analysis of three studies corroborated that
corticosteroids given for 28 days increase 1-month survival by 20% in severe
alcoholic hepatitis (Maddrey’s discriminant function >32) (Mathurin 2002). In these
studies Maddrey’s discriminant function >32 resembled a MELD score of >21. In
most studies prednisolone was given at 40 mg a day for 28 days. In some studies
prednisolone was stopped completely at 28 days (Mathurin 2003), while the dose
was gradually reduced in other studies (Imperiale 1990). Corticoids should not be
given in the presence of sepsis, severe infection, hepatorenal syndrome, chronic
hepatitis B, or gastrointestinal bleeding (O’Shea 2006).
The mechanisms by which corticosteroids improve short-term survival in severe
alcoholic hepatitis are not fully understood. In general, corticosteroids inhibit
various inflammatory processes by acting on activator protein 1 and NFkB (Barnes
1997). In some studies in patients with alcoholic hepatitis, the administration of
corticosteroids was associated with a decrease in circulating levels of
proinflammatory cytokines such as interleukin-8, TNF-α and others (Taieb 2000,
Spahr 2001).
Recent reviews and recommendations conclude that corticosteroids should not be
given to patients with a Maddrey’s discriminant function <32 or a MELD score <21
until further data can identify patients with a high short-term risk (Lucey 2009).
Corticosteroids are thus ineffective in a large group of patients with alcoholic
hepatitis and probably do not affect long-term outcome. There is also evidence that
corticosteroids can be discontinued after 7 days if there is no obvious improvement
in clinical signs and symptoms and in serum bilirubin (Maddrey 1978, Dunn 2005,
Forrest 2005, Louvet 2007).

Pentoxifylline
Pentoxyfilline (400 mg TID for 28 days) reduced short-term mortality in severe
alcoholic hepatitis (Maddrey’s discriminant function >32) in a randomized,
controlled trial; mortality was 24% in the pentoxifylline group and 46% in the
placebo group (p<0.01) (Akrivadis 2000). This trial did not include a group on
corticosteroid treatment. Although the phosphodiesterase inhibitor pentoxifylline
has been suggested to act as an anti-TNF agent, TNF-α concentrations did not differ
significantly between the two groups. Thus, the mechanisms by which
pentoxifylline may improve the prognosis in alcoholic hepatitis remains unknown.
Interestingly, almost all deaths (22 of 24; 92%) in the placebo group were
associated with hepatorenal syndrome while hepatorenal syndrome was considered
the cause of death in only 6 of 12 patients (50%) in the pentoxifylline group. Thus,
one might speculate that pentoxifylline may exert its beneficial effects by
preventing the development of hepatorenal syndrome. A recent study (De BK 2009)
compared the efficacy of pentoxifylline and prednisolone in the treatment of severe
alcoholic hepatitis. 68 patients with severe alcoholic hepatitis (Maddrey score >32)
received pentoxifylline (400 mg TID for 28 days) (n=34) or prednisolone (40 mg
QD for 28 days) (n=34) for 28 days in a randomized double-blind controlled study,
and subsequently in an open-label study (with a tapering dose of prednisolone) for a
total of 3 months, and were followed over a period of 12 months. Twelve patients in
the corticosteroid group died by the end of month 3 in contrast to five patients in the

Alcoholic Hepatitis 497
pentoxifylline group (mortality 35.3% vs 14.7%, p=0.04). Six patients in the
corticosteroid group but none in the pentoxifylline group developed hepatorenal
syndrome. Pentoxifylline was associated with a significantly lower MELD score at
the end of 28 days of therapy when compared to corticosteroids (15.5 ± 3.6 vs 17.8
± 4.6, p=0.04). Reduced mortality, improved risk:benefit profile and renoprotective
effects of pentoxifylline compared with prednisolone suggest that pentoxifylline is
superior to prednisolone for treatment of severe alcoholic hepatitis. Interestingly,
another recent study showed that long-term pentoxifylline therapy effectively
achieved sustained biochemical improvement and even histological improvement in
non-alcoholic steatohepatitis (Satapathy 2007).

N-acetyl cysteine
A multicentre, randomised, controlled trial (Nguyen-Khac 2009) analysed treatment
of severe acute alcoholic hepatitis via corticoids plus N-acetyl cysteine (C+NAC)
versus corticoids (C) alone. The background to this approach was the hypothesis
that the glutathione precursor NAC may rebuild antioxidant stocks in the
hepatocyte. Deaths were significantly lower in the C+NAC group than in the C
group at month 1 (n=7/85 (8.2%) vs. 21/89 (23.6%), p=0.005) and at month 2
(13/85 (15.3%) vs. 29/89 (32.6%), p=0.007) but not at month 3 (19/85 (22.4%) vs.
30/89 (33.7%), p=0.095) or at month 6 (23/85 (27.1%) vs. 34/89 (38.2%). NAC
may improve short-term survival. This improvement, however, is lost by month 3.

Anti-TNF-α therapy
Some smaller studies have shown beneficial results using the TNF-α receptor
antagonists infliximab and etanercept in patients with acute alcoholic hepatitis
(Spahr 2007, Mookerjee 2003, Tilg 2003, Menin 2004). A larger randomized,
controlled clinical trial compared the effects of infliximab plus prednisolone vs
placebo plus prednisolone in patients with severe alcoholic hepatitis (Maddrey’s
discriminant function >32) (Naveau 2004). The trial was stopped early by the safety
monitoring board because of a significant increase in severe infections and a
(nonsignificant) increase in deaths in the infliximab group. Similarly, etanercept
reduced 6-month survival when compared with placebo in a randomized, placebocontrolled trial (Boetticher 2008). Thus, TNF-α receptor antagonists should not be
used for clinical therapy of alcoholic hepatitis (Lucey 2009).

Nutritional support
Many patients with alcoholic hepatitis have signs of malnutrition associated with
high mortality (Mendenhall 1984, Mendenhall 1986, Stickel 2003). Parenteral and
enteral nutrition have been shown to improve malnutrition in alcoholic hepatitis but
has not improved survival (Mendenhall 1984). A randomised, controlled clinical
trial looked at the effects of enteral nutrition of 2000 kcal/day via tube feeding
versus treatment with 40 mg/day prednisolone for 28 days in severe alcoholic
hepatitis. Survival in both groups was similar after one month and one year. It may
be concluded that nutritional support is as effective as corticosteroids in some
patients (Cabre 2000). However, corticoids in many studies failed to improve longterm survival.

498 Hepatology 2012

Other pharmacologic treatments
The anabolic steroid oxandrolone failed to improve survival in patients with
alcoholic hepatitis (Mendenhall 1984). Numerous studies have shown that alcoholic
hepatitis is accompanied by oxidative stress. So far, all studies with antioxidants
such as vitamin E, silymarin (milk thistle) and others have failed to improve
survival in alcoholic hepatitis (Pares 1998, Mezey 2004). Older studies did show
that colchicine, propylthiouracil, insulin and glucagon failed to improve survival in
alcoholic hepatitis (Lucey 2009).

Liver transplantation
In current guidelines for liver transplantation, the patient needs to have at least a 6month period of alcohol abstinence before they can be evaluated for transplantation,
thus alcoholic hepatitis is usually a contraindication for liver transplantation (Lucey
1997, Everhardt 1997, Lucey 2007).

Figure 6. Treatment algorithm in alcoholic hepatitis.

Summary
Alcoholic hepatitis is a clinical diagnosis based on a history of heavy alcohol
consumption, jaundice, other signs of liver failure, and the absence of other causes
of hepatitis. A liver biopsy may be helpful but is not required either to determine the
diagnosis or prognosis. Abstinence from alcohol is the prerequisite for recovery.
Patients with signs of malnutrition should have adequate nutritional support.
Subjects with severe alcoholic hepatitis (Maddrey’s discriminant function >32 or
MELD score >21) who do not have sepsis or other corticosteroid contraindications

Alcoholic Hepatitis 499
may receive 40 mg prednisolone daily for 28 days (McCullough 1998, Lucey 2009).
A treatment algorithm is shown in Figure 6. After 7 days of corticosteroid treatment,
patients without obvious clinical benefit, without significant improvement of
jaundice and with a Lille score >0.45 may have disease that will not respond to
continued treatment with corticosteroids or an early switch to pentoxifylline (Louvet
2008). In situations where administration of corticosteroids appears to be risky,
pentoxifylline may be tried (Lucey 2009); this drug may decrease the risk of
hepatorenal syndrome that is often lethal in alcoholic hepatitis. Patients with less
severe alcoholic hepatitis have a good short-term survival of >90% and should not
be treated with corticosteroids or pentoxyfilline (Mathurin 2002).

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autoimmune hepatitis type 2. Proc. Natl. Acad. Sci. USA 1988;85:8256-60. (Abstract)
Zuchner D, Sternsdorf T, Szostecki C, et al. Prevalence, kinetics, and therapeutic modulation of
autoantibodies against Sp100 and promyelocytic leukemia protein in a large cohort of
patients with primary biliary cirrhosis. Hepatology 1997;26:1123-30. (Abstract)

Vascular Liver Disease 509

29. Vascular Liver Disease
Matthias J. Bahr

“It is impossible to explain or to understand the morbid appearances of the liver,
without referring to its intimate structure, and as some points relating to this have
been only lately made out, I shall commence with a short account of it.”
Georg Budd, Diseases of the Liver, 1853
Vascular liver diseases comprise a heterogeneous group of mostly rare hepatic
disorders – some of them exceedingly rare. This is why most of the evidence
regarding diagnosis and management results from retrospective and prospective
cohort studies rather than from randomized controlled trials.
Every single part of the hepatic vasculature may be affected, i.e., hepatic
sinusoids, portal vein, hepatic artery and liver veins. The clinical presentation varies
widely depending on the type of disease but also within the individual disease
entities. Vascular liver diseases may present as acute disorders or chronic liver
disease, as hepatocellular necrosis or cholestasis, as tumour-like lesions or portal
hypertension.
The spectrum of underlying causes is wide, and in many cases multiple risk
factors will result in the development of clinically significant disease (Table 1).

Disorders of the hepatic sinusoid
Hepatic sinusoidal disease may present as luminal obstruction (i.e., sinusoidal
obstruction syndrome), as luminal enlargement (i.e., peliosis hepatis) or as
perisinusoidal fibrosis. Whether the latter should be regarded as a separate disease
entity is debatable, as perisinusoidal fibrosis may also be observed as a histological
feature of common diseases such as steatohepatitis. Both sinusoidal obstruction
syndrome as well as peliosis hepatis are not strictly confined to the hepatic sinusoids
but may extend to the hepatic venous system.

