Number 1 MARCH 2006

HEPATITIS
CONTENTS
Preface xi
Robert C. Moellering, Jr
The Progression of Hepatitis B and CInfections

to Chronic Liver Disease and Hepatocellular Carcinoma:
Presentation, Diagnosis, Screening, Prevention, and
Treatment of Hepatocellular Carcinoma 1
Paul H. Hayashi and Adrian M. Di Bisceglie
Patients with or at risk for hepatocellular carcinoma (HCC) present
special challenges to the clinician. Despite improving understand-
ing of HCC, current guidelines and treatment algorithms are still
inadequate. Team coordination and expertise are highly important.
In this article, some of the challenging and controversial issues
regarding HCC detection, diagnosis, prevention, and care are re-
viewed, with particular emphasis on hepatitis B and Cassociated
HCC. Although not always evidenced-based, practice guidelines or
standard of care practices are summarized.
Hepatitis B Vaccines 27
Andy S. Yu, Ramsey C. Cheung, and Emmet B. Keeffe
Immunization is the most effective way to prevent transmission of
hepatitis B virus (HBV) and the development of acute or chronic
hepatitis B. The strategy to eliminate HBV transmission in the
United States is to vaccinate all newborn infants, children, and
adolescents, as well as high-risk adults. Postexposure prophylaxis
is advocated after a documented exposure, depending on vaccina-
tion history and antibody to hepatitis B surface antigen (antiHBs)
status. Seroprotection after hepatitis B vaccination, defined as anti
HBs 3 10 mIU/mL, is achieved in more than 95% of subjects.
Hepatitis B vaccines are very well tolerated with usually minimal
adverse effects. Increasing age, male gender, obesity, tobacco
smoking, and immunocompromising chronic diseases are predic-
tors of nonresponse to vaccination.
VOLUME 20 NUMBER 1 MARCH 2006 v

Serologic and Molecular Diagnosis of Hepatitis B Virus 47
Julie C. Servoss and Lawrence S. Friedman
Since the discovery in the 1960s of Australia antigen, subse-
quently determined to be hepatitis B surface antigen, serologic as-
says for hepatitis B virus (HBV) have expanded and evolved. More
recently, molecular biology-based techniques have allowed the de-
tection and quantification of HBV DNA in serum. In the past de-
cade, improvements in the sensitivity of these molecular assays
have allowed the detection of as few as 10 copies of HBV DNA
per milliliter of serum. The combined use of serologic and molecu-
lar assays for the diagnosis of HBV infection has resulted in more
precise definitions of chronic HBV infection. Molecular assays have
become essential to defining responses to antiviral treatment.
Assessment and Management of Chronic Hepatitis B 63

Marc G. Ghany and Edward C. Doo
Chronic hepatitis B (CHB) is a major worldwide cause of chronic liver
disease and a significant public health issue. Three predominant clin-
ical presentations are recognized: hepatitis B e antigen (HBeAg) or
typical CHB, HBeAg-negative or atypical CHB, and inactive CHB.
The natural history of CHB infection in an individual may be domi-
nated by one or a combination of these clinical presentations in a se-
quential fashion. These variations in clinical presentations reflect the
viral-host immunology dynamics that form the basis for the develop-
ment of liver disease. Therapy has been problematic in the past.
There are three licensed drugs available for therapy of CHB with var-
ied mechanisms of action. This has introduced the concept of tailored
therapy for the individual patient. Many promising new agents and
therapeutic approaches should become available in the near future.
Molecular Virology of the Hepatitis C Virus: Implication

for Novel Therapies 81
Jeffrey S. Glenn
The study of hepatitis C virus (HCV) molecular virology is helping
to shape the future of our anti-HCV strategies by identifying new
antiviral targets. With the advent of agents that specifically target
individual HCV proteins, HCV-specific therapy has arrived. Key
to these efforts is the development of high-efficiency HCV repli-
cons. The future effective pharmacologic control of HCV will likely
consist of a cocktail of simultaneously administered virus-specific
agents with independent targets, which should minimize the emer-
gence of resistance against any single agent. The way we treat HCV
should change dramatically over the next few years.
Antiviral Therapy for Treatment Nave Patients

with Hepatitis C Virus 99
Sangik Oh and Nezam H. Afdhal
This article focuses on the most recent therapies for patients with
hepatitis C virus (HCV) who are nave to therapy. The primary
vi CONTENTS
end point for the treatment of nave HCV patients is viral eradica-
tion or a sustained virological response, which is defined as the ab-
sence of HCV in the serum, as detected by a sensitive polymerase
chain reaction test, 24 weeks after stopping antiviral therapy. Re-
cent data suggest that an SVR can be equated with a biochemical,
virological, and histological response that is sustained for up to 5
years and is conceptually a cure of HCV in 90% of patients.
Approach to the Management of Patients with Chronic

Hepatitis C Who Failed to Achieve Sustained Virologic
Response 115
Amrita Sethi and Mitchell L. Shiffman
The combination of peginterferon and ribavirin is the most effec-
tive therapy for patients with chronic hepatitis C virus (HCV) infec-
tion. Although more than half of all patients are able to achieve a
sustained virologic response (SVR), a significant proportion of pa-
tients, particularly those with genotype 1, fail to have undetectable
HCV RNA during treatment or relapse after completing therapy
with return of detectable HCV RNA. The management of these pa-
tients creates a formidable challenge. This article outlines various
strategies for patients who have failed to achieve SVR and dis-
cusses the merits of different approaches to management.
Treatment of Relapsers After Combination Therapy

for Chronic Hepatitis C 137
Furqaan Ahmed and Ira M. Jacobson
A significant number of patients with chronic hepatitis C (CHC) re-
lapse after treatment. As therapy for CHC has improved over the
last decade, the issue of retreating patients who did not achieve a
sustained virologic response (SVR) with previous treatment regi-
mens frequently arises. Several studies have assessed the efficacy
of retreating patients who have previously relapsed to standard in-
terferon monotherapy or standard interferon and ribavirin combi-
nation therapy. Patients who have relapsed after therapy have
significantly higher SVR rates than those who are nonresponders
to therapy and should be considered candidates for retreatment.
Predictors of a favorable response to therapy in nave patients ap-
pear to also predict response to therapy in patients who have re-
lapsed previously.
Management of Recurrent Hepatitis C in Liver Transplant

Recipients 155
Scott W. Biggins and Norah A. Terrault
Chronic hepatitis C virus (HCV) infection is the most common indi-
cation for liver transplantation in the United States and Europe, and
more than 20,000 patients worldwide have undergone transplanta-
tion for complications of chronic hepatitis C. In North America,
HCV accounts for 15% to 50% of the liver transplants performed
CONTENTS vii
in United States transplant programs. To maximize the long-term
survival of liver transplant recipients who have HCV infection,
eradication of infection is the ultimate goal. Pretransplant antiviral
therapy with the goal of achieving viral eradication before trans-
plantation is a consideration in some patients, especially those
who have mildly decompensated liver disease. This article focuses
on the management of liver transplant recipients who have HCV in-
fection at the time of transplantation. Prophylactic and preemptive
therapies, as well as treatment of established recurrent disease, are
the strategies reviewed.
Index 175
viii CONTENTS
FORTHCOMING ISSUES
June 2006
Bioterrorism
Nancy Khardori, MD, Guest Editor
September 2006
Fungal Infections
Thomas F. Patterson, MD, FACP
Guest Editor
RECENT ISSUES
December 2005
Update on Musculoskeletal Infections
John J. Ross, MD, Guest Editor
September 2005
Pediatric Infectious Disease
Jeffrey L. Blumer, MD, PhD, and
Philip Toltzis, MD, Guest Editors
June 2005
Sexually Transmitted Infections
Jonathan M. Zenilman, MD, Guest Editor
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Infect Dis Clin N Am
20 (2006) xixii
Preface
Hepatitis
Several viruses are capable of causing hepatic inammation. These in-

clude the Epstein-Barr virus, cytomegalovirus, herpes simplex virus, mumps,
rubella, rubeola and varicella-zoster viruses, yellow fever virus, Coxsackie
viruses, and adenoviruses. In most cases, infection or inammation of the
liver is part of a systemic infection with the above agents. In addition, there
are a number of viruses that are primarily hepatotropic and are given alpha-
betical designations: hepatitis AE. Several other viruses that were initially
thought to cause posttransfusion hepatitis, including hepatitis G virus or
GBV-C and SEN viruses, are not currently believed to be human pathogens.
Of the truly hepatotropic viruses, hepatitis A and hepatitis E generally pro-
duce self-limited disease, although fulminant hepatic failure has been
reported in up to 1%2% of infected individuals. Hepatitis D virus (or
the delta agent) can produce coinfections in patients who are infected with
hepatitis B, but it appears to be incapable of causing serious disease in the
absence of hepatitis B virus. Hepatitis B and C viruses are arguably the most
important viruses of this group. In addition to acute infections, exposure to
these viruses frequently results in a chronic persistent infection that may
remain asymptomatic or lead to cirrhosis, liver failure, and hepatocellular
carcinoma. Chronic infections with hepatitis C virus are currently responsi-
ble for an increasing percentage of liver transplantations performed in the
United States and elsewhere. Because of the large number of patients world-
wide who suer from chronic infections with these two agents, they are
arguably the most important viral cause of carcinoma in the world. In recent
years, we have learned a great deal about the biology of hepatitis B and C
viruses and have gained increasing knowledge of the pathophysiology of the
disease they cause in the liver. Concomitant with our understanding of these
processes has been the development of a variety of therapeutic modalities,
ranging from relatively specic, classic antiviral agents (including lamivu-
dine, adefovir, tenofovir, ribavirin, and a number of new investigational
agents) to drugs such as interferon-a, which also have immunomodulatory
eects. Vaccine strategies likewise play an important role in preventing
hepatitis B. The rapidity of new developments in our understanding of the
pathophysiology and management of infections due to hepatitis B and C
0891-5520/06/$ - see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.idc.2006.01.008 id.theclinics.com
xii PREFACE
make it increasingly dicult for the practicing physician to keep up with

the eld.
For these reasons, we have assembled in this issue of the Infectious Dis-
ease Clinics of North America a series of articles written by experts in the
eld of hepatitis B and C infections. Taken in aggregate, they provide useful,
lucid, up-to-date information on the management of hepatitis B and C
infections.
Robert C. Moellering, Jr, MD

Harvard Medical School
Department of Medicine
Beth Israel Deaconess Medical Center
110 Francis Street, Suite 6A
Boston, MA 02215, USA
E-mail address: rmoeller@bidmc.harvard.edu
20 (2006) 125
The Progression of Hepatitis

B and CInfections to Chronic Liver
Disease and Hepatocellular Carcinoma:
Presentation, Diagnosis, Screening,
Prevention, and Treatment of
Hepatocellular Carcinoma
Paul H. Hayashi, MD*, Adrian M. Di Bisceglie, MD
Division of Gastroenterology and Hepatology, Department of Internal Medicine,
Saint Louis University Liver Center, 3635 Vista Avenue, St. Louis, MO 631100250, USA
Patients with, or at risk for, hepatocellular carcinoma (HCC) present

special challenges to the clinician. Despite improved understanding of
HCC, current guidelines and treatment algorithms are still inadequate.
The gastroenterologist or primary care physician may provide screening
and prevention, but clinical care after HCC has occurred is a specialized
task. A multidisciplinary team including a hepatologist, oncologist, trans-
plant surgeon, interventional radiologist, and liver histopathologist may
be needed. Team coordination and expertise is highly important, and
most available at tertiary care referral centers. In this article, some of the
challenging and controversial issues regarding HCC detection, diagnosis,
prevention, and care are reviewed, with particular emphasis on hepatitis
B and Cassociated HCC. Although not always evidenced-based, practice
guidelines or standard of care practices are summarized.
Clinical presentation
The clinical presentation of HCC ranges from catastrophic tumor rupture
into the peritoneum to incidental cancer diagnosed by abdominal imaging.
A version of this article originally appeared in the 89:2 issue of The Medical Clinics of
North America.
* Corresponding author.
E-mail address: hayaship@slu.edu (P.H. Hayashi).
2 HAYASHI & DI BISCEGLIE
The most common symptoms are abdominal pain (often right upper quad-
rant) and weight loss. These symptoms may be more common in areas of
endemic chronic hepatitis B and high incidence of HCC [1]. HCC may
also lead to hepatic insuciency by displacement of noncancerous hepato-
cytes. Cancer extension into the portal vein or its branches may lead to
thrombosis and increased portal hypertension. HCC should be suspected in
any cirrhotic patient presenting with worsening hepatic function manifested
by increased jaundice, encephalopathy, or portal hypertension (ie, ascites,
variceal bleeding).
Catastrophic bleeding from tumor rupture is rare. It usually arises from
an HCC lesion near the hepatic surface that has outgrown its blood supply
[2]. Paraneoplastic symptoms, such as diarrhea, polycythemia, hypercalce-
mia, hypoglycemia, and feminization, rarely occur. These cases presumably
arise from hormones produced by tumor cells. HCC can rarely grow into the
hepatic veins and right atrium producing cardiac symptoms [3].
Typical ndings on physical examination include cachexia, hepatomeg-
aly, and a rm hepatic edge. Signs of cirrhosis, such as clubbing, palmer
erythema, jaundice, and ascites, may occur. Because the blood supply to
HCC is arterial, a bruit may be auscultated over the liver. When the tumor
involves the liver capsule, a rub may be auscultated during inhalation and
exhalation. The rub is often low pitched and sounds like a rumble in con-
trast to the higher pitched pericardial rub.
Diagnosis
HCC in cirrhotic patients is diagnosed by (1) alpha fetoprotein (AFP)
level, (2) imaging studies, and (3) histologic diagnosis. The AFP level is nor-
mally less than 15 to 20 ng/mL in adults. The higher the AFP level, the more
specic it is for HCC. An AFP greater than 400 ng/mL in a cirrhotic with
a vascular hepatic mass on imaging is diagnostic. Unfortunately, many
HCC cases have only modestly elevated AFP values. Sensitivities are as low
as 45% even for a low cuto of 20 ng/mL [4]. Moreover, hepatocyte
regeneration during and after ares in chronic hepatitis may increase AFP
levels in the absence of HCC. Other serologic markers, such as des g-
carboxyprothrombin and glypican-3, have shown promise but are not
widely used clinically in the United States [5,6].
Imaging studies, including abdominal ultrasound (US), contrast-en-
hanced CT, and MRI, are useful. The latter two modalities rely on charac-
teristic vascular qualities to identify lesions. Virtually all HCCs are perfused
by the hepatic artery rather than the portal venous system. Large HCCs are
usually easy to identify, but small lesions (!23 cm) may have subtle
vascular markings. CT and MRI scans must include images taken before,
during, and after contrast administration (multiphased). An experienced
body-imaging radiologist is invaluable in interpreting these tests. Indeed,
the choice of CT or MRI for HCC diagnosis often depends on center
HBV & HCV: CHRONIC LIVER DISEASE & HCC 3
expertise and preference. US is also quite useful, but a lesion detected on US

generally requires an MRI or CT for better characterization. Nevertheless,
positive predictive values for CT and MRI can be as low as 37% to 69%,
particularly for lesions less than 1 to 2 cm [7,8]. Sonographic contrast
materials can be injected into a peripheral vein and cross pulmonary
microvasculature to produce air bubbles in the hepatic artery. This diag-
nostic technique is currently experimental [9,10]. Finally, invasive imaging
studies, such as hepatic artery angiogram, and CT and MRI angiography,
may increase accuracy [11,12] but are not widely used.
Some experts recommend needle biopsy under imaging guidance partly
because lymphoma and nonmalignant lesions can masquerade as HCC [13].
Tissue diagnosis, however, is neither always necessary nor feasible. Biopsies
entail some risk. Patients with HCC often have end-stage cirrhosis with
coagulopathy, thrombocytopenia, or ascites, all of which increase the risk
of bleeding. Small tumors may be dicult to reach by percutaneous biopsy.
Cancer cells can rarely seed along the biopsy needle track. Earlier large
studies reported a risk of about 0.003% to 0.009% of seeding [14], but more
recent studies report a 1% to 2% risk [15,16]. In some studies, the false-
negative rate of biopsies is as high as 10% to 40%, particularly for small
tumors, because of inadequate samples or sampling error [15,16]. The histo-
pathologic criterion separating dysplastic regenerative nodules from malig-
nancy can be ambiguous. Repeat biopsy does not signicantly decrease the
false-negative rate [13,16].
Generally accepted guidelines provide criteria for HCC, as published by
the European Association for the Study of Liver Disease in 2001 (Box 1)
[17]. Note that criteria 2 and 3 apply only in the setting of cirrhosis. The
United Network for Organ Sharing (UNOS) in the United States has similar
guidelines as described later in the liver transplantation section.
Screening
Screening for HCC remains controversial. HCC meets several criteria for
cost-eective screening, including an identiable risk group (cirrhotic
Box 1. European Association for the Study of Liver Disease

hepatocellular carcinoma diagnostic criteria
1. Cytohistopathologic diagnosis or
2. >2-cm arterial hypervascular lesion detected by two coincident
imaging techniques in the setting of cirrhosis or
3. >2-cm arterial hypervascular lesion detected by one imaging
technique with serum AFP >400 ng/mL in the setting of
cirrhosis
patients), a long latent phase, widely available screening tests (US and serum
AFP), and potentially curative treatment for early disease (resection, trans-
plantation). These very characteristics, however, have made it dicult to
perform a randomized, controlled study. Few patients or institutional re-
view boards accept a study in which controls do not undergo screening until
a proportion presents with symptomatic HCC. Patients with symptomatic
HCC typically have advanced disease and a poor prognosis. Although
screening for HCC makes intuitive sense, questions regarding inadequate
sensitivity, specicity, and positive predictive values of screening tests and
cost-eectiveness continue to be raised. No medical organization or associ-
ation has promulgated clear screening guidelines.
Inaccuracies in imaging and AFP interpretation raise concerns about
screening. Screening eectiveness depends greatly on the sensitivity and neg-
ative predictive value of the tests. Cost eectiveness also depends on the
specicity and positive predictive value because chasing false-positive results
incurs costs and potential morbidity. The currently available imaging mo-
dalities (eg, US or CT) and the serum AFP levels are inadequately sensitive.
US, CT, and MRI sensitivities are ne for large lesions (75%89%), but
poor for small lesions (28%43%) [18]. Because screening is most needed
to detect early, small tumors, US, CT, and MRI are inadequate screening
tools [4]. The AFP is considered a moderately sensitive test, with a 45% to
100% sensitivity at cutos of 10 to 19 ng/mL. Moreover, specicity remains
a problem at 70% [4].
Positive predictive value is highly pertinent to clinicians. For example, even
if an AFP test has a specicity and sensitivity of 90%, the positive predictive
value is only 50% in a population of cirrhotics with a 10% prevalence of HCC
(Fig. 1). A test with excellent sensitivity and specicity may be reduced to
a coin ip in clinical practice. Some have argued for the abandonment of
AFP screening based on this clinical problem [19]. Imaging studies have
similar problems. Lack of clearly eective screening tools and good outcomes
data results in cost estimates ranging widely from a reasonable $35,000 to
a prohibitive $284,000 per year of life saved by screening regimens [20,21].
Nevertheless, the evidence for screening is compelling. Cohort and case-
control studies show that patients with HCC detected by screening have sig-
nicantly earlier cancers at diagnosis, are more likely to undergo curative
therapy, and have increased survival time. McMahon et al [22] prospectively
analyzed their 16-year experience with twice annual screening of HBsAg-
positive Alaska natives using serum AFP. They detected 32 patients with
HCC in about 26,000 AFP tests. All but one had tumors less than 6 cm,
and 22 patients underwent attempted curative resection. Survival at 5 and
10 years from diagnosis was signicantly better than in historical controls
not enrolled in a screening program. In a retrospective cohort study of
308 patients with HCC from Hong Kong [23], the 142 patients who were
diagnosed through screening with AFP or US had signicantly smaller
tumors than the patients presenting with symptoms (3.5 cm versus 8.1 cm,
A HCC B HCC
(+) (-) (+) (-)
PPV 90%
81 9 (+) 9 9 PPV 50%
(+)
AFP AFP
(-) NPV 90% (-) 1 81 NPV 99%
9 81
Sensitivity Specificity Sensitivity Specificity

90% 90% 90% 90%
Fig. 1. Hypothetical example demonstrating a problem with using the serum AFP level as
a screening test for HCC. (A) Promising sensitivity and specicity in a cohort study translates
into (B) a low positive predictive value in the clinical setting when the prevalence of HCC is only
10%. AFP, alpha fetoprotein; NPV, negative predictive value; PPV, positive predictive value.
P ! .0001). Resectability and survival were also signicantly better in those

diagnosed through screening. In a study of 32 patients diagnosed with HCC
by screening with US and AFP among 602 patients with chronic viral hep-
atitis in Los Angeles [24], 18 had single, small tumors possibly amenable to
curative treatment. These studies are, however, subject to lead-time and
selection biases.
The recent change in priority of patients with HCC for liver transplantation
provides another argument for screening. In 2002, the Model for End-stage
Liver Disease (MELD) score became the basis for prioritizing patients. In
this scoring system, patients with HCC were assigned higher scores and the
rate of transplantation for these patients tripled in the rst year [25]. Liver
transplantation has become more available to patients with early HCC.
Screening has become accepted in clinical practice despite a lack of den-
itive data. In a survey, 84% of surveyed hepatologists in the United States
routinely screened cirrhotic patients, using AFP in 99.7% and US in 93%
[26]. An AFP and US done every 6 months for cirrhotic patients is an ac-
ceptable screening program; however, 25% in the previously mentioned sur-
vey preferred CT for screening. The screening practice usually does not
dier for patients with cirrhosis from hepatitis C versus hepatitis B versus
other causes of cirrhosis, including nonalcoholic fatty liver, alcohol, hemo-
chromatosis, or a1-antitrypsin deciency. Cirrhotic patients with disorders
carrying a lower risk of HCC (eg, Wilsons disease), however, might be
screened less aggressively [26]. Screening for patients with chronic hepatitis
C or B without advanced brosis or cirrhosis is not recommended.
Screening should be individualized. If a patients cirrhosis is so advanced
that no treatment would be oered, then screening is not indicated. Screening
is indicated only if treatment or intensive follow-up of detected lesions is rea-
sonable. Proposed guidelines for the management of detected lesions include
the following [17]: lesions less than 1 cm are usually followed with repeat
imaging in 3 months to determine lesion persistence or growth; lesions 1 to

2 cm are considered for biopsy or closely followed; and lesions more than
2 cm may or may not require a biopsy depending on the clinical setting, im-
aging characteristics, and AFP level. The protocol for elevated serum AFP
levels in the absence of any suspicious lesion is unclear. Indeed, the precise
cuto level triggering any action is undened. Some pursue imaging tests
(MRI, CT) to detect lesions for mild elevation of AFP (O25 ng/mL) [22].
Others might repeat the AFP in 1 month for mild elevations (eg, 2050 ng/
mL), especially if other reasons for a burst of hepatic regeneration exist
(eg, recent are of chronic hepatitis B or C).
Prevention
Primary prevention of hepatitis C and B virus infection through control
of high-risk behavior (eg, intravenous drug use), adequate screening of
blood products, and vaccination are likely to be highly eective if such pro-
grams and policies were to be widely implemented. The annual incidence of
hepatitis C infection has fallen in the United States since the 1970s, when
intravenous drug use was more widespread and screening tests for hepatitis
C were unavailable. The AIDS epidemic seems to have considerably de-
creased intravenous drug use. Posttransfusion hepatitis C has become ex-
ceedingly rare since the implementation of improved antihepatitis C
virus antibody testing by American blood banks in 1992. A vaccine for
hepatitis C remains elusive because of lack of host protective antibody and
viral immune escape mechanisms.
The incidence of hepatitis B fell with the decline in intravenous drug use,
and safer sexual practices caused by AIDS awareness. Available vaccine will
signicantly decrease the incidence of HCC related to chronic hepatitis B.
Data from Taiwan suggest a decrease in HCC incidence in children with
childhood vaccination against hepatitis B [27]. Presumably this will eventu-
ally produce a drop in the incidence of HCC in adults when the rst cohort
of vaccinated children reach adulthood.
Secondary prevention of HCC by control of viral replication and hepatic
injury may be vital for the millions already chronically infected with hepa-
titis C or B virus. Treatment to eradicate or control the viruses decreases the
chronic inammation and regeneration of hepatocytes. In the United States,
a large cohort has been infected with hepatitis C for 20 to 30 years; during
this time period the risk of cirrhosis and HCC increases signicantly. Treat-
ment has improved signicantly during the last several years. Combined
therapy with a long-acting pegylated interferon-a injected once a week
and ribavirin taken daily by mouth can achieve long-term eradication of
hepatitis C virus in 40% to 70% of cases, depending on the viral genotype.
Eradication of hepatitis C virus infection should prevent progression to
cirrhosis and decrease the risk of HCC. In a Japanese surveillance program
of nearly 3000 cases of chronic hepatitis C followed since 1994, treated
patients had a 50% reduction in HCC risk and patients with successful erad-
ication of the virus had an 80% reduction [28]. In this uncontrolled study,
treatment was signicantly associated with HCC risk reduction in a multi-
variate analysis. Other studies support a decrease risk of HCC with treat-
ment, and a meta-analysis by Camma et al [29] also points toward benet.
In another study, patients treated with interferon after HCC ablation had
a decrease in HCC recurrence [30]. In the Hepatitis C Antiviral Long-
Term Treatment against Cirrhosis multicenter trial, several hundred patients
who did not respond to standard interferon and ribavirin therapy are being
randomized to no treatment versus 42 months of interferon therapy for
chronic suppression. One of the measured outcomes is HCC occurrence [31].
Treatment of patients for hepatitis C virus is hampered in cirrhotics by
decreased tolerance to therapy, lower response rates, and unclear benet
in terms of lowering HCC risk [32,33]. Nonetheless, viral eradication may
even benet cirrhotics by slowing or preventing liver failure. Moreover,
eradication before liver transplantation prevents recurrence of infection
after transplantation [32]. Whether treatment or eradication in cirrhotics
lessens the risk of HCC is undetermined, but studies clearly demonstrate
that brosis and cirrhosis can regress after eradication [34].
Treatment for chronic hepatitis B continues to improve. Treatment op-
tions include interferon or the nucleoside analogues lamivudine and adefo-
vir. Lasting seroconversion (ie, development of anti-HBe antibody and loss
of HBeAg) may occur in 30% to 40% of patients treated with interferon for
4 to 6 months. Seroconversion is less likely and less stable with nucleoside
analogue treatment, but these agents generally suppress viral replication.
Even after viral mutation, which produces lamivudine resistance, viral rep-
lication can again be suppressed in 85% by adding adefovir [35]. Arguments
for benet and decreased risk of HCC are similar to those for hepatitis C
virus treatment. Suppression of hepatitis B virus presumably results in less
inammation and hepatic regeneration, and decreased risk of integration
of the viral genome into host DNA.
The available data suggest that hepatitis B virus treatment lowers the risk
of HCC. Natural history studies suggest that decreased viral replication is
associated with less risk of HCC. In one study of 11,893 Taiwanese men fol-
lowed for 8 years, the relative risk of HCC was 60.2 for patients with both
HBsAg and HBeAg in serum (indicating high viral replication) compared
with 9.6 for those with only HBsAg [36]. A randomized controlled trial of
interferon treatment in HBeAg-positive patients showed a signicant
decrease in HCC incidence in the treated arm during 1 to 12 years of
follow-up (1.5% versus 12%, P .04) [37]. A follow-up study of 165
HBeAg-positive patients treated with interferon reported a signicantly
lower relative risk of HCC in responders compared with nonresponders
[38]. A meta-analysis, however, failed to show clear benet [29]. Data on nu-
cleoside analogues and their eect on HCC risk are lacking, and placebo-
controlled trials will be dicult to perform because lamivudine has become
part of standard care. One placebo-controlled study was stopped on interim

analysis because lamivudine clearly slowed progression to cirrhosis [39]. Al-
though numbers were small, the hazard ratio for HCC development was
0.49 (95% condence interval: 0.220.90; P .05) for lamivudine treatment.
Because lamivudine or adefovir therapy is less likely to produce seroconver-
sion than interferon, long-term nucleoside analogue treatment may be nec-
essary to lower the risk of HCC.
Chemopreventive compounds and vitamins are being explored. Diets high in
carotenoid compounds may lower the risk of HCC [40,41]. Other compounds
that induce antioxidant enzymes (eg, dithiolethiones and isothiocyanates)
or bind toxins (eg, chlorophyllin binding of aatoxin) are under clinical
investigation [42].
Treatment
Introduction
Treatment for HCC depends on the stage of the tumor and of the chronic
liver disease or cirrhosis. The former dictates the chance of cure or response
to therapy, whereas the latter dictates the tolerability of therapy. Intolerabil-
ity to therapy is more common for HCC than for other cancers because
HCC typically evolves in the setting of cirrhosis, which increases both oper-
ative and chemotherapeutic risks. Moreover, advanced liver failure creates
signicant baseline mortality unrelated to HCC. Despite these problems, ag-
gressive therapy may be successful, particularly for patients with early stage
HCC and well-preserved hepatic function. The appropriate therapy is con-
troversial and can vary from center to center.
Surgical resection
Perioperative mortality for HCC resection used to be prohibitively high
(15%20%), but has decreased to less than 5% today because of improved
patient selection and surgical techniques [43,44]. Surgical resection may be
oered to patients with single-lesion HCC and well-preserved hepatic func-
tion (eg, Childs A cirrhosis). Patients with Childs B or C cirrhosis cannot
tolerate loss of surrounding nontumerous hepatic parenchyma during a local
resection. Even some patients with Childs A cirrhosis (eg, those with signs
of portal hypertension or hyperbilirubinemia) cannot tolerate and are not can-
didates for local resection. Metastatic disease and gross vascular spread into
the main portal vein or inferior vena cava (by the hepatic veins) are also exclu-
sion criteria. Large tumor size is not an exclusion criterion, but large tumors
mandate large resections leaving less functioning parenchyma behind. Liver
volume estimates based on CT or MRI scanning are used to predict whether
the hepatic parenchyma remaining after resection is adequate. In Japan,
hepatic clearance of indocyanine green is used for this purpose. Retention
of more than 14% of indocyanine green at 15 minutes indicates poor tolerabil-

ity of signicant resection [44]. Comorbidity, such as signicant coronary ar-
tery disease and diabetes mellitus, may also militate against resection [45,46].
Improved imaging technology allows for better planning of the extent of
resection. Some centers use intraoperative US to delineate the vascular sup-
ply to the tumor [44]. Intermittently clamping the portal vein and hepatic
artery signicantly decreases intraoperative blood loss [44]. Preoperative
embolization of the portal venous branches supplying the cancerous seg-
ments induces hypertrophy of the remaining lobes [47]. These renements
in selection criteria and surgical technique argue strongly for referral for sur-
gery to tertiary medical centers.
For patients meeting selection criteria, resection oers 5-year survival
rates of 50% to 70%, rates comparable with that provided by liver transplan-
tation, but about 60% to 100% have recurrent HCC at 5 years after resection
[48,49]. Close follow-up for recurrence of HCC with imaging and AFP levels
at least twice annually is necessary. Not all patients develop recurrent HCC,
however, and those who do may still be suitable for liver transplantation,
repeat local resection, or ablative therapy (see later). A Markov model sug-
gested that resection followed by liver transplantation, when necessary, may
be cost-eective [50]. Improvements in technique and patient selection have
made resection a primary therapeutic option for HCC.
Liver transplantation
Before 1995, the transplant community had lost enthusiasm for treating
HCC because of survival rates as low as 40% at 4 years. In 1996, Mazza-
ferro et al [51] published data showing that transplantation could have
long-term survival rates similar to non-HCC patients if the tumors were
few and small. Specically, patients with a single HCC less than or equal
to 5 cm or up to three HCCs each less than 3 cm on pretransplant imaging
had a 4-year survival rate of 75%. These criteria are now used by UNOS
in the United States [52]. These criteria constitute T2 stage in the tumor
nodemetastasis (TNM) staging system, with T1 being one lesion less than
or equal to 1.9 cm (Table 1). Patients with extrahepatic spread or vascular
involvement detected on imaging (eg, portal vein invasion) are excluded
from transplantation. Appropriate patient selection for transplantation is
critical for a successful outcome.
Poor organ availability hinders application of transplantation. Before
2002, prioritization of transplant candidates was based on Child-Pugh score.
Scores were gathered into four large UNOS status groups (status 1, 2A, 2B,
and 3) and time on the waiting list determined the ranking within each
group. Under this system, patients with less than or equal to T2 HCC
were moved into a higher priority group (status 2B), but waiting time within
the group remained a signicant factor. For example, a patient with a newly
diagnosed T2 HCC was listed as status 2B, but all 2B patients with more
Table 1
American Liver Tumor Study Group modied tumornodemetastasis staging classication
Classication Denition
TX, NX, MX Not assessed
T0, N0, M0 Not found
T1 1 nodule %1.9 cm
T2 1 nodule 25 cm; 2 or 3 nodules, all !3 cm
T3 1 nodule O5 cm; 2 or 3 nodules, at least 1 O3 cm
T4a 4 or more nodules, any size
T4b T2, T3, or T4a plus gross intrahepatic portal vein or hepatic vein
involvement as indicated by CT, MRI, or US
N1 Regional (portal hepatis) nodes, involved
M1 Metastatic disease, including extrahepatic portal or hepatic vein
involvement
Stage 1 T1
Stage II T2
Stage III T3
Stage IVA1 T4a
Stage IVA2 T4b
Stage IVB Any N1, any M1
Data from Bruix J, Sherman M, Llovet JM, et al. Clinical management of hepatocellular
carcinoma. Conclusions of the Barcelona-2000 EASL Conference. J Hepatol 2001;35:42130.
accrued waiting time were ranked higher. As waiting times lengthened

through the late 1990s, intention-to-treat survival for HCC patients listed
for transplant plummeted. In Italy, intention-to-treat 2-year survival de-
creased from 84% to 54% after 1996 when mean waiting time increased
from 62 to 162 days [53]. Many patients had tumor progression beyond
T2 while awaiting transplantation and were dropped from the waiting list.
In the United States, the cumulative probability of list drop out by HCC pa-
tients was 25% per year in 2001 [54].
In 2002, the MELD prioritization system replaced the UNOS status
groupings in part to minimize waiting time as a prioritization factor. The
MELD score is based on three laboratory values (international normalized
ratio, bilirubin, and creatinine). It had originally been designed to predict
short-term mortality after transhepatic portal systemic shunt placement,
but was subsequently shown to predict 3-month mortality for patients
awaiting liver transplantation. The MELD equation generates an integral
score from 6 to 40, with higher numbers indicating higher mortality and
ranking. Waiting time is considered only if two patients had identical scores
in their blood group. The MELD equation is complex, but on-line calculat-
ing is available through UNOS [55].
To accommodate the HCC patient, this system awarded 24 or 29 for pa-
tients with T1 or T2 lesions, respectively. Also, extra points are awarded
for every 90 days listed to represent a 10% increase in risk of list drop out.
The frequency of liver transplantation for HCC nearly tripled in the rst
year of the new priority system [56,57]. More than 85% of these HCC patients
waited less than 90 days for transplantation. The average waiting time de-
creased from 2.28 years before the MELD system to 0.69 years under MELD.
The new system favored patients with HCC. Non-HCC patients with
MELD scores of 24 to 29 had a signicantly greater chance of dying or
dropping o the list than HCC patients because they often had more ad-
vanced liver decompensation [57]. Also, the increase in transplantation for
HCC magnied the eect of inaccurate imaging diagnosis on organ alloca-
tion [58]. For example, 14% of transplants performed for HCC had no ev-
idence of HCC on explant histology in the rst 8 months of MELD [59].
This misallocation occurred more often for small, single lesions (ie, T1).
Moreover, retrospective data indicated that patients with T2 lesions were re-
sponsible for much of the poor intention-to-treat outcomes under the old
system. Patients with T1 lesions had less than a 10% risk of drop out in
the rst year of waiting [60]. For these reasons, the assigned MELD scores
were decreased to 20 and 24 for T1 and T2 lesions, respectively, in 2003.
This revision decreased the proportion of transplantations performed for
HCC from 21% to 14% [57]. The rate was only 8% before MELD.
Adult-to-adult, living donor related liver transplantation has increased in
the United States and Europe in response to the organ shortage [61]. Donors
are usually young, healthy individuals with a signicant relationship with
the recipient (eg, family member, friend). The right lobe or 60% of the do-
nor liver is removed and both the donated portion and the 40% remaining
in situ grows to more than 85% of the original volume within weeks to
months. Living donor related liver transplantation lls a vacuum in liver
transplantation. Patients with special circumstances that increase mortality
and morbidity, which are not reected in the MELD score, are well served
by living donor related liver transplantation. Patients with HCC beyond T2
stage can fall into this category. They have a 40% to 50% 5-year survival
after transplantation, which is an acceptable outcome in oncology. Argu-
ably, living donor related liver transplantation should be oered to an ap-
propriate recipient candidate if the donor is willing because a living donor
related liver transplantation does not take organs from the deceased donor
pool. Nevertheless, such cases must be weighed against the 1 in 300 mortal-
ity and the morbidity for young, healthy donors. Also, 5% to 10% of en-
grafted right lobes may initially fail (primary nonfunction). These cases
are relisted for a deceased donor organ and do impact this limited resource.
Currently, there is no consensus on living donor related liver transplantation
use for more advanced HCC. Decisions are made on a center-by-center and
case-by-case basis, but a multicenter National Institutes of Healthfunded
study is currently evaluating the indications, risks, and benets of living
donor related liver transplantation.
Emerging data suggest that hepatitis C virus patients have lower survival
after transplantation than other patients [62]. This might aect transplanta-
tion for HCC because most cases in the United States are associated with
hepatitis C. Hepatitis C infection uniformly recurs in the grafted organ.
Posttransplant hepatitis C has a highly variable course from rapid decline

with liver failure within the rst year to quiescent disease for more than 5
years. The course seems to have recently become worse, with approximately
20% to 30% progressing to cirrhosis within 5 years after transplant, possi-
bly because of the increasing age of deceased donors [63,64]. Older grafted
livers may poorly tolerate hepatitis C virus infection after transplantation.
This trend could adversely aect the cost-eectiveness of transplantation
for hepatitis Crelated HCC. Some data further suggest that retransplanta-
tion for recurrent hepatitis C cirrhosis has poorer outcomes than for other
indications for retransplantation [65].
The rules for organ allocation for patients with HCC continue to evolve
[59]. The following represents the current status of diagnostic criteria and
listing prioritization for patients with HCC [52]. UNOS diagnostic criteria
for HCC are satised by histopathologic ndings, but such tissue diagnosis
is not mandatory. Short of biopsy, the lesion or lesions must be detected by
US, CT, or MRI. In addition, at least one of the following must be present:
a vascular blush of the tumor on CT or MRI, a serum AFP level of greater
than 200 ng/mL, an hepatic arteriogram consistent with HCC, or prior local
ablation of the lesion. UNOS also accepts the diagnosis of HCC without bi-
opsy in a cirrhotic patient with an AFP level greater than or equal to 500
ng/mL, regardless of imaging ndings. If these diagnostic criteria are met,
then T2 cases may be upgraded to a score of 22. T1 cases no longer receive
upgrades. Patients with HCC at stage T3 or more may be listed under their
calculated MELD score without any upgrades. Centers rarely list such pa-
tients for deceased donor transplantation because of a higher risk of post-
transplant recurrence and mortality, but may consider living donor related
liver transplantation for these patients.
Percutaneous ethanol injection