Sinusoidal obstruction syndrome
Sinusoidal obstruction syndrome (SOS), previously referred to as hepatic
venooclusive disease (VOD), is a circulatory disorder primarily affecting the hepatic
sinusoids. Involvement of the hepatic central veins may occur, but studies after
conditioning for hematopoietic cell transplantation have demonstrated that in more

510 Hepatology 2012
than 40% of patients with SOS the hepatic venous system is not involved. The
proportion of sole sinusoidal affection falls to 25% in patients with severe SOS
(DeLeve 2009).
Table 1. Classification of predisposing factors for vascular liver disease.
Hereditary disorders

Congenital
malformations
Acquired cellular
defects
Inflammatory disease
& immune mediated
disorders

Infectious diseases
Miscellaneous

• Inherited thrombophilia, e.g., factor V Leiden mutation, mutations
of prothrombin, protein C, protein S, antithrombin III
• Hereditary hemorrhagic teleangiectasia
• Webs, shunts, aneurysms
•
•
•
•
•
•
•
•
•
•
•
•

Myeloproliferative disease
Paroxysmal nocturnal hemoglobinuria
Malignancy
Focal inflammatory lesions causing thrombosis, e.g., pancreatitis,
diverticulitis, appendicitis, cholecystitis, abscesses, inflammatory
bowel disease
Vasculitis, e.g., polyarteritis nodosa, Behçet syndrome
Rheumatic disease
Schistosomiasis
Bacillary angiomatosis (Bartonella h.)
Drugs, e.g., oral contraceptives, azathioprine, chemotherapy
Pregnancy
Cirrhosis
Radiation

Pathophysiology
Although many risk factors may be complicated by sinusoidal obstruction
syndrome, the by far most common cause in the Western world are myeloablative
regimens in the preparation for hematopoietic stem cell transplantation (HSCTx),
particularly when the transplant is for a malignancy. The proportion of patients with
SOS after HSCTx varies from the single-digit percentage range up to 50% if highly
toxic regimens are chosen. Apart from conditioning regimens for HSCTx (high-dose
chemotherapy plus total body irradiation), other drugs have been implicated in the
development of SOS (Table 2). Originally, the syndrome was described in
conjunction with the ingestion of herbal teas or foods containing pyrrolizidine
alkaloids.
Both the histopathological changes and the clinical picture of SOS were
experimentally studied in a rat model using monocrotaline, a pyrrolizidine alkaloid
that is directly toxic to sinusoidal endothelial cells. These experiments have
confirmed the primary sinusoidal damage infrequently followed by central venous
involvement (DeLeeve 1996).
Clinical presentation and diagnosis
The characteristic clinical presentation of patients with SOS is weight gainassociated or not with ascites, hepatomegaly with right upper quadrant pain, and
jaundice. The onset of symptoms usually occurs between day 10 and day 20 after
cyclophosphamide-containing regimens but can be delayed up to 1 month after
conditioning therapy if other therapies are used.
Severity of SOS varies from mild forms that just meet the diagnostic criteria to
rapidly progressing and eventually life-threatening disease (McDonald 1993). In

Vascular Liver Disease 511
patients not requiring treatment of fluid excess or hepatic pain, SOS is considered
mild and is associated with a self-limiting course. Treatment associated with a
complete remission within 100 days is considered moderate disease. If SOS does
not resolve by day 100, it is categorized as severe. This classification, however, is
retrospective and does not support clinical decision-making.
Table 2. Drugs associated with sinusoidal obstruction syndrome
•
•
•
•
•
•
•
•
•
•
•
•

6-mercaptopurine
6-thioguanine
Actinomycin D
Azathioprine
Busulfan*
Cytosine arabinoside
Cyclophosphamide*
Dacarbazine
Gemtuzumab-ozogamicin
Melphalan*
Oxaliplatin
Urethane

* Exclusively reported with conditioning regimens for HSCTx (modified
according to DeLeve 2009)

Primarily, SOS is a clinical diagnosis with the following characteristics: (1)
hepatotoxic conditioning regimen for HSCTx with an appropriate temporal relation
to the development of clinical signs and symptoms, (2) weight gain & hepatic pain
& jaundice and, (3) negative work-up for other causes. In patients meeting these
criteria, diagnosis can be made with reasonable certainty and solely based on
clinical judgement. Differential diagnoses comprise cholestatic jaundice due to
sepsis, drug-induced cholestasis, fluid overload due to renal failure or congestive
heart failure, liver involvement by viral or fungal infections, and acute graft-versushost disease. However, in up to 20% of patients the diagnosis of SOS cannot be
reliably made on clinical grounds (McDonald 1993 & 2004). This has promoted the
development of scoring systems such as the Seattle or the Baltimore Criteria (Jones
1987; McDonald 1993) (Table 3). However, up to 50% of patients not meeting the
Baltimore criteria may exhibit histological features of SOS (Shulman 1994).
Table 3. Clinical diagnosis of sinusoidal obstruction syndrome after HSCTx.
Seattle criteria (McDonald 1993)

Baltimore criteria (Jones 1987)

At least two of the following findings within 20 Hyperbilirubinemia >34.2 µmol/L (2 mg/dL)
days of transplantation:*
plus ≥2 additional criteria
● Bilirubin >34.2 µmol/L (2 mg/dL)
● Usually painful hepatomegaly
● Hepatomegaly or right upper quadrant pain of ● ≥5% weight gain
liver origin
● Ascites
● ≥2% weight gain due to fluid accumulation
* The 20-days rule applies to cyclophosphamide containing regimens, should be adjusted
according to the regimen actually used.

The gold standard to confirm SOS is based on the combination of hepatic
histology plus measurement of the wedged hepatic venous pressure gradient (HVPG

512 Hepatology 2012
>10 mmHg, specificity >90%, PPV >85%). Both can be achieved during a single
procedure via the transvenous route, especially as increased bleeding risk often
precludes percutaneous liver biopsy. However, histology may be negative due to the
sometimes patchy character of the disease. Imaging techniques are used to confirm
hepatomegaly or ascites and will help to rule out differential diagnoses such as
biliary obstruction. A more specific sign is the finding of hepatic inflow blockage
with reduced or reversed portal flow in colour Doppler ultrasound (Figure 1). In
addition, attenuation of hepatic venous flow or gallbladder wall edema may be
detected. Some authors suggest the use of composite imaging scores (Lassau 2002).

Figure 1. Doppler ultrasound in sinusoidal obstruction syndrome. Exemplary case
showing undulating portal venous flow in a jaundiced patient after HSCTx.

Management and prognosis
Taking into account that SOS is probably underdiagnosed solely employing clinical
criteria, case fatality rates of detected SOS vary between 15 and 20% (DeLeve
2009). Apart from deep jaundice additional signs of liver failure such as
coagulopathy or hepatic encephalopathy may be missing. In contrast, systemic

Vascular Liver Disease 513
complications leading to multiple organ failure (renal, pulmonary) are the main
reasons for death in these patients. This points to the necessity of a closely
supervised management concept. Highly toxic conditioning regimens should
possibly be avoided. Though commonly used, currently published data are too
scarce to endorse prophylactic therapy (e.g., ursodeoxycholic acid, heparin,
liposomal PGE2).
Several treatments have been suggested for established SOS, e.g., thrombolysis
using tPA or defibrotide. In addition, invasive strategies such as TIPS or liver
transplantation were evaluated. However, current knowledge is mainly based on
case reports or cohorts. Although current guidelines do not advise for or against
specific medical treatments, some recommendations can be made (DeLeve 2009).
First of all, fluid management should aim to control fluid overload (using diuretics,
paracentesis, hemofiltration/hemodialysis). Thrombolysis has not proved successful
and was associated with severe complications like bleeding. Several non-controlled
cohort studies suggested positive effects using defibrotide, a mixture of singlestranded oligodeoxyribonucleotides derived from porcine intestinal mucosa. Phase
II studies are completed (Richardson 2010) and Phase III studies are under way.
This compound can also be used in multiple organ failure without substantially
increasing the bleeding risk.
Unlike Budd-Chiari syndrome, decompression of portal hypertension using TIPS
does not improve SOS. For patients with favourable prognosis of the underlying
hematopoietic disorder after HSCTx, liver transplant might be considered.

Peliosis hepatis
Peliosis hepatis is a rare disorder characterized by single or multiple blood-filled
cystic cavities within the hepatic tissue. Prevalence may vary between 0.03% in
HIV infection, 0.2% in pulmonary tuberculosis and up to 20% after renal
transplantation. There is no favored localisation of the peliotic lesions and
appearance at all ages, including a fetal form, has been described. The size ranges
from submillimetres to centimetres but rarely exceeds 3 cm. The histopathological
appearance may show a missing endothelial cell lining with hepatocytes directly
serving as boundary (parenchymal type). In other cases the endothelium may be
preserved but the hepatic sinusoids appear dilated. The aneurysmal dilation may
extend to the central vein (phlebectatic type) (Yanoff 1964, Tsokos 2005).
Pathophysiology
Several risk factors have been accused to promote the development of peliosis
hepatis, e.g., infections, drugs or malignant disorders (Table 4). However, the exact
pathogenesis of peliosis hepatis is largely speculative. The histological appearance
suggests that endothelial damage leads to the destruction of the endothelial lining.
Other hypotheses favour an increased sinusoidal pressure resulting in the widening
of the sinusoidal lumen with consecutive destruction of the sinusoidal endothelium
or primary hepatocellular necrosis replaced by blood-filled cystic lesions. Fibrotic
changes and even liver cirrhosis as well as regenerative nodules may be found, but it
is unclear whether these features are directly linked to peliosis hepatis or whether
they are just coincidental.
Clinical presentation and diagnosis
In the majority of cases, peliosis hepatis is asymptomatic and incidentally detected
during hepatic imaging. On rare occasions, the peliotic cysts may rupture leading to

514 Hepatology 2012
intrahepatic or intraabdominal hemorrhage. Individual cases with overt liver disease
were reported, characterised by hepatomegaly, jaundice, ascites, portal hypertension
and liver failure. Extrahepatic manifestations may be found in organs of the
mononuclear phagocytic system (e.g., spleen, lymph nodes, bone marrow) but also
in lungs, kidneys, parathyroid or adrenal glands, or other parts of the gastrointestinal
tract.
Usually, peliosis hepatis is easily detected by imaging techniques. However,
discrimination between peliosis and other benign or malignant lesions may turn
difficult. Peliotic lesions miss a mass effect on the adjacent hepatic vasculature.
Blood flow within the lesion is slow, resulting in a hypodense appearance after
contrast application in CT. However, in some patients a ring-like accumulation of
contrast media may be present. Using MRI, low intensity is seen in T1-weighted
images while T2-weighted images show a high signal. Though imaging techniques
may assist the diagnosis of peliosis hepatis, a liver biopsy is often needed for final
confirmation. Wedged hepatic venography may also be diagnostic, but its use needs
a strong suspicion.
Table 4. Risk factors reported with peliosis hepatis.
Infections

Drugs, toxins

Malignant and
benign tumours
Miscellaneous

•
•
•
•
•
•
•
•
•
•
•
•

Human immunodeficiency virus
Bartonella spp. (bacillary angiomatosis)
Tuberculosis
Azathioprine, cyclosporine
Anabolic steroids, glucocorticoids, oral contraceptives,
tamoxifen
Vinyl chloride, arsenic, thorium oxide
Multiple myeloma, Waldenström disease
Hodgkin disease
Hepatocellular adenoma
Renal transplantation
Celiac disease, diabetes mellitus
No underlying disorder in up to 50%

Management and prognosis
In most cases, peliosis hepatis will not progress to symptomatic disease. Thus, in
these patients management has to concentrate on the identification and, if required,
treatment of the underlying disease. Causal treatment is the therapeutic mainstay
and leads to regression of the peliotic lesions in the majority of cases. However, in
individual cases surgery may be indicated if the risk of cyst rupture and consecutive
bleeding is estimated to be high. If liver failure and portal hypertension dominate
the clinical picture liver transplantation might be considered provided etiology does
not pose a contraindication.

Disorders of the hepatic artery
Pathologies involving the hepatic artery may lead to different clinical pictures
(Table 5, Figure 2).
Occlusion of the arterial lumen results in ischemia of the supplied tissue. Though
gross hepatocellular necrosis may follow, such as in ischemic hepatitis, preserved
portal venous oxygen supply often prevents the most devastating damage. In

Vascular Liver Disease 515
contrast to the hepatic parenchyma, the biliary system is exclusively supplied
arterially and, therefore, more susceptible to ischemic damage. Clinically, this may
present as an elevation of cholestasis-associated liver enzymes (e.g., gamma GT,
alkaline phosphatase). In more severe cases, structural damage to bile ducts may be
irreversible (i.e., ischemic cholangiopathy). Especially after orthotopic liver
transplantation ischemia type biliary lesions (ITBL) still pose a major challenge for
clinical management.
Table 5. Etiology of hepatic artery disease.
Obstruction or
destruction of the
hepatic artery

Aneurysms

Shunts

•
•
•
•
•
•
•
•
•
•

Hepatic artery embolism or thrombosis
Vasculitis
Sickle cell anemia
Hemolytic uremic syndrome
Chronic transplant rejection
Congenital malformations
Polyarteritis nodosa (PAN)
Focal inflammation, trauma
Congenital malformations
Hereditary hemorrhagic teleangiectasia

Figure 2. Spontaneous arterioportal shunt. Angiography in a patient with non-cirrhotic portal
hypertension. A small arterioportal shunt is detected by superselective catheterisation.

Apart from sequelae due to hepatic ischemia, hepatic artery disease may present
either as an aneurysm or as a shunt. Aneurysms of the hepatic artery are often
detected incidentally on imaging procedures. The majority are asymptomatic but
abdominal pain or – in rare cases – obstructive jaundice may develop. In a minority
of patients (about 20%) multiple aneurysms are present. Males are more often

516 Hepatology 2012
affected than women. The risk of rupture and subsequent hemorrhage is high and
may reach up to 80% – depending on the size of the aneurysm. Therefore, either
radiological intervention or surgery needs to be evaluated (Hulsberg 2011, Christie
2011).
In contrast to aneurysms, shunts involving the hepatic artery are predominantly
symptomatic. The spectrum of symptoms is wide including abdominal pain, portal
hypertension or signs of high-output heart failure. The therapeutic approach has to
be individualized including radiological interventions or surgical procedures.

Hereditary hemorrhagic teleangiectasia
(Osler-Weber-Rendu syndrome)
Hereditary hemorrhagic teleangiectasia (HHT) is a highly penetrant, autosomal
dominant disease showing a heterozygous prevalence between 1:5000 and 1:8000. It
is characterized by progressive and multivisceral development of arteriovenous
malformations (Govani 2009).
Mutations in several genes interacting with transforming growth factor (TGF)-β
receptor have been identified in HHT. According to the genes involved, different
subtypes can be discriminated:
• HHT 1 (ENG coding for endoglin, chromosome 9q33-q34.1),
• HHT 2 (ACVRL1 coding for activin A receptor type II-like kinase ALK-1,
chromosome 12q11-q14),
• HHT 3 (gene not yet identified, chromosome 5q31.3-q32),
• HHT 4 (gene not yet identified, chromosome 7p14),
• Juvenile polyposis/HHT (SMAD4, chromosome 18q21.1).
Liver involvement may be found in all subtypes but appears to be more frequent
in HHT 2. Though hereditary, HHT is characterized by marked intrafamilial
variation.
Clinical presentation and diagnosis
HHT is a multivisceral disease. Apart from the nasopharnyx and the gastrointestinal
tract, central nervous (~10%), pulmonary (~50%) and hepatic involvement occur at
high frequency. Accordingly, the spectrum of clinical disease is wide, e.g., anemia,
seizures, subarachnoid hemorrhage, paraplegia, transient ischemic attacks/stroke,
dyspnea, cyanosis, polycythemia, abdominal pain and hepatic abscesses. Symptoms
develop progressively throughout life. Telangiectasias appear before the age of 20
in half, before 40 in two-thirds of the patients. Thereafter it takes one or two
decades for the development of significant bleeding or symptomatic visceral
involvement (Plauchu 1989, Govani 2009).
The proportion of hepatic involvement in HHT is reported between 30 and 75%.
With the improvement of imaging technology over time, the reported incidence of
hepatic malformations increased. The clinical picture of liver involvement in HHT
depends on the predominant type of malformation (i.e., arterioportal vs.
arteriovenous shunts). Arteriovenous malformations increase cardiac output. In
individual cases up to 20 L/min may be reached. These patients suffer from high
output cardiac failure. In addition, symptoms of a mesenteric steal syndrome (e.g.,
postprandial abdominal pain) and signs of biliary ischemia (e.g., biliary abscesses)
may occur. As a consequence of ischemia, nodular regeneration of the liver

Vascular Liver Disease 517
develops (HHT-associated pseudocirrhosis). Arterioportal malformations will cause
portal hypertension with all its complications (Buscarini 2006, Garcia-Tsao 2000).
Diagnosis of HHT is made using the Curaçao criteria, 3 of 4 of which need to be
fulfilled (Shovlin 2000, Faughnan 2011):
− recurrent spontaneous epistaxis,
− telangiectasias, multiple and in typical localisation,
− positive family history,
− visceral arteriovenous malformations (lung, liver, brain, spine).
Every patient with HHT should be screened for hepatic vascular malformations.
Using Doppler ultrasound, screening is performed with high sensitivity and
specificity (Table 6) (Caselitz 2003). If hepatic involvement is confirmed, cardiac
output should be estimated (e.g., via echocardiography). Furthermore, patients must
be screened at regular intervals to detect complications such as development of
portal hypertension or biliary lesions.
Table 6. Ultrasound criteria for hepatic involvement in HHT*.
Major criteria
Minor criteria

Facultative findings

•
•
•
•
•
•
•
•
•
•

Dilated common hepatic artery >7 mm (inner diameter)
Intrahepatic arterial hypervascularization
Vmax of the proper hepatic artery >110 cm/s
RI of the proper hepatic artery <0.60
Vmax of the portal vein >25 cm/s
Tortuous course of the extrahepatic hepatic artery
Dilated portal vein >13 mm
Dilated liver veins >11 mm
Hepatomegaly >15 cm in midclavicular line
Nodular liver margin

* Two major criteria: definitive hepatic involvement in HHT, one major criterium
plus minor criteria: probable hepatic involvement (modified according to Caselitz
2003)

Management of hepatic involvement in HHT
Currently, no established medical therapy for HHT exists. In chronic GI bleeding
the use of hormonal therapy (estrogen-progesterone preparations, danacrine), antifibrinolytics (aminocaproic acid, tranexamic acid) and other experimental drugs
(tamoxifen, interferon, thalidomide, sirolimus) were suggested (Faughnan 2011).
However, no data supports the use of these drugs to treat hepatic vascular
malformations.
Limited data exist for the use of hepatic artery embolisation and liver
transplantation (Buscarini 2006, Chavan 2004). Due to the invasiveness and
complication rates of these approaches only patients with moderate to severe
symptoms should be regarded as candidates for therapeutic interventions. Hepatic
artery embolisation can be used to reduce shunt flow in patients with arteriovenous
hepatic shunts. Thus, a significant reduction of cardiac output with improvement of
associated symptoms can be achieved. However, complications such as hepatic and
biliary necrosis or acute cholecystitis have been described. Success of hepatic artery
embolisation very much depends on adequate patient selection. Current guidelines
do not endorse general use of embolisation outside of experienced centres but do
favour liver transplantation in advanced hepatic involvement of HHT.

518 Hepatology 2012

Disorders of the portal vein
In contrast to other disease entities affecting the hepatic vasculature, portal vein
thrombosis is a common disease. While portal vein thrombosis is located within the
main portal vein and its larger branches, rare forms of portal vein disease affecting
the medium-sized and preterminal portal venous branches have been identified. The
nomenclature for these diseases is inconsistent (e.g., obliterative portal venopathy,
hepatoportal sclerosis, idiopathic portal hypertension, nodular regenerative
hyperplasia).

Portal vein thrombosis
Portal vein thrombosis (PVT) is of heterogeneous aetiology. It is promoted by both
local and general risk factors (Tables 7 & 8). In about 20 to 30% of patients a local
risk factor can be identified. General risk factors are found in 50-70% (DeLeve
2009, Plessier 2010).
Table 7. Local risk factors for portal vein thrombosis.
•
•
Focal inflammation •
•
•
•
Portal venous injury •
•
•
•
Cirrhosis
•
Malignancy

Primary hepatic or abdominal cancer
Metastatic disease
Neonatal omphalitis, umbilical vein catheterisation
Pancreatitis, duodenal ulcer, cholecystitis
Diverticulitis, appendicitis, inflammatory bowel disease
Tuberculosis, CMV hepatitis
Cholecystectomy, splenectomy, colectomy, gastrectomy
Surgical portosystemic shunting, TIPS
Liver transplantation, hepatobiliary surgery
Abdominal trauma
Impaired hepatic inflow

Clinical presentation and diagnosis
Portal vein thrombosis may present as acute or chronic disease, representing
successive stages of the same disease. Special variants of PVT are malignant
thrombi resulting from tumours invading the portal venous circulation, septic
thrombi also known as acute pylephlebitis, and thrombi resulting from slowed portal
venous flow in liver cirrhosis (DeLeve 2009, Plessier 2010).
The typical clinical presentation of acute PVT includes abdominal or lumbar pain
of sudden onset or progressing over a few days. Depending on the extent of the
thrombosis the pain may be severe and colicky. The diminished mesenteric outflow
leads to intestinal congestion. Ileus may develop but without features of intestinal
obstruction. Moderate distension of the abdomen is common. However, peritoneal
signs are usually absent unless intestinal infarction develops. Fever and a marked
systemic inflammatory response may develop even without systemic infection. This
is accompanied by elevated laboratory markers of inflammation. In contrast, liver
function – apart from intermittent elevation of aminotransferases – is usually not
substantially affected by acute PVT unless significant liver damage pre-exists.
Clinical features should improve within 5-7 days. Otherwise transmural intestinal
ischemia has to be suspected.