Only 7% to 20% of HCC patients are candidates for curative surgical inter-
vention because of advanced tumor stage or comorbidities precluding surgery.
Local ablative therapies are important therapeutic alternatives in these other
patients. Small HCCs (!23 cm) can be locally ablated by percutaneous, US-
guided needle injection of ethyl alcohol. Alcohol causes dehydration and
coagulative necrosis of neoplastic cells. Ablation can be performed on out-
patients using local anesthesia. Needles are typically 22- or 21-gauge, with
a single end-hole or multiple side-holes. At each session from 1 to 10 mL
of 95% ethyl alcohol is injected. Some centers use general anesthesia when
more than 10 mL is injected at one session. Alcohol creates a hyperechoic
image allowing real time observation of the infused area.
The procedure is well-tolerated with very low complication rates. In a re-
view of 1008 reported cases, no deaths occurred and only 2.4% had minor
complications, most of which were treated conservatively [66]. The major
side eect is pain [67]. A disadvantage is the multiple sessions required; a
study from ve dierent Italian centers reported approximately six sessions

for 2-cm lesions, 10 sessions for 2- to 3.5-cm lesions, and 12 to 15 sessions for
3.5- to 5-cm lesions [68]. Sessions can be done once or twice weekly. More-
over, complete ablation of lesions larger than 2 to 3 cm is dicult. Although
no evidence of tumor on follow-up imaging is reported for lesions less than
3 cm, the rate of local recurrence rises to 7% to 27% when lesions under 4 to
5 cm are included [68,69]. On histologic analysis residual tumor is seen in
about 30% of lesions [70]. This therapy becomes inadvisable for lesions
larger than 5 cm because of the number of required sessions and poor
ecacy. Percutaneous ethanol injection (PEI) is generally recommended
only for lesions less than 2 to 3 cm.
PEI has been compared favorably with local resection for small tumors.
One Japanese cohort study reported similar 1-, 3-, and 5-year survivals for
PEI versus hepatic resection for lesions less than 3 cm (100%, 82%, and
59% for PEI versus 97%, 84%, and 62% for hepatic resection; P .96)
[71]. Disease-free survival gures were better for the hepatic resection group,
but did not reach statistical signicance (63%, 30%, and 10% for PEI versus
76%, 45%, and 26% for hepatic resection; P .10). Another cohort study
from France demonstrated similar survival gures, but showed signicantly
better disease-free survival with hepatic resection over PEI [67]. Prospective,
randomized, controlled data are needed to conrm these ndings.
Radiofrequency ablation
Radiofrequency ablation (RFA) has evolved rapidly in the last several
years because of promising clinical data and improved technology. HCC tu-
mors are destroyed by thermal injury from electromagnetic energy delivered
by RFA probes. The probes are signicantly wider (1417 gauge) than the
skinny 22- to 21-gauge needles used in PEI. The probes are connected to
an alternating current generator, which also has one or two large grounding
pads applied to the patients thighs. The probe is placed into the tumor un-
der US guidance. The current is concentrated in a small area around the
probe; tissue ions close to the probe move in alternating directions with
the alternating current creating frictional heat.
Heating above 50 to 60 C for 4 to 6 minutes causes irreversible damage,
but diusion and maintenance of this heat throughout the tumor takes lon-
ger. A typical session lasts 10 to 30 minutes with target temperatures ranging
from 55 to 100 C. Higher temperatures at the probe are necessary to diuse
sucient heat throughout the tumor. The high temperatures can produce
charring of tissue at the probe, which can hinder heat diusion. Recent
probes have cool tips with dual lumens that allow continuous infusion of sa-
line preventing overheating of tissues nearest the probe. Recent probes also
have several extendable prongs that are deployed circumferentially into the
tumor, arranged in an umbrella or ower pattern (Fig. 2) [72]. Each prong
applies current to signicantly enlarge the area of necrosis. Removal of heat
Fig. 2. Illustration of the StarBurst Xli-Enhanced RFA probe (Courtesy of RITA Medical Sys-
tems, Mountain View, California; with permission. Available at: http://www.ritamedical.com/
products/starburstxli_enh.shtml#. Accessed April 15, 2004.)
by intratumor and surrounding vasculature can create a signicant heat

sink, which limits thermal damage. Embolization of arteries supplying
the tumor, by femoral artery access, has shown promise in extending tumor
necrosis [73] but is not yet widely practiced.
Patient discomfort can be greater than for PEI because of the large probes
and the longer intrahepatic therapy. Conscious sedation and occasionally
general anesthesia are needed. The current and time settings are adjusted
to obtain the desired area of necrosis, including up to 0.5 to 1 cm beyond
the imaged border of the tumor. One capsular puncture of the liver is per-
formed per session. Some centers admit patients overnight for observation
postprocedure, whereas others release patients after several hours of observa-
tion. Complication rates are somewhat higher for RFA than PEI but are still
acceptable. A large multicenter study reported a mortality rate of 0.37% (6 of
1610 patients) and major complication rate of 2.2% [74]. Only 0.5% had
tumor seeding along the probe insertion track. Seeding may be prevented
by heating the track during probe withdrawal. Certain tumors, such as those
at the hepatic surface, have a higher risk of bleeding, peritonitis, or tumor
spread. RFA is applied with trepidation in these cases, if at all.
Advantages of RFA over PEI include fewer sessions and higher rates of
complete tumor necrosis. In a prospective study of tumors less than or equal
to 3 cm, 1.2 sessions per tumor were needed to obtain complete ablation in
90% of tumors using RFA, compared with 4.8 sessions and an 80% com-
plete ablation rate using PEI [75]. A randomized controlled trial from Japan
comparing RFA with PEI for lesions less than or equal to 3 cm showed sig-
nicantly fewer sessions required (2.1 versus 6.4) and fewer hospital days
(10.8 versus 26.1) for RFA, with no signicant dierences in adverse events
[76]. Two-year survival is excellent for both RFA and PEI (98% and 88%,
respectively; P .138), but local recurrence-free survival is better for RFA
(96% versus 62%; P .002) [77].
RFAs have higher complete ablation rates because of more uniform
tumor cell killing including a 0.5- to 1-cm rim of normal hepatocytes
surrounding the tumor. Ethyl alcohol tends to diuse unequally through
septated tumors. No margin of extended killing is obtained with PEI be-
cause ethyl alcohol tends not to diuse into the surrounding cirrhotic liver.
One study of biopsies of 33 tumors less than 3 cm, taken 4 days after RFA,
revealed complete tumor necrosis and normal hepatocyte killing within the
rimming margin for all specimens [78]. The only two cases of residual tumor
occurred in completely excised specimens taken 4 weeks later. The foci of

viable tumor cells were outside the RFA rim of ablation. Like PEI, RFA
has diculty ablating tumors greater than 3 cm, probably because of an in-
ability to maintain high temperatures further from the probe. One study
demonstrated complete necrosis by imaging in only 71% of 3- to 5-cm
lesions and only 25% of lesions greater than 5 cm [79].
RFA has become the most popular ablative therapy because of higher ef-
cacy in fewer sessions compared with PEI. Although no randomized con-
trolled trials have compared RFA with surgical resection, RFA like PEI
may yield comparable benet with less morbidity and mortality.
Transarterial chemoembolization
Transarterial chemoembolization (TACE) has a niche in treating unre-
sectable HCCs. It has higher ecacy with larger tumors (O35 cm).
TACE takes advantage of HCCs 90% to 100% arterial blood supply, as op-
posed to the rest of the liver, which derives most of its blood from the portal
venous system (only 20%25% arterial) [80]. In TACE, the hepatic artery is
accessed by the femoral artery under uoroscopic guidance. Iodinated dye is
administered to guide the catheter and provide a diagnostic angiogram.
Chemotherapeutic agents, including doxorubicin, cisplatin, epirubicin,
and mitomycin, are mixed with lipoidal and delivered transarterially to
the tumor. Some centers use a single agent, whereas others use a combina-
tion. The artery feeding the whole segment or subsegment harboring the
tumor is usually selected. No single agent or combination has shown ecacy
above others. Lipoidol is a poppy seed oil that preferentially stays in HCC
tissue for days to weeks, increasing chemotherapy contact time. Immediately
after delivery, the major feeding artery is embolized with substances, such as
absorbable gelatin sponge or polyvinyl alcohol particles, to produce ische-
mic damage to the tumor, permit better penetration of the chemotherapy
into cells, and to increase dwell time of the chemotherapy by reducing blood
ow. Patients are admitted to the hospital after the procedure for overnight
observation. Fever and a brisk rise in serum aminotransferase levels com-
monly occur the next day. If otherwise stable, patients are usually released
with close follow-up and oral analgesics. The therapy can be repeated 8 to
12 weeks later depending on patient tolerance, angiographic ndings, and
follow-up imaging results.
TACE is generally only oered to patients with Childs A or B cirrhosis
because the therapy injures surrounding hepatic parenchyma and results in
decreased hepatic function. Patients with poor performance status, renal
failure, poorly controlled ascites, or encephalopathy are often excluded.
Usually, a platelet count greater than or equal to 50,000/mL and an inter-
national normalized ratio less than 2 is required. Patients with portal vein
thrombosis are often excluded because of the risk of ischemia to large
hepatic areas. In marginal candidates, superselective arterial cannulation
of just the tumor-feeding artery and chemotherapy delivery without embo-

lization can be performed to limit collateral damage. Even with these
patient selection criteria and precautions, signicant complications, such
as hepatic decompensation and abscess, occur in 2.5% to 15% of cases
[60,81,82].
Like RFA and PEI, TACE has better success with smaller tumors (!4
5 cm). One study demonstrated a 33% local recurrence rate at 4 years for
tumors less than 4 cm [83]. Only 17% of tumors greater than 5 cm undergo
complete necrosis [84]. The periphery of large tumors may have neovascula-
rization with blood supply from other arteries (eg, phrenic, intercostals) or
the portal venous system. These areas are less aected by TACE. Neverthe-
less, TACE can induce tumor necrosis (O80%) and slow growth in most
large tumors [85]. Radiographic response rates can be as high as 80%.
This represents the most practical nonsurgical option for patients with large
HCC.
Until recently, studies showing improved survival after TACE were un-
available. In fact, a randomized controlled trial from France comparing
TACE with symptomatic care failed to show survival benet [86]. This study
was criticized for an unusually high survival in the control group, an unusu-
ally high rate of liver failure in the treated group, and low study power [87].
Lo et al [88] in Hong Kong, however, showed improved 1-, 2-, and 3-year
survival in the TACE group compared with untreated controls (57%,
31%, and 26% versus 32%, 11%, and 3%; P .002). A Spanish study
was truncated because of improved survival benet for the TACE group
on interim analysis (63% 2-year survival with TACE versus 27% 2-year sur-
vival with no treatment) [89]. TACE seems to improve survival based on
these studies and a recent meta-analysis [90].
TACE oers improved survival to patients with unresectable HCC and
mild to moderate hepatic decompensation. The procedure is performed by
interventional radiologists in conjunction with oncologists who prescribe
the chemotherapy. It is invasive, carries a higher risk than PEI or RFA,
and requires hospitalization. It provides an important option for patients
with tumors too large for RFA or PEI.
Systemic chemotherapy
Systemic chemotherapy for HCC has had disappointing results. HCC is
believed to be relatively drug resistant. The multidrug resistant gene
(MDR1) is active in HCC cells and hyperplastic hepatocytes [91]. HCC
patients usually have underlying cirrhosis that decreases tolerability of cyto-
toxic agents. For example, cirrhotic patients often have anemia, leukopenia,
or thrombocytopenia and are immunosuppressed and prone to infections.
HCC patients referred to the oncologist usually have hepatic decompensa-
tion or advanced HCC. Others may have advanced age, poor functional
status, or signicant comorbidities that disqualied them from other
therapies. It is not surprising that chemotherapy clinical trials have shown

disappointing results.
Several chemotherapeutic agents, including cisplatin, etoposide, and epi-
rubicin, have been tried for HCC. Doxorubicin (Adriamycin) is the closest
to a standard chemotherapeutic agent for HCC. As a single agent, it has
shown a modest survival advantage over no treatment [92], with a 25%
overall response rate and median survival of 4 months [93].
Interferon-a has antiviral and antitumor activities, but is not eective
as a single agent. It is often tried in combination with other agents. Of
50 patients with advanced unresectable HCC treated with a combination
of cisplatin, doxorubicin, 5-uorouracil, and interferon-a (abbreviated as
PIAF) 13% had a partial response demonstrated by follow-up imaging
[94]. Nine patients had sucient tumor shrinkage to qualify for surgical
resection. Of these nine, four had only necrotic tissue at the tumor site in
the resected specimen. Toxicities included signicant leukopenia in 34%
and two deaths related to neutropenic sepsis. Growth factors were not
administered, however, to increase the neutrophil counts. Overall median
survival was 8.9 months, but 94% of patients were HBsAg positive with
a median age of 47. Also, median Karnofsky score was 90%. There was
no control arm. Randomized controlled data and data on HCV-related
HCC are needed.
A phase III, multinational trial of thymitaq (nolatrexed dihydrochloride),
which inhibits thymidylate synthase for unresectable HCC, is ongoing. Ca-
pecitabine is an orally administered prodrug of 5-uorouracil. One prelim-
inary study demonstrated a 13% response rate in HCC patients. Many
patients (37%) developed hand-foot syndrome (palmar-plantar erythrody-
sesthesia), but therapy was otherwise well tolerated [95]. Other studied
agents or hormones, such as tamoxifen, octreotide, and thalidomide, have
less ecacy than doxorubicin.
Chemotherapy is theoretically appealing for the many patients who pres-
ent with advanced-stage HCC or have contraindications for other therapy.
Chemotherapy, however, remains disappointing. Indeed, supportive care
without therapy may be reasonable for patients with advanced cirrhosis
or poor functional status. Young patients with good functional status and
early cirrhosis presenting with large, unresectable tumors should be consid-
ered for chemotherapy.
Treatment summary
Stage of tumor, degree of hepatic dysfunction, comorbidities, and func-
tional status dictate which therapeutic options are reasonable. An overview
of options is graphically depicted with TNM tumor stage on the ordinate
axis and Child-Pugh stage on the abscissa (Fig. 3). Usually, curative options
are restricted to those with early stage HCC (T1 or T2). Transplantation is
the best option for T2 lesions. The Barcelona-Clinic Liver Cancer Group
Fig. 3. Recommended HCC treatments plotted against the TNM (tumor, node, metastases) tu-
mor stage and Child-Pugh status. Italicized treatments are potentially curative. PEI, percutane-
ous ethanol injection; RFA, radiofrequency ablation; TACE, transarterial chemoembolization.
provides a detailed therapeutic algorithm (Fig. 4) [96]. Patients are stratied

by Okuda stage, Child-Pugh class, or performance status. Okuda stages 1
through 3 are based on tumor size, serum albumin level, presence of ascites,
and serum bilirubin level, with 3 having more advanced disease [97]. Each
major group is then subclassied by size and number of lesions and by ex-
trahepatic spread of HCC.
Although treatment varies somewhat from center to center, the best
option for early HCC and mild to moderate hepatic dysfunction (eg, no
signicant portal hypertension or hyperbilirubinemia) is controversial.
Strong advocates for hepatic resection, RFA, and transplantation can be
found. Liver transplantation eliminates the high recurrence rate by remov-
ing the entire liver and the MELD system has made it somewhat easier for
HCC patients to be transplanted. Liver transplantation, however, brings
measurable morbidity from life-long immunosuppression. Resection has
an acceptable operative mortality and 3- to 5-year survival rates compara-
ble to liver transplantation. Recurrences may be retreated, even with liver
=
Fig. 4. Algorithm for HCC treatment according to the cancer stage and the level of hepatic
function. M1, metastatic disease; N1, regional (porta hepatis) node involvement; PEI, percuta-
neous ethanol injection; RFA, radiofrequency ablation; TACE, transarterial chemoemboliza-
tion. (Adapted from Llovet JM, Bru C, Bruix J. Prognosis of hepatocellular carcinoma: the
BCLC staging classication. Semin Liver Dis 1999:19;32938; with permission.)
Hepatocellular Carcinoma
Stage 0 Stage A-C Stage D
Good functional status, Child-Pugh A, Good-moderate functional status, Child-Pugh A-B, Poor functional status, Child-Pugh C,
Early stage HCC (Okuda 1) Moderate stage HCC (Okuda 1-2) Advanced stage HCC (Okuda 3)
HBV & HCV: CHRONIC LIVER DISEASE & HCC

Stage 0 Stage A Stage B Stage C Stage D
Single HCC <2cm Single or 3 nodules all <3 cm Multinodular HCC Multinodular HCC, Terminal stage
Portal invasion, N1, M1
Single 3 nodules all <3 cm
Portal invasion, N1, M1?

Portal pressure & Either abnormal Comordities?
bilirubin?
Both normal No Yes No Yes
Resection Transplantation RFA/PEI TACE New agents Supportive

Care
19
transplantation. Conversely, if recurrence presents at greater than stage T2,

then the opportunity for curative transplantation has been lost. RFA has
a lower procedure morbidity and mortality than resection. The choice be-
tween the three options largely depends on the center. It is hoped that ran-
domized clinical trials, particularly for RFA versus resection, will be
forthcoming.
Summary
Much information has been gained in the diagnosis and treatment of
HCC during the last 15 years. Ever improving imaging technology has
made nonhistologic diagnostic criteria possible, albeit controversial. Liver
transplantation, resection, and RFA are considered curative options. Yet,
HCC incidence is steadily rising because of limited progress on disease pre-
vention. Accurate and cost-eective screening is necessary. Presently, only
10% to 15% of HCC patients present with a curative stage of disease.
Because the eld of HCC is rapidly changing, patients with HCC should
be referred to liver centers with a full array of services, from surgical to on-
cologic. The prognosis for HCC patients will surely improve with a multidis-
ciplinary approach to care and further clinical research. Better screening and
prevention of recurrence should eventually improve survival. It is hoped
that antiviral treatment studies will lower the risk of HCC, and that these
changes will occur soon enough to help the many patients at risk for or di-
agnosed with HCC over the next several years.
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20 (2006) 2745
Hepatitis B Vaccines
Andy S. Yu, MDa, Ramsey C. Cheung, MDb,c,
Emmet B. Keee, MDc,*
a
Pacic Gastroenterology, 2101 Forest Avenue, Suite 106, San Jose, CA 95128, USA
b
Department of Hepatology. VA Palo Alto Health Care System, 3801 Miranda Avenue,
Palo Alto, CA 94304, USA
c
Division of Gastroenterology and Hepatology, Stanford University School of Medicine,
750 Welch Road, Suite 210, Palo Alto, CA 94304, USA
More than 350 million people worldwide are infected with the hepatitis B
virus (HBV), and more than 1 million of them die each year of liver failure
or hepatocellular carcinoma (HCC) [1]. The HBV prevalence varies widely
from 0.1% to 20% in dierent geographic regions in the world, depending
on the predominant age of infection and major mode of transmission [2].
There are currently 1.25 million HBV carriers in the United States, contrib-
uting to 17,000 hospitalizations and 5000 deaths annually [3]. As HBV is
transmitted by body uids, infection can be minimized by proper infection
control practices, risk-reduction counseling, virus deactivation of plasma-
derived products, and screening donors of blood, solid organs, and semen
[4]. However the most eective way to prevent transmission of HBV is by
immunization [5].
Indications
The national strategy to eliminate HBV transmission in the United States
focuses on four major categories of subjects, including: (1) neonates, (2) in-
fants, (3) adolescents, and (4) adults who are at increased risks for infection
[6]. Identication of pregnant women who test positive for hepatitis B sur-
face antigen (HBsAg) and timely postexposure prophylaxis of their new-
borns with hepatitis B vaccine can prevent most perinatal transmission
A version of this article originally appeared in the 8:2 issue of Clinics in Liver Disease.
* Corresponding authors. Liver Transplant Program, Stanford University Medical Center,
750 Welch Road, Suite 210, Palo Alto, CA 943041509.
E-mail address: ekeeffe@stanford.edu (E.B. Keeffe).
28 YU et al
[7]. Hepatitis B vaccine is now recommended for all newborns before hospi-
tal discharge, regardless of maternal HBsAg status. Neonates born to
mothers who are chronic HBsAg carriers should receive the rst dose of vac-
cine within 12 hours of birth, accompanied by hepatitis B immune globulin
(HBIG) at a dierent injection site. To further prevent transmission of
HBV, both universal vaccination of infants and catch-up vaccination of
all adolescents are now recommended in the United States (Box 1) [8]. It
is required by 33 states for entry to middle school or seventh grade, three
states for college entry, and by some colleges for matriculation. Juvenile cor-
rectional vaccination programs can also prevent infections among detainees
[9].
Hepatitis B vaccine should be administered to adult populations at high
risk for infection. High-risk ethnic groups include those who live in or em-
igrated from endemic areas, such as Asia and sub-Saharan Africa. Travelers
to endemic regions for a prolonged period, dened arbitrarily as 6 months,
should also be vaccinated. High-risk occupational groups include people
who may be in contact with blood or body uids. Workers in health care
Box 1. Indications for pre-exposure hepatitis B vaccination

Universal
All infants
All children and adolescents not previously vaccinated
On the basis of risk
Illicit injection drug users
Sexual partners of HBV carriers
Men having sex with men
More than one sexual partner in the previous 6 months;
history of sexually transmitted disease (STD); or
treatment in an STD clinic
Household contacts, including cellmates
People in occupational contact with blood or body fluids
Hemodialysis patients
Recipients of clotting factor concentrates
Inmates of long-term correctional facilities
Travel to endemic area > 6 months
Clients and staff of institutions for the developmentally
disabled
Adapted from Weinbaum C, Lyerla R, Margolis HS. Centers for Disease Control
and Prevention. Prevention and control of infections with hepatitis viruses in
correctional settings. Centers for Disease Control and Prevention. MMWR
2003;52(RR-1):136.
HEPATITIS B VACCINES 29
professions or institutions for inmates or mentally retarded people are typ-

ical examples. High-risk patients include those on hemodialysis or those
who are chronic recipients of blood products, such as patients with hemato-
logic disorders or bleeding diasthesis. Individuals with multiple sexual part-
ners should also be vaccinated. Other high-risk categories include illicit
injection drug users and household contacts of hepatitis B patients [9,10].
A number of studies suggest patients with chronic liver disease (eg,
chronic hepatitis C) may have a worse natural history when co-infected
with HBV [11]. Hepatitis B vaccines are safe and immunogenic in patients
with mild-to-moderate chronic liver diseases, although immunogenicity is
poor in patients with advanced cirrhosis [11]. Consensus panels convened
by the National Institutes of Health on the management of hepatitis C is-
sued the recommendation that patients with chronic hepatitis C receive vac-
cination with hepatitis B vaccine, as well as hepatitis A vaccine [12,13].
Although the Advisory Committee on Immunization Practices (ACIP) of
the Centers for Disease Control and Prevention (CDC) recommends immu-
nization of all people with chronic liver disease with hepatitis A vaccine [14],
the committee has not issued a recommendation for hepatitis B vaccine for
such individuals. The failure to recommend hepatitis B vaccination for these
people may have resulted from less convincing data supporting a more se-
vere outcome than is available in the case of superimposed hepatitis A in pa-
tients with chronic liver disease. However, as all patients with chronic liver
disease may be at risk of a more severe course if infected with either virus, it
seems reasonable to vaccinate these patients against both hepatitis A and B
early in the natural history of their liver disease. Despite the lack of a formal
recommendation from the CDC, this is common practice.
Besides newborn infants of HBsAgpositive mothers, other people ex-
posed to HBV infection should receive postexposure prophylaxis (Table 1).
Active immunization with intramuscular hepatitis B vaccine is the manage-
ment of choice for previously unvaccinated subjects and has at least 70%
ecacy in preventing HBV infection [15,16]. The rst of the three vaccine
doses should be administered within a relatively short period of time after
exposure. Concomitant passive immunization with one dose of HBIG, pref-
erably within 24 hours, may provide additional benet despite no rm data
substantiating this approach [17].
Postexposure prophylaxis varies according to the immune status of an ex-
posed patient who was previously vaccinated. Postvaccination testing for
antibody to HBsAg (antiHBs) is usually performed for selected vaccinees
1 to 2 months after the completion of immunization. It is only necessary
for those who are at increased risk of HBV infection as noted previously:
health care workers, hemodialysis patients, household contacts of hepatitis
B carriers, or infants born to HBV carrier mothers. It can also be considered
for those vaccinees likely to have poor antibody response. Testing for anti-
HBs is not recommended for follow-up after routine vaccination [18]. An ex-
posed person who never received postvaccination testing after previous
30 YU et al
Table 1
Postexposure prophylaxis for individuals exposed to hepatitis B virus
Vaccination and antiHBs
status of exposed person Treatment when source is found to be positive
Unvaccinated HBIG 1, and initiate hepatitis B vaccine series
Previously vaccinated
Known responder No treatment
Known nonresponder HBIG 2, or HBIG 2 and initiate vaccine series
AntiHBs response unknown Test exposed person for antiHBs:
If adequate, no treatment is necessary
If inadequate, administer HBIG 1 and vaccine
Booster, recheck antiHBs level in 1 month
Abbreviations: antiHBs, antibody to hepatitis B surface antigen; HBIG, hepatitis B im-
mune globulin.
Adapted from Weinbaum C, Lyerla R, Margolis HS. Centers for Disease Control and
Prevention. Prevention and control of infections with hepatitis viruses in correctional settings.
Recomm Rep MMWR 2003;52(RR-l):l36.
immunization should be tested for the presence of antiHBs. If antiHBs is

not detectable, immunoprophylaxis with HBIG and a second hepatitis B
vaccine series intramuscularly can then be initiated. The strategy to test
for antiHBs only after exposure was found to be more cost eective than
performing postvaccination testing on all high-risk vaccinees [19].
If an exposed individual is known to be a responder by postvaccination
testing, dened as having antiHBs R 10 mIU/mL, additional immunopro-
phylaxis is not required. The immunologic memory of a vaccine responder
for HBsAg appears to persist over a long period of time, independent of
antiHBs titer, after active immunization with hepatitis B vaccine [18]. On
the other hand, if the exposed subject is known previously to be a nonre-
sponder, immunoprophylaxis should be immediately initiated with one
dose of HBIG and a second series of hepatitis B vaccine intramuscularly.
For the exposed individual who was again a nonresponder after the second
course of hepatitis B vaccine, a dose of HBIG should be given, followed by
another dose in a month (Table 1) [8,20].
Prevaccination screening and isolated antibody to hepatitis B core antigen

Prevaccination screening may be cost eective when the expected preva-
lence of prior HBV infection exceeds 30% [10], such as in the high-risk adult
populations for whom hepatitis B vaccine is indicated. Occasionally poten-
tial recipients of hepatitis B vaccine test positive for antibody to hepatitis B
core antigen (antiHBc) but negative for both HBsAg and antiHBs. Iso-
lated antiHBc may be found due to suppression of HBV replication by
hepatitis C virus in co-infected patients [21,22], or in the setting of low level
infection with undetectable HBsAg but a positive HBV DNA by polymerase
chain reaction (PCR). Alternatively, an individual with isolated positive

antiHBc may have cleared HBsAg and had detectable antiHBc and
antiHBs, but subsequently lost antiHBs. Finally, an isolated antiHBc
may be a false-positive result especially in a low-risk population [23,24].
Anyone found to have isolated an antiHBc should be retested. A diag-
nostic algorithm can be used for those who are persistently positive for anti
HBc only [25,26]. To distinguish among the three possibilities mentioned
previously (low-level HBV infection, prior immunity without detectable
antiHBs, or false-positive result), the patient may be given a single dose
of hepatitis B vaccine with follow-up testing with a quantitative antiHBs
in 1 month. Should antiHBs become positive in high titer indicating an an-
amnestic response [27], a convalescent state is proven and no further vaccine
injections are necessary. On the other hand, if antiHBs remains negative
after the single dose of vaccine, HBV DNA should be tested by a PCR tech-
nique. If HBV DNA is detectable indicating a low level of viremia, the pa-
tient has HBV infection and further vaccination is not necessary. On the
other hand, a negative HBV DNA in combination with undetectable anti
HBs would establish that the patient is not infected nor has been previously
exposed, indicating the need to complete the three-shot vaccine series.
Immunogenicity
The HBsAg particle is the immunogen in both plasma-derived and re-
combinant hepatitis B vaccines. This envelope protein composed of several
allelic subtype determinants but only one common group-specic determi-
nant a, which allows cross-protectivity among dierent subtypes
[28,29]. Vaccinated subjects may have transiently detectable HBsAg in the
serum within the rst 24 hours. HBV vaccines stimulate active synthesis
of antiHBs conferring immunity. The rst commercially available hepatitis
B vaccine in the United States, licensed in 1981, was derived from chemically
treated or heat-inactivated sub-viral particles that were obtained from
plasma of chronic HBV carriers. Physician acceptance of these vaccines
was initially impeded by the unfounded concern of the product carrying
other blood-borne infectious agents [5]. Plasma-derived products still ac-
count for more than 80% of all hepatitis B vaccines used worldwide; how-
ever, in the United States the plasma-derived product has been completely
replaced by recombinant vaccines.
Recombinant hepatitis B vaccine consists of nonglycosylated HBsAg par-
ticles that are produced by cloning the S gene via the yeast Saccharomyces
cerevisiae. These newly synthesized HBsAg particles are then extracted
from disrupted yeast cells, physicochemically puried, adsorbed on alumi-
num hydroxide, and preserved with thimerosal [10]. The rst recombinant
vaccine became available in the United States in 1986, and there are cur-
rently two approved products, Recombivax HB (Merck & Co., West Point,
32 YU et al
Pennsylvania) and Engerix-B (Glaxo-SmithKline, Philadelphia, Pennsylva-

nia) (Table 2). In addition, Recombivax HB is available as combination
with hemophilus inuenzae type b (Hib) vaccine (Comvax) for children.
Furthermore, Engerix-B is available as combinations with hepatitis A vac-
cine (Twinrix) for adults, and with polio and diphtheria-tetanus-pertussis
(DTP) vaccines (Pediatrix) for children. Concurrent administration of
HBIG or other vaccines, such as Hib or DTP, does not interfere signicantly
with the antibody response to hepatitis B vaccine [10,30].
Only one among seven clinical series in the literature comparing the im-
munogenicity of Recombivax HB and Engerix-B is a prospective ran-
domized double-blind trial [31]. In this multicenter trial of 460 healthy
subjects between 39 and 70 years of age, either Recombivax HB 10 mcg or
Engerix-B 20 mcg was administered as three standard intramuscular doses
over a period of 6 months [32]. The geometric mean titer (GMT) of anti
HBs 2 months after the last vaccine dose was signicantly higher in the En-
gerix-B group (840 mIU/mL versus 340 mIU/mL). The seroprotection rate,
dened as antiHBs titer R 10 mIU/mL, was higher in the Engerix-B group
but did not reach statistical signicance (91% versus 85%). In a separate pro-
spective randomized but single-blind study, the superior immunogenicity of
Engerix-B was only demonstrated in subjects who were aged 40 or older
(87% versus 81%) [33].
The immunogenicity of other HBV antigens, including HBcAg, remains
ill dened. Addition of the pre-S components neither enhances the immuno-
gencity of HBV vaccines nor circumvents any immunologic unresponsive-
ness to the S protein [34]. In a single-blind multicenter trial where
volunteers were assigned to receive hepatitis B vaccines containing S protein
and pre-S2 of various doses, the titers of antiHBs that were achieved cor-
related with the dosage of S protein, and not of pre-S2. Furthermore, the
titers of antiHBs achieved in each vaccine group were higher in those
Table 2
Recommended dosages of licensed hepatitis B vaccines in the United States
Recombivax HB Engerix-B Twinrix
Age group (years) mcg mL mcg mL mcg mL
!19 5 0.5 10 0.5 d d
1115 10 1.0 d d d d
O20 10 1.0 20 1.0 20 1.0
Dialysis patients and other 40 1.0a 40 2.0b d d
immunocompromised hosts
a
Special formulation.
b
Two 1.0 mL doses administered at one site, in a 4-dose schedule at 0, 1, 2, and 6 months.
Adapted from Weinbaum C, Lyerla R, Margolis HS. Centers for Disease Control and Pre-
vention. Prevention and control of infections with hepatitis viruses in correctional settings. Cen-
ters for Disease Control and Prevention. MMWR 2003;52(RR-l):l36.
subjects who were younger (aged 20 to 39 years versus 40 to 59 years), sup-

porting the importance of age in achieving durable immunity [35].
Routes of vaccine administration

Hepatitis B vaccine is traditionally administered intramuscularly, using
a needle of 1.0 to 1.5 inches in length and 20- to 25-gauge in caliber. The
preferred injection site is the deltoid muscle in adults and anterolateral thigh
muscle in infants. Among 194 health care workers who received intramuscu-
lar buttock injections of hepatitis B vaccination, only 58% subsequently de-
veloped detectable antiHBs titers [36]. This nding was veried by
a prospective randomized trial where health care workers who received im-
munization in the arm achieved higher seroconversion rate of 93% and
GMT of 1454 mIU/mL, as compared with those who were injected in the
buttock. Among those who received buttock injection, the seroconversion
rate (83% versus 72%) and the GMT (387 mIU/mL versus 85 mIU/mL)
were higher with the use of 2-inch needles versus 1-inch needles [37]. Buttock
injection makes intramuscular delivery of the vaccine dicult and should be
avoided.
Intradermal injection of vaccine leads to very ecient antigen processing.
It requires only 10% of the dose used conventionally for intramuscular in-
jection and thus might oer a cost-eective advantage. However, data re-
garding the immunogenicity of intradermal vaccination, compared with
the intramuscular route, remain controversial, and the long-term immuno-
gencity of intradermal vaccine remains to be established [31]. Among 425
health care workers randomized to receive plasma-derived hepatitis B vac-
cines at doses of 2 mcg intradermally or 20 mcg intramuscularly, postvacci-
nation GMT was signicantly lower in the intradermal group [38]. In
a group of hospital employees who were retested 2 years after a documented
response to primary HBV vaccination, more subjects who had received in-
tramuscular inoculations as compared with those who received it by the in-
tradermal route maintained durable response (89% versus 64%), with GMT
of 66.4 mIU/mL and 20.7 mIU/mL, respectively [39]. In a study using an ac-
celerated scheme of hepatitis B vaccine over a 6-week period for post-
exposure prophylaxis, antiHBs seroconversion was achieved in signicantly
more patients who underwent the intramuscular than intradermal route
(77% versus 45%). The investigators in this latter report discouraged
the use of an accelerated low-dose intradermal schedule when rapid pro-
tection against HBV was desired [40].
In contrast, other clinical studies of intradermal hepatitis B vaccine
reported some encouraging results in subjects who were nonimmunized as
well as previous nonresponders to intramuscular immunization [41]. A trial
of 50 health care workers who were randomized to receive either intramus-
cular or intradermal vaccine series yielded comparable seroconversion rates
34 YU et al
of 100% and 96%, respectively [42]. In another study of 1400 health care
workers, protective antiHBs titer was achieved in only 68% of subjects af-
ter the third intradermal dose of hepatitis B vaccine, but an additional dose
of 2 mcg by the same route increased those with adequate protective titer to
89% [43]. Four doses of 2-mcg intradermal vaccines would still cost signif-
icantly less than three doses of 20-mcg intramuscular shots. Intradermal
vaccine was also explored for use as a booster injection in individuals whose
antiHBs decreased to below 10 mIU/mL 3 years after the primary vaccina-
tion series. It was equally eective as an intramuscular booster injection in
inducing an antibody response but was associated with more frequent local
reactions, such as pain and discoloration at the injection site (42% versus
17%) [44].
Predictors of nonresponse
An antiHBs level of R 10 mIU/mL is regarded as a protective serum
titer. Patients with certain clinical characteristics may have a lesser chance
to sero-convert than others. In a retrospective multicenter cohort study of
nearly 600 health care workers who underwent postvaccination testing for
antiHBs within 6 months after completion of vaccine series, ve indepen-
dent variables were identied by multivariate analysis as adverse prognostic
factors for sero-conversion [45], These predictors included increasing age,
male gender, obesity as reected by body mass index, cigarette smoking,
and use of Recombivax HB rather than Engerix-B. When subjects were
stratied by vaccine brands, the adverse predictors included only age,
body mass index, and smoking for the recipients of Recombivax HB. On
the other hand, male gender remained the only negative predictor for the re-
cipients of Engerix-B.
Other studies have conrmed the predictive roles of increasing age, male
gender, smoking status, and obesity that may be alternatively represented by
higher weight-height index [33,36,4649]. Medical conditions that compro-
mise the immune system may negatively aect the response to hepatitis B
vaccine. In a cohort of homosexual males, low antibody response of anti
HBs !10 mIU/mL or nonresponse to hepatitis B vaccine occurred in seven
of 16 patients infected with HIV compared with six of 68 HIVnegative vac-
cinees (P 0.002). Furthermore, the median antiHBs titers after immuni-
zation in the HIVnegative and HIVpositive responders were 205 and 15
sample ratio units, respectively [50]. Other risk factors for nonresponse in-
clude other conditions that may compromise the immune system, such as
chronic cardiopulmonary disorders, renal failure, hemodialysis, and prior
organ transplantation (Box 2) [51].
Nonresponse to hepatitis B vaccine may also be modulated by genetic
factors. When a vaccine series of three doses were repeated in a study
[48], all of the eight initial hyporesponders and eight of the 20 initial
Box 2. Risk factors associated with poor anti-HBs response to

hepatitis B vaccine
Increasing age
Male gender
Obesity
Tobacco smoking
Immunocompromised from chronic disease
nonresponders mounted an appreciable antiHBs titer. When nine of the 12

refractory nonresponders were sampled, six of them carried at least one of
two extended major histocompatibility complex (MHC) HLA haplotypes
B44-DR7-FC31 or B8-DR3-SC01. In contrast, only two of the 11 revaccin-
ees sampled who responded to the second vaccine series carried these hap-
lotypes, suggesting a genetic contribution to immuno-genicity [52]. In
a large series of nearly 600 subjects who received a full course of hepatitis
B vaccine, an analysis of HLA haplotypes among the 20 vaccinees with
the lowest antiHBs titer responses indicated a disproportionately higher
number of HLA-B8-DR3-SC01 [53]. When nine of the haplotype carriers
were given three additional doses of vaccine, four of the ve homozygotes
but none of the nine heterozygotes remained hyporesponders. These nd-
ings implicate a recessive MHClinked trait as a cause of hyporesponse to
hepatitis B vaccination [53].
Ecacy of hepatitis B vaccines

Seroprotective rate up to 95% or above has been demonstrated in multi-
ple placebo-controlled, randomized double-blind trials involving health care
workers [29] and homosexual males [5456]. This is dened as percentage of
vacinees achieving an anti-HBs titer O 10 mIU/mL. Infection rates in re-
ported studies were !4% among the vaccinees, in contrast with rates of
10% to 27% in the placebo arms. A signicant reduction of hepatitis B cases
within 75 days after randomization of subjects suggested the eectiveness of
vaccine even when given shortly after exposure to the virus [55].
Patients who develop seroprotective titers will continue to maintain high
levels of protection from clinical disease for at least a decade. In a large co-
hort of Yupik Eskimos who had a 94% rate of antiHBs seroconversion,
76% maintained titers R 10 mIU/mL after 10 years of follow-up. None be-
came chronically infected despite development of antiHBc in 10 vaccine re-
sponders, implicating subclinical exposure to HBV among vaccinees with
durable protection against the virus [57,58]. Long-term follow-up in similar
series have noted boosts in antiHBs titers despite no interim booster
36 YU et al
vaccine injection, probably attributable to immunologic protection against

viral exposure [59]. The maximal antiHBs response postvaccination is
strongly predictive of titer persistence and inversely correlated with the
risk of viral infection, as demonstrated by a longitudinal follow-up of nearly
800 homosexual males for 5 years after vaccination [60].
A two-dose vaccine regimen over a 6-month period may suce in pro-
ducing a comparable long-term protection against HBV in immunocompe-
tent hosts. In a group of young healthy adults who received Recombivax HB
doses of 10 mcg or 20 mcg, antiHBs seroconversion rates of 46% to 67%
were observed after the rst dose, but increased to 97% to 99% after the sec-
ond dose with booster responses in GMT. Furthermore, a sizable propor-
tion (75% to 89%) of vaccinees maintained titers R 10 mIU/mL 2 years
after the regimen was given, with 79% to 87% demonstrating rapid and vig-
orous anamnestic response after another booster dose [61]. Nevertheless, if
immediate short-term immunologic protection is also desired, a three-dose
regimen remains the preferred choice for individuals who are at high risk
for infection during the initial 6-month period.
A few investigators have reported that the interval between the second
and third vaccine doses is important in determining the ultimate antiHBs
titer. The CDC observed a signicant rise in antiHBs titers in those who re-
ceived the third vaccine dose late (O7 months after the rst dose) among the
1000 Yucpa Indian vaccinees [46]. Similar conclusion was drawn by an inter-
esting study comparing the ecacies of three vaccine regimens, given at 0, 1, 2,
12 months; 0, 1, 6 months; and 0, 1, 12 months [62]. Booster antiHBs re-
sponses were seen after the third dose for each of the last two regimens but
were not observed until after the fourth dose in the rst regimen. Furthermore,
the ultimate titers achieved by the rst and third regimens were comparable
but 20 fold higher than that achieved by the second regimen with the third
and last dose administered at 6 months. Hence, the antiHBs response after
a typical three-dose hepatitis B vaccine series depends on the time interval be-
tween the last two doses [62].
AntiHBs seroconversion is appreciably lower with vaccination of
immuno-compromised patients, such as those on hemodialysis, despite
early reports of excellent results [63]. Suboptimal results with intramuscular
double-dosing regimen prompt continued search for better solution. In a ran-
domized double-blind placebo-controlled trial of double-dosed hepatitis B
vaccine, only 63% of hemodialysis patients developed adequate antibody re-
sponse [64]. This gure, however, decreased to 50% after correction for pos-
sible transfer of antibodies by blood transfusion. When compared with the
placebo arm, there was no signicant reduction in HBV infection within
the rst two years among the vaccinees (6.4% versus 5.4%) [64]. Similarly,
the seroconversion rate with immunizing cirrhotic patients awaiting liver
transplantation was dismal. Vaccinating these patients with standard doses
produced seroconversion rate of 28%, compared with 97% achieved by
healthy controls [65]. Even when a double dose was given to cirrhotic patients,
the seroconversion rate was 44% and increased to only 62% with a repeat
series [66].
Perinatal immunoprophylaxis with hepatitis B vaccine is now routinely
given to newborn infants, with an additional early dose of HBIG for those
who are born to HBVinfected mothers. The use of HBIG alone does not
provide long-term protection against the infection [16]. The vaccine series
should be initiated within the rst 12 hours after birth for neonates born
to HBsAgpositive mothers but may be administered by a relatively exible
schedule to those born to HBsAgnegative women [67]. With immunization,
more than 95% of babies develop antiHBs titer R 10 mIU/mL [68,69].
Vaccinated infants of HBsAgpositive mothers should then be tested for
HBV status at 12 months of age. Without immunization, O90% would
be chronically infected due to vertical transmission of HBV [7]. As for adult
vaccinees, hepatitis B vaccine aords long-term protection for most infants
when initiated soon after birth [16,70], Even if children lose detectable anti
HBs later on in life, which occurs in up to half of individuals, a booster vac-
cine injection will almost always lead to a robust anam-nestic response and
resurgence of antiHBs [7173].
A delay in the initial dose of vaccine increases the risk to the child
for HBV infection [74], In addition, infants may fail immunoprophylaxis
due to in utero infection, genetically determined nonresponsiveness, or
vaccine-breakthough mutations in mother or in infants [5]. In utero infec-
tion of the fetus is relatively uncommon [75] but may result from placental
leakage [76,77]. Vaccine-breakthrough mutations are attributable to immune
pressure from either hepatitis B vaccine or HBIG [78] and may involve point
substitution from glycine to argi-nine at codon 145 (G145R), asparagine to
threonine at codon 126 (N126T), or asparagine to isoleucine substitution at
codon 126 (N1261) in the S gene [79], plus many others that were discovered
subsequently [80]. In general, the S gene mutation causes decreased binding
of antiHBs to determinant a, thus allowing the mutant virus to escape
neutralization [2].
The global impact of hepatitis B vaccination, including a decreased prev-
alence of chronic infection and associated complications, is clearly demon-
strated in recent studies. In July 1984, Taiwan became the rst country to
launch a universal newborn hepatitis B vaccination program. Over the sub-
sequent 15 years, a serologic survey of the prevalence of HBsAg among
youngsters of ages 15 years or less demonstrated a signicant reduction
from 9.8% to 0.7%. In addition, seropositivity for antiHBc, which reects
HBV infection and not vaccination, dropped from 20.6% to 2.9% [81]. A
retrospective review in 1997 showed a signicant decline in the average an-
nual incidence of HCC per 100,000 children of ages 6 to 14 years, with 0.70
between 1981 and 1986, 0.57 between 1986 and 1990, and 0.36 between 1990
and 1994 [82]. Furthermore, the incidence of HCC in children of ages 6 to 9
declined per 100,000 from 0.52 for those who were born between 1974 and
1984 to 0.13 for those who were born between 1984 and 1986 [82]. On the
38 YU et al
other hand, the prevalence of S gene mutations among HBsAgpositive chil-

dren has been increasing, from 7.8% in 1984 immediately before implemen-
tation of the universal vaccination program, to 19.6% in 1989 and 28.1% in
1994 [83]. It was not until 1991 in the United States that the ACIP recom-
mended universal hepatitis B vaccination of all infants [6]. Shortly afterward
the American Academy of Pediatrics and the American Academy of Family
Physicians published similar recommendations. Nevertheless, in 1999 the
proportion of children !3 years of age who were immunized against
HBV had not yet reached the target of 90% as originally proposed by the
Childhood Immunization Initiative [84].
Vaccine nonresponse and its management

Among individuals who do not respond to the initial series of hepatitis B
vaccine, half may convert after an additional one to three doses [60,85].
Hypo-responders are more likely to seroconvert than nonresponders [86].
The use of investigational mix-particle vaccines containing pre-Sl, pre-S2,
and S subunits does not enhance the seroconversion rate achieved by a stan-
dard S-unit vaccine of equivalent dosage [87]. Likewise, doubling adminis-
tered doses for revaccination does not confer any immunologic advantage
[88]. Nonresponders to a second series of vaccination are unlikely to develop
adequate antiHBs titers with further vaccine doses [5]. Another potential
alternative is the use of vaccine adjuvants to enhance the immunogenicity
of existing vaccines [89,90]. Clinical algorithms to reimmunize nonresponders
have been described [86].
The use of granulocyte-macrophage colony stimulating factor (GM-CSF)
as vaccine adjuvant operates by enhancing memory cell generation via both
T and B cell activation; activating macrophages and other eector cells; in-
creasing major histocompatibility complex (MHC) class II antigen expres-
sion on the surface of macrophages; promoting dendritic cell maturation
and migration, upregulating co-stimulatory molecules on dendritic cells; ac-
celerating the maturation of hematopoietic progenitor cells in bone marrow;
and inducing localized inammation at the site of injection [91,92]. Sixty
healthy nonresponders to hepatitis B vaccine were randomized to receive in-
tramuscularly high-dose Recombivax HB 40 mcg three doses alone versus
three injections of GM-CSF with each followed immediately in the same site
by standard Recombivax HB at 10 mcg. Each arm produced an equivalent
seroprotection rate of approximately 50% [93].
The nonresponse of renal failure patients to hepatitis B vaccine is due to
immunosuppression attributable to uremia, inadequate dialysis, use of low
biocompatibility dialysis material, hyperparathyroidism, anemia, iron over-
load, and malnutrition [94]. Ecient hemodialysis may signicantly improve
the response to vaccination [95]. In a pilot study of vaccine-na ve hemodial-
ysis patients randomized to receive GM-CSF followed 24 hours later by one
standard vaccine dose versus three monthly double doses of vaccine alone,
superior seroprotection rate was achieved in the GM-CSF group (87.5%
versus 25%) [96]. Impressive results of GM-CSF were noted in a separate
trial in which it was used as a high-dose 150-mcg adjuvant before a four-
dose hepatitis B vaccine series, resulting in a seroprotective rate of 100%
at 7 months [97]. When used in hemodialysis patients who were nonre-
sponders to previous vaccine series, GM-CSF as an adjuvant had variable
results, signicantly increasing the seroconversion rate and antiHBs titers
in some trials [98,99], but not in others [100]. Dose-dependent adverse events
were documented with GM-CSF, including fatigue, nausea, rigors, and hy-
potension [101]. It is possible that other adjuvants, such as MF59 that has
been used in inuenza vaccines, may also increase the immunogenicity of
hepatitis B vaccines.
Adverse eects of hepatitis B vaccines

Both plasma and recombinant vaccines are equally well tolerated
[5,10,102]. Transient tenderness or pain may occur in up to a fth of the vac-
cinees. Low-grade fever is found in ! 5% of the cases. Other much less
common adverse events, including fatigue, headache, malaise, nausea, dizzi-
ness, skin rash, arthralgia, myalgia, and respiratory distress, occur in ! 1%
of cases [5,103]. Anaphy-laxis may rarely occur, and epinephrine should be
available for immediate use [10]. Among 850,000 vaccinees monitored by the
CDC and Food and Drug Administration, 41 neurologic adverse events
were reported, including Bells palsy in 10, Guillain-Barre syndrome in
nine, convulsions in ve, lumbar radiculopathy in ve, optic neuritis in
ve, transverse myelitis in four, and brachial plexus neuropathy in three.
However, no conclusive epidemiologic association could be established be-
tween any neurologic events and the vaccine [104,105]. As animal reproduc-
tion studies have not been conducted with the vaccine, whether it can cause
fetal harm when administered to a pregnant woman is unknown.
Immunotherapy using hepatitis B vaccine

Hepatitis B vaccine may serve as an immunomodulator for suppressing
viral replication. In a study of the therapeutic ecacy of hepatitis B vaccine,
118 treatment-na ve patients with histologically and virologically proven
chronic hepatitis B were randomized to receive either placebo or intramus-
cular hepatitis B vaccine in the form of S vaccine alone or combined pre-S2
and S vaccine [106]. After ve vaccine injections were given, there was a sig-
nicant but transient 6-month decrease in viral load that did not occur in
controls, despite persistence of HBsAg in all patients. For the rst time, hep-
atitis B vaccine was shown to decrease viral replication, which may pave the
road for new therapies based on the concept of specic immunomodulation.
40 YU et al
Summary
Immunization is the most eective way to prevent transmission of HBV
and, hence, the development of acute or chronic hepatitis B. The national
strategy to eliminate transmission of the virus in the United States includes
vaccination of all newborn infants, children, adolescents, and high-risk
adults. Postexposure prophylaxis is also advocated, depending on the vacci-
nation and antiHBs status of the exposed person. Seroprotection after vac-
cination, dened as antiHBs R 10 mIU/mL, is achieved in over 95% of all
vaccinees. The hepatitis B vaccines are very well tolerated with usually min-
imal adverse eects. Predictors of non-response include increasing age, male
gender, obesity, tobacco smoking, and immunocompromising chronic dis-
ease. For those who remain nonresponders after the second series of vacci-
nation, adjuvants such as GM-CSF may be considered, but their results are
variable.
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20 (2006) 4761
Serologic and Molecular Diagnosis

of Hepatitis B Virus
Julie C. Servoss, MDa,b,
Lawrence S. Friedman, MDa,c,d,*
a
Department of Medicine, Harvard Medical School, Boston, MA 02114, USA
b
Gastrointestinal Unit, Blake 4, Massachusetts General Hospital, 55 Fruit Street, Boston,
MA 02114, USA
c
Department of Medicine, Newton-Wellesley Hospital, 2014 Washington Street, Newton,
MA 02462, USA
d
Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
Hepatitis B virus (HBV) is a hepadnavirus with a 3200-base-pair genome

that consists of partially double-stranded DNA (dsDNA) and a lipoprotein
outer envelope (Fig. 1). The HBV genome has four overlapping open read-
ing frames with four major genes designated pre-S/S, C, P, and X. The pre-S
genes (S1 and S2) code for the hepatocyte receptorbinding site, whereas the
S (surface) gene codes for hepatitis B surface antigen (HBsAg). The C (core)
gene codes for hepatitis B core antigen (HBcAg) and hepatitis B e antigen
(HBeAg), whereas the P (polymerase) gene encodes the HBV DNA poly-
merase. The X gene encodes a protein that transactivates transcriptional
promoters. Eight genotypes (AH) of HBV have been identied based on
nucleotide sequence divergences of at least 8%. HBV genotypes dier in
their predominant geographic occurrence and response to therapy with in-
terferon. Genotype A is the predominant genotype in the United States
and is more responsive to interferon than genotype D which predominates
in the Middle East and South Asia [1].
After a person is exposed to the virus, the serologic course of HBV infec-
tion begins approximately 6 to 10 weeks after exposure with the appearance
of HBsAg and HBeAg, a marker of active HBV replication. (HBV DNA
may be detectable in serum up to 21 days before the appearance of HBsAg
* Corresponding author. Department of Medicine, Newton-Wellesley Hospital, 2014
Washington Street, Newton, MA 02462.
E-mail address: lfriedman@partners.org (L.S. Friedman).
48 SERVOSS & FRIEDMAN
Fig. 1. Genomic organization of HBV Partially double-stranded DNA (complete minus strand
and partial plus strand); the major viral mRNA coded by these regions (wavy lines on the outer
circle); and resultant proteins (S, P, C, and X) from the four open reading frames (ORF). The
lled circle at the 59 end of the minus strand DNA represents the terminal protein; the wavy line
at the 59 end of the plus strand denotes the terminal RNA. DR1 and DR2 are the direct repeats,
which are important for the initiation of viral DNA synthesis. (From Ganem D. Hepadnaviri-
dae: the viruses and their replication. In: Fields BN, Knipe DM, Hawley PM, editors. Funda-
mental virology. 3rd edition. Philadelphia: Lippincott-Raven; 1996. p. 1199234; with
permission.)
[Fig. 2]). Patients then exhibit increased serum aminotransferase levels, usu-
ally R 500 U/L, with the serum alanine aminotransferase (ALT) typically
higher than the aspartate aminotransferase (AST) level. Approximately 10
weeks after exposure to HBV, patients may develop nonspecic symptoms
such as fatigue and malaise as well as right upper quadrant pain and jaun-
dice. At this time, antibody to hepatitis B core antigen (anti-HBc) of the
IgM class appears in the serum. In the recovery phase of HBV infection, se-
rum aminotransferase levels return to normal, HBeAg disappears and anti-
body to HBeAg (anti-HBe) appears in serum, and ultimately, HBsAg
seroconversion to antibody to HBsAg (anti-HBs) occurs. IgM anti-HBc
Fig. 2. Sequence of events after HBV infection. (A) Acute HBV infection with resolution. (B) Acute HBV infection progressing to chronic HBV infection. (From
Hoofnagle JH, DiBisceglie AM. Serologic diagnosis of acute and chronic viral hepatitis. Semin Liver Dis 1991;11:7383; with permission.)
levels begin to decline as levels of anti-HBc of the IgG class rise in serum.
Recovery from acute HBV infection is typically associated with undetectable
serum levels of HBV DNA. However, using polymerase chain reaction
(PCR) techniques (see later discussion), low levels (101 to 102 genome equiv-
alents/mL) of HBV DNA have been found in serum and peripheral blood
mononuclear cells of patients up to 21 years after clinical and serologic re-
covery from HBV infection [2].
The hallmark of progression to chronic HBV infection is the presence of
HBsAg for more than 6 months. Typically, in the early, or replicative, phase
of chronic HBV infection, markers of active viral replication, HBeAg and
serum HBV DNA levels O 105 copies/mL, are present. Patients may ulti-
mately enter a nonreplicative state characterized by seroconversion from
HBeAg to anti-HBe, serum HBV DNA levels ! 105 copies/mL, and nor-
malization of serum aminotransferase levels. This phase is also referred to
as the inactive carrier state. It is important to note that up to 20% of pa-
tients in the inactive carrier state may experience a reactivation to the rep-
licative state, or are, and may even cycle between the nonreplicative and
replicative state [3]. Such ares are characterized by an increase in serum
aminotransferase levels and serum HBV DNA levels to O 105 copies/mL,
with or without seroreversion to HBeAg. Reappearance of HBeAg may
or may not occur during reactivation (see later discussion).
Several clinically important mutations in the HBV genome have been
described. A subset of patients with chronic HBV infection has HBeAg-
negative chronic hepatitis B characterized by circulating HBV DNA, uctu-
ating serum aminotransferase levels, and, in some cases, severe hepatic
necroinammatory activity and even liver failure. This occurs as a result of
mutations in the precore or core region of HBV DNA. The most common
precore mutation is a single amino acid substitution of adenosine (A) for
guanine (G) at nucleotide position 1896 (G1896A) that results in a premature
stop codon that inhibits the production of HBeAg [4]. The most common
core mutations include single amino acid substitutions of threonine (T)
for adenosine (A) at position 1762 (A1762T) and adenosine (A) for guanine
(G) at position 1764 (G1764A) that result in decreased translation of HBeAg.
These core promoter variants have been associated with 10% of cases of ful-
minant HBV infection and 27% of cases of progressive chronic hepatitis B
(see also chapter in this issue by Drs. Wai and Fontana [5].
Other important mutations occur in the YMDD (tyrosine-methionine-
aspar-tate-aspartate) motif of the DNA polymerase gene and include sub-
stitutions of valine (V) for methionine (M) (M204V), isoleucine (I) for
methionine (M204I), or methionine for leucine (L) (L180M). These mutations
occur during treatment of chronic hepatitis B infection with lamivudine,
a nucleoside analogue, and result in the formation of bulky side chains
that inhibit the binding of lamivudine. The emergence of these lamivu-
dine-resistant mutants may be accompanied by HBeAg to anti-HBe sero-
conversion, a are of serum aminotransferase levels, or hepatic
SEROLOGIC AND MOLECULAR DIAGNOSIS OF HBV 51
decompensation. Mutations that lead to resistance occur less frequently dur-

ing the course of therapy with other nucleoside or nucleotide analogs, such
as adefovir, entecavir, and tenofovir [6].
Serologic diagnosis of hepatitis B virus

Serologic diagnosis of HBV can be accomplished by identifying virally
encoded antigens and their corresponding antibodies: HBsAg, anti-HBs,
HBeAg, anti-HBe, and anti-HBc. (HBcAg does not circulate freely in the
serum [Table 1].)
Acute hepatitis B virus infection

HBsAg, a product of the S gene, is part of the surface envelope of HBV
and also circulates in excess (w1013 particles/mL) in the serum as non-
virion associated spheres and tubules [7]. HBsAg is the rst marker of acute
HBV infection and appears as early as 1 week after initial exposure to HBV
and before the onset of symptoms or serum aminotransferase elevations (see
Fig. 2). Typically, HBsAg becomes detectable by 6 to 10 weeks after expo-
sure to the virus. In acute, resolving HBV infection, serum HBsAg levels
begin to fall 4 to 6 months after exposure, as anti-HBs levels increase.
For most patients, the presence of anti-HBs heralds resolving HBV infection
and subsequent lifelong immunity. HBsAg and anti-HBs can be detected in
Table 1
Interpretation of serologic and molecular markers of hepatitis B virus during dierent stages of
infection
Stage of HBV infection HBsAg Anti-HBs HBeAg Anti-HBe Anti-HBc HBV DNA
Acute HBV infection
Early IgM
Window period IgM /
Recovery IgG /a
Chronic HBV infection
Replicative IgG, IgMb O105 copies/mL
Nonreplicative/inactive IgG !105 copies/mL
carrier state
Reactivation HBV / IgM O105 copies/mL
HBeAg(-) chronic IgG O105 copies/mL
HBV (precore or
core mutant)
Abbreviations: anti-HBc, antibody to hepatitis B core antigen; anti-HBe, antibody to hepa-
titis B e antigen; anti-HBs, antibody to hepatitis B surface antigen; HBeAg, hepatitis B e anti-
gen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; IgG, IgG class of antibody;
IgM, IgM class of antibody.
a
Low levels (101102 genome equivalents/mL) may be detected in serum up to 21 years after
recovery from acute HBV infection.
b
Low levels may also be detected.
the serum using enzyme immunoassays (EIAs). The positive predictive value
of the assay for predicting an anti-HBs titer R 10 mIU/mL, which is asso-
ciated with immunity, is 97.6% [8].
When less sensitive assays for HBsAg and anti-HBs were used in the past,
patients with acute HBV infection were often noted to have a window pe-
riod during which neither HBsAg nor anti-HBs was detectable in the serum
(see Fig. 2); with contemporary assays, this window period is rarely, if ever,
observed [9]. During the window period, the presence of IgM anti-HBc was
used to diagnose acute HBV infection. IgM anti-HBc is still a reliable
marker of acute hepatitis B but may also be detected during ares of chronic
hepatitis B (see later discussion). HBcAg, a component of the nucleocapsid
protein, is associated with the intact virion and does not circulate freely in
the serum; therefore, HBcAg cannot be detected by standard assays. During
acute or recent HBV infection, IgM anti-HBc appears shortly after HBsAg
and persists for 6 to 24 months after exposure to HBV. During the course of
acute HBV infection, IgG anti-HBc appears and eventually replaces IgM
anti-HBc. The presence of IgG anti-HBc signies either resolved HBV infec-
tion (when detected with anti-HBs after clearance of HBsAg) or chronic
HBV infection (when detected in patients with persistent HBsAg). There
are commercially available EIAs for total anti-HBc and IgM anti-HBc.
The presence of IgG anti-HBc is inferred when total anti-HBc is present
but levels of IgM anti-HBc are undetectable.
Occasionally, people are found to have an isolated anti-HBc in the ab-
sence of HBsAg or anti-HBs. For example, up to 5% of healthy blood do-
nors have isolated anti-HBc in the serum. Among human immunodeciency
virus (HIV)infected people, the rate of isolated anti-HBc in serum is as high
as 42% [10]. Isolated anti-HBc can occur in four settings: (1) during the win-
dow period of acute HBV infection (see earlier discussion); (2) during
chronic HBV infection as levels of HBsAg become undetectable; (3) after re-
solved HBV infection in the remote past as anti-HBs levels fall below the
limits of detection; and (4) as a false-positive result. Up to 20% of people
with isolated anti-HBc in serum have circulating HBV DNA, signifying
chronic HBV infection [1113]. In the other 80% of cases, the isolated
anti-HBc usually represents a false-positive result or HBV infection in the
remote past. It is important to test for low levels of HBV DNA by molecular
assays in patients with isolated anti-HBc to identify occult chronic HBV in-
fection (see later discussion).
HBeAg is almost invariably detected during acute HBV infection, in
which case HBsAg and IgM anti-HBc are also usually present. During the
course of chronic HBV infection, detection of HBeAg generally signies ac-
tive viral replication and infectivity. HBeAg is translated from the C gene of
HBV. Unlike HBcAg, HBeAg is released in the serum and can be detected in
the serum by EIA. In the course of acute HBV infection, HBeAg is detect-
able between 6 and 12 weeks after exposure to HBV. For patients who suc-
cessfully clear the virus from serum, HBeAg levels decline with
seroconversion to anti-HBe in association with a marked decline in HBV

DNA levels in serum. Persistence of HBeAg in serum beyond the rst 3
or 4 months of acute HBV infection usually portends development of
chronic HBV infection.
Chronic hepatitis B virus infection

Chronic HBV infection is diagnosed by the presence of HBsAg for more
than 6 months, serum HBV DNA O 105 copies/mL, and persistent or inter-
mittent elevations of serum ALT or AST levels [14]. Chronic HBV infection
can be further divided into HBeAg-positive and HBeAg-negative chronic
HBV infection. The typical patient with chronic HBV infection has both
HBsAg and HBeAg in serum, reecting active viral replication, infectivity,
and hepatic inammation.
Three epidemiologic patterns of HBeAg-positive chronic HBV infection,
dened by mode of transmission, have been identied. Pattern 1 is typical of
chronic HBV infection resulting from perinatal transmission, in which
HBeAg seroconversion usually does not occur until adulthood [3], This pat-
tern is seen predominantly in Asia and Oceania, where patients present with
HBeAg positivity, high serum HBV DNA levels, and normal serum amino-
transferase levels, reecting an immune tolerant state [3]. People with Pat-
tern 1 chronic HBV infection may episodically exhibit elevated serum
aminotransferase levels and other evidence of ongoing liver injury.
Pattern 2 chronic HBV infection is seen primarily in sub-Saharan Africa,
Alaska, and Mediterranean countries where people become infected with
HBV during childhood through person-to-person contact [3]. Typically,
these people have HBeAg in serum, elevated serum HBV DNA levels, and
elevated serum aminotransferase levels. During the course of infection, sero-
conversion from HBeAg to anti-HBe may occur, often around puberty.
Pattern 3 chronic HBV infection, which is most common in the United
States, occurs among people who are infected with HBV through sexual
transmission [3]. As with pattern 2, aected people tend to have HBeAg
in serum, elevated serum HBV DNA levels, and elevated serum AST
or ALT levels (Fig. 2). These ndings indicate active hepatic
necroinammation.
Most people with HBeAg-positive chronic HBV infection who undergo
seroconversion to anti-HBe will subsequently have sustained control
of the HBV infection, with normal serum aminotransferase levels and
low (!105 copies/mL) or undetectable serum levels of HBV DNA, although
HBsAg remains detectable in serum. These people are known as inactive
carriers. However, some people who are HBeAg-negative and anti-HBe-
positive still have active chronic HBV infection. Despite the of circulating
HBeAg. suchjeopjejiave detectable HBV DNA (O105 copies/mL) and uc-
tuating serum ALT or AS levels [14]. The lack of HBeAg occurs as a result
of precore or core promoter region mutations in the HBV DNA that
halt production of HBeAg. As discussed earlier, the most commonly seen

precore mutation is the G1896A substitution that results in a stop codon.
This HBV mutant can be found in 10% to 15% of people with chronic
HBV infection in the United States and Europe and 40% to 80% of people
with chronic HBV infection in Southern Europe, the Middle East, and Asia
[15]. When compared with people with HBeAg-positive chronic HBV infec-
tion, the clinical course of people with HBeAg-negative chronic HBV infec-
tion typically is characterized by lower serum HBV DNA levels and
intermittent, rather than sustained, periods of necroinammatory activity
in the liver. In addition, long-term antiviral therapy is generally required
to maintain suppression of HBV replication.
Treatment of HBeAg-positive or HBeAg-negative chronic HBV infection
with lamivudine, a nucleoside analogue, is complicated by the development
of lamivudine-resistant HBV mutants, specically mutations in the YMDD
(tyrosine-methionine-aspartate-aspartate) motif of the HBV DNA polymer-
ase gene. These mutations occur in 17% of patients at 1 year of treatment
with lamivudine, 40% at 2 years, 55% at 3 years, and 67% at 4 years
[14]. A commercially available PCR test (Hepatitis B Virus GenotypR,
Roche Molecular Systems, Specialty Laboratories, Santa Monica, Califor-
nia) is available to identify the most common mutations in the DNA poly-
merase gene: M552V, M552I, or L528M.
Molecular diagnosis of hepatitis B virus

Clinical applications of hepatitis B virus DNA assays
The diagnosis of acute and chronic HBV infection can be made using the
serologic tests described previously. However, the ability to perform quan-
titative tests in serum for HBV DNA is useful in several settings: (1) to di-
agnose some cases of acute HBV infection; (2) to distinguish replicative
from non-replicative chronic HBV infection; and (3) to monitor a pa-
tients response to antiviral therapy. Determination of the HBV genotype
will likely be used in the future to help predict response to antiviral ther-
apy and can be performed with a commercially available line probe as-
say (INNO-LiPA HBV Genotyping Assay, Immunogenetics N.V., Ghent,
Belgium) [16].
In most instances, the diagnosis of acute hepatitis B can be made with se-
rologic testing for HBsAg. Coincident with the appearance of HBsAg in se-
rum, markers of active HBV replication and infectivity, specically HBeAg
and HBV DNA, appear. If the results of tests for HBsAg are equivocal, as-
says for HBV DNA in serum may be a useful adjunct in the diagnosis of
acute HBV infection. Moreover, HBV DNA can be detected approximately
21 days before HBsAg appears in the serum [17]. Thus, HBV DNA assays
may be used to diagnose acute HBV infection in those patients with high-
risk exposures, such as needlestick accidents in health care workers.
Assays for HBV DNA in serum are also used to characterize the replica-
tive state of chronic HBV infection. Patients with chronic HBV infection
may continue to display markers of active viral replication or may cycle be-
tween an active replicative and a nonreplicative state (see earlier discussion).
Patients who cycle from a nonreplicative state to an active replicative state
are said to have reactivated HBV infection. Reactivation of HBV infection
may occur with or without reappearance of HBeAg in serum. With the in-
creasing sensitivity of molecular assays that allows quantication of HBV
DNA levels, the threshold that distinguishes the replicative from the nonrep-
licative state has been dened as 105 copies of HBV DNA/mL. These assays
allow patients to be classied as having replicative, nonreplicative, or reac-
tivation HBV infection. Furthermore, in patients with precore or core mu-
tations resulting in HBeAg-negative chronic HBV infection, HBeAg cannot
be relied on as a marker of active viral replication, and detection of HBV
DNA is necessary for conrmation of an active replicative state.
In addition to characterizing the status of viral replication in patients
with chronic HBV infection, quantitative HBV DNA assays are useful in
monitoring response to antiviral treatment. Recently, the National Institute
of Diabetes and Digestive and Kidney Diseases and the American Gastro-
enterological Association proposed criteria to dene response to antiviral
therapy based on biochemical (BR), histologic (HR), and virologic response
(VR) [14]. BR refers to a decrease in serum ALT to the normal range, and
HR refers to a decrease in histologic activity index by at least 2 points com-
pared with ndings on a pretreatment liver biopsy. A critical component of
VR is undetectable HBV DNA levels (!105 copies/mL) with the use of an
unamplied assay (see later discussion) and loss of HBeAg in serum in pa-
tients who were initially HBeAg-positive. For patients with HBeAg-negative
chronic HBV infection, however, the only measure of virologic response is
loss of HBV DNA. For patients with HBV infection who are treated with
lamivudine, the emergence of a lamivudine-resistant strain is characterized
by the reappearance of HBV DNA in serum after an initial decline in level
or disappearance.
Molecular techniques for the detection and quantication of hepatitis B

virus DNA
There are both qualitative and quantitative assays for HBV DNA. How-
ever, the qualitative, PCR-based, assays are not necessary to assess the suc-
cess of treatment of HBV infection, characterized by suppression of HBV
DNA in serum, which can be adequately assessed by quantitative, non
PCR-based, HBV DNA assays. Quantication of HBV DNA in serum is
performed using either signal or target amplication techniques. Signal am-
plication techniques require the use of a specic capture oligonucleotide
probe that hybridizes to denatured DNA [17]. Then, the signal (radio-
isotope, chemi luminescence) from the probe-DNA hybrid is amplied for
detection and quantication [17], Target amplication requires amplica-

tion of the viral genome (amplicons); the amplicons are then detected and
quantied.
Assays using signal amplication include liquid hybridization (Genostics
assay, Abbott Laboratories, Chicago, Illinois), the Hybrid Capture System
(Digene Hybrid Capture II HBV DNA Test, Digene Corp., Gaithersburg,
Maryland), and a branched DNA (bDNA) assay (Bayer, Emeryville, Cali-
fornia). The liquid hybridization assay uses iodine 125-labeled nucleic acid
probes that hybridize to soluble, denatured HBV DNA [9]. After hybridiza-
tion, the radiolabeled probes are detected and quantied using a gamma
scintillation counter [18]. The sensitivity of liquid hybridization is 6 105
copies/mL or l2 pg/mL [9].
In the Hybrid Capture System [17,19,20], specic RNA probes are hybrid-
ized to the target HBV DNA to create RNADNA hybrids (Fig. 3). Then,
multiple RNADNA hybrids are captured onto microplate wells, using
Fig. 3. Principle of hybrid capture signal amplication assay. The target sequence is double-
stranded HBV DNA. Hybridization to specic RNA probes creates RNADNA hybrids, which
are captured on a solid phase (a tube in the rst-generation assay, a microplate in the second-
generation assay) by means of universal capture antibodies specic for RNADNA hybrids.
Detection is performed after signal amplication with multiple antibodies conjugated to a reve-
lation system based on chemiluminescence. Light emission is measured and compared with
a standard curve generated simultaneously with known standards. (From Pawlotsky J. Molec-
ular diagnosis of viral hepatitis. Gastroenterology 2002;122:155468; with permission from the
American Gastroenterological Association.)
universal capture antibodies specic for the hybrids. The captured RNA
DNA hybrids are then detected using multiple antibodies (creating signal
amplication) conjugated to alkaline phosphatase. The bound alkaline phos-
phatase is detected with a chemiluminescent dioxetane substrate that pro-
duces light, which is then measured. The signal can be amplied 3000 fold.
The sensitivity of the Hybrid Capture System is 4700 copies/mL [17].
The bDNA technique [17,21] involves the use of specic oligonucleotide
probes to hybridize HBV DNA to plastic microwells (Fig. 4). Signal
Fig. 4. Principle of branched DNA (bDNA) signal amplication assay. The target sequences
are captured on the wells of a microtiter plate by means of specic capture probes. Extender
probes are used to hybridize synthetic bDNA amplier molecules (in rst-generation HBV
DNA and hepatitis C virus (HCV) RNA assays, and in second-generation HCV RNA assays)
or, as shown in the gure, preamplier molecules that in turn hybridize bDNA molecules (third-
generation HCV and HBV assays). The multiple repeat sequences within each bDNA molecule
serve as sites for hybridization to alkaline phosphataseconjugated oligonucleotide probes. Al-
kaline phosphatase catalyzes chemiluminescence emission from a substrate, which is measured
and compared with a standard curve generated simultaneously with known standards. (From
Pawlotsky J. Molecular diagnosis of viral hepatitis. Gastroenterology 2002;122:155468; with
permission from the American Gastroenterological Association.)
amplication occurs when bDNA amplier molecules are hybridized to the

target HBV DNA hybrids in the microwell. Multiple repeat sequences
within the bDNA amplier molecule are then conjugated with an alkaline
phosphatasecatalyzed chemiluminescence probe similar to that used in
the Hybrid Capture System. The lower limit of detection for the bDNA as-
say is 7 105 DNA equivalents/mL [17].
Although the signal amplication techniques oer highly specic assays
to detect HBV DNA, they are unable to detect low levels of HBV DNA
(!w5,000 copies/mL). Target amplication techniques such as PCR-based
assays are highly sensitive with the ability to detect as few as 10 copies/mL
of HBV DNA (TaqMan-based PCR) [22]. PCR assays rely on the use of
specic primers that attach to each strand of target dsDNA, Then, new
DNA strands are synthesized and amplied behind the primer. This cycle
of DNA denaturing, primer annealing, and strand synthesis is repeated mul-
tiple times, thereby resulting in amplication of the target HBV DNA. The
most common primers used in HBV DNA PCR assays are complementary
to the precore or core region [9], Commercially available assays include the
Amplicor HBV Monitor Test, v2.0 (Roche Molecular Systems, Pleasanton,
California) and the Cobas Amplicor HBV Monitor Test, v2.0 (Roche Mo-
lecular Systems, Pleasanton, California). The ranges of HBV DNA detec-
tion are 1000 to 40,000,000 copies/mL and 200 to 200,000 copies/mL,
respectively. The Cobas Amplicor system is the more sensitive of the two as-
says and uses an Amplicor analyzer that automates the amplication and
detection process.
Recent advances in PCR technology include the development of real-
time PCR techniques to increase the sensitivity of the assay. Real-time
PCR refers to the simultaneous amplication and quantication of viral ge-
nomes, thereby obviating the need for post-PCR manipulations [23,24].
Real-time PCR can detect a wide range of HBV DNA levels and oers
a more rapid assay than conventional PCR techniques. In a recent study,
LightCycler (LC)-PCR (Roche Diagnostics, Pleasanton, California),
a real-time PCR technique, was compared with the Hybrid Capture II
HBV DNA test (Digene Corp., Gaithersburg, Maryland) [23], and LC-
PCR was found to be rapid (w2.5 hours) and 500 times more sensitive
than Hybrid Capture II, with an HBV DNA detection range of 250 to
2.5 109 copies/mL. Currently, real-time PCR using the TaqMan probe
is the most sensitive quantitative HBV DNA assay and is able to detect as
few as 10 copies/mL [22,25]. TaqMan technology uses a uorescent probe
annealed to target DNA sequences for quantication of DNA [26,27].
Although advances in PCR technology have permitted the detection of as
few as 10 copies/mL of HBV DNA, the clinical signicance of such low se-
rum levels of HBV DNA is unknown. An arbitrary value of 105 copies/mL
of HBV DNA has been selected as one of the diagnostic criteria for chronic
HBV infection. However, this denition is problematic for several reasons.
First, patients with chronic HBV infection can have HBV DNA levels that
Table 2
Commercially available hepatitis B virus DNA quantication assays
Assay Manufacturer Method Lower detection cuto Dynamic range of quantication
Signal amplication
SEROLOGIC AND MOLECULAR DIAGNOSIS OF HBV

Genostics Assay Abbott Labs, Liquid hybridization 12 pg/mL (w600,000 12 pg/mL w 800 pg/mL
Chicago, IL copies/mL) (600,000300,000,000 copies/mL)
Versant HBV DNA Bayer Corp, Manual branched DNA 700,000 genome 700,0005,000,000,000 genome
1.0 Assay Diagnostics (bDNA) signal amplication equivalents/mL equivalents/mL
Division,
Tarrytown, NY
HBV Hybrid-Capture I Digene Corp., Hybrid capture signal 700,000 copies/mL 700,000560,000,000 copies/mL
Gaithersburg, MD amplication in tubes
HBV Hybrid-Capture II Digene Corp., Hybrid capture signal 142,000 copies/mL 142,0001,700,000,000 copies/mL
Gaithersburg, MD amplication in microplates
Ultra-sensitive HBV Digene Corp., Hybrid capture signal 4700 copies/rnL 470057,000,000 copies/mL
Hybrid-Capture II Gaithersburg, MD amplication in microplates
after centrifugation
Target amplication
Amplicor HBV Roche Molecular Manual quantitative RT-PCR 1000 copies/mL 100040,000,000 copies/mL
Monitor Test v2.0 Systems, Pleasanton, CA
Cobas Amplicor HBV Roche Molecular Semi-automated quantitative 200 copies/mL 200200,000 copies/mL
Monitor Test v2.0 Systems, Pleasanton, CA RT-PCR
Adapted from Pawlotsky J. Molecular diagnosis of viral hepatitis. Gastroenterology 2002;122:155468; with permission from the American Gastroenterolo-
gical Association.)
59
uctuate and intermittently fall below 105 copies/mL. Second, the threshold
HBV DNA level associated with the development of hepatic brosis is un-
known. Third, currently available HBV DNA assays have not been stan-
dardized with respect to HBV DNA quantitative units [3] (Table 2). The
World Health Organization recently established an international standard
for HBV DNA assays [28]. Implementation of this standard will be essential
to dening clinically appropriate treatment guidelines based on serum HBV
DNA levels [17].
Summary
Serologic assays for HBV are the mainstay diagnostic tools for HBV in-
fection. However, the advent of molecular biologybased techniques has
added a new dimension to the diagnosis and treatment of patients with
chronic HBV infection. Over the past decade, improvements in molecular
technology, permitting detection of as few as 10 copies/mL of HBV DNA
in serum have led to redenitions of chronic HBV infection, as well as
thresholds for antiviral treatment. As the sensitivity of these molecular tech-
niques continues to improve, the challenge will be to standardize these as-
says as well as dene clinically signicant levels of HBV replication.
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20 (2006) 6379
Assessment and Management of Chronic