Vascular Liver Disease 519
Pylephlebitis is characterized by high, spiking fever with chills, a painful liver,
and sometimes shock. Blood cultures should be taken (usually Bacteroides spp. ±
other enteric species). Infected thrombi give rise to the development of hepatic
microabscesses (Kanellopoulou 2010).
Cases in whom acute portal vein thrombosis does not resolve, progress to chronic
portal vein thrombosis. The obstructed portal vein is replaced by collateral veins
bridging the thrombotic part, known as portal cavernoma. There is wide variation in
the clinical picture of portal cavernoma. It may rarely lead to obstruction of the
extrahepatic bile ducts – so-called portal biliopathy which may be associated with
marked jaundice. However, the leading symptom of chronic PVT are the facets of
portal hypertension (e.g., portosystemic collaterals such as gastric or esophageal
varices). As liver function is usually not impaired, complications such as hepatic
encephalopathy or ascites are substantially less frequent than in liver cirrhosis.
Hepatopulmonary syndrome may be found in up to 10% of patients.
PVT is a common complication of liver cirrhosis with an increasing prevalence in
more severe disease stages. It needs to be discriminated from portal venous
obstruction caused by hepatocellular carcinoma. Pathophysiologically, PVT in
cirrhosis arises as a consequence of the reduction in hepatic inflow leading to flow
reduction and eventually stasis within the portal vein. Therefore, thrombi are often
partial and development of portal cavernoma is rather unusual. In patients with
cirrhosis, a newly developed ascites or significant worsening of existing ascites
should trigger the search for PVT.
Both acute PVT and portal cavernoma are easily detected using sonography, CT
or MR imaging. Acute PVT presents as intraluminal hyperechoic material in
ultrasound, while Doppler imaging demonstrates a lack of blood flow (Figure 3).
Using contrast-enhanced ultrasound (CEUS), vascularisation of the thrombus may
be used to identify malignant thrombi. As PVT may extend to the mesenteric or
splenic veins, thorough assessment of the splanchnic tributaries is mandatory. For
detailed assessment of thrombus extension, CT or MR angiography are more
sensitive than Doppler sonography. Portal cavernoma presents as serpiginous vessel
structures, while the main portal vein or its branches are not visible. As a
compensatory mechanism hepatic arteries are usually enlarged. Depending on the
individual location and appearance of portal cavernoma it may be mistaken as part
of the surrounding organs or as tumour.
Management and prognosis
In acute PVT, recanalisation of the obstructed veins should be aspired. Causal
factors require correction where possible. If pylephlebitis is suspected antibiotic
therapy should be commenced immediately.
Spontaneous recanalisation without anticoagulation occurs infrequently (<20%).
Therefore, anticoagulation is the most commonly used therapeutic strategy to
reopen the obstructed portal vein. Though no controlled studies exist, prospective
data suggest this approach to be successful in about 40% of patients. The success
rate increases to about 60% if neither the splenic vein is involved nor ascites is
detectable. Anticoagulation should be initiated as early as possible - delay might be
associated with treatment failure. Major complications are reported in less than 5%
of treated patients. (DeLeve 2009, Plessier 2010, Hall 2011).
Experience with other treatment modalities is limited (e.g., systemic/local
thrombolysis, surgical thrombectomy, transjugular intrahepatic portosystemic stent

520 Hepatology 2012
[TIPS]). Systemic thrombolysis appears largely ineffective. Although performed
successfully in some centres, major procedure-related complications and even death
have been reported for local thrombolysis, which has to be regarded as
experimental. Emergency surgical intervention is indicated in suspected intestinal
infarction. In these cases, surgical thrombectomy can be performed.
(A)

(B)

Figure 3. Acute portal vein thrombosis. Ultrasound of patient with acute PVT. (A)
Hyperechoic material is located within the main portal vein. (B) Using the power mode for flow
detection, blood flow is limited to those parts of the portal vein without hyperechoic material.

The therapeutic approach is different in patients with PVT associated with liver
cirrhosis. Interventional therapy using TIPS appears to be highly effective (Luca

Vascular Liver Disease 521
2011). Preliminary data even support the use of systemic thrombolysis in these
patients (De Santis 2010).
If treatment is initiated early in acute PVT the outcome is favourable. Symptoms
may sometimes disappear within hours after start of therapy and portal hypertension
rarely develops. Overall mortality is well below 10% (DeLeve 2009, Plessier 2010).
In patients with portal cavernoma prevention of gastrointestinal bleeding due to
portal hypertension is a main focus of therapy. The use of non-selective betablockers is incompletely evaluated in portal cavernoma. However, an approach
similar to portal hypertension in liver cirrhosis is supported by current guidelines
and appears to improve prognosis (DeLeve 2009). Due to the variable genesis of
PVT, individual assessment for risk of recurrence of thrombosis and risk of bleeding
should be performed. Although data is scarce, anticoagulation seems to be
favourable for most patients.

Nodular regenerative hyperplasia
Occlusion of the medium-sized and preterminal portal venous branches induces
hypotrophy of the supplied tissue. As a compensatory reaction, regeneration of
appropriately perfused tissue gives rise to the development of regenerative nodules.
This combination of hypotrophic and hypertrophic liver tissue without signs of
fibrosis is the equivalent of nodular regenerative hyperplasia (Wanless 1990).
Nodular regenerative hyperplasia causes 14-27% of cases with non-cirrhotic
portal hypertension (Naber 1991, Nakanuma 1996). In autopsy studies the
prevalence is 3.1/100,000, one third of which are associated with portal
hypertension (Colina 1989). Nodular regenerative hyperplasia is associated with
autoimmune and hematologic disorders, e.g., rheumatoid arthritis, Felty’s
syndrome, other connective tissue disorders and myeloproliferative disease. It has
also been described in infective endocarditis, in conjunction with chemotherapy and
after kidney transplantation (Matsumoto 2000).
Clinically, nodular regenerative hyperplasia presents with complications of portal
hypertension. Liver function is usually not significantly impaired although
individual cases with liver failure have been described (Naber 1990, Blendis 1978,
Dumortier 2001). The prognosis depends on the underlying disorder and on the
control of portal hypertension.

Hepatoportal sclerosis
Similar to nodular regenerative hyperplasia, hepatoportal sclerosis affects the
smaller portal venous branches. In contrast to the former, portal veins are not just
destroyed but replaced by filiform fibrotic strands penetrating the hepatic tissue.
These fibrotic strands are strictly confined to the portal tracts and do not form
fibrotic septae (Nakanuma 2001). Several synonyms are used for hepatoportal
sclerosis, e.g., obliterative portal venopathy or idiopathic portal hypertension.
However, nomenclature is not well-defined and sometimes overlaps with nodular
regenerative hyperplasia.
Hepatoportal sclerosis is rarely found in the Western world but is more common
in Asia (e.g., India, Japan). Several risk factors are in discussion: (a) chronic
infections, (b) exposure to medication / toxins (e.g., arsenic, vinyl chloride,
azathioprine), (c) thrombophilia, (d) immune disease and (e) hereditary factors.
Infections and toxins appear to be more common in Asia, while Western patients

522 Hepatology 2012
suffer more often from thrombophilia (Schouten 2011). Also, HIV infection is
regarded as a risk factor for hepatoportal sclerosis.
Liver function as well as liver enzymes are usually unaffected by hepatoportal
sclerosis. Complications of portal hypertension pose the main clinical challenge.
Typically, the long-term clinical course of the disease is rather stable. Similarly to
nodular regenerative hyperplasia, prognosis depends on the underlying disorder and
on the control of portal hypertension (Schouten 2011).

Disorders of the hepatic veins
Budd-Chiari syndrome is the only defined entity of hepatic venous disease.
However, other disorders such as the sinusoidal obstruction syndrome or peliosis
hepatis may also affect the hepatic venous system. Furthermore, hepatic congestion
due to cardiac or pericardial disease shares clinical similarities with Budd-Chiari
syndrome.

Budd-Chiari syndrome
Budd-Chiari syndrome (BCS) is defined as hepatic venous outflow obstruction at
any level from the small hepatic veins to the junction of the inferior vena cava
(IVC) and the right atrium, regardless of the cause of obstruction (Janssen 2003).
Excluded from this definition are obstructions caused by sinusoidal obstruction
syndrome and cardiac or pericardial disorders.
Pathophysiology
Obstruction of the hepatic outflow may arise from endoluminal lesions, e.g.,
thrombosis, webs, endophlebitis (primary BCS) or from outside the venous system
by luminal invasion or by extrinsic compression, e.g., tumour, abscess, cysts
(secondary BCS) (Janssen 2003).
On rare occasions, BCS originates from congenital malformations, e.g., webs or
stenotic vessels (Ciesek 2010, Darwish Murad 2009). However, outflow obstruction
is usually caused by thrombosis. Prevalence of thrombophilic risk factors are given
in Table 8. Thrombi are exclusively located within the hepatic veins in 49% of
patients, exclusively within IVC in 2%, and as combined thrombosis of hepatic
veins and IVC in 49%. In about 18% a concomitant portal vein thrombosis is
identified (Darwish Murad 2009).
Obstruction of hepatic outflow leads to congestion of the drained tissue. Over
time this will induce hypotrophy of affected and consecutive regeneration of nonaffected parts of the liver. A typical area of hypertrophy is liver segment 1 (caudate
lobe), because it possesses its own separate venous drainage into the IVC.
Regenerative nodules may occasionally progress to hepatocellular carcinoma. In
addition, intrahepatic collaterals may develop.
Clinical presentation and diagnosis
Depending on the location of outflow obstruction, the number of vessels involved
and the temporal dynamics of BCS, the clinical presentation varies between light
symptoms, even sometimes subclinical disease and dramatic acute complaints
which may progress to acute liver failure. The disease might present with a
progressively relapsing course successively involving different hepatic veins.
Symptoms of hepatic congestion are ascites (>80% of patients), abdominal pain
(>60%) and esophageal varices (>50%). Disturbance of liver function is rather rare,