Hepatitis B
Marc G. Ghany, MDa,*, Edward C. Doo, MDb
a
Liver Diseases Section, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Building 10, Room 9B-06, 10 Center Drive, MSC 1800,
Bethesda, MD 29892-1800, USA
b
Liver Disease Research Branch, National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, 6707 Democracy Boulevard, MSC 5450,
Bethesda, MD 20892-5450, USA
Infection with the hepatitis B virus (HBV) is a signicant global public

health problem. Over one third of the world population has been exposed
to the virus, and an estimated 400 million people are chronically infected
[1,2]. Up to 40% of chronically infected individuals will be at risk for
cirrhosis, decompensated liver disease, and hepatocellular carcinoma
(HCC), and each year, an estimated 500,000 deaths occur from these
complications. Advances in molecular biology techniques have led to a better
understanding of the natural history and pathogenesis of HBV-related liver
disease, resulting in the development of potent antiviral agents. Some of
these agents can be used safely as maintenance therapy for patients who
fail to clear the virus following standard durations of treatment. Overall,
the advent of newer therapies has provided a wider range of therapeutic
options for chronic hepatitis B (CHB) infection. This article focuses on
the natural history of CHB-related liver disease, assessment and selection
of patients for therapy, and new therapeutic options. It is meant to provide
a succinct update on the management of CHB based on recent
advancements in knowledge of the disease.
A version of this article originally appeared in the 33:3 issue of Gastroenterology Clinics
of North America.
E-mail address: marcg@bldg10.niddk.nih.gov (M.G. Ghany).
64 GHANY & DOO
Natural history
The natural history of HBV infection is variable and inuenced by
a complex interplay between the host immune response and the replication
tness of the virus. Other factors impacting on the course of HBV infection
include age at time of exposure, integrity of the immune system, alcohol
consumption, obesity, and concurrent viral infections such as hepatitis
C virus (HCV), hepatitis D virus (HDV), and HIV. In addition, viral
load, viral variants, and perhaps HBV genotype may aect the clinical
course. Ultimately, the outcome of HBV infection is determined by the ro-
bustness of the immune response as highlighted by the dierent clinical
courses of perinatally and adult-acquired infection. Exposure at birth or
at a young age when the immune system is thought to be immature repre-
sents the highest risk for developing CHB, where 9095% of exposed infants
develop chronic infection. In stark contrast, 9095% of adult cases of HBV
infection resolve spontaneously.
The natural history of HBV can be divided into 3 phases, an immune tol-
erant phase, an immune active phase, and an inactive phase. The immune tol-
erant phase is generally absent in adult acquired infection. The immune
tolerant phase is characterized by a lack of symptoms, no or minimal eleva-
tion in serum aminotransferase levels and mild inammation on liver
biopsy. However, HBV DNA levels can be quite high and hepatitis B e antigen
(HBeAg), a surrogate marker of viral replication is found in serum [3]. Im-
mune tolerance is believed to be due to the inability of the host immune system
to fully recognize viral antigens and may persist for 10 to 30 years [3] (Fig. 1A).
During this period there is a low rate of spontaneous viral clearance.
The immune active phase is characterized by uctuating HBV DNA
levels, high serum aminotransferase levels, and hepatic necroinammation.
This phase is thought to be the result of incomplete attempts by the host im-
mune response to eradicate virally infected hepatocytes [4] (Fig. 1B). Clinical
manifestations of chronic liver disease may appear during repeated bouts of
immune-mediated aminotransferase ares and may accelerate the progres-
sion of hepatic brosis to cirrhosis [57]. During these ares, a small number
of patients (1015%) may lose HBeAg spontaneously, followed by the de-
velopment of antibody to HBeAg (anti-HBe), a so called HBeAg seroconver-
sion heralding a period of quiescence in liver disease and the inactive phase
[57]. This phase is associated with normal aminotransferase levels and less
hepatic inammation [6].
Attainment of the inactive phase has also been referred to as the transition
from a high to low viral replicative status due to the marked drop in HBV
DNA levels. Hepatitis B surface antigen (HBsAg), however, remains detect-
able in serum. The inactive phase may last for many years, and in the absence
of cirrhosis, there is diminished risk for disease progression or hepatocellular
carcinoma [8,9]. Thus the loss of HBeAg is an important clinical event asso-
ciated with a period of disease inactivity and improved prognosis, and it
ASSESSMENT AND MANAGEMENT OF CHRONIC HBV 65
HBeAg+/
A
HBeAg+ anti-HBe+ anti-HBe+
HBV DNA
ALT
Years
Immune Immune Non-replicative
tolerance phase clearance phase phase
HBeAg+ anti-HBe+
B
HBV DNA
ALT
Years
Replicative phase Non-replicative phase
Fig. 1. Natural history of chronic HBV infection. (dashed lines) Represent typical uctuations
that occur with HBV infection. (straight lines) Represent average trends for both HBV DNA
and ALT. (A) Course of perinatally acquired HBV infection. (B) Course of adult-acquired
HBV infection.
often is used as an endpoint of therapy [10]. During the inactive phase, 1% to

2% of individuals per year will clear HBsAg, but as many as 30% of people
may relapse back to the immunoactive phase.
Approximately 5% to 10% of individuals who lose HBeAg will continue
to have detectable HBV DNA, elevated aminotransferase levels, and active
66 GHANY & DOO
inammation on liver biopsy. These cases are dened as the HBeAg-nega-

tive or atypical form of CHB and remain in the immunoactive phase [11].
Based on this knowledge, the natural history of HBV infection can be
dened by three clinical, serologic, and virologic patterns:
1. HBeAg-positive or classic CHB. This is the most common pattern and is
dened by the presence of HBeAg and elevated HBV DNA in serum.
The prognosis is variable and is in part dependent upon whether the
infection is in the immunoactive phase (associated with elevated
aminotransaminase levels and chronic hepatitis on liver biopsy) or the
immunotolerant phase (associated with normal aminotransaminase
levels and minimal to absent inammation on liver biopsy).
2. HBeAg-negative or atypical CHB. This form of the infection is
characterized by the absence of HBeAg but with detectable HBV
DNA in serum and elevated aminotransferase levels. Features of
chronic hepatitis are seen on liver biopsy, and the disease is generally ac-
tive as dened by abnormal serum aminotransferase levels and liver his-
tology [12,13]. Molecular analysis of HBV has identied the cause of
this clinical presentation. Mutations in the HBV precore and core
gene lead to an abrogation or reduction of HBeAg synthesis [12,13].
The clinical course can be quite severe, with more frequent episodes
of acute aminotransferase ares that may lead to a greater number of
liver-related complications [14]. Patients presenting with this pattern
of disease should have other forms of chronic hepatitis excluded, such
as hepatitis C and D infections and autoimmune and drug-induced liver
disease.
3. The inactive carrier state. This is typied by the presence of anti-HBe in
serum, normal alanine aminotransferase (ALT) levels, undetectable
HBV DNA by hybridization assays, and minimal changes on liver bi-
opsy. Viral replication is low in these individuals. Patients in this phase
of the disease usually have a favorable prognosis and are at low risk for
liver disease progression [8,15]. Occasionally, spontaneous reactivation
may occur.
Assessment and monitoring

The primary goals of assessment are to determine the level of disease
(active or inactive) and to stage the disease as mild, moderate, or severe.
The initial evaluation of the patient should include a complete history and
physical examination with particular attention directed toward eliciting
signs, symptoms, and risk factors for chronic liver disease. A family history
of liver disease should be sought and a detailed alcohol history obtained.
Routine blood tests should include standard biochemical and hematological
proles and specic viral serologic assays for HBsAg, HBeAg, antibody to
hepatitis B core antigen (anti-HBc), anti-HBe, antibody to hepatitis B
surface antigen (anti-HBs), and quantitation of HBV DNA [16,17]. Which

HBV DNA assay to use in clinical practice is an unresolved issue. Quanti-
tative assays vary signicantly in their sensitivity, with the lower limit of de-
tection varying by as much as 103 log copies per mL. Testing for hepatitis A,
C, and D and HIV should be performed routinely, as most of these viruses
share similar routes of transmission as HBV, and coinfection with another
virus may modify the course of HBV. Other causes of chronic liver disease
should also be excluded. A baseline ultrasound examination is recom-
mended to exclude abnormal masses or anatomical variants. A liver biopsy
provides information on the grade (severity of inammation) and the stage
(severity of brosis) of the disease. A liver biopsy, however, can conrm the
diagnosis, provide prognostic information for the patient, and assist in
determining the need for therapy. Although the information gained from
liver biopsy is helpful in clinical decision making, it is not an absolute re-
quirement before treatment and should be individualized in each patient.
Patients should be informed of other factors that might exacerbate progres-
sion of underlying liver disease, notably alcohol consumption, use of hepa-
totoxic medications, and immunosuppressants. Counseling on reducing the
risk of transmission should be undertaken, especially for teenagers, young
adults, and injection drug users, populations among whom the risk of sexual
and parenteral transmission is high. Close family, household, and sexual
contacts should be vaccinated against HBV. Individuals who do not have
immunity against hepatitis A virus (HAV) should be oered hepatitis A vac-
cination. If bridging brosis or cirrhosis is found on liver biopsy, or if there
is clinical evidence of advanced liver disease, a screening esophagogastro-
duodenoscopy is advisable to assess for portal venous hypertension via
the presence of esophageal varices. Attention should be given to initiating
a screening program for HCC in patients with established cirrhosis and par-
ticularly in males, older individuals, and those with a strong family history
of liver cancer.
Selection of patients for treatment

There is no consensus on how to treat chronic HBV; however, several
treatment guidelines have been published [1619]. Minor dierences exist
in the recommendations. Identifying patients who require treatment may
appear to be a straightforward task, but it is usually quite complicated in
practice. There are many factors to consider in the decision-making process,
including the age of the patient, stage of disease, pattern of liver disease, the
patients willingness to be treated, coinfection with other hepatotrophic
viruses (HCV, HDV) and HIV, presence of other comorbid conditions
and adverse eects of treatment. CHB often has a uctuating course; there-
fore, after an initial assessment patients should be monitored with liver
chemistries for a period of at least 6 months to assess the pattern of disease
before initiating therapy (Fig. 2). Patients with inactive CHB are not
68 GHANY & DOO
HBsAg +
HBeAg Decompensated
Inactive carrier
Liver disease
Anti-HBe+; Pos Neg
HBV DNA neg;
Normal ALT HBV DNA <105 HBV DNA >105
copies / ml; normal copies / ml; elevated
ALT ALT for 6 months
No treatment Liver biopsy

Consider
Monitor q 3-
antiviral
6 months Mild Moderate to severe chronic hepatitis /
therapy /
hepatitis compensated cirrhosis
refer for
OLT
Consider
antiviral therapy
Fig. 2. Algorithm for selection of patients for therapy.
candidates for therapy. Those with mild disease as evidenced by a brosis

score of 2 or less on the Ishak and Metavir scales or 1 on the Knodell scale
and serum ALT levels persistently less than twice the upper limit of normal
can defer therapy safely until more eective treatments or agents with better
long-term resistance proles become available. These patients should con-
tinue to be monitored by biochemical and serologic tests every 3 to 6 months
for any evidence of acceleration of the disease. Patients with ALT elevations
greater than twice the upper limit of normal, HBV DNA levels greater than
105 copies per mL, or with evidence of moderate-to-severe chronic hepatitis
on liver biopsy are candidates for therapy. HBeAg status should be deter-
mined, as therapeutic options dier between HBeAg-positive and -negative
patients. Patients with decompensated liver disease are managed best at
a liver transplant center by an experienced hepatologist.
Role of hepatitis B virus genotypes

Traditionally, HBV isolates have been distinguished by serotyping based
on three antigenic determinants of the HBsAg. Common to all isolates is the
a epitope and two pairs that are mutually exclusive to each other, (d or y)
and (r or w), thus giving rise to four major serotypes: adr, adw, ayr, and
ayw. With the advent of genetic sequencing, HBV was classied into seven
genotypes (A to G) based on a nucleotide divergence in the entire genome of
at least 8% [2022]. Genotypes also can be identied by analysis of the small
envelope gene of HBsAg (s-gene), where the inter-genotype divergence is of
the order of 4%.
The seven genotypes have a characteristic geographical distribution, but
most have a worldwide prevalence because of human migration. Of the
seven genotypes, types A through D are the most common worldwide. Ge-
notype A is found predominantly in North America, northwest Europe, and
central Africa; genotypes B and C prevail in China, Japan, and Southeast
Asia. Genotype D is found primarily in the Mediterranean, Middle East,
and Indian subcontinent (Table 1). A recent study reported that the pre-
dominant United States genotypes were types A and C, suggesting a change
in the prevalence of genotypes in the United States population. However,
over 50% of patients in the study were of Asian ethnicity. Additionally,
genotype A was more common in American-born patients, whereas geno-
type C was more common among Asian-born patients. This suggests that
the increased prevalence of HBV genotype C in the United States popula-
tion was most likely caused by immigration from endemic regions of the
world [23].
With HCV, genotyping has been found to have an important role in man-
aging infection. It is one of the strongest predictors of response to therapy
and inuences the therapeutic duration. There have been several recent stud-
ies attempting to correlate HBV genotypes with clinical parameters such as
the rate of HBeAg seroconversion, development of clinically important mu-
tations, severity of liver disease, and response to treatment.
Several studies have suggested that genotype may correlate with disease
activity. When compared with genotype B, genotype C was associated
with higher ALT levels and a higher prevalence of cirrhosis [24]. HBV
genotype B was shown to be associated with an earlier time to HBeAg sero-
conversion, which may help to explain the lower disease activity reported in
Asian patients [2426], and with a higher rate of hepatocellular carcinoma in
younger individuals [27]. A recent study from India found that patients with
genotype D have more severe disease and a higher rate of HCC compared
Table 1
Geographical distribution and clinical relevance of hepatitis B virus genotypes
HBV
genotype Geographical distribution Clinical relevance
A North America, North western Europe, Better response to peginterferon
Central Africa
B China, Indonesia, Vietnam, Taiwan Lower disease activity
Associated with HCC in
noncirrhotic individuals
Improved response to interferon
and lamivudine
C East Asia, Korea, China, Taiwan, Japan, Associated with more severe
Polynesia, Vietnam liver disease
D Mediterranean, Middle East, India Associated with precore
mutation and HBeAg status
E Nigeria, West Africa Unknown
F Alaska, Polynesia Unknown
G North America, France Unknown
70 GHANY & DOO
with patients with genotype A [28]. Given the heterogeneity of the data, it is
clear that the role of HBV genotypes in relation to clinical outcome requires
further investigation.
A relationship between viral burden and HBV genotype also has been
suggested. Analysis of a large cohort of patients enrolled in phase III trials
of adefovir showed that in HBeAg-positive CHB patients, HBV DNA levels
were signicantly higher in genotypes A, B, and D compared with geno-
type C. In contrast, among HBeAg-negative patients, signicantly higher
HBV DNA levels were found in those with genotype D.
The two predominant mutations that lead to HBeAg-negative CHB
appear to be associated with specic HBV genotypes [24,29]. The precore
mutation and the basic core promoter mutation are found more com-
monly with HBV genotypes D and A, respectively.
Lastly, genotype may aect the response to antiviral treatment. In Asian
patients infected with genotypes B or C, interferon (IFN) was shown to be
less eective in genotype C compared with genotype B infections [30]. A re-
cent study reported a higher rate of HBeAg loss in HBV genotype A patients
treated with peginterferon a2B [31]. A small European trial suggested that
the response to lamivudine was better in patients with serotype ayw (corre-
sponding to genotype D) compared with serotype adw (corresponding to ge-
notype A) [32]. Furthermore, the rate of resistance was higher in patients
with serotype adw [32]. A recent analysis suggested that genotype B was
associated with a higher sustained loss of HBeAg in lamivudine-treated pa-
tients [33]. No association between genotype and response to treatment,
however, was found in a larger cohort of adefovir-treated patients [34].
Much of the current data regarding the clinical implications of HBV ge-
notypes should be viewed with some prudence. Many of the studies were
small and cross-sectional, comparing two of the major genotypes with
each other, either B with C or A with D, and may have been susceptible
to referral bias. These issues serve to limit the generalizability of their
respective ndings and highlight the need for further studies to help dene
the clinical implications of HBV genotypes. At present, the data are
insucient to merit genotype testing as part of the evaluation of patients
with CHB.
Screening for hepatocellular carcinoma

Hepatocellular carcinoma is a signicant cause of morbidity and mortal-
ity in patients with CHB. There is no eective therapy for HCC. The ratio-
nale for HCC screening is to detect small, solitary lesions that may be more
amenable to surgical resection or liver transplantation [35]. The benet
of this approach has not been substantiated. The best data to support
a screening program in patients with HBV come from a population-based
screening and surveillance study using periodic alpha fetoprotein (aFP) test-
ing in 1487 HBsAg-positive Alaskan natives [36]. Rates of cancer in the
screened group were compared with historical controls from a national can-
cer registry. During a 16-year period, 100 aFP elevations were detected, and
32 cancers were diagnosed; 22 patients underwent successful resection. The
comparison group was comprised of 12 historical controls diagnosed before
institution of the screening program. Survival rates were improved signi-
cantly in the screened population versus historical controls at 5 years
(42% versus 0%) and at 10 years (30% versus 0%) [37]. Thus in this pop-
ulation, screening appeared to be eective in reducing mortality from HCC.
There have been no randomized controlled trials of screening for HCC.
The two most widely employed modalities for screening are aFP testing
and periodic ultrasound exams of the liver [36]. There are several limitations
to both options. In the case of aFP, dening a standard cut-o and optimal
frequency of testing is a matter of intense debate. Sensitivity of ultrasound is
poor, ranging from 35% to 84%. It is highly operator-dependent, and like
aFP testing, there is no consensus on how frequently the procedure should
be performed. HCC has widely variable growth rates, with a doubling time
of 1 to 19 months, with a median of 6 months. Thus, most authorities rec-
ommend a screening interval of 6 months.
Among histologically proven cases of HCC, 30% do not present with
serum aFP elevations, and 30% of tumors smaller than 2 cm may not be
detected by ultrasound. Thus, in clinical practice, both tests are used, adding
to the cost of screening. Specic guidelines for screening are dicult to for-
mulate in part because of the expense of current screening modalities, the
absence of a test with suitable sensitivity and specicity, and the lack of ef-
fective therapy. Two recent conferences held on the management of CHB
recommended that aFP and/or ultrasound should be performed every 6
months in patients with cirrhosis, patients older than 40 years, and those
with a family history of HCC [16,17].
Treatment goals
The objectives of therapy can be viewed in terms of short- and long-term
goals. Short-term objectives are to reduce viral burden, improve serum ami-
notransferase levels, and reduce hepatic necroinammation. Improvement
of these parameters would be expected to slow the progression of brosis.
Long-term goals of therapy are to prevent progression to cirrhosis and
HCC and ultimately improve survival. Once a decision is made to treat,
the dilemmas facing the clinician are choosing which drug to use, deciding
upon the optimal duration of treatment, and how to assess response to treat-
ment. There are ve approved therapies for CHB: IFN-a, peginterferon,
lamivudine, adefovir dipivoxil, and entecavir. Any of these agents can be
used as rst-line therapy, and each is discussed in detail in separate articles
within this issue. When recommending treatment, the benets and adverse
eects of each drug should be discussed, as well as the situations where
one agent is favored over another. The advantages of IFN-a include a nite
72 GHANY & DOO
period of administration, lack of drug resistance and, if attained, a durable

response. Patients opting for nucleoside therapy should be informed of
the potential need for long-term treatment and the development of drug
resistance.
Traditionally, the endpoint of therapy was loss of HBeAg and gain of
anti-HBe, because seroconversion was associated with a favorable long-
term prognosis after successful IFN-a treatment. This endpoint may not
be suitable to assess the ecacy of nucleoside/nucleotide analog therapy
because of reported relapse rates that range from 30% to 67% 2 to 3 years
after therapy cessation [3840]. At a recent NIH workshop on the manage-
ment of CHB, the need for standardized denitions of response to antiviral
therapy to allow for comparison of responses among dierent therapies was
highlighted. It was recommended that response incorporate biochemical,
serological, virological, and histological parameters and dened in the
context of timing of treatment (ie, at initiation, during, at termination, or
after termination of treatment) [16]. The remainder of this article will be
devoted to a discussion on new investigative agents, highlighting those
that appear to be most promising (Table 2).
Promising new hepatitis B agents

Tenofovir
Tenofovir disoproxil fumarate is the prodrug of tenofovir and is a nucle-
oside analog approved as an antiretroviral agent. Tenofovir has been shown
to exhibit antiviral activity against HBV [41]. In patients coinfected with
HIV and HBV where tenofovir was added as a component of antiretroviral
therapy, HBV viral load decreased by approximately 4 log after 1 year of
therapy [42]. Tenofovir appears to remain eective against the lamivudine
HBV polymerase mutant [43,44]. Additionally, in patients with HIV/HBV
coinfection who were HBeAg negative, short-term treatment with tenofovir
led to a reduction of HBV viral titers [45]. Thus, tenofovir may be a potential
adjunctive therapeutic agent against CHB infection in patients coinfected
with HIV. Additional studies are needed to evaluate the potential of tenofo-
vir as monotherapy for CHB infection. Renal toxicity has been reported
with tenofovir.
Emtricitabine
Emtricitabine (FTC, 2939-dideoxy-59uoro-39-thiacytidine) is a nucleoside
analog that is structurally similar to lamivudine. An initial in vitro study
demonstrated inhibition of HBV replication by emtricitabine [47].
Signicant reductions in woodchuck hepatitis virus (WHV) titers with
emtricitabine were observed in chronically infected woodchucks [48]. The
antiviral eect also was observed in people in a phase I clinical trial [49].
Table 2
New compounds in development for treatment of chronic hepatitis B infection
ASSESSMENT AND MANAGEMENT OF CHRONIC HBV

Activity against
Phase of lamivudine resistant
Generic name Molecular structure development strains Resistance reported
Clevudine 29-uoro-5-methyl-L-arabinofuranosyl Phase II No Yes
uracil nucleoside analogue
Emtricitabine 2939dideoxy-59uoro-39-thiacytidine ?Phase III No Yes
nucleoside analogue
Lobucavir Carbocyclic analogue of oxetanocin Phase I., studies Unknown Unknown
terminated because to
toxicity in rodents
Tenofovir Adenosine nucleotide analogue Phase III Yes No
Amdoxovir D-2,6-diaminopurine dioxolane Phase I Unclear Unknown
Beta-L-nucleosides Beta-L-thymidine Phase I Unknown
Heteroaryldihydropyrimidines Same Unknown Unknown
73
74 GHANY & DOO
Preliminary results from a placebo-controlled phase 3 study in treatment

na ve patients has demonstrated that 48 weeks of emtricitabine (200 mg
daily) reduces serum HBV DNA by a median of 310 copies per mL and sig-
nicantly improves liver histology [50]. The drug is structurally similar to
lamiveudine and shares similar mutational sites and rate of resistance.
The incidence of resistance is 96% at week 96 [51]. Given these ndings
it is unlikely this drug will play a major role in the management of chronic
HBV.
Clevudine
Clevudine (L-FMAU, 29-uoro-5-methyl-b-L-arabinofuranosyl uracil) is
a pyrimidine nucleoside analog that has been demonstrated to have antiviral
eects in short-term studies with woodchucks chronically infected with
WHV [52]. In chronically infected woodchucks, however, prolonged therapy
with L-FMAU resulted in viral rebound cause by the development of resis-
tant WHV polymerase mutants [53]. In a small human pilot study evaluating
four doses of clevudine (10 mg, 50 mg, 100 mg, and 200 mg) for 4 weeks, the
mean reduction in HBV DNA level was 2.5 to 3.0 log for all doses tested.
The reduction in HBV DNA was sustained up to 24 weeks after cessation of
treatment and was highest in the 100 mg dose group (2.7 logs). Of treated
patients, 19% had loss of HBeAg, and few adverse eects were reported. No
mitochondrial toxicity was observed. A phase III trial of clevudine 3 mg
orally daily for 24 weeks followed by 24 weeks of followup showed 59%
of undetectable virus and 68% with ALT normalization. No viral break-
through was noted during therapy. Final results are pending.
Beta-L-nucleosides
Beta-L-nucleosides are potential therapeutic antiviral agents against
HBV being evaluated in duck hepatitis virus and WHV models. There are
three of these analogs under investigation: LdC (beta-L-29-deoxycytidine;
valtorcitabine), LdT (beta-L-thymidine, telbivudine), and LdA (beta-L-29-
deoxyadenosine). All have demonstrated specicity for the HBV polymer-
ase, and LdT is being evaluated in clinical trials [54].
Telbivudine
Telbivudine (Ldt) is a beta-L-nucleoside that is currently in Phase III tri-
als. Initial studies demonstrated the potent activity of LdT against HBV in
in vitro studies. Results of a Phase II trial with LdT in varying doses as
monotherapy and in combination with lamivudine demonstrated signi-
cantly more reduction in HBV DNA in treatment arms that contained
LdT than with lamivudine monotherapy. The rate of HBV genotypic resis-
tance to LdT is not insignicant and was reported to be 4.5% after one year
and rose to 18.2% after 96 weeks of therapy.
Antiviral agents requiring further investigation

Lobucavir
Lobucavir is a carbocyclic analog of oxetanocin shown to be an eective
antiviral agent against HBV [46]. Further studies were terminated when he-
patic and foregut tumors emerged during long-term rodent carcinogenesis
studies.
Amdoxovir
Amdoxovir (DAPD, b-D-2,6-diaminopurine dioxolane) is a prodrug of
1-b-D-dioxolane (DXG), which has antiretroviral activity against HIV-1
[55]. In vitro studies of amdoxovir sensitivity among lamivudine-resistant
HBV polymerase mutant strains are inconsistent, and additional studies
will be needed to clarify DAPDs utility in this setting [56]. Amdoxovir is
currently being evaluated in phase I/II studies of HIV-positive patients.
Pradefovir
Pradefovir mesylate (PDV) is a cyclodiester prodrug of adefovir that is
biotransformed in heptocytes by the cytochrome P450 3A4 into the active
compound. The premise of this approach is to use higher doses that
concentrate into infected hepatocytes while sparing renal tubule toxicity. In-
terim results at 24 weeks of therapy of a Phase II study in patients with lam-
ivudine resistant HBV, pradefovir at a dose of 30 mg appeared to be
equivalent to standard adefovir. The caveat is that the number of patients
analyzed was small and the results will need to be reassessed once the study
matures.
Heteroaryldihydropyrimidines
Heteroaryldihydropyrimidines (HAPs) are compounds that eectively in-
hibit HBV replication at the level of viral capsid assembly. Critical molecu-
lar events in the life cycle of HBV occur within the milieu of the assembled
capsid, including synthesis of the genome [57]. Initial in vitro studies have
demonstrated that HAPs disrupt the formation of viral capsids by enhanc-
ing proteasome-mediated degradation of capsid monomers [58]. Future
studies with these compounds are awaited, as they may represent a novel ap-
proach to HBV treatment.
Other novel approaches

Several novel approaches are being developed for treating hepatitis B.
Several immunotherapeutic strategies have been tried in an attempt to mod-
ulate the immune response either alone or in combination with antiviral
agents [59]. HBsAg vaccination, in combination with lamivudine and
76 GHANY & DOO
a lipopeptide-based T-cell vaccine designed to induce a core-specic cyto-

toxic T-lymphocyte (CTL) response, has been used in clinical trials, albeit
without much success. Another novel approach to inhibit viral replication
has been through the development of antisense oligonucleotides and ham-
merhead ribosomes. Both of these agents form hybridization duplexes
with specic target viral mRNA sequences, ultimately leading to viral
RNA degradation either through activation of host RNaseH or intrinsic
catalytic activity. Several technical issues hinder practical use of this technol-
ogy in the treatment of HBV infection, such as transport into target cells,
absence of target tissue specicity, degradation by host enzymes, and poor
bioavailability. Most recently, small interfering RNAs have been shown in
cell culture to reduce the level of viral transcripts and proteins and replica-
tive forms [60]. Many technical issues will need to be addressed before these
potential therapeutic modalities become feasible.
Summary
An understanding of the natural history of CHB is critical for the man-
agement of the liver disease. Three clinical patterns with dierent clinical
outcomes are recognized: HBeAg-positive CHB, HBeAg-negative CHB,
and inactive CHB. Patients with elevated aminotransferase levels and
HBV DNA greater than 105 viral copies per mL in serum and with features
of chronic hepatitis on liver biopsy are candidates for therapy regardless of
HBeAg status. Multiple host and viral factors and safety proles of current
therapies need to be considered carefully before recommending therapy.
There appears to be no role for HBV genotyping in the management of pa-
tients. Three antiviral agents are approved for use against CHB infection:
IFN-a, lamivudine, and adefovir. Ecacy is moderate at best and is limited
by the poor tolerability of IFN and the development of resistance, coupled
with concerns regarding the long-term safety with nucleoside analogs. Sev-
eral new nucleoside and nucleotide analogs and novel agents are at various
stages of development as potential therapies for CHB. The ideal compound
would be one that is active against all replicative intermediates of the virus
and has a low toxicity prole. Despite current shortcomings, the future of
therapy for HBV is promising, as newer therapeutic options are being
developed based on an understanding of the HBV life cycle.
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20 (2006) 8198
Molecular Virology of the Hepatitis C

Virus: Implication for Novel Therapies
Jerey S. Glenn, MD, PhD
Division of Gastroenterology and Hepatology, Stanford University School of Medicine
and Palo Alto Veterans Administration Medical Center, CCSR Building, Room 3115,
269 Campus Drive, Palo Alto, CA 94305-5187, USA
Hepatitis C virus (HCV) is a signicant cause of morbidity and mortality,

infecting over 150 million people worldwide [1,2]. In spite of recent progress,
current therapies remain inadequate for the majority of patients [35]. The
study of HCV molecular virology is providing an increasing number of new
anti-HCV targets. These are being translated into the development of new
drugs that oer the prospect of more eective antiviral therapies. This arti-
cle provides a concise review and update of the major highlights in these
eorts.
Overview of life cycle

HCV is a positive, single-stranded, RNA virus. Its 9.6-kb genome en-
codes a single w3000 amino acid polyprotein that is proteolytically pro-
cessed by a combination of cellular and viral proteinases into structural
(components of the mature virus) and nonstructural (elements proposed
to help replicate new virions) proteins (Fig. 1) [68]. Flanking this long
protein-encoding sequence are conserved nontranslated regions at the 59 and
39 ends of the RNA genome. These contain highly structured elements that
represent important cis-acting signals for initiating replication and transla-
tion of the viral genome.
The structural proteins include the core and envelope proteins. The core
protein is thought to serve as a nucleocapsid protein. It also seems to inter-
act with several host cell signaling pathways and has been implicated in stea-
tosis. The core protein and the viral RNA genome are encapsulated in
E-mail address: jeffrey.glenn@stanford.edu
82 GLENN
IRES
ARFP
p 4
5' C E1 E2 7 NS2 NS3 A NS4B NS5A NS5B (U/UC) 3'
core
envelope
protease
?
helicase polymerase
structural non-structural (NS)

Fig. 1. Schematic of HCV genome. The 9.6-kb () strand RNA genome is translated beginning
from an IRES into a single polyprotein (indicated by a box), 59 and 39 non-coding regions con-
tain RNA elements required for genome replication. Individual proteins are liberated by a com-
bination of host signal peptidase (N-terminal cleavages) and viral protease activity (indicated by
arrows above polyprotein). Known activities of selected proteins are indicated below them.
a lipid envelope in which the two HCV envelope proteins, El and E2, are
embedded. A characteristic of all viruses that, like HCV, replicate via an
RNA-dependent RNA polymerase, is a relatively high rate of spontaneous
mutations (presumably due to the lack of an editing function of the poly-
merase). The resulting genetic heterogeneity means that the virus present
at any given time in an individual is best thought of as a population of re-
lated but slightly dierent and ever-changing genomes. This pool, or quasi-
species, provides the virus with an increased ability to respond to changing
selective pressures that arise from the hosts immune response or the admin-
istration of antiviral drugs. The heterogeneity is particularly pronounced in
certain regions of the genome, such as the hypervariable region of E2, and
can pose special challenges in the areas of vaccine development and drug
resistance.
Our understanding of the function of individual nonstructural proteins is
better for some than others. For example, based in part on the presence of
characteristic amino acid sequence motifs, enzymatic activities for NS3 and
NS5B were deduced early on. Thus, NS3 has a protease activity responsible
for liberating itself and the downstream nonstructural (NS) proteins from
the polyprotein precursor, and NS5B encodes the catalytic activity for
RNA-directed RNA polymerization. The precise roles of other NS proteins,
such as NS4B and NS5A, in the HCV life cycle have been less well dened.
However, they have some interesting properties, are critical for RNA repli-
cation, and are yielding attractive new targets for anti-HCV strategies.
A simplied schematic of the viral life cycle is presented in Fig. 2. Even
after 15 years of intensive research since the rst molecular cloning of the
HCV genome, many critical details remain to be lled in. The precise mech-
anism of viral entry has not been dened, although several candidate
MOLECULAR VIROLOGY OF HCV 83
Fig. 2. Schematic of HCV life cycle. An HCV particle attaches and gains entrance to its host
cell. This is thought to involve a specic receptor(s) and membrane fusion event. The use of dot-
ted lines for various stages of the life cycle is meant to convey that many mechanistic details
remain to be claried.
receptors, or co-receptors, have been proposed [912]. Upon entry, uncoat-

ing of the viral genome occurs and it gains access to the host cells transla-
tional machinery by use of an internal ribosome entry site (IRES) near the 59
end of the genome. Initial translation and protcolytic processing occur on
the endoplasmic reticulum. A novel intracellular membrane structure
termed the membranous web is established and seems to be the platform
upon which RNA replication occurs [13]. A minus strand RNA is made rst
that serves as template for the transcription of progeny plus strand RNA ge-
nomes, which are packaged and released in the form of new virions. The ma-
jor steps in these processes occur in the host cells cytoplasm. Several viral
proteins localize to lipid droplets and, under certain circumstances, may
be able to gain access to the cells nucleus, although the signicance of these
events for the viral life cycle is not clear.
Molecular virology and antiviral implications

Current therapy for HCV consists of interferon (IFN) and ribavirin
(RBV). These agents were already developed for other indications before
HCV was rst identied. In that sense they are not specic for HCV. Al-
though IFN has anti-HCV activity by itself, the benet of RBV is achieved
84 GLENN
only when it is used in combination with IFN. In light of their nonspecic

nature, it is somewhat remarkable that impressive improvements in response
rates have been achieved with these agents. This can be mainly attributed to
the optimization of formulations and regimens and to the use of combina-
tion therapy. This therapy remains expensive, lengthy, and with signicant
side eectsdall of which combine to limit its application to many patients.
More importantly, current therapy is frequently ineective.
Ultimate pharmacologic control of HCV is likely best achieved by a cock-
tail consisting of multiple agents against independent, virus-specic targets.
Improved understanding of HCV molecular virology can play a critical role
in helping to develop this more eective therapy. Such knowledge can be
translated into new antiviral strategies by increasing the repertoire of poten-
tial targets that may serve as the basis for novel therapies.
One can consider three generations of anti-HCV therapy (Fig. 3). The
rst consists of currently approved (and nonspecic) drugs (eg, IFN and
RBV), The second generation of therapy includes agents that for the rst
time are designed against HCV-specic targets. Several such compounds
are the subject of active preclinical drug development programs. Finally,
an increasing number of promising new targets are being identied at an ex-
citing pace. These are expected to form the basis of a third generation of
therapydtherapies of the future. In the following section, a survey of indi-
vidual viral elements is presented, focusing on potential implications for the
design of novel anti-HCV strategies.
Fig. 3. Three generations of anti-HCV therapy can be dened. First generation therapies are
those that are currently approved, consist of interferon and ribavirin, were available before
the identication of the HCV genome, and are thus not really specic for HCV. Second gener-
ation agents are those that are specically designed against HCV targets (such as the protease
and polymerase) and are in various stages of advanced development. Third generation agents
consist of the therapies of the future, and increased knowledge of HCV molecular virology is
expected to expand their number. Ultimate eective pharmacologic control of HCV is likely
to result from combination therapy with a cocktail of multiple drugs, each targeting an indepen-
dent virus-specic function.
Core protein
The rst product to be liberated from the HCV polyprotein is the core
protein. The latter has been extensively studied in a variety of heterologous
expression systems and has been shown to interact with a number of impor-
tant host cell intraccllular signaling pathways [1418]. The precise role of
these interactions in HCV pathogenesis and replication needs to be further
claried, but this raises the possibility that specic inhibition of cores inter-
face with these signaling pathways may have important therapeutic eects.
Another core function is its presumed role in assembly. Recombinant
core protein has the ability to assemble into higher-order particles [19,20],
suggesting the existence of an ordered capsid structure. By analogy with
HBV [21], agents capable of interfering with core particle assembly can be
imagined, although much needs to be learned about natural HCV particle
morphogenesis.
Many key proteins involved in intracellular signaling are localized to
a specialized subdomain of host cell membranes termed lipid rafts. These
lipid rafts, or detergent-resistant membranes (DRMs), are dened by their
enrichment in cholesterol and resistance to solubilization with cold, non-
ionic detergents. Not only do lipid rafts serve as a platform for intracellular
signaling, but a variety of viruses exploit lipid rafts to help mediate their as-
sembly [22]. Recently, it was demonstrated that a subpopulation of HCV
core protein in cells harboring full-length HCV replicons is biochemically
associated with DRM in a manner similar to markers of classical lipid rafts
[23]. This nding provides a basis for how core may accomplish its signaling
and assembly functions. Moreover, the mechanism by which core protein
associates with its lipid raft domain might oer a new target for antiviral
intervention.
Alternate reading frame protein