Vascular Liver Disease 523
e.g., hepatic encephalopathy (<10%), as is involvement of extrahepatic organs, e.g.,
hepatorenal syndrome (<10%) (Darwish Murad 2009).
In the majority of cases, diagnosis of BCS can be obtained using Doppler
ultrasound. If technical difficulties obviate diagnosis by ultrasound, MRI is the
imaging method of choice. Only in rare cases, liver biopsy or hepatic venography
are required to confirm the diagnosis (Janssen 2003). Ultrasound characteristics of
BCS are clearly defined (Boozari 2008). They comprise specific signs such as direct
visualisation of thrombi, stenoses, webs, replacement of hepatic veins by fibrotic
strands or reversed flow in hepatic veins or IVC. Suggestive signs are hepatic
collaterals that may be interposed between hepatic veins or may be located on the
hepatic capsula. Widening of the caudate vein (>3 mm) is also regarded as
suggestive for BCS. These signs serve in the diagnosis of BCS and may be
accompanied by a myriad of non-specific changes (e.g., ascites, regenerative
nodules, splenomegaly).
Table 8. Prevalence of thrombophilic risk factors in acute and chronic portal vein
thrombosis and in primary Budd-Chiari syndrome*.
Risk factor

Portal vein
thrombosis

Budd-Chiari
syndrome

Myeloproliferative disorders
Atypical
Classical
Paroxysmal nocturnal hemoglobinuria
Antiphospholipid syndrome

21% - 40%
14%
17%
0% - 2%
6% - 19%

40% - 50%
25% - 35%
10% - 25%
0% - 19%
4% - 25%

Factor V Leiden mutation

3% - 32%

6% - 32%

Factor II (prothrombin) mutation

14% - 40%

3% - 7%

Protein C deficiency

0% - 26%

4% - 30%

Protein S deficiency

2% - 30%

3% - 20%

Antithrombin deficiency

0% - 26%

0% - 23%

Plasminogen deficiency

0% - 6%

0% - 4%

Hyperhomocysteinemia
TT677 MTHFR genotype
Recent pregnancy
Recent oral contraceptive use

11% - 22%
11% - 50%
6% - 40%
12% - 44%

22% - 37%
12% - 22%
6% - 12%
6% - 60%

Behçet’s disease

0% - 31%

0% - 33%

Connective tissue disease

4%

10%

* Adult patients without malignancy or cirrhosis (according to DeLeve 2009, Darwish Murad
2009, Plessier 2010).

Management and prognosis
Treatment strategy in BCS has to be adjusted to the etiology of BCS and the
severity of the clinical picture. If BCS is caused by congenital malformations such
as webs, radiological interventions using balloon catheter-assisted dilation may be
sufficient to solve the problem.
In case of a primary thrombotic event, anticoagulation is the mainstay of therapy
(Janssen 2003, DeLeve 2009, Darwish Murad 2009). However, in medium-term
follow-up less than one third of patients will be solely treated with anticoagulation

524 Hepatology 2012
and remain free of further interventions (Darwish Murad 2009). Therefore,
interventional techniques (e.g., TIPS, recanalisation) should be evaluated early,
especially in patients with moderate to severe symptoms. With the advent of TIPS,
the necessity for liver transplantation in BCS has declined sharply. Success rates of
TIPS – both in the short-term and in the long-term – are high. Thus, surgical
procedures (e.g., surgical shunt, liver transplantation) are only rarely performed.
With this approach, actual data show that survival in BCS is above 80% after 2
years (Darwish Murad 2009).

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526 Hepatology 2012

30. Acute Liver Failure
Akif Altinbas, Lars P. Bechmann, Hikmet Akkiz, Guido Gerken, Ali Canbay

Introduction and definition
Acute liver failure (ALF) is a devastating clinical syndrome, occurring in previously
healthy individuals, which is characterized by hepatocellular death and dysfunction
(O'Grady 2005). ALF is characterized by onset of coagulopathy (International
Normalized Ratio, INR ≥1.5) and hepatic encephalopathy within 26 weeks of
symptom appearance in a previously healthy subject (Larson 2010). Exclusion of an
underlying liver disease (alcoholic hepatitis, chronic HBV and HCV, autoimmune
hepatitis) is mandatory, as management of acute-on-chronic liver failure differs
from ALF treatment. The most common causes of ALF in Europe and the US are
acetaminophen intoxication, acute hepatitis B (HBV) infection and nonacetaminophen drug-induced liver injury (Bernal 2010). With progressive loss of
hepatic function, ALF leads to hepatic encephalopathy, coagulopathy, and
multiorgan failure within a short period of time. Established specific therapy
regimens and the introduction of liver transplantation (LTx) improves the prognosis
for some etiologies. However, the overall mortality rate remains high (Bernal 2010).
ALF accounts for approximately six to eight percent of LTx procedures in the US
and Europe (Lee 2008). The accurate and timely diagnosis of ALF, rapid
identification of the underlying cause, transfer of the patient to a specialised
transplant center and, if applicable, initiation of a specific therapy and evaluation for
LTx are crucial for modern ALF management. Therefore, we focus here on
epidemiology, pathophysiology, diagnosis and treatment of ALF, including a brief
overview of different etiologies and specific treatment options as well as novel tools
to predict prognosis.

Epidemiology and etiologies
ALF is a rare disease based on multiple causes and varying clinical courses, and
exact epidemiologic data is scarce. The overall incidence of ALF is assumed as one
to six cases per million people each year (Bernal 2010). Recent data from the US
(Ostapowicz 2002), the UK (Bernal 2004), Sweden (Wei 2007), and Germany
(Canbay 2009) reveal drug toxicity as the main cause of ALF, followed by viral
hepatitis followed by unknown etiology. In contrast, in the Mediterranean, Asia, and

Acute Liver Failure 527
Africa, viral hepatitis is the main cause of ALF (Escorsell 2007, Koskinas 2008,
Mudawi 2007, Oketani 2011).
Table 1. Etiologies of ALF.
Intoxication
Viral hepatitis

Direct, idiosyncratic, paracetamol, ecstasy, amanita, phenprocoumon,
tetracycline, halothane, isoniazid, anabolic drugs
HBV, HAV, HEV, HBV+HDV, CMV, EBV, HSV

Immunological

Autoimmune, GVHD

Metabolic

Wilson’s disease, alpha 1 antitrypsin deficiency, hemacromatosis

Vascular

Budd-Chiari syndrome, ischemic, venoocclusive disease

Pregnancy-induced

HELLP syndrome

Intoxication
Drug-induced liver injury
Drug toxicity is the main cause of ALF in Western societies. Although the incidence
of drug-induced liver injury (DILI) in the general population was estimated at 1-2
cases per 100,000 person years (de Abajo 2004), in Germany DILI accounts for
approximately 40% of patients with ALF (Canbay 2009). As a structured medical
history may be difficult in some cases, a standardised clinical management to
identify the cause of DILI and optimize specific treatment has been proposed
(Fontana 2010). This includes assessment of clinical and laboratory features,
determining the type of liver injury (hepatocellular vs. cholestatic), the clinical
course after cessation of the suspected drug, assessment of risk factors (age, sex,
alcohol consumption, obesity), exclusion of underlying liver diseases, previous
episodes of DILI, liver biopsy and in some cases rechallenge to identify the drug.
Furthermore, to identify a cause, one must distinguish between a direct (intrinsic;
dose-dependent) and an idiosyncratic (immune-mediated hypersensitivity or
metabolic injury) type of liver injury (Larson 2010). Acetaminophen intoxication, as
discussed in detail below, is the prototype of a direct, dose-dependant intoxication
with acute hepatocellular necrosis. However, most cases of DILI are due to
idiosyncratic reactions with a latency period of up to one year after initiation of
treatment. Drugs that induce idiosyncratic DILI include narcotics (halothane),
antibiotics (amoxicilline/clavulanate; macrolides, nitrofurantoine, isoniazid),
antihypertensive drugs (methyldopa) and anticonvulsants and antipsychotic drugs
(valproic acid, chlorpromazine) and many others, including herbal medicine.
Demonstrating the need for new algorithms and biomarkers of liver injury, the
observation by Hy Zimmerman, that elevation of transaminase levels above three
times the upper limit of normal indicates early DILI, is still in use to assess the risk
of DILI in drugs in development since the 1970s (Reuben 2004).
Acetaminophen intoxication
In a recent study, more than seventy percent of the patients with acetaminopheninduced ALF were reported as suicidal intents, the rest as accidents (Canbay 2009).
The presence of any ALF risk in the recommended dose range of acetaminophen is
controversial. However, the presence of risk factors, particularly obesity and alcohol
abuse seem to increase the risk of ALF in patients that use acetaminophen (Canbay
2005, Krahenbuhl 2007). Acetaminophen serum concentrations above 300 μg/mL
four hours after the ingestion is a predictor for severe hepatic necrosis. With high

528 Hepatology 2012
doses of acetaminophen its metabolite N-acetyl-p-benzoquinone imine (NAPQI)
accumulates in hepatocytes and induces hepatocellular necrosis. In the presence of
glutathione, NAPQI is rapidly metabolized to non-toxic products and excreted via
the bile (Bessems 2001). In acetaminophen intoxication, the glutathione pool is
rapidly diminished, but could easily be restored by N-acetylcysteine therapy (see
below).
Table 2. Clinical determination of the cause of ALF.
Etiology

Subtype

Investigation

Intoxication

Drug

Drug concentrations in serum

Amanita

History

Idiosyncratic drug toxicity
HAV

Drug concentrations in serum/
eosinophil count
IgM HAV

HBV

HBsAg, IgM anti-core, HBV DNA

HBV/HDV

IHBsAg, gM HDV, HDV RNA

HCV

Anti-HCV, HCV RNA

HEV

Anti-HEV

Autoimmune

ANA, LKM, SLA, ASMA, IgG

GVHD

Biopsy

Wilson’s disease

Urinary copper, coeruloplasmin in
serum, slit-lamp examination
AT level in serum, AT genotyping

Viral hepatitis

Immunological
Metabolic

AT deficiency
Hemacromatosis
Vascular

Budd-Chiari syndrome
Ischemic
Veno-occlusive disease

Pregnancy-induced

HELLP syndrome

Ferritin in serum, transferrin
saturation
Ultrasound (Doppler)
Ultrasound (Doppler),
echocardiography (ECO)
Ultrasound (Doppler)
Hematocrit test, peripheral blood
smear, platelet count

N/A, not available; ANA, anti-nuclear antibody; ASMA, anti-smooth muscle antibody; IgM,
immunoglobulin M; IgG, immunoglobulin G; HBsAg, hepatitis B surface antigen.

Amanita intoxication
The spectrum of mushroom poisoning varies from acute gastroenteritis to ALF.
Even though the mortality rate of all mushroom poisoning cases is low, the
mortality rate of those patients who develop ALF is extremely high, despite the
improvement in intensive care management (Broussard 2001). Amanita phalloides,
the wild mushroom, is attributed to the deadly mushroom poisoning, which occurs
mostly in spring and early summer. Amanita toxin has a dose-dependant, direct
hepatotoxic effect and distrupts hepatocyte mRNA synthesis (Kaufmann 2007).

Acute Liver Failure 529

Viral hepatitis
Historically the most common cause of ALF in Europe and nowadays still the most
prevalent etiologies in developing countries is fulminant viral hepatitis (Larson
2010). Hepatitis A and E (HAV and HEV), both transmitted via the fecal-oral route
are endemic in countries with poor sanitation, tropical and subtropical countries.
HEV was determined as the main cause of ALF in some Asian countries. The
clinical presentation of HAV is more severe in adults than in children, and HEV is
more common in pregnant women, especially in the third trimester (Dalton 2008).
Fulminant HBV, transmitted vertically or by infected blood and body fluids is the
most predominant viral cause of ALF in Western countries (Bernal 2010, Canbay
2009). The incidence of fulminant HBV is decreasing with the implementation of
routine vaccination. Super-infection with hepatitis D virus in HBV infection is
associated with higher risk to develop ALF. HBV infection and treatment is
discussed in detail elsewhere. Acute cytomegalovirus, Epstein-Barr virus,
parvovirus B19, and herpes simplex virus type 1 and 2 are less frequently associated
with ALF.

Immunologic etiologies
Autoimmune hepatitis
In rare cases autoimmune hepatitis (AIH) may induce ALF. The acute onset of ALF
and its potentially rapid progression causes a diagnostic dilemma since exclusion of
other liver diseases might be too time-consuming in patients with ALF secondary to
AIH. Thus, IgG elevation and positive ANA titer, combined with typical
histological features might be sufficient to induce specific therapy in this cohort
(Suzuki 2011). However, as DILI might perfectly mimic AIH, detailed history
taking is the key to adequate therapy in all ALF patients with features of AIH
(Bjornsson 2010).
Graft versus host disease
With the development of new options of donor leukocyte infusion, nonmyeloablative methods and umbilical cord blood transplantation, the indications of
allogenic hematopoietic stem cell transplantation have been expanding in recent
years (Ferrara 2009). Therefore, any hepatopathy in patients who have undergone
bone marrow transplant is suspicious for Graft versus Host Disease (GVHD). On
the other hand, chemotherapy and myeloablation themselves are hepatotoxic and
might induce reactivation of HBV infection, leading to fulminant liver failure.

Wilson’s Disease
Wilson’s Disease (WD), the autosomal recessive disorder of copper metabolism, is
a rare cause of ALF. The prognosis of WD patients presenting with ALF is
devastating, and almost all die without LTx (Lee 2008). Very high serum bilirubin
and low alkaline phosphatase are typical laboratory constellations, and renal failure
is a common clinical feature in WD.

Vascular disorders
Acute systemic hypotension secondary to heart failure or systemic shock syndromes
may induce acute liver injury (Canbay 2009). Occlusion of at least two liver veins in
Budd-Chiari syndrome or venoocclusive disease is a rare cause of ALF.
Anticoagulatory or lysis therapy is the management of choice; in severe cases,

530 Hepatology 2012
emergency TIPSS or surgical shunt placement may be indicated, as well as a
thorough workup to identify any underlying prothrombotic conditions (Fox 2011).

Pregnancy-induced liver injury
Besides acute fatty liver of pregnancy (AFLP), which usually occurs in the third
trimester of pregnancy, HELLP syndrome (hemolysis, elevated liver enzymes, low
platelet level) is a rare complication of pregnancy and presents with ALF. HELLP
syndrome typically presents with LDH, ALT and bilirubin elevation and
thrombocytopenia. Hepatopathy usually completely reverses after termination of
pregnancy. Patients are at increased risk for complications in future pregnancies
(Hay 2008, Westbrook 2010).

Undetermined
Despite dramatic improvements in diagnostic tests in approximately twenty percent
of patients with ALF, the etiology remains undetermined (Canbay 2009, Hadem
2008).

Molecular mechanisms and clinical presentation
As mentioned above, ALF occurs on the basis of acute hepatocellular injury caused
by toxic, viral or metabolic stress or hypotension. However, regardless of the initial
type of liver injury, ALF propels a series of events inducing hepatocellular necrosis
and apoptosis and reducing the regeneration capacity of the liver. Massive loss of
hepatocytes reduces the functional capacity of the liver for glucose, lipid and protein
metabolism, biotransformation, synthesis of coagulation factors, leading to
encephalopathy, coagulopathy, hypoglycemia, infections, renal and multi-organ
failure. In fact, even the pattern of hepatic cell death might be of clinical
importance, as necrosis or apoptosis seem to be specific for different causes and are
associated with clinical outcome (Bechmann 2008, Volkmann 2008).
Apoptosis, programmed cell death, is a process in which ATP-dependant
processes lead to activation of caspases that induce a cascade of events, which ends
in the breakdown of the nucleus into chromatin bodies, interruption of membrane
integrity and finally total breakdown of the cell into small vesicles, called apoptotic
bodies. Upon massive cell injury, ATP depletion leads to necrosis with typical
swelling of the cytoplasm, disruption of the cell membrane, imbalance of electrolyte
homeostasis and karyolysis. Necrosis typically leads to local inflammation,
induction of cytokine expression and migration of inflammatory cells. However,
apoptosis itself might induce mechanisms that lead to necrosis and the ratio of
apoptosis vs. necrosis seems to play an important role in liver injury rather than the
individual events (Canbay 2004). This hypothesis is supported by observations that
a death receptor agonist triggers massive necrosis secondary to the induction of
apoptosis (Rodriguez 1996).
The rates of apoptosis or necrosis in ongoing ALF processes seem to be different
according to the underlying etiologies (Bechmann 2010). The degree of apoptosis
and necrosis, assessed by specific ELISA assays were significantly increased in
amanita intoxication compared to other causes. Apoptosis is the predominant type
of cell death in HBV and amanita-related ALF, vs. necrosis in acetaminophen and
congestive heart failure. Furthermore, entecavir treatment of fulminant HBV

Acute Liver Failure 531
significantly reduces serum cell death markers and improves clinical outcome
(Jochum 2009).
The regenerative capacity of the liver depends on the patient’s gender, age, weight
and previous history of liver diseases. Important mediators of liver regeneration
include cytokines, growth factors and metabolic pathways for energy supply. In the
adult liver, most hepatocytes are in the G0-phase of the cell cycle and nonproliferating. Upon stimulation with the proinflammatory cytokines tumour necrosis
factor α (TNFα) and interleukin- (IL-) 6, growth factors like transforming-growth
factor α (TGFα), epidermal growth factor (EGF) and hepatocyte growth factor
(HGF) are able to induce hepatocyte proliferation. TNF and IL-6 also induce
downstream pathways related to NFκB and STAT3 signaling. Both transcription
factors are mandatory for coordination of the inflammatory response to liver injury
and hepatocyte proliferation (Dierssen 2008). Emerging data supports an important
role for hepatic progenitor and oval cells as well as vascular endothelial growth
factor (VEGF) mediated angiogenesis in liver regeneration (Ding 2010, Dolle
2010).
TNFα, IL-1 and IL-6 are also important mediators of hyperdynamic circulation by
alterations of nitric oxide synthesis in ALF (Larson 2010). Renal failure, hepatic
encephalopathy, and brain edema are the results of these pathophysiologic changes.
Hyperammonemia correlates with brain edema and survival (Clemmesen 1999).
Decreased hepatic urea synthesis, renal insufficiency, the catabolic state of the
musculoskeletal system and impaired blood-brain barrier leads to ammonia
accumulation and alterations in local perfusion, which induces brain edema in ALF.
Interestingly, brain edema is a presentation of ALF rather than cirrhosis, and the risk
of brain edema increases with the grade of hepatic encephalopathy. After acute and
massive hepatic cell death, the release of proinflammatory cytokines and
intracellular material result in low systemic blood pressure leading to impairment of
splanchnic circulation. Indeed, renal failure in ALF patients is common, up to 70%
(Larsen 2011). Reduced qualitative and quantitative functions of platelets and
inadequate synthesis of prothrombotic factors are the causes of coagulopathy.
Leukopenia and impaired synthesis of complement factors in ALF patients increases
the risk for infections, which might result in sepsis. Infections increase the duration
of ICU stays and the mortality rate in ALF dramatically. With the impairment of
hepatic gluconeogenesis, hypoglycemia is a frequent feature of patients with ALF
(Canbay 2011).

532 Hepatology 2012
Table 3. Grade of hepatic encephalopathy (West Haven criteria).
Grade

Clinical findings

I

Triphasic waves

III

Changes in behavior, euphoria, +/–
depression, mild confusion
Inappropriate behavior, lethargy, +
moderate confusion
Marked confusion, somnolence +

IV

Coma

Delta waves

II

Asterixes

–

EEG

Triphasic waves
Triphasic waves

Prognosis
With persistently high, although variable, mortality rates from ten to ninety percent,
accurate prediction of the clinical course is crucial for accurate management and
decision-making. Most importantly, identification of the underlying etiology
improves prognosis and opens the door for specific treatment. The degree of hepatic
encephalopathy is traditionally considered an important indicator of prognosis
(O'Grady 1989). Cerebral edema and renal failure worsen the prognosis
dramatically. In some studies, the INR was determined as a strongest single
parameter to predict the prognosis of ALF. Another interesting point is that the
presence of hepatic encephalopathy means a poor prognosis for acetaminophen
induced ALF, which in contrast has little meaning for mushroom poisoning. LTx is
the ultimate treatment option in patients with ALF, in which conservative treatment
options fail and a lethal outcome is imminent. Therefore, assessment of likelihood
of the individual patient to undergo a fatal course is important for timely listing of
the patient. Standardised prognosis scores, based on reproducible criteria are
important in times of donor organ shortage and to avoid LTx in patients that might
fully recover without LTx (Canbay 2011).
King’s college criteria (KCC) was established in the 1990s based on findings
from a cohort of 588 patients with ALF (O'Grady 1989). The authors also
introduced a classification based on the onset of encephalopathy after an initial rise
in bilirubin levels into hyperacute (<7 days), acute (8-28 days) and sub-acute (5-12
weeks) liver failure (O'Grady 1993). KCC includes assessment of encephalopathy,
coagulopathy (INR), acid homeostasis (pH), bilirubin and age. For patients with
acetaminophen-induced ALF, another KCC formula was implied from that in
patients with non-acetaminophen-induced liver injury. Clichy criteria were
introduced for patients with fulminant HBV infection and include the degree of
encephalopathy and factor V fraction as a measure for hepatic synthesis (Bernuau
1986). The model for end stage liver disease (MELD) was designed to predict the
likelihood of survival after transjugular portacaval shunt (TIPS) in cirrhotic patients.
However, it has recently been established as an allocation tool for LTx in patients
with cirrhosis in the US and Europe. It has been tested as a model for prediction of
ALF and was found to be superior to KCC and Clichy criteria in independent
studies (Schmidt 2007, Yantorno 2007). Novel approaches that include mechanistic
characteristics of ALF like the CK-18 modified MELD, which includes novel
markers for hepatocellular death or lactate are promising, but need validation in
prospective cohorts (Bechmann 2010, Hadem 2008).