All of the HCV proteins mentioned so far are translated o of one long
open reading frame. It has recently been shown that at least one protein can
also be translated in a second reading frame o the same HCV genomic
RNA template. This later protein, termed alternate reading frame protein
(ARFP), comes about as a result of a frameshift event in the core region of
the genome [24,25]. Antibodies and evidence of cell-mediated immunity [26]
against it have been found in HCV-infected patient sera. The function of the
ARFP in the HCV life cycledand validation of its requirement for produc-
tive infectiondawait further denition before ARFP can be considered a po-
tential antiviral target.
E1/E2
The HCV envelope proteins El and E2 are liberated from the viral poly-
protein by host signal peptidase and undergo extensive glycosylation. The
86 GLENN
two envelope proteins form a noncovalent heterodimer [27]. The membrane

translocation signal sequences for El and E2 are contained within the C-
terminal ends of core and El, respectively [28,29]. Upon cleavage by signal
peptidase, the El and E2 C-termini undergo a reorientation in the endoplas-
mic reticulum (ER) membrane toward the cytosol [29].
The surface envelope proteins of viruses have frequently been used as
classic elements of traditional vaccines. Eective, broad-spectrum, neutraliz-
ing antibodies against HCV using this traditional approach have been di-
cult to obtain. This may be related to a high rate of viral mutation of
epitopes targeted by the host humoral response. Research in this area has
been hampered by the lack of an ecient culture system capable of produc-
ing, and being infected with, HCV virions. The ability to make chimeric, or
so-called pseudotyped, viruses having a surface coat of HCV envelope
proteins and inner replication machinery of a retrovirus [30] should help ac-
celerate the search for neutralizing therapeutics. The latter may include
drugs designed to inhibit the initial stages of viral attachment and entry. Ef-
fective vaccines of the future may also include other viral immunogens, in-
cluding ones selected from conserved epitopes in the NS proteins. This may
be important for stimulating optimal cellular immunity.
p7
This small (63-amino-acid) protein lies between the structural and non-
structural proteins. Similar to the viroporin M2 of inuenza A virus,
HCV p7 seems to function as an ion channel and is sensitive to inhibition
by amantadine [31]. As such, p7 may be well suited for small molecule in-
hibitors, especially because its function seems to be essential for in vivo
infectivity [32].
NS2
NS2 participates in the proteolytic cleavage reaction between itself and
NS3 [33]. It is not required for RNA replication in model culture systems
[34,35], but it may play a role in assembly. The protease reaction in which
NS2 participates is a potential target for inhibition whose importance
may be better appreciated with improved HCV culture systems capable of
producing infectious particles. In addition, NS2 may have distinct functions
when in a partially versus completely processed state. Such incompletely
processed precursors are known to play important roles in other positive-
strand polyprotein viruses.
NS3
NS3 is a 630-amino-acid protein that has two domains with dierent en-
zymatic activities that are thought to be essential for the HCV life cycle.
The amino terminal segment has a serine protease activity, whereas the
remaining two thirds have an RNA helicase activity. The protease activity is
most ecient when combined with NS4A, which serves as a noncovalent
cofactor and promotes the membrane association of NS3. This complex
is responsible for liberating the downstream NS proteins from the initial
polyprotein precursor.
The helicase activity is presumably required to help facilitate the unwind-
ing of duplex RNA during replication of the viral genome. The crystal struc-
tures of each isolated NS3 domain and the complete molecule with and
without the NS4A cofactor segment have been solved [3639]. This has
greatly facilitated the ability to perform rational drug design and optimiza-
tion. These atomic structures have also revealed some potential challenges
for obtaining potent inhibitors. For example, the active site of the protease
is a relatively shallow cleft without prominent features that readily lend them-
selves to facile design of highly selective complementary chemical entities.
Several companies have NS3 protease inhibitors in advanced develop-
ment. Two pharmacologically eective approaches have been taken. One
is based on the nding that the products of the NS3 protease reaction are
inhibitory to the enzyme. This has driven a peptidomimetric strategy
whereby the inhibitor resembles a peptide within the active site of the
NS3 protease. An oral-optimized inhibitor of this type was shown to de-
crease HCV titers by up to several logs in patients, providing successful
proof-of-concept [40]. Further clinical development of this compound has
been halted due to unanticipated cardiotoxicity in animal studies [41]. Al-
though this toxicity may not be related to NS3 inhibition per se but rather
to a compound-specic phenotype, it is likely that other NS3 protease inhib-
itors will be required to undergo cardiotoxicity screens.
A second strategy taken to develop NS3 protease inhibitors exploits the
fact that this viral protease is in the class of serine proteases. Here, a so-
called serine trap moiety, or warhead, capable of covalently binding the
catalytic serine, is incorporated into the nal compound [42].
Although the rst two NS3 protease inhibitors to enter human trials may
not be those to enter clinical practice, the relatively rapid development of
these compounds highlights the value that detailed structural knowledge
of the target can provide. This structural information should continue to
guide the development of new types of inhibitors, and it will be useful in
helping us to understand the nature of potential resistance to future
compounds.
The NS3 helicase function might also be considered an antiviral target
[43], assuming specicity for the viral enzyme can be achieved with respect
to related host cell enzymes [44].
NS4A
NS4A is small (54-amino-acid) HCV protein containing three distinct do-
mains. Its central domain mediates the above-mentioned role as a cofactor
88 GLENN
for NS3s protease activity. The hydrophobic N-terminal segment is respon-

sible for NS4As membrane targeting and the recruitment of NS3 to mem-
branes. The acidic C-terminal domain has recently been found to harbor
amino acids that are important for establishing RNA replication [45] and
that may dene an interaction with NS3 that might be targetable by rational
drug design. Finally, NS4A may have another function in the context of
a precursor protein in which NS4A remains linked to NS4B. Expression
of this precursor inhibited general ER to Golgi trac, including the trans-
port of newly synthesized MHC-I to the cell surface [46].
NS4B
NS4B is an HCV NS protein that until recently was characterized mainly
by its lack of known function. Individual reports have suggested that NS4B
might inhibit translation, modulate NS5B function, recruit other NS pro-
teins into a protected intracellular compartment with characteristics of lipid
rafts, or mediate cellular transformation [4750]. Perhaps most critical for
the integrity of the HCV life cycle is NS4Bs ability to induce the novel in-
tracellular membrane structures, termed membranous webs, that represent
the candidate sites for replication complex assembly (Fig. 4) [51]. NS4B
thus seems to play a key role in membrane-associated RNA replication. At-
tempts to understand the mechanisms underlying its function have led to the
identication of new potential targets for drug development.
Fig. 4. Electron micrograph of the membranous web. The membranous web is a novel intracel-
lular membrane structure that is induced by HCV and seems to be the platform upon which
replication occurs. The viral RNA and individual viral proteins involved in its replication local-
ize to this apparent collection of vesicles clustered together within a web of membranes. The
gure is an enlarged segment of a cell harboring the HCV polyprotein.
NS4B is tightly associated with intracellular membranes, and several pre-

dicted transmembrane domains have been proposed to account for this [52].
NS4B also contains a predicted N-terminal amphipathic helix (Fig. 5) that
can mediate membrane association in the absence of all predicted transmem-
brane domains. Moreover, the NS4B amphipathic helix (AH) seems to help
assemble the components of the HCV replication complex, and disrupting
the function of this AH abrogates HCV RNA replication [53].
NS4B has recently been shown to harbor a nucleotide binding motif
(NBM). This NBM mediates GTP binding and GTPase activity. The
GTPase activity seems to be essential for HCV replication [54]. Because
the NS4B NBM has some unique features that distinguish it from host
cell GTP-binding proteins, it represents an attractive target for drug devel-
opment. In addition, it may be amenable to small molecule inhibitor design.
Fig. 5. The N-terminus of NS4B contains an amphipathic alpha helix. This region of NS4B is
predicted to adopt an alpha helical secondary structure. It is depicted above in a helix net dia-
gram wherein the cylindrical alpha-helical segment is cut longitudinally along one face and
then attened into the plane of the page. The amino acid sequence of NS4B from amino acids
6 to 29 in the N-terminal to C-terminal direction is shown. Hydrophobic amino acids in the am-
phipathic helix are shaded. Note the long, continuous stretch of such amino acids along one
face of the helix, dening its amphipathic nature.
90 GLENN
NS5A
NS5A has been implicated in interactions with a variety of host cell intra-
cellular signaling and antiviral pathways. A lot of attention has focused on
its possible role in modulating the response to IFN treatment. As these in-
teractions become better dened and if they are found to be generalized
across HCV infections, they may yield new targets for therapy. A more im-
mediate target has emerged from studies focused on the mechanism of
NS5As membrane association and its role in HCV RNA replication.
All positive-strand RNA viruses studied to date have a common feature
of their replication strategy. The RNA-directed RNA replication occurs in
association with a specialized subset of intracellular membranes. For some
viruses, the latter are pre-existing membrane compartments such as endo-
somes, whereas other viruses induce novel membrane structures that become
their platform for replication. Because this is a feature critical for the virus
but not its host cell, this is a promising area for antiviral intervention.
Studying the mechanistic details of how HCV induces and maintains its
membrane-associated replication complex has begun to identify interesting
new potential anti-HCV strategies.
That NS5A might play a role in membrane-associated RNA replication
was suggested by the proteins ability to localize to a subset of host cell in-
tracellular membranes. Intriguingly, however, NS5A possesses no classical
transmembrane domains to explain how it localizes to membranes. The an-
swer to this puzzle turns out to reside in a small stretch of amino acids near
the proteins N-terminus. As found in NS4B, this segment consists of an am-
phipathic alpha helix. Unlike NS4B, NS5A has no other predicted trans-
membrane domains to account for its membrane association, and the
NS5A amphipathic helix has been proposed to anchor the protein by insert-
ing the hydrophobic face of the helix into the plane of the membrane [55]. If
the hydrophobic face is genetically disrupted, NS5A membrane association
is lost, and HCV RNA replication is abolished [56]. Pharmacologic inhibi-
tors that can similarly interfere with the ability of the AH to mediate mem-
brane association would oer a new approach to anti-HCV therapy. One
type of such inhibitors is a peptide mimic of the NS5A amphipathic helix
and is designed to compete with the viral NS5A for its membrane-binding
site [56]. Proof-of-concept for another type of peptide inhibitordone that
is a direct ligand of the NS5A amphipathic helixdhas been recently obtained
(Cheong KH, Smith R, Nolan GP, et al, unpublished observations).
NS5B
NS5B is another target for drug development because its RNA-depen-
dent RNA polymerization activity is a virus-specic feature; our cells are
not supposed to have such enzymes, and therefore the potential for selectiv-
ity exists. Moreover, like inhibitors of the viral protease, there is precedence
for a collection of drugs successfully targeting polymerases in other viruses,
such as HBV and HIV. The crystal structure of NS5B has also been solved
[57]. It shares several similarities to other viral RNA-dependent RNA
polymerases, although it has some unique features. Such detailed knowledge
of the proteins structure is invaluable to rational drug design and
optimization.
Nucleoside [58,59] and non-nucleosidc inhibitors [6063] have been devel-
oped. Structural and resistance-mapping studies have revealed that current
inhibitors fall into two distinct classes: those that directly target the en-
zymes active site and those that bind elsewhere on the protein and function
as allo-steric inhibitors.
Although NS5B encodes the catalytic activity for RNA polymerization,
the overall HCV replication complex is likely composed of other NS proteins
as well. Thus, inhibitors developed solely against puried NS5B may prove
disappointing when evaluated against actively replicating virus in cells.
Cis-acting elements
Specic sequence elements in the HCV RNA genome are being consid-
ered as potential antiviral targets. These so-called cis-acting elements
include the IRES and conserved RNA structural motifs in the HCV non-
translated regions (NTRs). In addition, at least one cis-acting sequence ele-
ment has been described within the coding region of NS5B [64]. Because
these are RNA targets, antisense and ribozyme-based strategies for drug de-
velopment have been initiated. siRNA technology [65] may also be suitable
for these targets, especially if the RNA targets are accessible enough during
the viral replication cycle. Achieving adequate delivery of nucleic acid-based
drugs may be the biggest obstacle. Small molecule or peptide ligands of
these RNA targets may also be considered.
Host targets
Recent work suggests that prenylation inhibitors may have activity
against HCV. Such compounds target the enzyme(s) responsible for attach-
ing post-translational prenyl lipid modications to proteins. The addition of
prenyl lipidsdor prenylationdis essential for the function of a variety of
proteins. When added to cells harboring HCV replicons, the prenylation in-
hibitor GGTI-286 prevented the formation of HCV replication complexes
and RNA replication, although the precise protein whose inhibited prenyla-
tion is responsible for this eect was not claried [66]. This represents an ex-
ample of a new approach to antiviral therapy [67]. In contrast to classical
antivirals that target a virus-specic enzyme, prenylation inhibitors target
a host cell enzyme and thereby seek to deprive the virus access to a host
cell function. One interesting consequence may be to make the development
of resistance a more dicult task because the targeted locus is not under ge-
netic control of the virus.
92 GLENN
Individual genes capable of modulating host antiviral mechanisms may

yield new targets for drag development. For example, IRF-3 has been shown
to be at a key intersection of the virushost response interface [68]. Drugs
designed to promote signaling through the IRF-3 pathway in HCV-infected
cells may help stimulate the host antiviral response. Immunomodulatory
therapy [69] and the use of growth factors are other areas of potential anti-
viral development, and the latter is covered in great detail in the article by
Curry and Afdhal elsewhere in this issue.
The above approaches and other agents which target host cell functions
are not necessarily specic for just HCV. In that sense, they take us partially
full circle, sharing features with current rst-generation agents.
Hepatitis C virus replicons

When considering eorts aimed at better understanding the HCV life cy-
cle and the identication of targets for drug development, it is imperative to
at least briey review the development of HCV replicons. The latter repre-
sent one of the most important advances since the rst cloning of the virus
and are becoming a standard feature of research in the eld.
The study of HCV replication has been hampered by the lack of a conve-
nient cell culture system. A major problem has been that neither virus
isolated from serum nor in vitro-transcribed RNA corresponding to the
full-length genome is capable of initiating ecient replication in cultured
cells. The rst breakthrough was provided by Lohman and colleagues [35].
They constructed a bicistronic RNA molecule derived from HCV in which
the virus structural genes were removed and replaced with a drug resistance
gene (neomycinphosphotransferase, or neo, which confers resistance
to the drug G418) (Fig. 6). This so-called subgenomic HCV replicon
(Fig. 6B) contains the HCV NS proteins thought to constitute the viral rep-
lication machinery and the conserved 59and 39 NTR regions at the ends of
the HCV genomic RNA, which are presumed recognition sequences for
the viral replication complex. Delivery of such a replicon to the human liver
tumor-derived Huh-7 cell line, followed by selection in G418, led to colonies
composed of individual cells each harboring replicating replicons that pro-
vided enough neomycin resistance to allow growth in the presence of the
selecting drug, G418. The eciency of colony formation was quite low,
approximately one colony for every 106 electroporated cells (Fig. 7A).
This was sucient, however, to permit the next important advancement.
Formally, the colonies obtained in such assays could result from a muta-
tion in the host Huh-7 cells, which confers resistance to G418 or allows in-
creased replicon replication. An example of the latter has recently been
described [70]. Many of the colonies are the result of a mutation that has
occurred in the replicon. This was rst shown by Blight and colleagues
[34], who isolated and sequenced the replicon RNA from individual colonies
Fig. 6. Schematic of HCV sub-genomic replicons. (A) The full-length HCV genome. (B) HCV
subgenomic replicon in which the HCV structural genes have been replaced with the neomycin
phosphotransferase gene, which confers resistance to the drug G418 (E-IRES, encephalomyo-
carditis internal ribosome entry site). (C) HCV subgenomic replicons are delivered to Huh-7
cells, and G418 selection is applied. Cells that do not take up replicons or in which the replicons
do not replicate (left and middle of gure) die in the presence of G418. Cells in which the repli-
cons do replicate, and thereby provide a source of neomycin phosphotransferase, survive and
multiply to yield a colony (right of gure).
and found that specic so-called adaptive mutations had occurred that al-
lowed the replicons to replicate with much greater eciency. By incorporat-
ing selected adaptive mutations into the original replicon of Lohman and
colleagues [35] and repeating the original experiment, they observed up to
Fig. 7. Isolation of high-eciency HCV subgenomic replicons. (A) The original HCV replicon
from Lohman and colleagues [35] was delivered to Huh-7 cells, and G418 selection was applied.
Rare colonies were obtained. (B) Sequencing of the replicons in the colony reveals a new mu-
tation (white dot). The new mutation is incorporated into a new stock of HCV replicons, and the
experiment is repeated. This time, colony formation eciency is increased by 4 logs [34].
94 GLENN
4 logs increase in colony formation eciency [34] (Fig. 7B). Although they
are not a complete substitute for a culture system permitting infection with
natural HCV virions, these second-generation, high-eciency replicons are
an important breakthrough. They open the prospect of performing detailed
molecular genetic studies and are ideal for the evaluation of candidate
drugs. An example of such genetic studies is shown in Fig. 8, where the eect
of disrupting the amphipathic helix of NS5A is tested. A replicon harboring
a mutated amphipathic helix (Fig. 8B) is compared with a wild-type replicon
(Fig. 8A). The dramatic eect of such mutation is readily apparent by the
lack of colonies on the mutant plate. One can imagine how a drug with po-
tential anti-HCV activity can be assayed in such a system.
Recent improvements in replicon technology include the incorporation of
the coding region of the structural proteins to yield so-called full-length
replicons [71] and the expansion of the range of host cells capable of harbor-
ing HCV replicons [72,73]. Finally, the recent reports of full-length genomes
capable of generating secreted HCV particles which can re-infect cells repre-
sent a potentially dramatic breakthrough, as they should enable an array of
exciting new research and drug development [7476].
Fig. 8. Eect of disrupting the NS5A amphipathic helix on HCV RNA replication. (A) A wild-
type, high-eciency HCV replicon (diagrammed at the top of the gure) was delivered to Huh-7
cells, and G4I8 selection was applied in a standard colony formation assay, the basis of which is
outlined in Figs. 6 and 7. The plate was stained with cresyl violet to reveal the presence of col-
onies. Numerous colonies were obtained, indicating good HCV genome replication (lower
panel). (B) A mutant replicon (diagrammed at the top of the gure) was constructed by intro-
ducing mutations (indicated by X) into the region of the high-eciency HCV replicon, which
encodes for the hydrophobic face of the NS5A amphipathic helix. This mutant replicon was
subjected to the same type of colony formation assay as in (A). As shown in the lower panel,
no colonies were obtained, indicating that HCV RNA replication was abrogated (lower panel).
These results genetically validate the NS5A amphipathic helix as a potential new antiviral target
(see Ref. [56] for additional details).
Summary
With the advent of second-generation agents that for the rst time specif-
ically target individual HCV proteins, HCV-specic therapy has arrived.
The study of HCV molecular virology has helped make this possible and
is helping us to identify additional new antiviral targets that will be targeted
by third-generation drugs. Key to these eorts is the development of high-
eciency HCV replicons. The future eective pharmacologic control of
HCV will likely consist of a cocktail of simultaneously administered virus-
specic agents with independent targets. This should minimize the emer-
gence of resistance against any single agent. The way we treat HCV should
change dramatically over the next few years.
Acknowledgments
This work was supported in part by R01-DK066793, R01-DK064223,
a Burroughs Wellcome Fund Career Development Award, and a Burroughs
Wellcome Fund Clinical Scientist Award in Translational Research.
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20 (2006) 99113
Antiviral Therapy for Treatment Na ve

Patients with Hepatitis C Virus
Sangik Oh, MD, MMSca,b, Nezam H. Afdhal, MDa,b,*
a
Department of Medicine, Harvard Medical School, 25 Shattack Street,
Boston, MA 02115, USA
b
The Liver Center, Beth Israel Deaconess Medical Center, 110 Francis Street,
Suite 8E, Boston, MA 02215, USA
Chronic hepatitis C virus (HCV) infection is recognized as a global health

problem, with 170 to 200 million people estimated to be infected worldwide.
In the United States, chronic HCV is the most common cause of end-stage
liver disease, hepatocellular cancer, and the most frequent indication for
liver transplantation [1]. Data from the third National Health and Nutrition
Examination Survey (NHANES III) estimated that 2.7 million Americans
have active HCV infection [2]. This gure probably underestimates the
true prevalence of chronic HCV infection, however, as the study excluded
high-risk groups such as prisoners and homeless people. HCV infection gen-
erally is regarded as a disease of decades, as the most signicant clinical
consequences occur 20 to 30 years after the initial exposure. Approximately
10,000 HCV related deaths occur each year, mostly resulting from end-stage
liver disease and development of hepatocellular carcinoma (HCC) [3]. The
NHANES study also revealed that HCV prevalence was the highest in per-
sons 30 to 49 years of age. Because of its slowly progressive natural history,
the Centers for Disease Control and Prevention (CDC) predict that HCV-
associated mortality might double or triple over the next 10 to 20 years
[4]. Davis et al estimated that the need for liver transplantation will increase
by 528%, and liver-related death might increase by 223% by 2008 [5]. One
logical solution is early detection and aggressive antiviral treatment to erad-
icate HCV or to halt disease progression.
of North America.
E-mail address: nafdhal@bidmc.harvard.edu (N.H. Afdhal).
100 OH & AFDHAL
This article focuses on the most recent therapies for patients with HCV
who are na ve to therapy. The primary end point for the treatment of na ve
HCV patients is viral eradication or a sustained virological response (SVR),
which is dened as the absence of HCV in the serum, as detected by a sensi-
tive polymerase chain reaction (PCR) test, 24 weeks after stopping antiviral
therapy. Recent data suggest that an SVR can be equated with a biochemi-
cal, virological, and histological response that is sustained for up to 5 years
and is conceptually a cure of HCV in 90% of patients [6]. Some patients fail
to clear HCV by PCR while on treatment, and these are classied as nonre-
sponders (NR). Additionally, there is a nal group of relapsers, who are able
to clear HCV on treatment, but in whom HCV reappears within 24 weeks of
stopping treatment. These denitions of patient responses are used in the
clinical trials discussed in this article.
Patient selection
The indications for therapy for na ve patients have undergone many re-
visions over the last 10 years (Box 1). Patients who are HCV-positive by
PCR test with an elevated alanine aminotransferase (ALT) and necro-
Box 1. Indications and contraindications to therapy

Indications
Elevated/normal ALT
Necro-inflammation and fibrosis on liver biopsy
Hepatitis C virus RNA positive
Extrahepatic manifestations (renal disease, cryoglobulinemia,
porphyria cutanea tarda)
Patient preference
Contraindications
Absolute
Severe uncontrolled depression
Unstable cardiac disease
Uncontrolled concomitant disease
Pregnancy
Hemolytic diseases
Renal disease; creatinine greater than 2 mg/dL
Seizure disorder
Relative
Active drug and alcohol use
Moderate depression/situational suicidal ideation
Decompensated liver disease
Autoimmune diseases
ANTIVIRAL THERAPY FOR TREATMENT NAIVE PATIENTS 101
inammation and brosis on liver biopsy are obvious candidates for treat-
ment. At the National Institutes of Health (NIH) consensus conference in
2002, however, there was a strong movement toward expanding the number
of potential treatment candidates to include people with controlled depres-
sive illness, methadone users, and patients who had stopped using alcohol
recently. The other controversial area is whether to treat patients with per-
sistently normal ALT. In the era of interferon (IFN) monotherapy, several
case series suggested that patients with normal ALT might experience ALT
ares and have a reduced SVR rate if treated with IFN [7,8]. Larger studies
with IFN and ribavirin (RBV), however, have shown that ALT ares are
not common and that the SVR rate is similar in patients with normal
ALT compared with those with elevated ALT [9,10]. The current recom-
mendation is that treatment of patients with normal ALT be considered
on an individual basis, and it is believed that these patients will respond
equally well to IFN and RBV combination therapy.
There remain some absolute contraindications to treatment, and these are
listed in Box 1. In particular, therapy should not be undertaken in patients
with decompensated liver disease and with active manic depressive disease
or recent suicidal ideation. RBV is contraindicated specically in patients
with hemolytic disease, unstable cardiac disease, renal disease with a creati-
nine above 2.0 mg/dL, and in patients who are pregnant or contemplating
pregnancy.
Historical perspective
The ecacy of alpha interferon for treatment of HCV rst was recog-
nized when Hoofnagle et al published their preliminary ndings in 1986
when HCV was known as non-A, non-B hepatitis [11]. They treated 10 pa-
tients with chronic non-A, non-B hepatitis with varying doses (0.5 to 5 mil-
lion U) up to 12 months. The aminotransferases levels improved in 8 of 10
patients and in 3 patients who had follow-up biopsies done after 1 year of
therapy showing marked improvement in liver histology. Several subsequent
studies, including a large, multi-center, randomized clinical trial in the
United States by Davis et al conrmed the initial nding that long-term
IFN therapy improved liver function tests and liver histology [12]. The
US Food and Drug Administration (FDA) approved alpha interferon
monotherapy for the treatment of chronic HCV infection in 1993. In
1997, The NIH released the consensus statement recommending alpha inter-
feron monotherapy at a dose of 3 million U three times weekly for 48 weeks
as the standard of care for patients with chronic HCV infection. There are
three IFNs approved for monotherapy in the United States, including IFN
alfa 2b, IFN alfa 2a, and consensus IFN (CIFN). Multiple clinical trials of
IFN monotherapy have been published using dierent doses and schedules,
and overall there have been no major clinical dierences in SVR between the
dierent IFNs. Overall, the rates of SVR have been limited to 6% to 16%.
102 OH & AFDHAL
The optimal duration of treatment is 48 weeks for IFN monotherapy. In

view of this relatively low response rate, higher doses, longer duration, in-
duction doses, and dose escalation have been tried to increase SVR, but
none of these variations has been shown prospectively to signicantly in-
crease response.
Combination therapy of interferon with ribavirin

A major advance occurred when IFN was combined with RBV in the piv-
otal studies of McHutchison et al [13], and SVR was increased from 13% to
38%. RBV is a nucleoside analog that putatively acts by means of inhibition
of RNA-dependent RNA polymerase, depletion of intracellular guanosine
triphosphate pools, and by altering Th1 and Th2 cytokine balance in the
liver. Initial studies of RBV monotherapy in HCV resulted in biochemical
but not histological response [1416]. Two large randomized controlled clin-
ical trials comparing IFN/RBV combination therapy with IFN monother-
apy in HCV infection demonstrated a signicant improvement in SVR for
combination therapy (Fig. 1) [13,17]. Combination IFN/RBV therapy be-
came the standard of care for na ve patients, with a 24-week course of
70
60
50
S 40
Geno 1
V 30 Geno Non-1
R 20
10
0
IFN 24 IFN 48wks IFN/RBV IFN/RBV
wks 24wks 48wks
Fig. 1. SVR rates according to genotype for na ve patients receiving IFN alfa-2b versus com-
bination IFN alfa-2b plus 1000/1200 mg ribavirin daily. In genotype non-1 patients, 24 weeks of
combination IFN/RBV was equivalent to 48 weeks with a 62% SVR. For genotype 1 patients,
SVR was 29% with 48 weeks of combination therapy (P less than 0.008 compared with IFN
monotherapy for 48 weeks). (Data from McHutchison JG, Gordon SC, Schi ER, Shiman
ML, Lee WM, Rustgi VK, et al. Interferon alfa-2b alone or in combination with ribavirin as
initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group. N Engl J
Med 1998;339(21):148592; Poynard T, Marcellin P, Lee SS, Niederau C, Minuk GS, Ideo G,
et al. Randomised trial of interferon alfa-2b plus ribavirin for 48 weeks or for 24 weeks versus
interferon alfa-2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C
virus. International Hepatitis Interventional Therapy Group (IHIT). Lancet 1998;
352(9138):142632.)
treatment for genotyped 2 and 3 patients and a 48-week course for genotype
1 patients.
Pegylated alfa interferon monotherapy

Pegylation is a process by which a polyethylene glycol molecule is at-
tached to a protein or drug to decrease renal clearance and increase bioavail-
ability and ecacy. The renal clearance and short half-life of alfa interferons
necessitated their administration three times a week and made them an ex-
cellent target molecule for pegylation. A critical concept with pegylation of
biologically active proteins is the need to achieve a balance between increas-
ing the half-life while maintaining biological activity. Increasing the size of
the pegylated component can decrease renal clearance but also reduces the
biological activity of the IFN component. There are two forms of peginter-
ferons that have been approved by the FDA for use in chronic HCV; PEG-
IFN 2a (Pegasys) IFN and PEG-IFN 2b (PEG-INTRON). PEG-IFN 2a
has a 40kDa branched chain PEG attached to IFN alfa 2a, and PEG-
IFN 2b has a 12kDa straight chain PEG attached to IFN alfa 2b. Both
PEG-IFNs retain reduced but eective biological activity while having a fa-
vorable pharmacological prole that allows for once weekly administration.
These PEG-IFNs have replaced IFN as the preferred form of IFN for use in
treatment of HCV infection.
Clinical trials have conrmed a twofold increase in ecacy of PEG-IFNs
compared with standard IFN in HCV treatment-na ve patients (Table 1).
Zeuzem et al showed that in the SVR was 39% in patients treated with
PEG-IFN 2a at a dose of 180 mg for 48 weeks compared with 19% in pa-
tients taking standard interferon 2a at a dose of 6 mU three times weekly
for 12 weeks then 3 mU three times weekly for 36 weeks [18]. In this trial,
62% of the subjects had genotype 1 HCV, and 7% had cirrhosis. In their
multivariate analysis, use of PEG-IFN 2a, younger age, smaller body sur-
face area, absence of cirrhosis or bridging brosis, a lower HCV RNA level,
and a nongenotype 1 infection were associated with higher SVR. Subsequent
studies using PEG-IFN 2a have shown a somewhat reduced SVR of 29%.
The results are similar in a study from Lindsay et al comparing there
doses of PEG-IFN 2b (0.5, 1.0 and 1.5 mg/kg per week) with standard
IFN alfa 2b at 3 mU three times weekly for 48 weeks [19]. The SVR was
only 12% with standard IFN but 18%, 25%, and 23%, respectively with es-
calating doses of PEG-IFN 2b. In this study, two pretreatment variables
were associated with higher SVR when a multivariate analysis was per-
formed: nongenotype 1 and HCV RNA less than 2 million copies/mL.
In a dicult-to-treat population of na ve patients with advanced brosis,
dened as bridging brosis or cirrhosis, PEG-IFN 2a again demonstrated
superior SVR compared with IFN alfa 2a. Heathcote et al randomized
271 patients with cirrhosis or bridging brosis into one of three groups:
standard IFN alpha-2a at a dose of 3 mU three times weekly; PEG-IFN
104
Table 1
Randomized controlled trials of peginterferon monotherapy
Study Zeuzem et al, 2000 Heathcote et al, 2000 Lindsay et al, 2001
Type of IFN IFN a-2a PegIFN a-2a IFN a-2a PegIFN a-2a PegIFN a-2a IFN a-2b PegIFN a-2b PegIFN a-2b PegIFN a-2b
Dose and 6 mU for 180 mg for 3 mU for 90 mg for 180 mg for 3 mU for 0.5 mg/kg/wk 1.0 mg/kg/wk 1.5 mg/kg/wk
duration 12 weeks, 48 weeks 48 weeks 48 weeks 48 weeks 48 weeks
OH & AFDHAL
then 3 mU
for 36 weeks
Number of 264 267 88 96 87 303 315 297 304
patients
Sustained 19% (14 39% (33% 7% (4 15% (9 30% (21% 12% (9% 18% (14% 25% (20 23% (19%
virological to 24%) to 45%) to 16%) to 23%) to 40%) to 16%) to 23%) to 30%) to 28%)
response
(95% CI)
2a at a dose of 90 mg; and PEG-IFN 2a at a dose of 180 mg for 48 weeks [20].

The SVR was 30% with PEG-IFN 2a at 180 mg compared with 8% for stan-
dard IFN alfa 2a.
In summary, the pegylation of interferons has resulted in improving the
pharmacokinetic (PK) prole, allowing for once weekly administration
while improving the biological activity and doubling the SVR rates for
IFN alfa 2a and 2b.
Pegylated interferon and ribavirin combination therapy

A logical extension of the PEG-IFN monotherapy trials was to combine
PEG-IFN with RBV, and two large prospective studies have compared
combination PEG-IFN/RBV with IFN/RBV. Both trials have demon-
strated a signicant benet of the PEG-IFN/RBV combination over stan-
dard combination therapy, and PEG-IFN/RBV is the standard of care for
na ve chronic HCV patients currently.
Manns et al conducted a randomized clinical trial with PEG-IFN 2b in-
volving 1530 HCV patients from Europe, North America, and Australia
[21]. They were randomized into one of three groups: IFN alfa 2b (3 mU
three times weekly) with RBV (1000 to 1200 mg daily based on body weight)
for 48 weeks, PEG-IFN alfa-2b at 1.5 mg/kg/week with RBV 800 mg daily
for 48 weeks, or PEG-IFN alfa-2b at 1.5 mg/kg/week for 4 weeks and then
0.5 mg/kg/week for 44 weeks with standard doses of RBV. The group receiv-
ing highest dose of PEG-IFN alfa-2b with RBV achieved highest rate of
SVR (54%) when compared with the group that received lower dose of
PEG-IFN (47%) and standard IFN (47%) combined with RBV (Table 2).
Fried et al conducted a comparable study with PEG-IFN alfa-2a in com-
bination with RBV [22]. A total of 1121 patients were assigned randomly to
one of three groups: standard IFN alfa-2b (3 mU three times weekly) with
RBV (1000 to 1200 mg daily based on body weight), PEG-IFN alfa-2a (180
mg weekly) with RBV (1000 to 1200 mg daily based on body weight), or
Table 2
Results from the two clinical trials of peg-interferon and ribavirin
Study Manns et al 2001 Fried et al 2002
Interferon INF a-2b PegIFN PegIFN IFN a-2b PegIFN PegIFN
Regimen 3 mU tiw a-2b 1.5 mg/ 1.5 mg/ 3 mU tiw a-2a a-2a 180 mg
kg/wk kg/wk 180 mg
Ribavirin 1000 or 1000 or 800 mg 1000 or Placebo 1000 or
1200 mg 1200 mg 1200 mg 1200 mg
Number of 505 514 511 444 224 453
patients
Sustained viral 47% 47% 54% 44% 29% 56%
response (42 (43% (49% (40% (24% (52%
(95% CI) to 51%) to 52%) to 58%) to 49%) to 36%) to 61%)
Abbreviation: tiw, three times weekly.
106 OH & AFDHAL
PEG-IFN alfa-2a (180 mg weekly) with placebo. All patients were treated for
48 weeks. The SVR was the highest in the group that received PEG-IFN in
combination with RBV (56%) compared with IFN alfa 2b and RBV (45%)
and PEG-IFN alfa 2a alone (30%) (Table 2).
A third large randomized controlled study has been performed by Had-
ziyannis et al [23]. The study design was to examine the eect of duration of
therapy (24 versus 48 weeks) and of RBV dose (800 mg versus 1000 or 1200
mg) on SVR using xed-dose PEG-IFN alfa 2a. A total of 1284 patients
were stratied, based on their genotype and pretreatment viral load, and
randomized into one of four groups: PEG-IFN alfa-2a (180 mg weekly)
and RBV at 800 mg daily for 24 weeks, PEG-IFN alfa-2a (180 mg weekly)
and RBV at 1000 or 1200 mg daily for 24 weeks, PEG-IFN alfa-2a (180 mg
weekly) and RBV at 800 mg daily for 48 weeks or PEG-IFN alfa-2a (180 mg
weekly) and RBV at 1000 to 1200 mg daily for 48 weeks. The genotype-specic
SVR is shown in Fig. 2 for the four groups. In patients with genotype 1,
the highest SVR was obtained in the group that was treated with highest
dose of RBV for the longer duration. In contrast, the SVR did not dier
signicantly among patients with genotypes 2 or 3 regardless of dose of
RBV or the duration of therapy. These ndings suggest that patients with
genotypes 2 and 3 can be treated eectively with PEG-IFN and lower doses
of RBV (800 mg) for 24 weeks, whereas patients with genotype 1 should be
treated with higher doses of ribavirin (1000 to 1200 mg) for a longer duration.
Pretreatment predictors of response

Analysis of the clinical trials has identied pretreatment predictors of re-
sponse to PEG-IFN and RBV (Box 2). The major predictor is genotype,
with genotype 1 patients having a markedly reduced response (42% to
46%) compared with patients with genotypes 2 and 3 (76% to 82%). After
genotype, multiple other factors have lesser predictive value but can still be
clinically important when one is individualizing therapy. Clinically relevant
predictors that indicate a more dicult-to-treat patient include viral load
greater than 2 million by PCR test, presence of advanced brosis or cirrho-
sis, high body mass index, and African American race. Posthoc analysis of
the clinical trials has suggested that PEG-IFN alfa 2b, which is dosed on
a weight basis at 1.5 mg per kg, may be more eective in patients with
a body weight of greater than 75 kg. Similar analyses have suggested that
PEG-IFN alfa 2a may be better for genotype 1 patients with high viral
load. All analyses suggest that a weight-based dose of RBV should be
used for genotype 1 patients, and the authors recommend 12 to 15 mg of
RBV per kilogram.
Early virological response

Although useful, the baseline predictors highlighted are a general guide-
line for discussion with patients as to the likelihood of response to
Genotype 1
SVR %
100
80 24 Weeks 48 Weeks
Treatment Treatment
60
51
41 40
40
29
N=101 N=118 N=250 N=271
20
0
PEG-IFN -2a PEG-IFN -2a PEG-IFN -2a PEG-IFN -2a
180 g + RBV 180 g + RBV 180 g + RBV 180 g + RBV
800 mg 1000-1200 mg 800 mg 1000-1200 mg
Genotype 2 and 3
SVR %
100 24 Weeks 48 Weeks
Treatment Treatment
82 81
80 76 76
60
40
20
N=96 N=144 N=99 N=153
0
PEG-IFN -2a PEG-IFN -2a PEG-IFN -2a PEG-IFN -2a
180 g + RBV 180 g + RBV 180 g + RBV 180 g + RBV
800 mg 1000-1200 mg 800 mg 1000-1200 mg
Fig. 2. SVR for four dierent treatment regimens of PEG-IFN alfa-2a with either RBV 800 mg
or 1000/1200 mg given for 24 or 48 weeks. Optimal SVR for genotype 1 is seen with 48 weeks
treatment with RBV 1000/1200mg daily. For genotypes 2 and 3, 24 weeks with 800 mg of RBV
are equivalent to longer duration of therapy and higher RBV doses. (Data from Hadziyannis SJ,
Cheinquer H, Morgan T, Diago M, Jensen DM, Sette H. Peginterferon alfa 2-a (40kD)
(PEGASYS) in combination with ribavirin (RBV): ecacy and safety results from a phase III
randomized double-blind multi-centre study examining eect of duration of treatment and
RBV dose. J Hepatol 2002;36(Suppl 1):3.)
108 OH & AFDHAL
Box 2. Factors predicting increased SVR