Acute Liver Failure 533
Table 4. Scoring systems in patients with ALF for emergent LTx.
Scoring System

Prognostic factors

King’s College
Criteria

Paracetamol
intoxication

Clichy Criteria

Non-paracetamol INR >6.5 and hepatic encephalapathy or
any of these three: INR >3.5 and bilirubin >300
μmol/L and age >40 years and unfavorable
etiology (undetermined or drug-induced)
HBV
Hepatic encephalopathy grade 3-4 and factor V
<20% (for <30 years old); <30% (for >30 years
old)
10x(0.957xInserum creatinine +0.378x Intotal
bilirubin +1.12xInINR+0.643)

MELD

Arterial pH <7.3 or
INR >6.5 and creatinine >300 μmol/L and
hepatic encephalapathy grade 3-4

CK-18 modified
MELD

10x(0.957xInserum creatinine +0.378x
InCK18/M65 +1.12xInINR+0.643)

Bilirubin-LactateEtiology Score (BILE
score)

Bilirubin (μmol/)/ 100+ Lactate (mmol/L)
+4 (for cryptogenic ALF, Budd-Chiari or
Phenprocoumon induced)
-2 (for acetaminophen-induced)
+0 (for other causes)

Adapted from Canbay 2011; INR, International Normalized Ratio; MELD, model of end stage
liver disease.

Treatment
General management
Given the high risk of deterioration and development of hepatic coma, immediate
transfer of the patient presenting with ALF to the ICU is mandatory. Early referral
or at least consultation of an experienced transplant center is indicated in any ALF
patient, since LTx is the ultimate treatment for ALF in case conservative therapy
fails. The cause of ALF should be determined as soon as possible. Besides specific
detailed history taking, laboratory and radiologic tests need to be done in order to
establish the diagnosis of ALF and identify the underlying cause. Diagnostic studies
include, but are not limited to, arterial blood gas analysis, glucose, electrolytes,
bilirubin, ammoniac, lactate, protein, albumin, C-reactive protein (CRP),
procalcitonin (PCT), urine electrolytes, urinalysis, and chest X-ray, cranial
computed tomography (CT) in patients with advanced hepatic encephalopathy as
well as assessment of intracranial pressure (ICP) in some cases. Additional to
specific diagnostic studies (HBV serology, ceruloplasmin, urine copper
concentration, etc.) transjugular or laparoscopic liver biopsy might be indicated to
identify the underlying disease (Canbay 2011).

Hepatic encephalopathy
In general in patients with hepatic encephalopathy, sedative agents should be
avoided and if necessary restricted to short-acting benzodiazepines or propofol, as it
might decrease intracranial pressure (Wijdicks 2002). Some studies favor utilization
of ICP monitoring, especially in patients with hepatic encephalopathy grade III/IV,

534 Hepatology 2012
and clinical signs of brain edema. Mannitol therapy (0.5-1 g/kg) might be beneficial
in some patients. Head elevation, induction of hypothermia and hyperventilation are
recommended by some experts in patients with increased ICP. With worsening of
brain edema, the patients present with systemic hypertension and bradycardia
(Cushing reflex), dilated and fixed pupils, and in the end respiratory arrest. The
target ICP should remain below 20 mmHg, with cerebral perfusion pressure above
70 mmHg and jugular venous saturation of 55-80%. Phenytoin is the drug of choice
for treatment of seizures and hypertonic sodium chloride might be beneficial on ICP
(Larsen 2011). Symptomatic treatment of encephalopathy includes bowel
decontamination with neomycin or rifaximin, induction of diarrhea and reduction of
colonic pH and thus reduction of ammonia absorption by lactulose as well as
treatment with branched-chain aminoacids to improve peripheral ammonia
metabolism, although large, randomized clinical trials have failed to show clinical
improvement (Larson 2010, Nguyen 2011).

Coagulopathy
In general, without clinical signs of bleeding, fresh frozen plasma (FFP) or
individual coagulation factor treatment is not indicated. To exclude vitamin K
deficiency, vitamin K challenge should be performed. Platelets and recombinant
activated factor VII are indicated in case of bleeding or before invasive procedures.

Liver Transplantation
LTx, is the therapy of choice for ALF in those individuals with insufficient
regeneration capacity and an otherwise fatal prognosis. In patients without
contraindications to LTx, the one-year survival rate is as high as 80-90% with a
five-year survival of 55%. As mentioned above, with LTx available as the most
favorable therapy, the accurate assessment of the patient’s prognosis is crucial to
initiate evaluation of the patient for LTx and decision making in this clinical setting.
The underlying disease, the clinical condition and the status of the graft influence
the patient’s prognosis after the transplant. In times of general organ shortage, the
graft pool might be extended by utilisation of living-donor transplants, split liver
surgery or transplantation of livers in reduced conditions (Canbay 2011).

Extracorporal liver support systems
Extracorporal systems include support devices or bioreactors, which provide
individual or a combination of functions that are insufficiently performed by the
diseased liver. The scientific and clinical aim of the introduction of these novel
techniques is to stabilize the patient until a donor organ is available or ideally until
the liver completely recovers. However, adequately powered, randomized studies to
establish these techniques in the treatment of ALF are either lacking or have failed
to show any benefit over conventional therapy. Thus, treatment with these devices
most likely remains a part of a bridging to transplantation strategy within an
academic setting. The same accounts for novel stem cell and adult hepatocyte
transplant approaches (Canbay 2011).

Acute Liver Failure 535

Specific treatment options
Table 5. Specific treatments for the causes of ALF.
Causes

Medication

Doses

Acetaminophen

Activated oral charcoal

1 g/kg

N-acetyl cysteine (oral/IV)

Mushroom

Silibin

150 mg/kg loading dose,
50 mg/kg for 4h,
100 mg/kg for 20h
20-50 mg/kg/day

Acute HBV

Lamivudine

100-300 mg/day

Entecavir

0.5-1 mg/day

Tenofovir
Delivery

245 mg/day

Pregnancy
Autoimmune

Prednisolone

1-2 mg/kg/day

Budd-Chiari syndrome

TIPS/surgical shunt

HSV

Acyclovir

3x10 mg/kg/day

Acetaminophen Poisoning
Activated oral charcoal (1 g/kg) might be indicated if administered up to four hours
after acetaminophen ingestion. N-acetyl cysteine infusion to restore glutathione
should be administered until as late as 24-36 hours after ingestion, and continued for
20 hours or longer. Monitoring of blood acetaminophen levels might help in
decision-making regarding the duration or initiation of treatment. N-acetyl cysteine
should be started as soon as possible, even in patients with a low probability of
acetaminophen overdose or even in patients with non-paracetamol drug-induced
ALF (Lee 2009). Moreover, steroid and ursodeoxycholic acid combination seems to
be effective in drug-induced severe liver injury (Wree 2011).
Mushroom poisoning
Silibin, with its cytoprotective affects against amatoxin is used despite a lack of the
controlled trials (Broussard 2001).
Acute HBV infection
Antiviral therapy with lamivudine or entecavir has proven as efficient and safe in
fulminant HBV infection (Tillmann 2006). Moreover, with initiation of entecavir
within the first days of admission, HBsAg concentrations and cell death were
significantly reduced (Jochum 2009).
Pregnancy related
Immediate delivery and abortion are the available causal treatments. With early
delivery, the rates of fetal death remain high, however the mortality rate of the
mother decreases significantly (Westbrook 2010).
Autoimmune hepatitis
Steroid treatment should be initiated and if started in time might help to avoid the
need for LTx. With improvement of liver function, prednisone might be tapered and
azathioprine treatment added to the regimens. Recent studies identified the topical

536 Hepatology 2012
steroid budesonide as a potential substitute for systemic prednisone therapy
(Schramm 2010).

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Acute Liver Failure 539

540 Hepatology 2012

541

Index

A

B

Abacavir 308
Abbott RealTime 192
Abstinence 495
ABT-072 240
ABT-333 240
ABT-450 240
Acetaminophen 535
Acetaminophen intoxication 527
ACH-1625 240
Acute hepatitis
hepatitis B 36
hepatitis C 47
hepatitis C treatment 226
hepatitis E 59
Acute Liver Failure 526
Adefovir 137, 138, 142, 164, 295, 297,
323
Adherence 220, 269
ADV 138
Adverse drug reactions see Side effects
Albinterferon 256
Alcoholic Hepatitis 488
Alisporivir 240
Amanita intoxication 528
Aminotransferase levels 227
Amplicor 192
ANA598 240
Antidepressants 266
Anti-TNF α antibodies 465
Anti-TNF-α therapy 497
Antiviral resistance 206
ARFP 92
Arterial chemoembolisation 343
Arthralgia 262
Arthropathy 417
Asthenia 263
Asunaprevir 239
Autoimmune hemolytic anemia 273, 281
Autoimmune hepatitis 453, 529
Autoimmune thyroidopathies 273
Azathioprin 471
Azathioprine 458

Baraclude 137
BI201335 239
BI207127 240
BILB 1941 240
Biochemical response 136
Blood transfusion 45
hepatitis B virus 34
BMS-650032 239
BMS-790052 240, 251
BMS-824393 240, 251
Boceprevir 205, 208, 239, 244, 253, 308
adverse effects 268
Breakthrough 204
Budd-Chiari syndrome 522
Budesonide 462

C
Cardiac arrhythmias 416
Cardiomyopathy 273, 416
Celgosivir 240
Ceruloplasmin 440
cEVR 204
Cholestyramine 470
Chronic hepatitis
hepatitis B 37
hepatitis C 48
Hepatitis D 174
Ciluprevir 239, 243
Cirrhosis
HBV/HCV coinfection 322
hepatitis C 49
Cirrhosis complications
end-stage liver disease 389
Clevudine 181
Cobas Amplicor HCV 193
Cobas Ampliprep 192
Cognitive disturbances 262
Coinfection
HBV/HCV 318
Coinfections 230
Colchicine 471
Competitive PCR 193
Complete Early Virological Response 204
Consensus Interferon 205