Fixed
Genotype 2,3
Low viral load (less than 2 million copies/mL)
No or minimal fibrosis
Achieving EVR
Female gender
Short duration of disease
Race (non-African American)
Variable
Increasing physician experience
PEG-IFN/RBV therapy (weight-based dosing; 24 weeks for
Genotype 2,3; 48 weeks for Genotype 1)
Adherence to therapy
treatment. Once treatment starts, however, one can use viral kinetic re-
sponses in the early stages of treatment to predict response. This is applica-
ble to genotype 1 patients where early virological response (EVR) has been
used to predict who is unlikely to have an SVR, a so-called stop rule. The
advantages of EVR are that it reduces the morbidity and cost of treatment
by stopping treatment in patients who are unlikely to respond [24]. To use
viral kinetics, however, one needs to have reproducible and quantitative
measurement of HCV RNA with no more than 0.5 log10 variability. Recent
PCR tests are able to provide this accurate level of HCV RNA quantitation
enabling physicians to use EVR for treatment decisions. The other impor-
tant factor is to determine the primary goal of treatment, which is whether
SVR alone is the endpoint of treatment or whether secondary benets of
therapy such as slowing disease progression or improving liver histology
are applicable in the individual patient.
Early virological response is dened for treatment with both PEG-IFNs
and RBV as either a 2 log10 unit reduction or loss of detectable HCV RNA
at 12 weeks. When EVR does not occur, the chance of SVR being achieved
by continuing the same treatment for 48 weeks is reduced to 1% to 3%, and
an individualized decision can be made as to whether to stop treatment. For
those patients who achieve EVR, there is an increased likelihood (65% to
70%) that they will have an SVR (Table 3).
Adherence to treatment
Another factor that has been reported to eect outcome is whether pa-
tients are able to tolerate therapy and are adherent to the planned treatment
dose and duration. McHutchison looked at the eect of adherence in
Table 3
Eect of achieving EVR on hepatitis C virus treatment response
Treatment response
Early virological response (n) SVR NR
PEG-IFN alfa2b RBV
EVR ACHEIVED (388) 70% 30%
No EVR (124) 1% 99%
PEG-IFN alfa2a RBV
EVR ACHIEVED (390) 65% 35%
NO EVR (63) 3% 97%
patients treated with IFN/RBV and used the simple adherence parameters
of whether patients were able to take 80% of their IFN, 80% of their
RBV for 80% of the time, the 80/80/80 rule [25]. Approximately 30% of pa-
tients were unable to adhere to the treatment regimen, usually secondary to
adverse eects of therapy. Inability to comply with therapy resulted in an
overall loss of response that was more marked in genotype 1 patients, where
SVR in the adherent group was 51% and fell to 34% in the patients who
were dose-reduced or stopped therapy prematurely (Fig. 3). Factors that af-
fect adherence include patient education and motivation, physician experi-
ence with adverse eect management, and positive reinforcement by
physicians to the patient. The most successful centers treating HCV patients
have incorporated these factors into overall management and often use phy-
sician extenders successfully in the role of primary caregivers to manage
HCV adverse eects.
Fig. 3. Eect of adherence on SVR in patients treated with Peg-IFN alfa 2b 1.5 mg/kg and RBV
800 mg. The left panel shows the SVR for patients on intention-to-treat analysis (ITT), with the
gray bars representing all patients and the black bars representing genotype 1 patients. Patients
compliant to treatment by 80/80/80 rule had an increased SVR. All patients 54% to 63%,
p 0.01; genotype 1 patients 42% to 51%, P greater than 0.03. (Data from McHutchison JG,
Manns M, Patel K, Poynard T, Lindsay KL, Trepo C, et al. Adherence to combination therapy
enhances sustained response in genotype-1-infected patients with chronic hepatitis C. Gastroen-
terology 2002;123(4):10619.)
110 OH & AFDHAL
Adverse eect management

The adverse eects of IFN and RBV are shown in Box 3, although a de-
tailed discussion is beyond the scope of this article. One of the most frequent
causes of dose reduction is the cytopenias associated with combination ther-
apy. Thrombocytopenia with platelet counts of less than 50,000 usually is
treated with dose reduction of IFN and is only seen in 1% to 4% of patients
[26]. Neutropenia with neutrophils less than 750 per cc is seen in up to 20%
of patients. No increased risk of bacterial infections has been reported with
neutropenia, but dose reduction of IFN should be considered. Alternatively,
use of growth factors such as granulocyte colony stimulating factor may be
considered. Finally, anemia is common with combination therapy, with
50% of patients having a reduction of 3 g/dL in hemoglobin or a fall in he-
moglobin to less than 12 g/dL [27]. Recent studies have shown that epoetin
alfa at 40,000 U weekly can maintain RBV dose, improve hemoglobin by
Box 3. Common adverse effects of interferon/ribavirin therapy

Interferon
Bone marrow suppression
Anemia
Thrombocytopenia
Neutropenia
Neuropsychiatric syndromes
Depression
Mood swings
Anxiety/mania
Neurocognitive dysfunction
Flulike syndrome
Nausea/gastrointestinal upset
Thyroid disease /thyroiditis
Alopecia
Neuropathy
Retinopathy
Injection site reactions
Ribavirin
Hemolytic anemia
Teratogenic
Dyspnea/cough
Rash/pruritis
Insomnia
Nausea/gastrointestinal upset
a mean of 2 g/dL, and improve quality of life in patients with IFN/RBV-in-

duced anemia [28,29].
Interferon also is associated with several neuropsychiatric syndromes, in-
cluding depression, mood swings, mania, anxiety, and neurocognitive dys-
function. Aggressive management of these side eects is necessary to
maintain compliance to therapy.
In summary, remarkable improvements have been achieved in ecacy of
treatment of na ve patients with HCV over the last decade, with SVR rates
going from 10% to 50% with the current recommended PEG-IFN/RBV
combination therapy. The success of therapy is dependent on many vari-
ables, and physicians who treat HCV should become very familiar with ad-
verse eect management to achieve the results in clinical practice that have
been demonstrated in the clinical trials to date.
Benets and cost eectiveness of antiviral therapy

Follow-up of patients who have had an SVR for up to 10 years suggests
that there is a persistent biochemical, virological, and histological remission
in 95% of patients. This probably represents a true cure of HCV. It remains
unknown if this translates into a reduction in the development of cirrhosis,
liver failure, and HCC or translates into an improvement in survival. Eco-
nomic and cost-eectiveness analyses have suggested that treatment of
HCV has future cost savings comparable with many other accepted medical
practices such as screening for colorectal cancer or hypertension and coro-
nary artery bypass graft [30]. Finally, patients who have an SVR also have
been shown to have signicant improvements in health related quality of
life, representing another strong argument for treating patients with HCV
[31].
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20 (2006) 115135
Approach to the Management of Patients

with Chronic Hepatitis C Who Failed to
Achieve Sustained Virologic Response
Amrita Sethi, MD, Mitchell L. Shiman, MD*
Hepatology Section, Virginia Commonwealth University Medical Center, Box 980341,
Richmond, VA 23298. USA
Therapy for chronic hepatitis C virus (HCV) infection has improved dra-
matically since interferon (IFN) was rst introduced for treatment of non-A,
non-B hepatitis over 15 years ago [13]. Historically, standard IFN mono-
therapy yielded a sustained virologic response (SVR) in less than 15% of pa-
tients. The addition of ribavirin (RBV) [4,5], and later the substitution of
peginterferon (PEGIFN) for standard IFN [69], led to dramatic improve-
ments in SVR rates, which can now be achieved in 45% to 50% of patients
who have HCV genotype 1 and approximately 80% of patients who have
genotypes 2 or 3 [1012]. As each new improvement in HCV therapy has
emerged, many patients who had failed to achieve SVR with previous,
less eective therapy have been retreated. Recently, several large multicenter
clinical trials have demonstrated the impact of retreating such patients with
PEGIFN/RBV [1316]. In the largest of these trials, 18% of patients who
had a nonresponse (NR) after treatment with IFN or IFN/RBV achieved
SVR after being retreated with PEGIFN/RBV. Certain subgroups, particu-
larly those with previous relapse, HCV genotypes 2 and 3, without cirrhosis,
or with low serum HCV RNA levels, faired better and seemed to be excel-
lent candidates for retreatment (Table 1). In contrast, African Americans
had a SVR of only 6%, and patients with multiple poor prognostic factors
(HCV genotype 1, high serum HCV RNA levels, cirrhosis, and previous NR
to IFN/RBV therapy) had poor SVR rates [13]. Given these ndings, a thor-
ough discussion regarding the pros and cons of retreatment should be
Supported by NIH contract N01-DK-9-2322.
E-mail address: mlshiffm@vcu.edu (M.L. Shiffman).
116 SETHI & SHIFFMAN
Table 1
Response to retreatment with peginterferon and ribavirin in patients who failed to achieve sus-
tained virologic response after treatment with interferon and ribavirin
Sustained virologic response
a
Prior relapse to IFN/RBV 4050%
Prior nonresponse to 1FN or IFN/RBVb
Race
Caucasian 20%
African American 6%
Genotype
1 14%
non-1 60%
Serum HCV RNA level:
!l.5 million IU/mL 31%
O1.5 million IU/mL 6%
Cirrhosis
Yes 11%
No 23%
Abbreviations: IFN, interferon; RBV, ribavarin.
a
Data from Ref. [14].
b
Data from Refs. [1315].
undertaken with each patient who failed to achieve SVR with IFN or IFN/
RBV.
The majority of patients who have received therapy for chronic hepatitis
C have been treated with PEGIFN/RBV as initial therapy or as retreatment
after previous NR or relapse. No alternative with proven superiority is
available for patients who have failed to achieve SVR after treatment with
PEGIFN/RBV. Although the management of these patients represents a for-
midable challenge, several approaches are available that have the potential
to yield SVR during retreatment or reduce the rate of disease progression.
This article outlines an approach for managing patients who have failed
to achieve SVR and reviews preliminary results of promising new therapies
for these patients.
Identifying reasons for treatment failure

A number of factors may have contributed to the development of relapse
or NR during previous therapy with PEGIFN and RBV. One possibility is
that the patient was responding to treatment, had a marked decline in serum
HCV RNA, and achieved early virologic response (EVR) or became HCV
RNA undetectable (ie, the patient had a virologic response [VR], but this
response was not recognized by the treating physician who subsequently dis-
continued therapy). Another possibility is that the physician signicantly re-
duced the dosage of PEGIFN or RBV in the rst several months of
instituting treatment in response to the development of various side eects
MANAGEMENT OF NONRESPONDERS 117
related to these medications. Finally, the patient may have been participat-
ing in behaviors that reduced the eectiveness of therapy. We refer to these
three categories as correctable factors; the most important aspect of as-
sessing a patient who has NR is to look for these factors because they can
often be corrected before or during retreatment (Box 1). The successful cor-
rection of one or more of these factors has the potential to result in SVR
during retreatment.
Failure to recognize virologic response

The use of testing for HCV RNA is essential to the diagnosis of HCV in-
fection and to monitoring patients during therapy. Three basic methods are
available to assess serum HCV RNA: polymerase chain reaction (PCR),
branched chain DNA (b-DNA), and transmission mediated amplication
(TMA). These assays, their limitations, and the proper manner by which vi-
rologic assays should be used to monitor HCV RNA during therapy were re-
cently reviewed [17]. VR cannot be identied and patients cannot be
appropriately monitored if the various HCV RNA response patterns that
occur during therapy are not recognized. The denitions of VR were
Box 1. Factors that may increase the likelihood of sustained

virologic response if corrected before or during retreatment
Failure to recognize virologic response
HCV RNA not assessed during treatment
HCV RNA not measured at the correct time during treatment
Miscalculating the changes in HCV RNA from baseline during
treatment
Failure to account for natural variation in HCV RNA assays
Overly aggressive dose reduction
Significantly reducing the doses of PEGIFN and RBV during the
first 24 weeks of treatment or stopping ribavirin before therapy
is complete for the following reasons:
Anemia
Neutropenia
Thrombocytopenia
Severe flu-like symptoms
Neuropsychiatric side effects
Thyroid disease
Noncompliant behavior
Continued alcohol use during treatment
Continued illicit narcotic drug use during treatment
Missed doses secondary to work or family/personal issues
developed during two National Institutes of Health (NIH) Consensus De-

velopment Conferences in 1997 and 2001 [18,19]; a graphic depiction of
these denitions is provided in Fig. 1. Dierentiating EVR, the null re-
sponse, and partial VR is particularly important. EVR is dened as a
2-log (100-fold) or greater decline in the level of serum HCV RNA from
the pretreatment baseline level or an undetectable HCV RNA 12 weeks after
initiating IFN therapy. Virtually all patients with genotype 2 or 3 and 80%
of treatment-naive patients with genotype 1 achieve EVR during PEGIFN/
RBV therapy (Ferenci et al, submitted for publication, 2004) [20]. Approx-
imately 65% of patients with an EVR achieve SVR. In contrast, SVR rarely,
if ever, occurs in the absence of EVR.
VR occurs when a patient has an undetectable serum HCV RNA at any
time during treatment. VR occurs in over 95% of patients who have geno-
types 2 or 3 and in approximately 65% to 70% of patients who have geno-
type 1 by week 24 (Ferenci et al, submitted for publication, 2004). Only
patients who achieve VR have the ability to develop SVR. However, ap-
proximately 10% of patients who have partial VR and detectable HCV
RNA at treatment week 24 can become HCV RNA undetectable and
achieve VR by week 48 if treatment is continued (Ferenci et al, submitted
for publication, 2004). Although none of these patients will achieve SVR,
the ability to become HCV RNA undetectable is potentially important for
patients with chronic hepatitis C and advanced hepatic brosis or cirrhosis.
The use of maintenance therapy for such patients is discussed below.
Null response is dened as the failure to achieve a 2-log decline in serum
HCV RNA during the rst 12 weeks of therapy. Patients with a null re-
sponse rarely if ever have further decline in serum HCV RNA with contin-
ued therapy and are unlikely to respond during retreatment. Partial VR is
dened by a 2-log or greater decline in serum HCV RNA from the pretreat-
ment baseline value within 12 weeks but failure to become HCV RNA neg-
ative by treatment week 24. As opposed to the null response, patients who
have a partial VR are excellent candidates for retreatment particularly if the
8
7 Peginterferon/Ribavirin
HCV RNA (IU/ml)
Relapse
6
5 Null Response
2-log decline
4
3 Partial Response
Limit of
2 Detection
Virologic response SVR
1
0
-6 0 6 12 18 24 30 36 42 48 54 60 66 72 78
WEEKS
Fig. 1. Patterns of serum HCV RNA response during treatment.
factor or factors responsible for the failure to achieve VR can be identied

and corrected.
In 1999, a universal standard for reporting serum HCV RNA was devel-
oped and implemented [17,21]. When reporting in international units, the
most commonly used assays seem to be consistent with each other in their
ability to measure serum HCV RNA [22,23]. However, like any other test,
the values reported by any of these HCV RNA assays are subject to variabil-
ity. The accuracy of the commercially available HCV RNA assays is re-
ported to be G0.5 log units [17,24,25], and this variation must be taken
into account when assessing patients for EVR, as illustrated in Fig. 2. In
this example, the patient had a baseline HCV RNA value of log 6 IU/mL
(1 million IU/mL). Given the variability of the assay (G0.5 log units), the
HCV RNA level may have been as high as log 6.5 IU/mL (3,162,278
IU/mL) or as low log 5.5 IU/mL (316,228 IU/mL). At 12 weeks of treat-
ment, the HCV RNA level had declined by 2 log units to log 4 IU/mL
(10,000 IU/mL), but, given the assay variability, the actual HCV RNA level
could have been anywhere between log 4.5 IU/mL (31,623 IU/mL) and log
3.5 IU/mL (3162 IU/mL). Thus, EVR or a 2-log decline in the HCV RNA
level from baseline could have occurred with a decline in HCV RNA of as
little as 1 log unit, from log 5.5 IU/mL (316,000 IU/mL) to log 4.5 IU/mL
(31,600 IU/ml), or as great as 3 log units, from log 6.5 IU/ml (3.16 million
IU/ml) to log 3.5 IU/ml (31,600 IU/mL). Failure to recognize the variability
in HCV RNA assays may lead the treating physician (or the patients insur-
ance carrier) to discontinue the patients therapy at week 12 for falling just
short of the 2-log decline in HCV RNA from the pretreatment baseline
value (Fig. 3A). This scenario is one of the important correctable factors
to look for when assessing patients who have NR.
10,000,000
1,000,000
HCV RNA (IU/ml)
1 log 2 log 3 log

100,000
10,000
1,000
100 Limit of detection
10
Baseline Week 12
Fig. 2. Variations in serum HCV RNA results and how this can aect the ability to identify
EVR. HCV RNA assays vary by G0.5 log units, the range of which is highlighted by the shaded
circles within the rectangular boxes. Because of this variability, a 2-log decline in the HCV RNA
level may have occurred with only a 1-log decline and up to a 3-log unit decline in serum HCV
RNA. This variability needs to be accounted for when assessing a patient for EVR during
treatment.
A B
7 7
Log HCV RNA (IU/ml)
Log HCV RNA (IU/ml)

6 6
5 5
4 4
3 3
Limit of
2 2 detection
Limit of detection
1 1
0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Time Time
Fig. 3. Example of how more frequent testing of serum HCV RNA, particularly during the rst
3 to 6 months of treatment, can improve recognition of EVR, VR, and the null response.
(A) This patient was having a stepwise decline in HCV RNA at 1 and 2 months, but the value
at 3 months was slightly higher and fell just short of a 2-log decline from baseline (arrow). By
measuring serum HCV RNA monthly during the rst 3 months of treatment, it was recognized
that the patient already had a 2-log decline in serum HCV RNA at month 2 and that the ele-
vation at 3 months could have represented assay variation. Treatment was continued, and the
patient achieved VR by month 6. If only one HCV RNA value had been obtained at month 3
(arrow), treatment would have been discontinued, and the patient would have been labeled as an
NR. (B) This patient had become HCV RNA undetectable by treatment month 2 but was HCV
RNA positive at treatment month 6 (arrow). If HCV RNA had not been measured at monthly
intervals, the patient might have been mischaracterized as an NR at month 6 and treatment dis-
continued. Because HCV RNA had been measured at monthly intervals, VR had been recog-
nized. The positive HCV RNA value at month 6 was a discordant value, and treatment was
continued.
Another important nuance of HCV RNA assays is the risk of false-

positive and false-negative values. Recent studies have suggested that this
may occur in up to 1% to 2% of samples assayed and is independent of
the assay used [22,23]. Although false-negative results may be related to
the lower limit of sensitivity in some assays [17,24], most discordant results
seem to stem from sample processing errors that occur between the time
blood samples are obtained, frozen, shipped to the reference laboratory,
thawed, and pipetted into the amplication device used to measure HCV
RNA. The reporting of a false-positive result when the sample is HCV
RNA negative is another reason EVR or VR may not be recognized at
weeks 12 and 24, respectively (Fig. 3B).
The recent recommendations to measure serum HCV RNA at baseline
and week 12 in a patient with genotype 1 and to use only these values to de-
termine EVR and whether treatment should be terminated or continued for
48 weeks may be insucient to assess for variations in the HCV RNA assay
or for the possibility of discordant results [26]. Examples of how more fre-
quent monitoring of serum HCV RNA, particular during the rst 3 to 6
months of therapy, can help the physician recognize EVR, VR, null
response, and discordant results are provided in Fig. 3. The inability to rec-
ognize EVR or VR because HCV RNA was assessed too infrequently and
treatment was discontinued prematurely is another potentially correctable
factor when assessing patients who were thought to have NR.
Dose reduction and premature discontinuation of therapy

Approximately 20% to 30% of patients treated with IFN or PEGIFN
and RBV develop adverse events sucient to warrant dose reduction in
one or both of these agents [1012]. The major reasons to dose reduce
PEGIFN include severe u-like symptoms, neutropenia, thrombocytopenia,
thyroid disorders, and psychiatric manifestations (eg, irritability and depres-
sion). The major reasons to dose reduce RBV include anemia, nausea, and
rash. Aggressive management of these side eects has the potential to limit
the number of patients who dose reduce or prematurely discontinue therapy
[27].
Reducing the dose of PEGIFN or RBV seems to signicantly impair the
patients ability to achieve EVR and SVR, particularly in patients with ge-
notype 1 [13,2830]. The degree by which the dose of PEGIFN or RBV can
be reduced without adversely aecting SVR is controversial. In the rst
study to describe this association, SVR declined from 51% to 34% when
PEGIFN or RBV was reduced to !80% of the starting dose at any time
during the 48-week treatment course or if either of these agents was discon-
tinued before the patient received 80% of the prescribed duration of therapy
(ie, 36 weeks) [28]. This study assumed all dose reductions were equal and
did not evaluate the impact of dose reducing PEGIFN or RBV indepen-
dently or separate patients who dose reduced from those who prematurely
discontinued treatment. The most critical time period for dose reduction
seemed to be within the rst 12 and 20 weeks of therapy. Patients who
had a dose reduction before week 12 had a decline in SVR from 62% to
34%, whereas dose reduction after week 12 reduced SVR to only 51%
[28]. In other studies, dose reducing PEGIFN or RBV after week 20 in pa-
tients who were HCV RNA undetectable had no impact on SVR as long as
these agents were not stopped [13,29,30]. Preliminary data from a study of
over 1100 patients with previous NR to IFN or IFN/RBV undergoing re-
treatment with PEGIFN/RBV demonstrated that the RBV dose could be re-
duced signicantly during the rst 20 weeks without aecting SVR as long
as RBV was not discontinued and the PEGIFN dose was also not reduced
[30].
Hematologic toxicities are the most common adverse events experienced
by patients receiving PEGIFN and RBV, and anemia is the most common
reason to dose reduce or discontinue RBV alone or both agents [1012,27].
Anemia seems to be secondary to a combination of RBV-induced hemolysis
and IFN-induced bone marrow suppression [31,32]. This most commonly
occurs within the rst 12 weeks of therapy, which is the most critical time
for achieving EVR and is a time at which RBV use seems to be critical for
enhancing SVR [13,2730]. The mean decline in hemoglobin observed in pa-
tients treated with PEGIFN/RBV is 3 g, but declines of 4 g are routinely ob-
served in over 20% of patients [33,34].
Identifying patients with relapse or NR who signicantly reduced the
dose or prematurely discontinued PEGIFN or RBV secondary to anemia
is important when assessing patients with NR or relapse because this is a fac-
tor that could potentially be corrected or prevented during retreatment. Two
studies have demonstrated that IFN/RBV-induced anemia can be corrected
with epoetin alfa, which is also associated with a signicant improvement
in quality of life [3537]. An example of how this agent can be used to
prevent anemia and the need to dose reduce RBV in a patient with previous
relapse is illustrated in Fig. 4. In this example, the patient developed severe
anemia and had reduction in the doses of PEGIFN and RBV within the rst
12 weeks. During retreatment, epoetin alfa was instituted early before the pa-
tient developed severe anemia, and the doses of PEGIFN and RBV were not
reduced at any time during treatment, with the result that the patient
achieved an SVR during retreatment.
Severe neutropenia can be corrected by using treatment with granulocyte
colony-stimulating factor (GCSF), although no controlled trials of this
agent have been performed in patients with chronic hepatitis C undergoing
IFN therapy. Preliminary data have suggested that patients who have HCV
who develop neutropenia are not at increased risk for bacterial or other in-
fections [38]. As a result, the need for GCSF can probably be limited to
PEGIFN PEGIFN 1200

(mg/day)
Ribavirin
EPO 600
16 0
Hemoglobin
12
Log HCV RNA
6
(g/dl)
5 8
(IU/ml)
4
3
2 4
UD
0
-6 0 6 12 18 24 30 36 42
MONTHS
Fig. 4. Treatment of patient who had HCV with PEGIFN/RBV. During the rst course of
therapy, this patient developed severe anemia (hemoglobin !10 g/dL) and required discontin-
uation of RBV. The patient had already achieved EVR and was HCV RNA undetectable before
RBV was discontinued and the PEGIFN dose reduced. As the hemoglobin rose, RBV was re-
instituted, but at a lower dose (600 mg/d). The patient relapsed after 48 weeks of treatment. The
patient was retreated with PEGIFN/RBV, and epoetin alfa (40,000 lU/week) was initiated as
soon as the hemoglobin began to fall. No signicant anemia developed. The patient remained
on full-dose PEGIFN and RBV for 48 weeks and achieved SVR. UD: HCV RNA undetectable.
patients with a theoretical increased risk for bacterial infection. This in-
cludes patients with advanced cirrhosis awaiting liver transplantation, liver
transplant recipients, and those with HIV coinfection. A reduction in the
platelet count to values below 20,000/mL is rarely associated with spontane-
ous bleeding [39]. However, values this low are rarely observed during treat-
ment with PEGIFN even in patients with cirrhosis. As a result, severe
thrombocytopenia is an infrequent reason to dose reduce or discontinue
treatment for chronic hepatitis C.
PEGINF may also need to be dose reduced or prematurely discontinued
secondary to psychiatric side eects [10,11,27]. Psychiatric side eects severe
enough to warrant intervention occur in about 20% to 33% of patients re-
ceiving PEGIFN/RBV, and these adverse events can frequently be treated
with various antidepressant or antianxiety agents [40,41]. In contrast,
some patients develop depression or irritability that is so severe and so rapid
in its onset that dose reduction or discontinuation of treatment is necessary.
Identifying such patients is important when assessing the reason for NR be-
cause this is a potentially correctable factor. Retreatment of these patients
after their psychiatric disorder has been properly diagnosed and treated
can be successful in achieving SVR.
Noncompliant patients
Many patients who begin IFN-based therapy for chronic HCV infection
are not aware of the side eects of treatment or how to manage such side
eects or regularly miss doses of PEGINF or RBV because of personal or
work-related activities. Recent studies have demonstrated that the SVR
rate may be up to 20% lower when patients are treated outside of organized
clinical trials or by physicians who are relatively inexperienced at managing
adverse events or who do not spend enough time educating patients regard-
ing how to manage the side eects of PEGIFN and RBV [34]. Another type
of noncompliance is consumption of large amounts of alcohol on a regular
basis while receiving IFN therapy. Previous studies have demonstrated that
alcohol use signicantly reduces the SVR rate in patients being treated with
IFN [4244]. Similarly, ongoing or a recent history of injection drug use may
aect the ability of patients to achieve SVR during treatment for chronic
hepatitis C [45,46]. This NR seems to be secondary to a high rate of psychi-
atric side eects and noncompliance with the treatment regimen rather than
to any specic antiviral eect of ongoing injection drug use. It is also possi-
ble that patients with ongoing drug abuse may become reinfected with a new
strain of HCV during therapy.
Recognizing that noncompliance may have contributed to NR is impor-
tant particularly if these factors can be addressed and corrected. Improving
education and awareness regarding the side eects of PEGIFN and RBV,
having a stronger commitment to therapy, or seeking care from a more ex-
perienced or attentive IFN provider may be useful for some patients. With
proper counseling and support, some patients who consumed alcohol or

used illicit drugs during previous therapy may achieve long-term abstinence.
Noncompliance for any of these reasons is therefore an important correct-
able factor to address with patients who have failed to achieve SVR during
previous therapy.
Therapies under investigation for the treatment of nonresponders

The list of new pharmaceutical agents being developed and evaluated as
a possible treatment for patients with chronic HCV infection and NR to
PEGIFN/RBV therapy was recently reviewed [47]. Some of the agents being
evaluated in phase I and phase II clinical trials are listed in Table 2. By the
time this issue of Infectious Disease Clinics is printed, it is likely that some
of these agents will have failed clinical trials and no longer be under evalu-
ation. It is equally likely that other agents will have completed preclinical
development and will have entered the clinical trials arena as a potential
treatment for patients with chronic HCV infection and previous NR or
relapse. Numerous compounds in preclinical development are not listed in
Table 2 because it is impossible to know which of these agents will enter
Table 2
Investigational agents for chronic hepatitis C nonresponders being investigated in clinical trials
Agent Company Description
CPG 10101 Coley Stimulates production of many dierent endogenous IFNs
Eect on serum HCV RNA unknown
NM283 Idenix HCV protease inhibitor being evaluated as combination
therapy with PEGIFN
1DN-I566 Idun Caspase inhibitor reduces apoptosis
Has no eect on serum HCV RNA level but seems to
reduce serum ALT
ISIS 14803 ISIS Oligonucleotide being evaluated as combination therapy
with PEGIFN
Maximine Maxim Histamine analog being evaluated as combination therapy
with PEGIFN
Thymosin alfa-1 Sciclone Immune modulatory agent being evaluated as combination
therapy with PEGIFN
Viramadine Valaent Ribavirin analog that does not cause hemolysis
Being evaluated as combination therapy with PEGIFN
VX-950 HCV Ns3*4A protease inhibitor being evaluated as
monotherapy
Signicant reduction in HCV RNA and serum ALT levels
SCH 503034 HCV protease inhibitor being evaluated in combination
therapy with PEG-IFN. Seems to reduce HCV RNA
Albuferon Genetically fused interferon alpha with serum human albumin
Increased dosing interval of two to four weeks
Abbreviations: ALT, alanine aminotransferase; HCV, hepatitis C virus; IFN, interferon;
IMPDH, inosine monophosphate dehydrogenase; PEGIFN, peginterferon; RBV, ribavirin.
clinical trials or be discarded during preclinical testing. Pharmaceutical com-

panies, clinical research organizations, and governmental web sites can help
patients and physicians to remain abreast of drug development and emerg-
ing clinical trials with new agents for patients with chronic hepatitis C.
Several agents available to physicians are being evaluated in clinical trials
as a potential treatment for patients with chronic HCV infection and NR to
previous PEGIFN/RBV therapy. Other trials are investigating the ecacy
of higher doses of IFN or PEGIFN or a longer duration of therapy with
these agents. Several of these strategies are discussed here because they
may potentially be used by physicians for their patients with chronic hepa-
titis C. Only preliminary data are available regarding the use of these agents
in this o-label manner. Discussing these approaches in this article does
not advocate their use or suggest that they may be eective for treatment
of patients who have HCV infection and NR to PEGIFN/RBV. Safety
and ecacy will be determined when the results of these ongoing trials be-
come available.
Protease inhibitors
Anti-viral therapy with rst generation protease inhibitors has been un-
dergoing evaluation for the past few years. Initially, signicant results
were seen with BILN 2061 used as monotherapy with 3 log reductions in
HCV RNA. Studies were halted, however, after the development of cardiac
toxicities in animal models [48]. The enthusiasm for this class of agents has
been renewed with the development of three new drugs, NM283, VX-950,
and SCH 503034. Each of these agents have been shown to signicantly sup-
press HCV RNA when used either alone or in combination with PEGIFN
[4951]. Given prior experience with these agents, and the early phase of
their trials, therapy with anti-HCV protease inhibitors should be approached
with caution. Further studies of their use in combination therapy with inter-
feron or triple therapy with interferon and ribavirin need to be evaluated.
Consensus interferon and ribavirin

Consensus interferon (CIFN) is a synthetic IFN product with an amino
acid sequence that reects all a-IFNs. CIFN has been shown to be eective
in the retreatment of patients who failed to achieve SVR after 24 weeks of
treatment with standard IFN monotherapy [52]. In that study, SVR was
achieved in 58% of patients with previous relapse and in 13% with previous
NR. High-dose daily CIFN using an induction approach has recently
been evaluated for patients who have NR after treatment with PEGIFN/
RBV in two small, single-center trials [53,54]. In one of these studies,
CIFN was administered at doses of 27 or 18 mg daily for 4 weeks followed
by 18 or 9 mg daily for 8 weeks, followed by 9 mg daily plus RBV (1000
1200 mg/d) for an additional 36 weeks. In the other study, CIFN was dosed
at 15 mg/d plus RBV (10001200 mg/d) for 12 weeks followed by CIFN
15 mg TIW and RBV (10001200 mg/d) for 36 weeks. Preliminary data from
these studies suggested that 37% to 42% of patients with previous
PEGIFN/RVN could achieve SVR with these high-dose daily CIFN treat-
ment regimens. One of these studies has suggested that this approach may
be particularly helpful in African Americans [54], a group with a signicantly
lower SVR to PEGIFN/RBV therapy [55]. Based upon these single-center
preliminary studies, a large, multicenter, clinical trial has been initiated in
which CIFN (15 or 9 mg/d) plus RBV (10001200 mg/d) is being evaluated
for the treatment of HCV patients with NR to previous PEGIFN/RBV ther-
apy. Until this trial has been completed and this approach has been shown
to have a positive impact, the use of high-dose daily CIFN along with RBV
should be considered o-label and investigational.
Higher doses and longer duration of peginterferon and ribavirin

Another approach to the retreatment of patients who have NR to
PEGIFN/RBV is to use these same agents but at a higher dose. In a prelim-
inary study, patients with previous NR to IFN/RBV were retreated with
180, 270, or 360 mg of PEGIFNa-2a and RBV (10001200 mg/d). Although
VR at the end of treatment was similar in all three groups, SVR increased
stepwise from 21% to 46% in the highest PEGIFN dose group [56]. The in-
crease in SVRs observed with higher doses of PEGIFN/RBV was therefore
the result a marked reduction in relapse after discontinuation of this ther-
apy. These data have led to the initiation of multicenter trials in which
higher doses of PEGIFN and RBV are being used in patients who have
NR to previous treatment with PEGIFN/RBV.
It has been hypothesized that patients who relapse after treatment
with PEGIFN/RBV may have not been treated long enough to achieve
long-lasting viral suppression. Preliminary results from a treatment trial in
which patients were randomized to receive 48 or 72 weeks of therapy has
demonstrated that relapse was reduced from 48% to 13% in patients treated
for a longer period of time [57]. This study suggested that genotype 1 pa-
tients who relapsed after a standard course of PEGIFN/RBV may fare bet-
ter if retreated for a longer period of time. The duration of retreatment and
doses necessary to optimize SVR using this approach remains to be dened.
In contrast, current data suggest that retreating patients with genotype 2 or
3 for a longer duration does not aect SVR because 24 and 48 weeks of ther-
apy yield similar rates of SVR [12].
Peginterferon, ribavirin, and amantadine

Amantadine is an antiviral agent initially used for the treatment of inu-
enza A [58]. Over the past several years, studies have investigated the possi-
ble role of amantadine for the treatment of chronic HCV infection.
Amantadine has been used alone [59], with IFN [60], and as triple therapy
with IFN and RBV [61,62] as initial therapy and for retreatment of patients
with previous NR. Although most of these studies have yielded conicting
results, a recent meta-analysis has demonstrated that SVR might be about
5% to 7% higher in patients who received amantadine as part of triple ther-
apy compared with IFN/RBV [63]. Preliminary reports evaluating amanta-
dine, PEGIFN, and RBV triple therapy in patients with chronic HCV and
previous NR remain inconclusive [64,65]. It is unclear what role amantadine
will play in the management of patients who have failed to achieve SVR
with PEGIFN/RBV.
Maintenance therapy
It is well established that patients who achieve SVR have an improvement
in liver histology, a reduction in hepatic brosis [6668], a reduced risk for
developing hepatocellular carcinoma [69,70], and prolonged survival [70,71].
In contrast, it is doubtful that patients with NR achieve such benet. Al-
though it does seem that some nonresponders can achieve histologic im-
provement, this is probably limited to patients with partial VR and
a marked decline in serum HCV RNA from the pretreatment baseline level
[66,67,72]. Furthermore, the improvement in liver histology associated with
partial VR seems to be limited to changes in hepatic inammation. An over-
all improvement in liver brosis has not been convincingly demonstrated to
occur in patients with NR after a single course of IFN [66,72]. Although one
study implied that marked regression in brosis may occur in some patients
with NR, this is unlikely because an equal percentage of patients in this
study had brosis progression, over 60% of patients had no change in bro-
sis, and the mean brosis scores from before and after treatment were not
signicantly dierent [68]. It is therefore, most likely that sampling variation
accounted for the improvement in brosis reported to occur in a subset of
patients who had NR.
Fibrosis progression in patients who have chronic HCV is mediated by
hepatic inammation [73,74]. As a result, it is possible that a reduction in
hepatic inammation may reduce the rate of brosis progression or allow
brosis to resolve. This hypothesis forms the basis for the concept of main-
tenance therapy.
Maintenance therapy using interferon

Maintenance therapy using standard IFN was evaluated in a controlled
trial of patients who achieved partial VR and a reduction in hepatic inam-
mation after an initial course of IFN therapy [72]. These patients were ran-
domly assigned to continue IFN for an additional 2 years or to stop
treatment and be followed as a control group. Patients who remained on
IFN (3 MU tiw) therapy maintained the initial reduction in serum HCV
RNA and improvement in hepatic inammation observed after initial
therapy. In contrast, serum HCV RNA and hepatic inammation returned

to the pretreatrnent baseline in patients randomized to stop therapy.
Changes in mean brosis scores after 2.5 years in these two groups of pa-
tients were small and not statistically signicant. Nevertheless, these ndings
were encouraging and led to the initiation of a several large, multicenter
clinical trials to investigate the possibility that long-term maintenance ther-
apy using PEGIFN could prevent brosis progression, reduce hepatic de-
compensation, lower the incidence of hepatocellular carcinoma, reduce the
need for liver transplantation, and improve survival in patients who have
HCV with NR and advanced brosis or cirrhosis. The HALT-C trial is
the largest and most well publicized of these studies [75].
Preliminary results from another trial of maintenance PEGIFN (Co-
Pilot) have recently been reported [76]. After 2 years of maintenance therapy
(PEGIFNa-2b 0.5 mg/kg/wk), a signicant reduction in the incidence of var-
iceal hemorrhage was observed compared with patients treated with colchi-
cine. No reduction in the incidence of liver failure, the development of
hepatocellular carcinoma, the need for liver transplantation, or death was
observed. This suggests that continuing PEGIFN as maintenance therapy
may selectively reduce portal pressure but may not aect the natural pro-
gression of chronic hepatitis C in patients who have advanced brosis or
cirrhosis.
Final results from the various clinical trials evaluating PEGIFN mainte-
nance therapy will not be available for several years. Based upon available
data, it is possible to consider maintenance therapy for patients with ad-
vanced brosis or stable cirrhosis who have relapsed after treatment with
PEGIFN/RVN and who can tolerate the side eects of long-term treatment.
Initiating PEGIFN at full dose and gradually tapering the dosage every
3 months to the lowest dose that maintains HCV RNA undetectable is a ratio-
nal approach to patients with chronic HCV infection and advanced brosis
or cirrhosis. In contrast, maintenance therapy should probably not be con-
sidered at this time in patients who have already developed complications of
cirrhosis, in patients who do not achieve VR during treatment with PEGIFN
and RBV, or in patients who do not remain HCV RNA undetectable with
maintenance PEGIFN monotherapy. There are no data to suggest that pa-
tients who remain HCV RNA positive while on maintenance therapy achieve
signicant benet from ongoing treatment.
Maintenance therapy using ribavirin

One study has evaluated the benet of continuing RBV as maintenance
therapy in patients who have HCV with NR [77]. Although RBV monother-
apy does not reduce serum HCV RNA levels, serum alanine aminotransfer-
ase (ALT) declines signicantly [78]. This implies that RBV may act as an
anti-inammatory agent to reduce hepatic inammation and serve as a basis
for the use of RBV as maintenance therapy. In this pilot trial, 38 patients
who had NR to IFN/RBV were randomized to discontinue IFN and remain

on RBV (10001200 mg/d) or placebo. After discontinuation of IFN, serum
HCV RNA levels rose back to the pretreatment baseline levels in both
groups. Although mean serum ALT levels also rose after discontinuation
of IFN, patients who remained on RBV had a lower mean serum ALT levels
compared with placebo-treated patients. After 48 weeks of RBV mainte-
nance therapy, repeat liver biopsy demonstrated that 47% of patients had
a signicant decline in inammation, whereas this was not observed in the
placebo group. Although no signicant improvement in brosis was ob-
served with RBV maintenance therapy, the decline in hepatic inammation
suggests that this approach could be useful for the management of patients
who have HCV with NR. It remains unknown which subgroup of patients
who have NR could benet from RBV maintenance therapy and if this de-
cline in inammation could be maintained without suppressing serum HCV
RNA levels. As a result, the use of RBV as maintenance therapy should be
considered un-proven at this time.
Modifying lifestyle factors associated with brosis progression

Many patients with chronic HCV infection and NR to PEGIFN/RBV
have no, mild, or moderate brosis. Many of these patients are not at risk
for developing brosis progression to cirrhosis for 5 to 15 years or longer
[73,79,80]. It is estimated that 25% to 33% of patients who have chronic
HCV infection will never develop brosis. It is therefore rational for
many patients who have chronic HCV infection to be observed at periodic
intervals until denitive improvements in therapy have been established.
During this period, a focus on modifying lifestyle factors that seem to en-
hance brosis progression seems rational.
Several studies have demonstrated that patients who have HCV who con-
sume alcohol on a regular basis have an increased rate of brosis progres-
sion to cirrhosis and a higher percentage of cirrhosis than patients who
consume ethanol rarely or not at alt [42,43,79,81,82]. The minimum amount
of alcohol that seems to be safe and not associated with an enhanced rate of
brosis progression in HCV patients remains undened. It is therefore pru-
dent to counsel HCV patients not to consume alcohol except on rare
occasions.
Another factor that seems to be associated with more rapid brosis pro-
gression to cirrhosis is hepatic steatosis secondary to nonalcoholic fatty liver
disease (NAFLD). Several recent studies have suggested that patients who
have chronic hepatitis C and NAFLD, or more importantly nonalcoholic
steatohepatitis, have more brosis and a higher prevalence of cirrhosis
than patients who have HCV infection without fatty liver [83,84]. NAFLD
is strongly associated with obesity, insulin resistance, and hyperlipidemia
and seems to be improved by weight loss, improved control of diabetes
mellitus, and lowering of serum lipids [85,86], Such recommendations are

strongly encouraged for patients with chronic HCV infection and co-
existent NAFLD.
Patients who have chronic HCV infection should be vaccinated to pre-
vent other forms of viral hepatitis [87,88]. A recent study has demonstrated
that patients with chronic hepatitis C who acquire acute hepatitis A virus
(HAV) are at increased risk for developing fulminant hepatic failure [89].
Although this same study did not nd an increased risk of liver failure after
acute infection with hepatitis B virus (HBV), patients coinfected with HCV
and HBV seem to progress to cirrhosis at a faster rate [90]. Because exposure
to HAV is sporadic and cannot be predicted, it is recommended that all pa-
tients with chronic HCV infection be vaccinated against HAV [87,88]. In
contrast, the great majority of adults with acute hepatitis B acquired this in-
fection by participating in some sort of risk behavior, most commonly sex-
ual activity or illicit drug use. It is not always possible to predict which HCV
patients might indulge in these risk behaviors. As a result, vaccination of pa-
tients with chronic HCV and NR against HAV and HBV seems prudent.
Summary
The combination of PEGIFN and RBV is the most eective therapy for
patients with chronic hepatitis C. Although more than half of all patients are
able to achieve SVR, a signicant proportion of patients, particularly those
with genotype 1, fail to have undetectable HCV RNA during treatment or
relapse after completing therapy with return of detectable HCV RNA. An
approach in the management of these patients is to identify factors that
could have led to the NR or relapse and that could be corrected before or
during a second course of therapy. Because brosis progression occurs
slowly over decades for many patients with chronic hepatitis C, avoiding al-
cohol or other factors that could lead to brosis progression may be su-
cient for the vast majority of patients. Other options that could be
considered in patients who have more advanced disease include retreating
with one of several new antiviral agents; retreating with higher doses of
IFN or PEGIFN and RBV; or using IFN, PEGIFN, or RBV monotherapy
long-term as maintenance therapy. The safety and ecacy of these ap-
proaches is being evaluated in numerous clinical trials.
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20 (2006) 137153
Treatment of Relapsers after

Combination Therapy
for Chronic Hepatitis C
Furqaan Ahmed, MD, Ira M. Jacobson, MD*
Division of Hepatology and Gastroenterology, Weill Medical College of Cornell University,
450 East 69th Street, New York, NY 10021, USA
The treatment for chronic hepatitis C (CHC) has evolved over the last de-
cade from standard interferon (IFN) monotherapy to combination therapy
with standard IFN and ribavirin (RBV) and more recently, pegylated (PEG)
IFN and RBV combination therapy. As each successive regimen has led to
improved sustained virologic response (SVR) rates, the issue of retreating
those patients who did not achieve a sustained response with previous ther-
apy arises. A relapse after therapy is dened as viral clearance with a nega-
tive hepatitis C virus (HCV) RNA by polymerase chain reaction (PCR)
during therapy and reappearance of the virus after treatment is discontin-
ued. This article focuses on the treatment of patients who have relapsed after
being treated with IFN or a combination of IFN and RBV. Relapse to PEG
IFN and RBV will also be discussed.
Rates of relapse
Studies with standard IFN monotherapy at 3 million units three times
weekly have demonstrated SVR rates after 24 and 48 weeks of therapy to
be 6% and 13% to 19%, respectively [1,2]. RBV was approved by the US
Food and Drug Administration (FDA) for the treatment of CHC in combi-
nation with IFN in 1998. RBVs mechanism of action, a nucleoside analog,
has still not been elucidated rmly. Postulated mechanisms include
Dr. Jacobson is a consultant and has received research support from Schering-Plough.
of North America.
E-mail address: imj2001@med.cornell.edu (I.M. Jacobson).
138 AHMED & JACOBSON
inhibition of host inosine monophosphate dehydrogenase, enhancement of

host T-cell mediated immunity against HCV, direct HCV inhibition, and in-
creased HCV RNA mutagenesis leading to error catastrophe [3,4]. Recent
viral kinetic studies favor one or more antiviral rather than immunomodu-
latory mechanisms [5]. Although ineective in the treatment of CHC as
monotherapy [68], when administered with IFN, RBV increases SVR rates
by increasing response rates and, even more notably, by decreasing the rate
of post-treatment relapse in patients who become HCV RNA-negative on
therapy. The combination of standard IFN and RBV therapy for 24 weeks
in na ve patients results in end-of-treatment viral clearance rates of 53% to
57% and SVR rates of 31% to 35% [1,2]. Combination therapy for 48 weeks
results in end-of-treatment response rates of 50% to 52% and SVR rates of
38% to 43% [1,2]. Prolongation of combination standard interferon and ri-
bavirin therapy to 72 weeks does not result in signicantly higher SVR rates
(43%) than that achieved with 48 weeks of therapy [9]. The incremental ef-
cacy of 48 weeks or 72 weeks versus 24 weeks of therapy appears to be lim-
ited to patients with HCV genotype 1 infection.
Pegylated IFNs, developed by the covalent attachment of a polyethylene
glycol molecule to standard IFN, have a longer half-life and increased thera-
peutic ecacy. PEG IFN alfa-2b and alfa-2a have been approved by the FDA
for the treatment of CHC in combination with RBV, and this therapy repre-
sents the current standard of care for CHC. Two landmark studies evaluated
the ecacy of PEG IFN alfa-2a and alfa-2b and RBV treatment. PEG IFN
alfa-2b at 1.5 mg/kg per week and RBV (800 mg per day) in na ve patients
led to end-of-treatment response rates of 65% and SVR rates of 54% [10].
PEG IFN alfa-2a at 180 mg per week and RBV (1000 to 1200 mg) resulted
in end-of-treatment response rates of 69% and SVR rates of 56% [11]. Even
with these newer treatment regimens that result in higher SVR rates, a signif-
icant number of patients relapse when therapy is stopped. The relapse rates are
lower than they were with older forms of therapy, however.
Clearance of HCV viremia with antiviral therapy followed by relapse is
associated with improvements in liver histology and reductions in the devel-
opment of liver complications that are in-between those seen in patients who
achieve SVR and those who are nonresponders to anitiviral therapy [12,13].
Mechanisms of relapse
The precise reason for HCV relapse is likely multifactorial and may result
from host-, viral-, and treatment-related factors. Dierences in HCV geno-
types inuence response to therapy. Genotype 1 is more resistant to therapy
than genotypes 2 and 3, and patients with genotype 1 who achieve a response
on treatment have slightly higher rates of relapse after discontinuation of
therapy, even with PEG IFN alfa-2b and RBV [14]. Similarly, patients
with advanced brosis have higher rates of relapse, particularly with IFN
monotherapy [14].
TREATMENT OF RELAPSERS AFTER COMBINATION THERAPY 139
The host immune response plays a pivotal role in determining the ulti-
mate outcome of therapy. Also, factors including age, ethnicity, and the
degree of liver brosis inuence treatment outcome. The development of
anti-IFN neutralizing antibodies may play a role for some patients who
have viral breakthrough during therapy [15,16]. The importance of these an-
tibodies in patients who relapse after therapy is discontinued is doubtful,
however. Other host factors not yet identied also may predispose some pa-
tients to fail to clear the virus completely from the liver.
A two-phase model of viral clearance after initiation of IFN therapy has
been described [17]. The rst phase, consisting of a rapid decline in vire-
mia, results from IFN-induced inhibition of HCV virion production. The
second and more gradual decline in viremia represents HCV-infected cell
death (Fig. 1). This model suggests that relapsers are patients who have
not cleared all virus-infected hepatocytes at the time their treatment is
stopped. This hypothesis is supported by studies using transcription medi-
ated amplication, a recently developed sensitive viral assay (O5 IU/mL),
which detected low levels of HCV viremia in retrospectively analyzed
stored serum samples of up to 36% of patients who were classied as
end-of-treatment responders by conventional PCR tests but subsequently
relapsed [18].
Because of the practical obstacles to serial liver biopsy and the chal-
lenges in quantifying intrahepatic virus, it is unknown whether there is pro-
gressive attrition of infected cells or whether, in some patients, the process
achieves a plateau beyond which further clearance does not occur. Simi-
larly, it is unknown whether the mechanism of intrahepatic viral persis-
tence represents some form of latency or simply very low viral
replication undetectable in serum. As a avivirus, however, it is presumed
that HCV is not capable of genomic integration, as is the case with HIV
and hepatitis B virus (HBV).
Fig. 1. Two phase model of viral clearance with interferon therapy. The rst, steeper phase is
thought to be related to inhibition of viral production, while the second, more gradual phase,
represents clearance of infected hepatocytes. (Courtesy of Gerond Lake-Bakaar, MD.)
Retreatment of interferon monotherapy relapsers with interferon alone

or with combination interferon and ribavirin therapy
Several studies have evaluated retreatment of patients who relapsed after
IFN monotherapy. Retreating patients who relapsed after a course of stan-
dard IFN at 3 million units three times weekly for 24 weeks with the same
regimen has not been shown to be ecacious, with SVR rates of only 0% to
5% [1921], although a few studies have reported higher SVR rates [22,23].
Retreatment with standard IFN at 3 million units three times weekly but for
a longer duration results in higher SVR rates. Forty-eight weeks of therapy
has led to SVR rates of 29% to 36% [22,23]. Treatment for 96 weeks has
resulted in SVR rates of 68% [22].
Other investigators have retreated relapsers with higher doses of standard
IFN for 24 weeks. IFN doses of 4.5 to 5 million units three times weekly for
24 weeks were reported to induce an SVR in 35% to 38% of monotherapy
relapsers [19,24]. Studies using even higher doses of IFN, however, have re-
ported lower SVR rates. One study using 6 million units three times weekly
resulted in an SVR in 5% of patients [25], and a dose of 10 million units
resulted in an SVR of 18.5% [23]. Cavaletto et al treated 25 patients with
an 8-week induction phase of 6 million units three times weekly, followed
by 3 million units for 24 weeks and reported an SVR rate of 16% [26]. It
is unclear why the highest doses of IFN therapy resulted in lower SVR rates.
Chow et al combined both approaches to the retreatment of patients who
have relapsed to prior therapy using both a higher dose of IFN and a longer
duration of therapy [19]. They treated 11 patients with 5 million units of
IFN three times weekly for 48 weeks and reported an SVR rate of 27%,
which was lower than the SVR rate in patients treated with 5 million units
of IFN for 24 weeks (35%).
Retreatment of IFN monotherapy relapsers with combination standard
IFN and RBV results in higher SVR rates than retreatment with IFN alone.
Davis et al retreated patients with IFN (3 million units three times weekly)
and RBV for 24 weeks and reported an SVR rate of 49%, markedly higher
than SVR rates in patients who were retreated with IFN monotherapy for
24 weeks (5%) [20]. Similar SVR rates of 25% to 40% have been reported
in most studies retreating IFN monotherapy relapsers with IFN and RBV
combination therapy [25,2729]. One study reported an SVR rate of 75%
in 20 patients treated with this regimen [30]. Similar or slightly higher rates
of SVR (29% to 49%) have been reported when patients are treated with
higher doses of IFN (5 to 6 million units three times weekly) and RBV
for 24 weeks [24,29,31].
Reported SVR rates after 48 weeks of IFN at 3 million units three times
weekly and RBV range from 40% to 67% [27,29,32]. Forty-eight weeks of
high-dose (5 to 6 million units) IFN and RBV therapy resulted in SVR rates
of 47% to 72% [29,31,32]. Cavaletto treated 25 patients with an 8-week in-
duction phase of 6 million units three times weekly, followed by 3 million
units of standard IFN in addition to RBV (1000 to 1200 mg per day) for
24 weeks and reported an SVR rate of 44% [26]. In another study, biochem-
ical relapsers were retreated with a 24 week course of high dose IFN (6 mil-
lion units three times weekly) followed by 3 million units three times weekly
for 24 weeks with or without ribavirin (for 24 or 48 weeks) [33]. SVR rates in
patients with low HCV viral load (less than 2 million copies/mL, n 89)
were signicantly greater in patients who also received RBV (600 mg daily
for the rst 24 weeks) compared with IFN monotherapy alone (59.6% ver-
sus 37.7%, P ! 0.05). In patients with a high viral load (greater than 2 mil-
lion copies/mL, n 208), SVR rates were 44.7% and 51.4% in patients
treated with RBV (600 mg daily) for 24 weeks and 48 weeks, respectively.
Genotype non-1, Knodell score of less than 6 on liver biopsy, and ALT level
greater than three times the upper limit of normal were associated with
a greater likelihood of achieving an SVR.
Shiman et al randomized patients with an end-of-treatment virologic re-
sponse after 24 weeks of IFN and RBV to either discontinue therapy or con-
tinue RBV monotherapy for 24 additional weeks [30]. Of the 46 patients
with an end-of-treatment response, SVR rates were 75% in the group that
discontinued therapy and 50% in the group treated with RBV mono-
therapy, indicating that continuing RBV monotherapy after achieving a
virologic response does not increase the likelihood of an SVR. Krawitt
et al treated 17 IFN monotherapy relapsers with PEG IFN alfa-2b (100
to 150 mg per week) and RBV (1000 mg daily); 53% had an SVR [34].
In conclusion, retreatment with the same treatment regimen for the same
duration of therapy has not been shown to result in signicant SVR rates in
patients who have relapsed after a previous course of therapy previously. In
contrast, higher doses, and, more importantly, longer duration of therapy
lead to signicant SVR rates in these groups. It is also important to remem-
ber that when IFN monotherapy relapsers are treated with standard IFN
and RBV or PEG IFN and RBV therapy, much of the increase in SVR rates
undoubtedly reects the incremental increase in SVR associated with com-
bination therapy regardless of whether treatment duration is extended.
Treatment of patients who have relapsed after interferon and ribavirin

combination therapy
Data on the retreatment of patients who have relapsed after combination
therapy with standard IFN and RBV are more limited. There are no data on
retreating these patients with regimens of standard IFN and RBV. In a few
studies mostly reported only in abstract form, combination relapsers have
been retreated with PEG IFN and RBV (Table 1).
Jacobson et al randomized patients who had relapsed after IFN and RBV
therapy to receive 48 weeks of either PEG IFN alfa-2b 1.0 mg/kg per week
and RBV 1000 to 1200 mg per day or PEG IFN alfa-2b 1.5 mg/kg per week
and RBV 800 mg per day [35]. Twenty ve patients received the higher dose
142
Table 1
Combination peginterferon and ribavirin for relapses to standard interferon and ribavirin
Overall end Genotype
Patient of treatment Overall Genotype 1 non-1
Author number Treatment regimen response (%) SVR (%) SVR (%) SVR (%)
Krawitt [34] 68 PEG IFN a-2b (100150 mg/week) RBV 1000 mg daily 48 weeksa 66 54 55 53
Jacobson [35] 25 PEG IFN a-2b 1.0 mg/kg/week RBV 10001200 mg daily 60 32 27 67
48 weeksa
30 PEG IFN a-2b 1.5 mg/kg/week RBV 800 mg daily 48 weeksa 77 50 48 60
Gaglio [36] 128 PEG IFN a-2b 1.5 mg/kg/week RBV 8001400 mg daily 78 56 50 60
48 weeks
Portal [38] 24 PEG IFN a-2b 1.5 mg/kg/week RBV 8001000 mg daily 94 69 69 67
AHMED & JACOBSON

8 weeks, then PEG IFN a-2b 0.7 mg/kg/week RBV
8001000 mg daily 40 weeks
22 PEG IFN a-2b 0.7 mg/kg/week RBV 8001000 mg 88 67 73 50
48 weeks
Lawitz [39] 60 PEG IFN a-2b 1.5 mg/kg/week RBV 10001200 mg daily 55 30 30 NA
12 weeks, then PEG IFN a-2b 1.0 mg/kg/week RBV 800 mg
daily 36 weeks
65 PEG IFN a-2b 1.0 mg/kg/week RBV 800 mg daily 48 weeks 58 37 36 NA
Freilich [45] 12 PEG IFN a-2b 1.0 mg/kg/week RBV 1000 mg daily AMD 200 mg NA 50 25 100
daily 48 weeks
11 PEG IFN a-2b 1.0 mg/kg/week RBV 1000 mg daily 48 weeks NA 36 40 33
Herrine [43] 32 PEG IFN a-2a 180 mg/week RBV 8001000 mg daily 48 weeks 59 38 28 71
29 PEG IFN a-2a 180 mg/week MMF 2000 mg daily 48 weeks 72 17 17 17
31 PEG IFN a-2a 180 mg/week AMD 200 mg daily 48 weeks 42 10 4 20
31 PEG IFN a-2a 180 mg daily RBV 8001000 mg daily AMD 71 45 40 67
200 mg daily 48 weeks
Abbreviations: AMD, amantadine; MMF, mycophenolate mofetil.
a
HCV RNA by PCR was determined at 24 weeks of therapy. If PCR was positive, treatment was discontinued; if PCR was negative, treatment was
continued for 48 weeks.
of RBV, and 30 patients were in the group that received the higher dose of
IFN. Overall, 69% (38/55) of patients had an end-of-treatment response.
Only 42% (23/55), however, had SVR. Thirty-nine percent (15/38) of pa-
tients relapsed once therapy was discontinued. There was a trend toward
higher SVR rates in patients who received a higher dose of PEG IFN
(50% versus 32%), although this dierence did not reach statistical signi-
cance (P 0.18). SVR rates were lower in genotype 1 patients than in those
with other genotypes (38% versus 63%). In this study, patients who were
nonresponders to IFN monotherapy or combination IFN and RBV therapy
also were treated. SVR rates in prior nonresponders were signicantly lower
than in patients who had relapsed to therapy previously (8% vs 42%). In
a multivariate logistic regression analysis, genotype non-1 and weight
greater than 75 kg were associated with high rates of SVR; however, this
analysis including both nonresponders and relapsers.
Krawitt et al retreated 68 combination standard IFN and RBV relaps-
ers with a regimen of PEG IFN alfa-2b (100 to 150 mg per week) and
ribavirin (1000 mg per day) [34]. In this group of patients, the overall
end-of-treatment response rate was 66% (45/68), and the SVR rate was
54% (37/68). Genotype 1 and genotype non-1 patients had similar end-of-
treatment (67% and 65%, respectively) and sustained response (55% and
53%) rates. SVR rates were higher in patients who had previously relapsed
to therapy than in those who were previous nonresponders. Age and geno-
type were associated with response rate in previous non-responders but not
in relapsers in this study.
Gaglio et al treated patients with PEG IFN alfa-2b (1.5 mg/kg per week)
and xed-dose (800 mg) or weight-based (800 to 1400 mg) RBV for 48 weeks
[36]. Of the patients who have completed therapy and reached 24 weeks of
follow-up, 78% had an end-of-treatment response, and 56% achieved an
SVR. The SVR rate was lower in genotype 1 patients than in those with
other genotypes (50% versus 60%). Recently, preliminary data from the
lead-in phase of EPIC3, a multicenter study on maintenance therapy with
PEG IFN a-2b showed SVR in 39% of relapsers to standard IFN and
RBV who were retreated with PEG IFN a-2b (1.5 mg/kg weekly) and
weight-based RBV (800 to 1400 mg daily) [37].
In another study by Portal et al in 46 patients, the ecacy of an induction
dose of PEG IFN and RBV was compared with a xed lower dose of PEG
IFN and RBV [38]. One group of patients received PEG IFN alfa-2b 1.5 at
mg/kg per week plus RBV (800 to 1000 mg per day) for 8 weeks followed by
PEG IFN alfa-2b 0.7 mg/kg per week and RBV therapy for 40 weeks. The
second group of patients received low-dose PEG IFN alfa-2b 0.7 at mg/
kg/week and ribavirin (800 to 100 mg per day) for 48 weeks. The end-of-
treatment response rate was 94.1% in the induction group and 88.2% in
the group that did not receive induction therapy. SVR rates were similar
in both groups (68.7% and 66.7% in the induction and noninduction
groups, respectively). SVR rates in this study were unexpectedly higher in
patients with genotypes 1 and 4 (69.2% to 72.7%) than in patients with ge-
notypes 2 and 3 (50% to 66.7%). The authors reported that tolerance was
similar in both groups, although u-like symptoms were more frequent in
the induction group. The degree of liver brosis, as measured by Metavir
staging, decreased in approximately 50% percent of patients regardless of
the treatment regimen or the ultimate outcome of treatment. In this study,
low-dose PEG IFN and RBV therapy was as ecacious as induction-dose
therapy.
The ecacy of induction versus xed-dose PEG IFN and RBV therapy
was assessed by Lawitz et al in 125 patients [39]. One group of patients re-
ceived PEG IFN alfa-2b at 1.5 mg/kg per week and RBV (1000 to 1200 mg
per day) for 12 weeks followed by PEG IFN alfa-2b 1.0 mg/kg per week and
a reduced dose of RBV (800 mg per day) for 36 weeks. The second group of
patients received PEG IFN alfa-2b 1.0 mg/kg per week and RBV (800 mg
per day) for 48 weeks. End-of-treatment (58% versus 55%) and sustained
virologic response (37% versus 30%) rates were slightly but not signicantly
higher in the patients receiving induction dosing. Overall SVR rates, as well
as SVR rates in patients with genotype 1 with advanced brosis (Metavir
stage 3-4) were higher in patients who had relapsed after a prior course of
therapy than those who were nonresponders to IFN monotherapy or com-
bination IFN and RBV therapy.
Factors predicting sustained virologic response in prior relapsers

In HCV treatment-na ve patients, SVR has been associated with age
younger than 40, genotypes 2 and 3, non-African American race, low serum
HCV RNA levels (less than 2 million copies/mL), the absence of insulin re-
sistance or steatosis, the absence of bridging brosis or cirrhosis, and the ab-
sence of coinfection with HIV [40]. During treatment, adherence to therapy
and a decline in HCV RNA by greater than 2 logs at 12 weeks of therapy
also are associated with achieving an SVR [41,42].
A smaller body of data suggests similar predictors of response in prior
relapsers (Box 1). Data on favorable predictors of SVR in prior relapsers
are available mostly from patients in studies on relapsers to IFN monother-
apy given combination therapy with IFN and RBV; further data on predic-
tors of SVR in combination therapy relapsers given PEG IFN and RBV
have recently become available.
In relapsers to IFN monotherapy who are treated, genotype 1 clearly is
associated with lower response rates in most studies [20,23,24,29,31,33].
Low pretreatment viral load (less than 2 million copies/mL) [20,23,30,31]
and lesser degrees of brosis on liver biopsy [23,29,30,33] are predictors of
a favorable treatment response, although not all investigators have shown
this [19,20,25,27,32]. Older age at treatment and African-American race
are associated with poorer response rates [30,31]. Some studies have shown
that an elevated ALT level at baseline predicts a favorable response to
Box 1. Predictors of response in prior relapsers:

To standard interferon
Genotype non-1
Low viral load
No or mild fibrosis
Age greater than 40
Non-African-American ethnicity
Early virologic response to therapy
To standard interferon and ribavirin
Genotype non-1
Low viral load
Early virologic response to therapy
antiviral theapy [33]. An early virologic response is also a strong predictor of

a virologic cure [20,25].
Longer duration of treatment (24 versus 48 weeks) [22,23,31], particularly
for genotype 1 patients with a high viral load, is associated with a better
chance for SVR [27,29] in IFN mono-therapy relapsers given IFN and
RBV. Higher doses of IFN have not been associated with higher response
rates in most studies [23,29,32]. Saracco et al found that the cumulative
dose of IFN during the initial course of therapy or retreatment did not pre-
dict a favorable response to therapy [29]. Increased dose or duration of ther-
apy in patients with genotypes 2 and 3 is not associated with higher SVR
rates [27,29,31].
Similar predictors of response have been recently reported in relapsers to
combination standard IFN and RBV therapy who are retreated with PEG
IFN and RBV. Genotype non-1 and low viral load are predictors of a favor-
able response [43,44]. In one study, the absence of HCV viremia at week 6,
as determined by the transcription-mediated amplication assay, was highly
predictive of SVR (80% in the overall study population, 92% in genotype
non-1 patients) [44].
Adjunctive therapies for patients who have relapsed after interferon

and ribavirin combination therapy
In an eort to improve SVR rates in this population, some investigators
have evaluated adjunctive therapies in addition to PEG IFN a2b and RBV.
Freilich et al assessed the benet of adding amantadine to PEG IFN and
RBV therapy in treating patients who previously had relapsed to therapy
[45]. They compared PEG IFN (1.0 mg/kg per week) and RBV (1000 mg
per day) with a regimen of PEG IFN (1.0 mg/kg per week) and RBV
(1000 mg per day) plus amantadine (200 mg per day). The overall SVR
rate presented from interim data in 23 patients was 43%. The addition of
amantadine was not benecial in genotype 1 relapsers. The SVR rate in ge-
notype 1 relapsers was 25% (2/8) in patients treated with amantadine and
40% (2/5) in those treated with standard PEG IFN and RBV combination
therapy. In contrast, the addition of amantadine may have led to superior
treatment ecacy in nongenotype 1 patients. All (4/4) of the genotype
non-1 patients treated with amantadine had an SVR, compared with only
33% (two of six) of patients treated with PEG IFN and RBV alone, but nal
data from this study are not available, and the number of patients is too
small to draw any denitive conclusions.
In another study, 37 relapsers (to standard IFN monotherapy or standard
IFN and RBV combination therapy) were retreated with an induction dose of
standard IFN (18 MU daily) in combination with ribavirin (1000 to 1200 mg
daily) and amantadine (100 mg daily) for two weeks and then randomized to
22 weeks of continued ribavirin and amantadine with or without IFN (6 MU
three times weekly) [44]. The 10 patients who discontinued interferon after the
2 week induction course all relapsed within two weeks. Of the patients who re-
ceived 24 weeks of interferon, the end-of-treatment response was 63% (17/27)
and the SVR rate was 44% (12/27). Twenty nine percent (5/17) of patients with
an end-of-treatment response subsequently relapsed.
Herrine et al assessed the ecacy of mycophenolate mofetil and amanta-
dine in addition to PEG IFN and RBV in a pilot study [43]. They treated 124
patients (106 relapsers, 18 breakthrough relapsers) who were randomized to
receive PEG IFN alfa-2a 180 mg once weekly in addition to one of four fol-
lowing treatment arms: (1) RBV at 800 to 1000 mg daily, (2) mycophenolate
at 1000 mg twice daily, (3) amantadine at 100 mg twice daily, or (4) amanta-
dine at 100 mg twice daily and RBV at 800 to 1000 mg daily. End-of-treat-
ment response rates were highest in patients who were treated with PEG
IFN and mycophenolate (72.4%) and PEG IFN, RBV, and amantadine
(71%). Patients who received PEG IFN, RBV, and amantadine had the high-
est SVR rates (45%), followed by those who received PEG IFN and RBV
(38%). However, the dierence in response rates in the two groups did not
reach statistical signicance. Treatment with PEG IFN and either amanta-
dine or mycophenolate alone resulted in lower SVR rates (10% and 17%, re-
spectively), as in other trials. Thus, RBV appears to exact its benet by
preventing relapse. There was a trend towards higher SVR rates in patients
with genotype non-1 and low HCV viral load. Tolerance of the medications
was similar in the dierent treatment groups, except for the more signicant
decline in hemoglobin seen in patients who received ribavirin.
Approach to the retreatment of patients who relapse after interferon

Patients who relapse after standard IFN and RBV should be considered
strongly for retreatment with PEG IFN and RBV because of their
signicant chance of SVR (30% to 70%). The available data are derived
from studies in which PEG IFN and RBV were administered for 48 weeks.
In these studies, however, relapse even after PEG IFN and RBV occurred in
signicant numbers of patients. Current viral kinetic models suggest that
a gradual second phase of HCV-infected cell death occurs, ultimately lead-
ing to viral eradication in patients who achieve an SVR [17]. It is thought
that patients who relapse may have a more prolonged period of elimination
of infected cells, which may not have been completed when a standard
course of IFN and RBV therapy ends. Therefore, in these patients with
a previously demonstrated proclivity to relapse, longer courses of therapy
than the standard duration of 48 weeks may be worth consideration in
the retreatment of selected patients, with a goal of maximizing the chance
of eventual eradication of all virally infected cells. Such patients might in-
clude those with genotype 1, high viral load, and advanced brosis, or those
with a delayed response to therapy. However, controlled trials demonstrat-
ing the ecacy of such prolonged therapy have not been reported.
Adherence to optimal doses of PEG IFN and RBV during prior therapy
also should be considered. The superior ecacy (44% versus 41%,
P 0.02) of weight-based (800 mg to 1400 mg) versus xed (800 mg)
RBV dosing has been recently demonstrated in a multicenter, randomized,
community based trial of 4913 patients [46]. In treatment na ve patients,
dose reductions of either drug, and particularly both drugs, have a signi-
cant impact on attainment of early virologic response (EVR) after 12 weeks
(80% EVR with full doses versus 60% to 70% and 33% with dose reduc-
tion of either or both drugs, respectively) [47]. In patients who attain
EVR, the chance of SVR is adversely aected by dose reductions or discon-
tinuation of therapy beyond the 12-week timepoint. Although comparable
data do not exist for the relapse population, these same principles may
well apply. Accordingly, every eort must be made to maintain optimal
dosing during the retreatment of patients who have relapsed to a prior
course of therapy. Patients who underwent dose reduction during prior
therapy for depression, anemia, or neutropenia may be considered for anti-
depressants, granulocyte colony stimulating factor therapy, or erythropoie-
tin, respectively, as appropriate during retreatment. It must be recognized,
however, that while recent studies on erythropoietin show an eect on suc-
cessful maintenance of ribavirin dose, improvements in hemoglobin levels
and quality of life [48], no prospective trials have demonstrated the use of
adjunctive factors to enhance SVR.
Treatment of patients who have relapsed after pegylated interferon

Patients who have relapsed after a course of PEG IFN and RBV pose
a dicult problem, because more eective therapy is not available. There
are no data on the ecacy of higher doses of drugs, leaving a repeat course
of more prolonged therapy as the major option. Goncales et al treated na ve

patients with either 24 or 48 weeks of combination PEG IFN alfa-2a and
RBV. Patients who received 24 weeks of therapy and relapsed were eligible
to enter a retreatment study consisting of PEG IFN alfa-2a (maximum dose
of 180 mg per week) and RBV (1000 to 12000 mg per day) for 48 weeks [49].
PEG IFN and RBV doses were adjusted by the investigators according to
the patients tolerance of the medications during the initial course of ther-
apy. Preliminary data from this study on 59 patients indicate high end of
treatment virologic response rates of 90% (88% in genotype 1 patients;
93% in patients with genotype 2 and 3) with retreatment. SVR rates from
this study are awaited.
Because most patients who relapse after a course of PEG IFN and RBV
will have received 48 weeks of therapy, the only realistic option is to con-
sider a repeat course of treatment for a longer duration (eg, 72 to 96 weeks).
In apparent conformity with concepts of the kinetics of viral clearance, pa-
tients who clear HCV RNA on their initial course of PEG IFN and RBV
have an enhanced risk of post-treatment relapse if clearance of HCV
RNA requires longer than 12 weeks of therapy [41]. The concept of modi-
fying the duration of therapy according to a patients virologic response pat-
tern to PEG IFN and RBV therapy has been evaluated in a preliminary
study of nine HCV genotype 1, treatment-na ve patients [50]. These nine pa-
tients all had delayed virologic clearance; they were positive for HCV RNA
by PCR but had a 2 log decline in HCV RNA by week 12 of therapy and
had cleared virus by week 24. Patients were treated for 72 weeks, and
88% (7/8, with one lost to follow-up) had an SVR. As in patients with re-
lapse after a course of standard IFN and RBV, this approach to therapy
needs to be evaluated further in patients who have relapsed after a prior
course of PEG IFN and RBV, particularly patients with genotype 1, high
viral load, and cirrhosis, or patients in whom clearance of HCV RNA
was delayed beyond 12 weeks.
Preliminary data with consensus interferon, a novel recombinant type 1
interferon, suggests sustained virologic response rates of 2337% in previ-
ous nonresponders to pegylated interferon and ribavirin [51,52]. However,
data from large multicenter studies is needed to conrm these ndings in
prior nonresponders. Studies evaluating the ecacy of consensus interferon
in prior relapsers to PEG IFN and RBV are currently in progress, including
at our center.
Future directions
In addition to the options of PEG IFN and RBV for relapsers to previous
combination therapy, or prolonged courses of PEG IFN and RBV in re-
lapsers to previous PEG IFN and RBV therapy, new therapies on the hori-
zon for treatment-na ve patients with chronic hepatitis C include genome
sequence-based therapies, viral enzyme inhibitors, and immunomodulatory
agents may be ecacious in patients who have relapsed to previous therapy

also [9,53]. Most promising are the protease and polymerase inhibitors
which are currently in Phase I and Phase II trials. Preliminary data from
both treatment-na ve HCV-infected patients and previous nonresponders
have demonstrated marked declines in HCV viremia with these novel com-
pounds [5457]. Patients with mild liver disease or those who had great dif-
culty tolerating interferon-based therapy may wish to defer retreatment as
novel therapies are awaited. In the authors practice, however, it is felt that
most patients with relapse after standard IFN and RBV should be oered
a course of PEG IFN and RBV.
Summary
Sustained virologic response rates are signicantly higher in patients who
have relapsed after a previous course of therapy compared with patients
who did not respond. A meta-analysis of combination therapy in patients
who failed IFN monotherapy reported SVR rates of 52% in relapsers to prior
therapy and 16% in nonresponders [12]. Similarly, relapsers after combina-
tion standard IFN and RBV therapy have higher SVR rates than combination
of therapy nonresponders when treated with pegylated interferon and ribavi-
rin [34,3539]. For this reason, patients who relapse after a previous course of
therapy should be considered potential candidates for retreatment. Factors
that have been associated with SVR in these patients include genotype
non-1, low viral loads, and lesser degrees of brosis. The course of treatment
in all patients who have relapsed after prior therapy should be reviewed to
identify possible reasons for failure to achieve an SVR. In particular, optimal
dosing of PEG IFN and RBV and the occurrence and timing of treatment dose
reductions during prior therapy should be reviewed. The reasons for dose
reduction should be addressed before initiating another course of therapy
in an eort to optimize the chance for a SVR. Patients who had dose reduc-
tion for depression, anemia, or neutropenia, should be considered for antide-
pressants, erythropoietin, or, if neutropenia is severe, granulocyte colony
stimulating factor therapy, respectively, during retreatment. Prolongation
of therapy beyond 48 weeks in patients with relapse after a standard course
of PEG IFN and RBV may oer a chance of SVR. Novel agents currently in
development, including protease and polymerase inhibitors, may prove to be
therapeutic options for these patients in the future.
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20 (2006) 155174
Management of Recurrent Hepatitis C

in Liver Transplant Recipients
Scott W. Biggins, MD, Norah A. Terrault, MD, MPH*
Division of Gastroenterology, Department of Medicine, University of California,
San Francisco, 513 Parnassus Ave, S357, Box 0538 San Francisco, CA 94143, USA
Chronic hepatitis C virus (HCV) infection is the most common indication

for liver transplantation in the United States and Europe, and more than
20,000 patients worldwide have undergone transplantation for complica-
tions of chronic hepatitis C. In North America, HCV accounts for 15%
to 50% of all liver transplants performed [1]. Available prevalence data pre-
dict that this proportion will likely increase as the number of persons with
chronic hepatitis C developing cirrhosis and hepatocellular carcinoma in
North America rises over the next 1 to 2 decades [2,3].
Recurrent HCV infection is universal in those with viremia before trans-
plantation, and the rate of histologic disease progression after transplanta-
tion is more rapid. The risk of death and allograft failure is increased in
HCV-positive transplant recipients (hazard ratio [HR] 1.23 and 1.30) com-
pared with HCV-negative recipients [4]. The risk of cirrhosis is as high as
30% within 5 to 10 years after transplantation [57]. Retransplantation
for recurrent hepatitis C is an option for only a limited number of patients,
and with a 5-year survival rate of % 50%, it is likely to remain a controver-
sial indication for retransplantation [8].
To maximize the long-term survival of liver transplant recipients who
have HCV infection, eradication of infection is the ultimate goal. Pretrans-
plant antiviral therapy with the goal of achieving viral eradication before
transplantation is a consideration in some patients, especially those who
have mildly decompensated liver disease [9]. This article focuses on the man-
agement of liver transplant recipients who have HCV infection at the time of
transplantation. This group of patients is at risk for recurrent and progres-
sive disease after transplantation. There are several dierent time points for
E-mail address: Norah.Terrault@ucsf.edu (N.A. Terrault).
156 BIGGINS & TERRAULT
considering treatment interventions (Fig. 1): (1) Prophylactic therapy is

started prior to or at the time of transplantation and continued for variable
periods post-transplant. The goal of prophylaxis is to prevent reinfection of
the graft. (2) Preemptive therapy of recurrent HCV infection refers to initi-
ation of therapy early in the post-transplant period, typically within the rst
4 to 8 weeks after transplantation, and before biochemical and histologic
disease are manifest. (3) Therapy for established recurrent disease refers
to the use of antiviral treatment when there is biochemical and histologic
evidence of recurrent disease. Treatment of established recurrent disease
has been the most frequently used strategy for the management of post-
transplant HCV infection.
Natural history of recurrent hepatitis C virus after transplantation