542
Copegus 205
Copper 441
Corticosteroids 495
Cryoglobulinemic vasculitis 273
Cyclophilin B inhibitors 252
Cyclophosphamide 462, 464
Cyclosporin 462, 471
Cyclosporine 464

D
Daclatasvir 240
Danoprevir 239
Debio-025 240
Deferasirox 418
Deflazacort 463
Delirium 262
Delta hepatitis see Hepatitis D
Depressive episodes 262
Diabetes mellitus 273, 416
Dimercaprol 443
Direct sequence analysis 197
Disease progression
hepatitis C 50
Disorders of the hepatic artery 514
Disorders of the hepatic veins 522
Disorders of the portal vein 518
D-penicillamine 471
Drug abuse 230
Drug interactions 225
Drug-induced liver injury 527
Dyspnea 264

E
Early Virological Response 204
Efavirenz 308
Emtricitabine 164, 296, 308
End-stage liver disease
HIV infection 386
Entecavir 137, 138, 143, 161, 297, 323
Epidemiology
hepatitis A 27
hepatitis C 44
hepatitis D 176
eRVR 204
ESLD see End-stage liver disease
ETV 138
EVR 204
Extended Rapid Virological Response
204

Extracorporal liver support systems 534
Extrahepatic manifestations
hepatitis A 29
hepatitis B 41
hepatitis C 49, 230
hepatitis E 61

F
Famcyclovir 181
Fatigue 262
Ferroportin Disease 420
Filibuvir 240
Flu-like symptoms 262

G
Gastrointestinal disorders 263
Genotypes
hepatitis C virus 86
GH-insufficiency 273
Glomerulonephritis 41, 282
Graft versus host disease 529
GS-5885 240, 251
GS-9190 240
GS-9451 240

H
HAART
liver transplantation 391
Haemochromatosis 405
juvenile hereditary 419
secondary 421
TFR2-related 419
Hair loss 267
Hashimoto thyroiditis 273
HBcAg 120
HBeAg 121
HBeAg loss 149
HBeAg seroconversion 133
HBsAg 120
treatment response 150
HBsAg loss 134
HBV 65
animal models 75
serological tests 119
HBV DNA
treatment response 147
HBV genotypes 122

543
treatment response 147
HBV Virology 65
HBV/HCV Coinfection 318
HBV/HIV coinfection 291
HCC see Hepatocellular carcinoma
HCV See also Hepatitis C virus
HCV genotype 1 207
HCV genotypes 2 and 3 215
HCV genotypes 4, 5, and 6 218
HCV genotyping 196
HCV life cycle 240
HCV replicon systems 97
HCV SuperQuant 192
HCV/HBV Coinfection 318
HCV/HBV management
liver transplantation 390
HCV/HIV Coinfection 302
HCV-796 240
HCV-associated thrombocytopenia 281
HCVcc 98
HCVpp 98
HDV see Hepatitis D
Hemodialysis 230
transmission of HCV 47
Hemophilia 230
Hepadnaviridae 65
Hepatic fibrosis 326
Hepatitis A 27
prophylaxis 108
vaccination 110
Hepatitis B 32
chronic infection 124
coinfection with HCV 318
diagnostic tests 119
drug resistance 160
hepatitis C coinfection 40
hepatitis D coinfection 40
immunosuppression 154
liver biopsy 125
occult infection 125, 320
past infection 123
post-exposure prophylaxis 113
pregnancy 153
prognostic factors 147
prophylaxis 109
serum transaminases 124
superinfection 320
treatment 128
treatment guidelines 130
vaccination 111
Hepatitis C 44

coinfection with HBV 318
diagnostic Tests 189
endocrine manifestations 283
extrahepatic manifestations 272
lifecycle 93
prophylaxis 109
serologic assays 190
superinfection 319
treatment 202
vaccination 114
virology 85
Hepatitis D
iagnostic procedures 174
prophylaxis 109
treatment 174
virology 175
Hepatitis E 55
virology 55
Hepatocellular carcinoma 338, 398
curative therapy 341
HBV/HCV coinfection 322
palliative therapy 343
Hepatoportal sclerosis 521
Hepsera 137
Hereditary hemorrhagic teleangiectasia
516
HEV See Hepatitis E virus
Histologic response 136
HIV
hepatitis E 60
HIV/HCV Coinfection 302
HIV/HVB coinfection
end-stage liver disease 397
Horizontal transmission
hepatitis B virus 34

I
Idiopathic pulmonal fibrosis 273
IDX184 240
IDX320 240
IDX375 240
IL28B 160
Immune thrombocytopenic purpura 273
Incivek 244
Incivo 244
Infergen 205
Injection drug use 45
Insulin resistance 273
Interferon lambda 1 256
Interferon α 182

544
Interferon α-2a 137, 205
Interferon α-2b 137, 205
Interferons 139
Intron 137, 205
Iron overload 405
Irritability 262

L
LAM 138
Lamivudine 137, 138, 142, 161, 296, 308
LdT 138
Lead-In 204
Lichen planus 273
Liver biopsy 327
Liver cancer
prophylaxis 345
Liver cirrhosis
compensated 228
Liver fibrosis
surrogate markers 329
Liver retransplantation
HIV infection 398
Liver transplantation 349
AIH 466
hepatitis delta 183
HIV infection 386
NASH 433
PSC 478

M
Malignant lymphoproliferative disorders
276
Membrano-proliferative
glomerulonephritis 273
Mericitabine 240, 248, 250
Methotrexate 471
Miravirsen 240
Mixed cryoglobulinemia 272
treatment 277
MK-3281 240
MK-5172 239
MK-7009 239
Molecular-targeted therapeutic
strategies 344
Monoclonal gammopathies 273
Mushroom poisoning 535
Myalgia 262
Mycophenolate mofetil 462
Mycophenolic acid 464

Myopathy 273

N
N-acetyl cysteine 497
NAFLD 427
Narlaprevir 239
NASH 427
Natural history
hepatitis B 36
hepatitis C 49
Needlestick injury
transmission of HCV 47
Nephropathy 41
Nexavar 344
NIM811 240
Nitazoxanide 240, 253
NM283 240
Nodular regenerative hyperplasia 521
Non-Hodgkin lymphomas 273
Nonresponse 204
Nosocomial infection
hepatitis B 35
NS2 91
NS3 91
NS3-4A protease inhibitors 241
NS4A 91
NS4B 91
NS5A 92
NS5A inhibitors 251
NS5B 92
NS5B polymerase inhibitors 248
Nucleic acid testing for HCV 191
Nucleos(t)ide analogs 138, 141
Nucleoside analogs
delta hepatitis 181
Null response 204

O
Opioid antagonists 470
Organ transplantation
HCV transmission 46
hepatitis B virus 36
hepatitis E virus 60
Orthotopic liver transplant 392
outcome 394
Osler-Weber-Rendu syndrome 516

545

P
p7 protein 91
Partial Response 204
Pathogenesis
hepatitis D 178
Pegasys 137, 205
PEG-IFN lambda 1 256
PEG-IFN maintenance therapy 215
PEG-Intron 205
Pegylated interferon alpha 182
Peliosis hepatis 513
Penicillamine 443, 444
Pentoxifylline 496
Perinatal transmission
hepatitis B virus 34
hepatitis C virus 46
Peripheral neuropathy 273, 275
treatment 278
PF-00868554 240
Phlebotomy 418
PHX1766 239
Polyarteritis nodosa 41
Porphyria cutanea tarda 273
Portal vein thrombosis 518
Postexposure prophylaxis
hepatitis B infection 36
PPI-461 240, 251
Prednisone 458
Pregnancy-induced liver injury 530
Primary biliary cirrhosis 467
Primary sclerosing cholangitis 473
Prognosis
hepatitis B 39
Prophylaxis
viral hepatitis 108
PSI-7977 250
PSI-938 240, 250
Psychosis 262

Q
Quadruple therapy 254
Qualitative assays for HCV RNA detection
192
Qualitative RT-PCR 192
Quantitative HCV RNA detection 193

R
R1626 240

R7128 240
R7227 239
Radiotherapy 344
Raltegravir 308
Rapamycin 462
Rapid Virological Response 204
RealTime HCV test 196
Real-time PCR technology 197
Real-time PCR-based HCV RNA detection
assays 194
Rebetol 205
Recombinant interferon alpha
delta hepatitis 182
Relapse 204
Renal impairment 275
Reverse hybridising assay 197
Rheumatoid arthralgias 273
Rheumatoid factor positivity 273
ribavirin 306
Ribavirin 181, 205, 223, 322
Rifampicin 470
Roferon 137, 205
RVR 204

S
SCH503034 239
SCH900518 239
Sebivo 137
Serotonin antagonists 470
Serum HBV DNA assays 121
Sexual impotence 417
Sexual transmission
hepatitis B virus 33
hepatitis C virus 46
Sicca syndrome 273
Side effects 262
management 221
Simeprevir 239
Sinusoidal obstruction syndrome 509
Skin disorders 267
Sleep disturbances 262, 266
Sorafenib 344
Suicidal syndrome 262
Sulindac 472
Sustained Virological Response 204
SVR 204
SVR-12 204
Systemic vasculitis 275
treatment 278

546

T
Tacrolimus 462, 464
Taxonomy
hepatitis C virus 86
TDF 138
Telaprevir 205, 210, 239, 244, 253, 308
adverse effects 268
Telbivudine 137, 138, 143, 161, 297, 323
Tenofovir 137, 138, 144, 161, 296, 323
Tetrathiomolybdate 443, 444, 446
TMC435350 239
TMC647055 240
Transcription-mediated 192
Transferrin receptor 2 419
Transient elastography 330
Transmission
hepatitis A virus 27
hepatitis B virus 33
hepatitis C virus 45
hepatitis E virus 58
Transplantation 349
Treatment failure 212
Treatment response 206
Trientine 443, 445

U
Ulcerative colitis 478
Ursodeoxycholic acid 465, 470, 472, 477

V
Vaccination 108
hepatitis B 111
hepatitis C 114
hepatitis E 61, 115
Valopicitabine 240, 249
Vaniprevir 239
Vascular liver disease 509
VCH222 240
VCH759 240
VCH916 240
Versant 192, 194
Victrelis 244
Viread 137
Virologic response 136
Virology
HDV 175
hepatitis B 65
hepatitis C 85
Vitamin E 446

W
Weight loss 263
Wilson’s Disease 437

Z
Zeffix 137
Zinc 445
Zinc acetate 444