Recurrent infection, dened by the presence of detectable HCV RNA in
serum and liver, occurs in essentially all patients who are viremic pretrans-
plant. Kinetic studies of the early virologic events after transplantation
have shown that HCV RNA levels drop abruptly during the anhepatie phase
and again 8 to 24 hours after reperfusion [10]. In general, viral levels increase
beginning 24 to 72 hours post-transplant, but there is considerable interpa-
tient variability in the rate of increase in viral levels during the rst postoper-
ative week [10]. Thereafter, a progressive increase in viral load occurs in the
majority of patients, with the peak viral level occurring between months
1 and 4 [11]. At 1 year post-transplant, HCV RNA levels are on average
1-log10 higher than pretransplant levels [12,13]. Pretransplant viral liters have
been inconsistently correlated with post-transplant HCV RNA levels [13].
High pretransplant viral levels have been associated with reduced overall
Fig. 1. This identies the potential time points for intervening with therapy to prevent or erad-
icate HCV infection or slow disease progression. (Courtesy of M Berenguer, MD, Valencia,
Spain.)
POSTTRANSPLANT HEPATITIS C MANAGEMENT 157
patient and graft survival in one study [14], but whether this reects the eects
of HCV recurrence per se or other transplant complications to which HCV-
infected persons with high pretransplant levels of HCV are more susceptible
is unknown, HCV genotype has not been found to be predictive of post-
transplant HCV RNA levels. Multiple studies have shown post-transplant
HCV viral liters to correlate poorly with histologic severity of disease [1517].
Histologic manifestations of recurrent hepatitis C are variable, but the
majority of patients have evidence of chronic hepatitis on liver biopsy within
the rst year. Approximately 70% develop acute hepatitis, usually within
the rst 3 to 6 months after transplantation, which evolves to chronic hep-
atitis. Chronic hepatitis without a preceding acute hepatitis occurs in about
20% of patients and may have a more favorable outcome in terms of disease
progression. A severe and rapidly progressive form of recurrent disease,
termed cholestatic hepatitis, is associated with a histologic pattern of bal-
looned hepatocytes, conuent necrosis, bile duct proliferation, and cholesta-
sis, but with minimal inammation [18]. Another hallmark of cholestatic
hepatitis is high serum HCV RNA levels (O106 IU/mL) [19]. Historically,
the outcome of cholestatic hepatitis was dismal, with mortality rates of
50% within the rst year. This severe manifestation of recurrent disease oc-
curs in 10% or less of HCV-infected transplant recipients, and antiviral ther-
apy has been recently shown to result in disease stabilization and prolonged
survival in some recipients with this condition [20,21].
Fibrosis progression in liver transplant recipients is more rapid than in
nontransplant patients. In untreated patients, 8% to 44% develop recurrent
cirrhosis within 5 to 7 years of transplantation [57,2224]. Once cirrhosis
develops, the risk of decompensation and graft loss are high, with one study
reporting 42% mortality within 1 year [25]. Another study suggested the rate
of brosis progression was not linear. Mean brosis scores were 1.2 after
1 year, 1.7 after 3 years, 1.9 after 5 years, 2.1 after 7 years, and 2.2 after
10 years [26]. Several studies indicate that early aggressive disease carries
a worse prognosis [13,23,26]. Patients with high histologic activity within
the rst year post-transplant are at greatest risk of progressive brosis
and cirrhosis. One study found the presence of conuent necrosis on early
biopsy to be highly predictive of risk of cirrhosis [26]. In a recent study,
39 recipients with brosis stages 3 or 4 at the 1-year biopsy had a signi-
cantly reduced survival compared with those with stages 0 to 2 at 1 year [23].
Factors associated with severity of hepatitis C virus recurrence

Numerous studies have sought to identify recipient, donor, viral, and
other factors that are associated with severe and progressive recurrent hep-
atitis C. End points for these studies dier, with some studies focused on -
brosis progression or incidence of cirrhosis and others using graft or patient
survival as a marker of severe disease outcome (Table 1). Analysis of
survival rather than histology may lead to overestimation of graft and pa-
tient losses due to HCV because other causes of graft loss (eg, chronic rejec-
tion) and nonliver-related causes of death (eg, sepsis) are included.
Additionally, the predictors of all causes graft survival may not mirror
those of progressive HCV disease leading to graft loss.
One of the more controversial factors linked with more severe HCV dis-
ease is the use of a living donor. Theoretically, the rapid regeneration of the
living donor graft in the postoperative period may alter early virologic or
immunologic events related to HCV recurrence such that disease progres-
sion is more rapid. Live donors are also more likely to share human leuko-
cyte antigens, which may alter the natural history of post-transplant
recurrence. Studies using graft survival as the primary outcome are dicult
Table 1
Possible factors associated with increased severity of hepatitis C virus recurrence
Donor Recipient HCV-specic
Study factors factors factors Other factors
Worse graft and patient survival
Charlton, 1998 [14] d d HCV RNA d
Ol MEq/mL at
transplantation
Berenguer, 2002 [37] Donor age Female d Year of LT
gender OKT3 induction
Burak, 2002 [34] d d d CMV infection
Sanchez-Fueyo, d d Biochemical CMV disease
2002 [6] hepatitis
Neumann, 2004 [23] Donor age d Genotype 1 and 4 Number of
steroid boluses
Thuluvath, 2004 [27] LD d d d
Garcia-Retortillo, LD d d d
2004 [32]
More advanced histological brosis
Berenguer, 2000 [5] d Non-white HCV RNA at LT Year of LT
race Number of
corticosteroid
boluses
Papatheodoridis, d d d Triple or dual
2001 [35] versus single
initial IMS
Berenguer, 2002 [37] Donor age d Acute recurrent OKT3 induction
hepatitis TAC induction
Burak, 2002 [34] Donor age Recipient d Year of LT
age CMV infection
MMF use
Guido, 2002 [26] d d Severity of d
necroinammation
within rst year
Abbreviations: CMV, cytomegalovirus; HCV, hepatitis C virus; IMS, immunosuppression;
LD, live donor graft; LT, liver transplantation; MMF, mycophenolate mofetil; TAC,
tacrolimus.
to interpret because the complications related to live donor liver transplan-

tation per se may be the cause of worse outcome rather than more severe
recurrent hepatitis C. This was shown in a study of 764 live donor liver
transplant (LDLT) and 1470 matched deceased donor liver transplant
(DDLT) recipients, with 2-year graft survival rates of 64% and 73%, respec-
tively (P ! .001) [27]. Among LDLT recipients, the risk of graft failure was
60% higher (HR 1.6, 95% condence interval [CI] 1.12.5) after adjusting
for baseline dierences in the groups such as serum creatinine, United Net-
work of Organ Sharing (UNOS) status, and need for life support [27]. Sim-
ilar to the overall analysis, a subanalysis of HCV-positive patients found
graft survival to be lower in LDLT compared with DDLT recipients. A sec-
ond study of UNOS data by Russo and colleagues [28] found no signicant
dierence in survival between 279 LDLT and 3955 DDLT recipients who
were HCV positive, with 2-year graft survival rates of 72% and 75%, respec-
tively (P .11). Six studies have been published on this issue, with no dif-
ferences in outcome between LDLT and DDLT recipients reported in four
of six studies (Table 2) [29]. The rate of cholestatic hepatitis was signicantly
higher (17% versus 0%) in live donor versus deceased donor recipients in
one single-center study [30], but the time to recurrence and overall severity
of disease was not dierent between groups. Only two studies have used pro-
tocol biopsies to assess for HCV disease recurrence and progression and are
most informative in terms of the eect of donor type on HCV natural his-
tory. In a United States study of 23 LDLT and 53 DDLT patients, patient
and graft survival was not dierent, and there was no patient with cirrhosis
in either group at the end of a median 40 months follow-up [31]. In a Spanish
study of similar study design, results were dierent. Cirrhosis or liver de-
compensation occurred in 44% of LDLT patients, compared with 29% of
DDLT patients (P .019) [32]. With these divergent results, no denite con-
clusion can be drawn. Although there is no rationale for HCV-positive pa-
tients to be denied access to a timely transplant if a liver donor is available,
physicians should discuss with patients the unclear impact of LDLT on
HCV disease progression.
The factors most consistently associated with a high risk of progression
to cirrhosis and graft loss due to HCV infection include treated acute rejec-
tion, cytomegalovirus infection, nonwhite race, and early severe or acute re-
currence. Corticosteroid boluses, cumulative corticosteroid doses, and the
use of lymphocyte-depleting agents have been associated with increased
HCV RNA levels and are linked with progressive HCV disease in some
studies [33].
The relationship between other immunosuppressive agents and severity of
HCV disease progression after transplantation are even less clear (Table 3).
Steroid-free immunosuppressive regimens have shown some benets in
HCV-infected recipients [34]. Retrospective analyses of patients receiving ta-
crolimus versus cyclosporine-based immunosuppression show no dierence
in disease severity between calcineurin inhibitor groups [35,36,37]. The impact
Table 2
Comparison of live donor and deceased donor liver transplant recipients with hepatitis C
Mean
Study follow-up
Study population n (LD/DD) time (mo) Outcomes
Gaglio, Single center 23/45 26 No dierence in graft
2003 [30] survival, time to recurrence,
or incidence of recurrence
Higher rate of cholestatic
hepatitis in LDLT versus
DDLT (17% versus 0%)
Shiman, Single center 23/53 40 No dierence in graft survival:
2004 [31] 82% and 82% at 2 yr
Russo, UNOS 279/3955 d No dierence in graft survival
2004 [28] at 2 yr, LDLT versus
Thuluvath, UNOS 764/1470 d Worse graft survival at 2 yr
2004 [27] for LDLT versus DDLT
(64% versus 73%) (OR 1.6,
95% CI 1.12.5)
Bozorgzadeh, Single center 35/65 25 No dierence in graft survival at
2004 [29] 39 months for LDLT versus
Garcia-Retortillo, Single center 22/95 22 Increased probability of severe
2004 [32] recurrence (decompensation
or cirrhosis) at 2 yr for LDLT
versus DDLT (45%
versus 22%) (OR 2.5, 95%
CI 1.15.7)
Abbreviations: CI, condence interval; DDLT, deceased donor liver transplant; LDLT, live
donor liver transplant; OR, odds ratio; UNOS, United Network of Organ Sharing.
of mycophenolate mofetil on HCV disease severity is uncertain. In a prospec-

tive study comparing tacrolimus, mycophenolate mofetil, and prednisone
to tacrolimus and prednisone alone, there were no dierences in graft loss
or rate of HCV recurrence (although protocol biopsies were not performed)
[38]. A recent study found that HCV viral loads increased in patients on a sta-
ble regime of cyclosporine and low-dose prednisone when they were changed
from azathioprine to mycophenolate mofetil [39]. In a third retrospective
study of patients who had recurrent HCV infection, tapering of calcineurin
inhibitor therapy with the addition of myeophenolate mofetil was associated
with less progression of liver disease compared with patients treated with cal-
cineurin inhibitor tapering only [40]. An initial study of antibody induction
therapy suggested a negative impact on HCV disease progression [40], but
subsequent studies have not conrmed this nding [41,42]. Sirolimus has
not been studied suciently to draw conclusions.
A trend reported by some authors is the decreasing graft and patient sur-
vival rates over time for patients who have HCV [4,43], which contrasts with
Table 3
Immunosuppression and hepatitis C virus recurrence
Drag Viral replication Graft hepatitis
Steroids Increase Progression
Cyclosporine Decreasea or stable No eect
Tacrolimus Unknown No eect
MMF Increase or stable Controversial
OKT3 Unknown Controversial
ATG Unknown Unknown
Anti-IL2 Unknown Unknown
Combinations (three or four drags) Increase Progression
a
In cell culture systems.
Data from Refs. [39,45].
the increasing survival rates seen for non-HCV indications during the same
time period. There are two changes in clinical practice that have been impli-
cated as the cause for this trend in patients with HCV: (1) the use of newer
and more potent immunosuppressive agents [5,35,44] and (2) the increasing
use of older donors [23,43,46,47].
Interferon treatment and rejection

In contrast to liver transplant recipients without HCV infection, in whom
an episode of acute cellular rejection is associated with improved survival,
HCV-infected recipients have a 2.4-fold increase in mortality after an epi-
sode of acute rejection [48]. The histologic features of early recurrent
HCV infection and acute cellular rejection overlap and make the diagnosis
of acute cellular rejection in the setting of recurrent HCV disease dicult
[49]. Treatment of recurrent HCV with increased antirejection therapy
would be predicted to increase the risk of disease progression, so the issue
of misdiagnosis is signicant. In a retrospective study of 285 liver transplant
recipients, HCV infection was associated with an increased risk of early (!6
months after transplant) acute cellular rejection compared with other trans-
plant indications (HR 1.7) [50]. In this same study, 49% of transplant recip-
ients with HCV infection had at least one episode of early acute cellular
rejection. In light of the diculty in distinguishing mild acute rejection
and recurrent HCV from recurrent HCV alone and due to the reported as-
sociated between treatment of acute rejection and increased risk of HCV dis-
ease progression, some experts have recommended that only acute rejection
of moderate or severe degree be treated and that corticosteroid boluses and
antibody therapy be used judiciously [51].
Because interferon (IFN) has immunomodulatory eects, specically the
upregulation of HLA expression on bile duct epithelia, there has been con-
cern regarding the risk of rejection related to the use of IFN in liver trans-
plant recipients. This risk of acute rejection may be greater, theoretically, if
IFN is used preemptively rather than later post-transplantation because the

risk of acute rejection is highest in the rst 3 months after transplantation.
Controlled studies of preemptive antiviral treatment strategies have reported
similar rates of acute rejection between IFN-treated and control groups [52].
In studies using IFN or peginterferon (PEGIFN) plus ribavirin (RBV) to
treat recurrent HCV disease, treatment is initiated typically at least 6
months after transplantation. Two recent uncontrolled retrospective studies
of IFN (standard and PEGIFN) plus RBV for the treatment of recurrent
hepatitis C showed rates of acute cellular rejection of 11% and 30%, respec-
tively [53,54]. These rates are signicantly higher than a previous random-
ized controlled study [55] and multiple other uncontrolled studies that
reported acute rejection rates of 0% to 5% [20,5663]. Possible explanations
for this discrepancy are bias from lack of control subjects, the extended half-
life of PEGIFN, heterogeneity in immunosuppression from variable regi-
mens, reduced drug levels, or lower doses of RBV. Chronic rejection rates
associated with IFN treatment have ranged from 0% to 4% in controlled
and uncontrolled studies, with the exception of one early study reporting
a chronic rejection in 5 of 14 IFN-treated subjects [64].
Prevention and treatment of recurrent hepatitis C virus infection

To prevent HCV infection after transplantation, therapies must be given
prior to or at the time of transplantation. This prophylactic approach has
been successful in the prevention of recurrent HBV infection among trans-
plant recipients [65]. Hepatitis B immune globulin (HBIG) combined with
nucleoside analogs can eectively prevent recurrent HBV infection in O 90%
of patients. A similarly ecacious therapy has not been identied, however,
for HCV-infected patients. In the absence of an eective prophylactic ther-
apy, the focus of studies has been the use of IFN (conventional or PEGIFN)
with and without RBV to treat transplant patients preemptively (ie, early
in the post-transplant period before clinical onset of recurrent HCV) or
later when recurrent and progressive histologic disease is apparent. The latter
approach has been the mainstay of management to date, but success has
been modest at best (Table 4).
The limited tolerability of IFN and RBV is a major impediment in the
treatment of HCV infection in the post-transplant setting. Dose reductions
are more common in transplant patients receiving antiviral therapy for re-
current hepatitis C than in nontransplant populations. Dose reductions
likely contribute to the lower response rates observed. Cytopenias are fre-
quently due to the concurrent use of immunosuppressive agents and alter-
ations in renal function related to calcineurin inhibitor therapy or other
post-transplant factors aecting drug pharmacokinetics. The optimal dose
of RBV in transplant patients has not been determined. Perhaps more
than in any other treatment population, growth factors to manage
Table 4
Preemptive treatment of hepatitis C virus infection in liver transplant recipients
Dose
Study Treatment Time to Histologic reduction F/U AR (Rx/
Study type n regimen Rx (wk) SVR response or D/C (mo) no Rx)
Singh, RCT 12 treated, IFN, 3MU tiw 6 2 0% in both No dierence 50% 29 6/5
1998 [73] 12 control mo versus no groups in severity
treatment
POSTTRANSPLANT HEPATITIS C MANAGEMENT

Sheiner, RCT 35 IFN, IFN 3MU tiw 6 3 17% Fewer with 28% drug D/C 22 17/23
1998 [71] 46 no Rx mo versus no IFN; 5% recurrence
treatment no Rx at 1 and 2 yr
in IFN group
Mazzaferro, UC 36 IFN 3MU tiw 3 33% Normal biopsy 47% 52 0/
2001 [74] RBV 10 mg/kg/d in virologic
for 12 mo responders
Shergill, RCT (two 44 IFN/PEGIFN alone %6 9% NA 85% dose 18 18/
2004 [70] dierent versus IFN/ reductions,
Rx arms) PEGIFN RBV 37% D/C
12 mo
Sugawara, UC 21 (all IFN 3MU Median 43% Lower HAI 62% dose 26 6/
2004 [75] LDLT) tiw RBV 4.3 score in reductions or
400 mg/d responders than discontinuation
increased to IFN non-responders
6MU tiw RBV
600 mg/d 12 mo
Chalasani, RCT 26 treated, PegIFNa2a 180 3 8% (0% in No dierence 31% drug 18 3/6
2005 [82] 28 control mg/wk 12 mo controls) in severity discontinuation
Abbreviations: AR, acute rejection; D/C, discontinuation: F/U, follow-up time; HAI, hepatic activity index; IFN, interferon; LDLT, living donor liver
transplant recipient; MU, million units; PEGIFN, pegylated interferon; RBV, ribavirin; RCT, randomized controlled trial; Rx, treatment; SVR, sustained
virologic response; UN, uncontrolled trial.
163
cytopenias and maintain optimal doses of antiviral treatment are likely to be

important in maximizing responses to therapy.
Prophylactic therapy
Preclinical and retrospective cohort data suggest that HCV antibodies
have neutralizing eects and may attenuate the risk of HCV infection
[66,67]. In a retrospective observational study of transplant recipients with
HBV and HCV receiving HBIG, the prevalence of recurrent HCV disease
after transplantation was lower in those who received HBIG before screen-
ing blood donors for HCV compared with those who received HBIG after
routine screening of blood donors for antibody to HCV began [66]. These
results suggest that the HCV antibodies in HBIG before screening the do-
nors for HCV infection reduced the risk of recurrent disease. In a study
of two chimpanzees treated with high-dose hepatitis C immune globulin
(HCIG) immediately before and after HCV inoculation, both animals
were initially viremic but subsequently had undetectable HCV RNA and
had no evidence of biochemical hepatitis [67].
At least three HCV antibody preparations are being studied in prospec-
tive clinical trials [68]. A preliminary report of the Canadian HCIG study
found no benet, with HCV RNA detectable in all treated patients post-
transplant and no appreciable dierence in the level of viremia or histologic
disease in patients receiving HCIG prophylaxis versus control subjects [69].
A second study in the United States evaluated 18 patients (six high-dose
HCIG, six low-dose HCIG, and six untreated control subjects) with chronic
HCV infection undergoing liver transplantation [70]. Treatment began in
the anhepatic phase, and a total of 17 infusions were given over 14 weeks.
During treatment, alanine aminotransferase (ALT) levels were lower in
treated (especially the high-dose group) than untreated patients, suggesting
a possible antiinammatory eect. There were no dierences in serum HCV
RNA levels between groups; however, a lower (but not signicantly re-
duced) liver HCV RNA level was noted at month 1 post-transplant in the
high-dose group compared with control subjects. In these studies, the
HCIG product was produced from anti-HCV positive donors. The studies
diered in terms of the doses used and schedule of administration; however,
in both studies, reinfection occurred in all patients. A third study, using fully
human monoclonal HCV antibodies, is underway, and results are expected
in 2005. Based on the data available, however, there is no established role
for prophylactic antibody therapy in the management of patients with
HCV infection undergoing liver transplantation at this time.
Preemptive antiviral therapy

Preemptive therapy refers to initiation of therapy in the early post-trans-
plant period when HCV viral loads are lower than pretransplant levels, and
histologic disease is absent or minimal. These characteristics of the early

post-transplant period may enhance virologic responses with antiviral ther-
apy, as has been shown in nontransplant patients. Studies have shown that
preemptive antiviral therapy can lead to eradication of HCV infection, but
tolerability of therapy is a limitation. Patients with high pretransplant
Model for End-stage Liver Disease (MELD) and Child-Turcotte-Pugh
scores were less suitable for this therapeutic approach [52]. Unlike nontrans-
plant and acutely infected patients, liver transplant recipients are typically
on multiple immunosuppressive drugs and prophylactic antimicrobial
agents, have variable renal function, and are recovering from a major oper-
ative procedure in the setting of advanced liver disease. All these factors may
aect candidacy for and tolerability of HCV therapy. In a study of 110
consecutive patients transplanted for chronic hepatitis C at a single center
in the United States, only 60% were found to be candidates for IFN and
RBV therapy at 8 weeks post-transplant [52]. Reasons for ineligibility in-
cluded persistent anemia, renal dysfunction, cardiopuimonary complica-
tions, active psychiatric or neurologic diseases, and others reective of
a slower postoperative recovery among patients with advanced liver disease
pretransplantation.
Clinical trials using preemptive IFN monotherapy, although generally
positive, demonstrated a modest benet, primarily in delaying time to his-
tologic recurrence. A sustained virologic response (SVR) was infrequent,
occurring in 0% to 22% [52,7173]. A recent study of PegIFNa2a mono-
therapy for 12 months reported a SVR rate of 8% with high rates of
withdrawal due to intolerance (31%) [74]. Studies using IFN and RBV
suggest combination therapy is more ecacious. An uncontrolled study
from Italy of 36 patients (83% genotype Ib) treated with combination
standard IFNa-2b 3 million units (MU) thrice weekly and RBV 10
mg/kg/d within 3 weeks after transplantation and continued for 52 weeks,
reported a SVR of 33%, and all patients had normal serum ALT levels
and histology after completion of therapy [75]. Viral clearance was more
frequent in patients with non-1 HCV genotypes (100%) compared with
those with genotype 1b (20%). In contrast to the Italian experience, a
United States study of preemptive standard IFN or PEGIFNa-2b 3 MU
thrice weekly or 1.5 mg/kg/wk, alone or in combination with RBV 600 mg
daily increasing to 1.0 to 1.2 g daily, for a total of 48 weeks, showed
an SVR in only 9%, with most responders in the combination group.
Dose reductions and discontinuations were required in 85% and 37% of
patients, respectively. The inability to achieve full-dose therapy may explain
the low SVR rate. The dierences in applicability and tolerability of pre-
emptive therapy in the Italian and United States studies may reect the
clinical status of the patients in the early post-transplant period. Whether
treating HCV disease preemptively results in better long-term outcomes
than treatment initiated only when recurrent disease is established has not
been evaluated.
Living donor liver graft recipients may be good candidates for preemp-
tive HCV treatment because they are generally less sick pretransplantation
than deceased donor recipients. In an uncontrolled study of 21 HCV-posi-
tive recipients of LDLT grafts given IFNa-2b and RBV starting 1 month
after transplantation, the SVR was 43% and patient survival was 85%.
Six patients (29%) were withdrawn from treatment due to intolerance [76].
Antiviral therapy for recurrent hepatitis C virus disease

In the treatment of established recurrent HCV disease, study protocols
have generally initiated treatment between 6 to 24 months after transplanta-
tion (Table 5). Initiating treatment after recurrent disease is clinically or his-
tologically apparent, rather than as preemptive treatment, has potential
advantages that include lower doses of immunosuppressive drugs (which
may enhance the response to antiviral therapy), improved clinical status
to better tolerate treatment, lower risk of acute rejection, and cost savings
because only those with progressive disease are treated. The potential dis-
advantages of the approach of wait and treat if there is progressive his-
tologic disease include a higher viral load and more advanced stage of
brosis at the time of treatment, which are factors that may reduce the like-
lihood of achieving a SVR.
Results with IFN or RBV monotherapy have been disappointing. IFN
plus RBV achieves superior results compared to those reported with mono-
therapy [20,5563], with histologic improvement reported in both responders
and non-responders (Table 5). The majority of the studies evaluating combi-
nation therapy for recurrent HCV disease are uncontrolled, use variable
doses and duration of IFN and RBV therapy, and are of a sample size too
limited to perform multivariate analyses to identify predictors of response.
The doses of IFN used in these studies ranged from 1.5 to 5 MU thrice
weekly, and the doses of RBV used ranged from 400 to 1200 mg daily; in
most studies, the duration of therapy was 48 weeks. End-of-treatment bio-
chemical and virologic responses ranged from 25% to 100% and 9% to
64%, respectively. There was also a wide range of reported SVR rates (as-
sessed at 6 months after completion of therapy) ranging from 5% to 45%.
Adverse events during combination treatment are common with withdrawal
of treatment, which is required in up to 50% of patients. Anemia, leukope-
nia, and thrombocytopenia are the most common reasons cited for treatment
discontinuation and dose reduction. The SVR rates using PEGIFN plus
RBV are typically higher than with standard IFN plus RBV, ranging from
26% to 37.5%. Despite titrated regimens using low initial doses of PEGIFN
and RBV, dose reductions and discontinuations are frequently required.
The optimal duration of treatment for recurrent HCV disease is un-
known. In immunosuppressed liver transplant recipients, longer treatment
periods may be necessary to enhance the SVR. In nontransplant patients
who have HCV infection who fail to clear HCV after combination IFN
and RBV therapy, maintenance IFN therapy may be benecial in preventing

disease progression and complications of cirrhosis. Although several ongo-
ing clinical trials (HALT-C, EPIC, and CO-PILOT) are investigating this
treatment strategy, maintenance therapy has not been evaluated fully as
a means of preventing disease progression in liver transplant recipients.
The cost-eectiveness of antiviral therapy for HCV disease in liver trans-
plant recipients has been examined in only one study. Using a Markov-based
decision analytic model to simulate costs and health outcomes, treatment of
recurrent HCV infection with antiviral therapy (IFN and RBV) was found to
be cost eective. Treatment of 100 men, 55 years of age, with recurrent hep-
atitis C prevented 29 cases of cirrhosis, prevented seven deaths, and had an
incremental cost eectiveness ratio of $29,100 per year of life saved [77].
Retransplantation for recurrent hepatitis C virus

The only denitive treatment for patients with advanced recurrent HCV-
related liver disease is a repeat liver transplant. Over the next decade, the
number of patients with recurrent hepatitis C seeking retransplantation is
expected to increase severalfold [78]. In the United States, 8% to 9% of
transplants performed are retransplants, with recurrent HCV infection ac-
counting for approximately 40%. Compared with primary transplantation,
retransplantation for all indications is associated with longer hospital stays,
40% greater cost, and a 10% to 20% reduction in survival [8]. Outcomes of
retransplantation for recurrent HCV disease are reported to be interior to
results of retransplantation for other indications. In a large study of over
10,000 patients in the UNOS database, anti-HCVpositive retransplant re-
cipients had reduced survival compared with anti-HCVnegative retrans-
plant recipients, with 5-year survival rates of 54% and 61%, respectively
[79]. Studies specically evaluating retransplantation for recurrent HCV dis-
ease (not just HCV-positive status) are smaller and from single centers but
show a similar picture. Predictors of poor outcome after retransplantation
included recipient age over 50 years, serum creatinine O2.0 mg/dL, serum
bilirubin O10 mg/dL, and poor physical condition [80,81]. In general, out-
comes are predicted to be best if retransplantation occurs before the onset of
liver decompensation or signicant renal dysfunction. In a study modeling
outcomes of retransplantation, the optimal graft allocation utility function,
dened as the product of outcome (predicted post-transplantation survival)
and urgency (MELD score), occurred at lower MELD scores for HCV
(MELD 21) compared with non-HCV (MELD 24) patients [82]. Addi-
tionally, those with a longer period of time from rst to second transplant
seem to have a better outcome [81]. Retransplantation for recurrent HCV
disease remains a controversial issue. Most transplant programs use a selec-
tive approach, choosing HCV-infected patients with favorable clinical char-
acteristics for retransplantation. Some have advocated that organ allocation
168
Table 5
Treatment of established recurrent hepatitis C virus disease in liver transplant recipientsa
Average
Study duration time W/D EOTR SVR F/U AR (Rx/ CR (Rx/
Study design Treatment n LT to Rx (mo) N (%) N (%) N (%) (mo) no Rx) no Rx)
IFN and RBV
Firpi, UC IFN 3 MU tiw RBV 54 31 7 (13) 21 (38) 16 (30) 18 3/ 1/
BIGGINS & TERRAULT

2002 [20] 8001000 mg/d 48 wk
Shakil, UC IFN 3 MU tiw RBV 38 23 16 (42) 5 (13) 2 (5) 18 0/ 0/
2002 [61] 800 mg/d 48 wk
Lavezzo, UC IFN 3 MU tiw RBV
2002 [62] 800 mg/kg
6 mo 27 9 2 (4) 9 (33) 6 (22) 18 1/ 0/
12 m 30 7 (30) 5 (17)
Menon, UC IFN 3 MU tiw RBV 26 15 13 (50) 9 (35) 6 (23) 0/ 0/
2002 [63] 800100 mg/d R 12 mo
Bizollon, UC IFN 3 MU tiw RBV 54 13 6 (11) 14 (26) 54 0/ 0/
2003 [56] 1000 mg/d 6 mo then
RBV 600800 mg/d
alone 12 mo
Samuel, RCT IFN 3 MU tiw RBV 54 (28 54 12 (43) 9 (32) 6 (21) 18 0/0 1/0
2003 [55]b 10001200 mg/d 48 wk treated)
Chalasani, RCT PegIFN2a 180 mg/wk 48 wk 67 (34 25 10 (30) 4 (15)c 3 (12)c 18 4/0 NA
2005 [74]b untreated)
Castells, CT PegIFN2b 1.5 mg/kg/wk RBV 48 (24 3.8 3 (13) 15 (63)c 8 (35)c 18 1/2 NA
2005 [83] 400800 mg/d 48 wk untreated)
Giostra, UC RBV 10 mg/kg 12 wk then 31 18 9 (29) 14 (45) 9 (31) 18 0/ 1/
2004 [57] IFN 3 MU tiw RBV
10 mg/kg 48 wk
PEGIFN and RBV
Abdelmalek, UC IFN 3 MU tiw or PEGIFN a-2b 90 NA NA NA 28 (31) 36 0/ 2/
2004 [58] 1.5 mg/kg/wk or PEGIFN 90
a-2a mg/wk plus RBV
6001000 mg/d 48 wk
Stravitz, UC IFN 3 MU tiw or PEGIFN 23 42 NA 11 (48) 8 (35) 33 7/ 1/
2004 [53] 1.0 mg/kg/wk RBV 1000
POSTTRANSPLANT HEPATITIS C MANAGEMENT

mg/d 48 wk
Saab, UC IFN or PEGIFN and RIB 44 42 23 (52) NA 3 (7) NA 5/ 0/
2004 [54] Doses NA
Mukherjee, UC PEGIFN a-2b 1.5 mg/kg/wk 39 20 17 (44) 15 (39) 12 (31) 18 NA NA
2003 [59] RBV 800/d 48 wk
Dumortier, UC PEGIFN a-2b 1.0 mg/kg/wk 20 28 4 (20) 11 (55) 9 (45) 18 0/ 0/
2004 [60] RBV 1000 mg/d 48 wk
Abbreviations: AR, acute rejection; CR, chronic rejection; CT, controlled trail; F/U, follow-up time; IFN, interferon; MU, million units; N, number;
NA, not available; PEGIFN, pegylated interferon; RBV, ribavirin; RCT, randomized controlled trail; UC, uncontrolled trail.
a
Limited to studies with R20 patients.
b
Only randomized study with untreated control group.
c
0 in untreated group.
169
for retransplantation for HCV and non-HCV etiologies include outcome

measures, such as predicted survival after retransplantation, rather than
solely wait-list mortality (eg, MELD score) [51,82].
Summary
Recurrent HCV infection is universal in liver transplant recipients who
are viremic pretransplant. The rate of histologic disease progression after
transplantation is more rapid, and the risk of cirrhosis by 5 to 10 years is
about 30%. Several donor, recipient, and viral factors have been associated
with worse post-transplant outcomes in recipients with recurrent hepatitis C.
Whether or not HCV-infected recipients of live donor grafts have worse out-
comes compared with deceased donor graft recipients is controversial. To
maximize the long-term survival of recipients with HCV infection, eradica-
tion of infection is the ultimate goal. Treatment of recurrent HCV after liver
transplantation can be undertaken at several dierent time points: (1) pro-
phylactically, at the time of transplantation; (2) preemptively, in the early
post-transplant period; and (3) after established recurrent histologic disease
is present. Prophylactic therapy for HCV infection has no established role at
present, but studies are ongoing. Preemptive therapy using IFN and RBV
has resulted in variable SVR rates (9%43%) and is generally poorly toler-
ated, especially if the patient has advanced liver disease pretransplantation.
Treatment of established recurrent HCV disease with combination PEGIFN
and RBV is associated with a SVR in about 30% to 35% of patients overall
but is limited by high rates of dose reduction or drug discontinuation. In
conclusion, successful HCV eradication in the post-transplant setting is dif-
cult with current treatment options, but it is possible. Determination of
the optimal doses of antiviral drugs in transplant patients and improvements
in drug tolerability may be important rst steps in achieving enhanced
response rates. There is a need for new drugs in this population that have
greater ecacy and a better safety prole.
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20 (2006) 175178
Index
Note: Page numbers of article titles are in boldface type.
A Core protein
Alternate reading frame protein (ARFP) implications for design of novel
dened, 85 anti-HCV strategies, 85
implications for design of novel
anti-HCV strategies, 85 E
Amantadine/PEGIFN/ribavirin E1/E2
for nonresponders to chronic HCV implications for design of novel
management, 126127 anti-HCV strategies, 8586
Amdoxovir Emtricitabine
for HBV infection, 75 for HBV infection, 7274
Antiviral agents
for naive patients with HCV infection, G
99113. See also Hepatitis C virus
(HCV) infection, naive patients Genotype(s)
with, treatment of, antiviral HBV
therapy in role of, 6870
Antiviral therapy
preemptive H
for recurrent HCV infection in HAPs. See Heteroaryldihydropyrimidines
liver transplant patients, (HAPs)
164167
HCC. See Hepatocellular carcinoma (HCC)
ARFP. See Alternate reading frame protein
(ARFP) HCV infection. See Hepatitis C virus
(HCV) infection
Hepatitis. See also under Hepatitis C virus
C
(HCV) infection; specic type, e.g.,
Chemoembolization Hepatitis B virus (HBV) infection
transarterial types of, 12
for HCC, 1516
Hepatitis B virus (HBV), 4761
Chemotherapy described, 4750
systemic DNA assays of
for HCC, 1617 clinical applications of, 5455
CIFN. See Consensus interferon (CIFN) detection and quantication of
molecular techniques for,
Cis-acting elements 5560
anti-HCV strategies, 93 Hepatitis B virus (HBV) core antigen
isolated antibody to
Clevudine screening for
for HBV infection, 74 before HBV immunization,
3031
Consensus interferon (CIFN)/ribavirin
for nonresponders to chronic HCV Hepatitis B virus (HBV) genotypes
management, 125126 role of, 6870
doi:10.1016/S0891-5520(06)00028-6 id.theclinics.com
176 INDEX
Hepatitis B virus (HBV) infection modifying lifestyle factors

acute, 5153 associated with,
chronic, 5354, 6379 129130
assessment of, 6667 management of
monitoring of, 6667 failure of
natural history of, 6366 reasons for, 116124
prevalence of, 63 in patients who failed to
progression to, 5051 achieve sustained
treatment of virologic response
amdoxovir in, 75 therapies under
clevudine in, 74 investigation, 124127
emtricitabine in, 7274 maintenance, 127129
goals in, 7172 interferon in, 127128
HAPs in, 75 ribavirin in, 128129
lobucavir in, 75 HCC and, 125. See also
new agents in, 7274 Hepatocellular carcinoma (HCC)
-L-nucleosides in, 74 molecular virology of
patient selection for, 6768 antiviral implications of, 8392
pradefovir in, 75 novel antiviral strategies, 8198
telbivudine in, 74 ARFP, 85
tenofovir in, 72 cis-acting elements, 93
HCC and, 125. See also core protein, 85
Hepatocellular carcinoma (HCC) E1/E2, 8586
molecular diagnosis of, 5460 host targets, 9192
prevalence of, 27 NS2, 86
serologic diagnosis of, 5154 NS3, 8687
vaccines for, 2745 NS4A, 8788
adverse eects of, 39 NS4B, 8889
ecacy of, 3538 NS5A, 90
immunogenicity of, 3133 NS5B, 9091
immunotherapy using, 39 p7, 86
indications for, 2730 naive patients with
nonresponse to treatment of
management of, 3839 antiviral therapy in, 99113
predictors of, 3435 adherence to, 108109
prevaccination screening in, adverse eects of, 110111
3031 benets of, 111
routes of administration of, combination therapy of
3334 interferon with
screening for isolated antibody to ribavirin, 102103
HBV core antigen in, cost eectiveness of, 111
3031 early virological response
to, 106108
Hepatitis C virus (HCV) infection historical perspective of,
chronic 101102
combination therapy for
patient selection for,
relapse after
100101
future directions in,
PEGIFN monotherapy,
148149
103105
mechanisms of, 138139 PEGIFN/ribavirin
prediction of sustained therapy, 105106
virologic response to,
pretreatment predictors of
144145
response to, 106
rates of, 137138
recurrence of
treatment of, 137153. See
in liver transplant patients
also specic agents, management of, 155174
e.g., Interferon/ interferons in, 161162
ribavirin
preemptive antiviral
brosis progression in
therapy in, 164167
INDEX 177
natural history of, 156157 Interferon(s) (IFNs)/ribavirin

prevention of, 162167 for retreatment of interferon
prevalence of, 155 monotherapy relapsers in chronic
retransplantation for, 167170 HCV infection treatment,
severity of 140144
factors associated with,
157161 L
Hepatitis C virus (HCV) replicons, 9294 Life cycle
Hepatocellular carcinoma (HCC), 125 overview of, 8183
clinical presentation of, 12 Lifestyle modications
diagnosis of, 23 brosis progression and, 129130
HBVassociated, 125
HCVassociated, 125 Liver transplantation
mortality due to, 27 for HCC, 912
prevention of, 68 recurrent HCV infection after
screening for, 36, 7071 management of, 155174. See
treatment of, 820 also Hepatitis C virus
introduction to, 8 (HCV) infection, recurrence
liver transplantation in, 912 of, in liver transplant
percutaneous ethanol injection patients, management of
in, 1213 natural history of, 156157
radiofrequency ablation in, Lobucavir
1315 for HBV infection, 75
surgical resection in, 89
systemic chemotherapy in,
1617 M
TACE in, 1516 Molecular virology
of HCV infection
Heteroaryldihydropyrimidines (HAPs)
implications for novel therapies,
for HBV infection, 75
8198. See also Hepatitis C
Host targets virus (HCV) infection,
implications for design of novel anti- molecular virology of, novel
HCV strategies, 9192 antiviral strategies
N
I
NS2
IFNs. See Interferon(s) (IFNs)
Immunogenicity anti-HCV strategies, 86
of HBV vaccines, 3133
NS3
Immunotherapy implications for design of novel
HBV vaccines and, 39 anti-HCV strategies, 8687
Interferon(s) (IFNs) NS4A
in maintenance management of HCV implications for design of novel
infection in patients who failed to anti-HCV strategies, 8788
achieve sustained virologic
NS4B
response, 127128
ribavirin with
anti-HCV strategies, 8889
for naive patients with HCV
infection, 102103 NS5A
Interferon(s) (INFs)
anti-HCV strategies, 90
for recurrent HCV infection in liver
transplant patients, 161162 NS5B
for retreatment of interferon implications for design of novel
monotherapy relapsers in chronic anti-HCV strategies, 9091
HCV infection treatment, -L-Nucleoside(s)
140144 for HBV infection, 74
178 INDEX
P achieve sustained virologic

p7 response, 128129
implications for design of novel Ribavirin/CIFN
anti-HCV strategies, 86 for nonresponders to chronic HCV
PEGIFN. See Peginterferon (PEGIFN) management, 125126
Peginterferon (PEGIFN) Ribavirin/IFN

alfa for naive patients with HCV infection,
for naive patients with HCV 102103
infection, 103105 Ribavirin/PEGIFN
Peginterferon (PEGIFN)/ribavirin for naive patients with HCV infection,
for chronic HCV infection 105106
relapse after for nonresponders to chronic HCV
treatment of, 147148 management, 126
for naive patients with HCV infection, Ribavirin/PEGIFN/amantadine
105106 for nonresponders to chronic HCV
higher doses and longer duration of management,
for nonresponders to chronic 126127
HCV management, 126
Peginterferon (PEGIFN)/ribavirin/
amantadine S
for nonresponders to chronic HCV Systemic chemotherapy
management, 126127 for HCC, 1617
Percutaneous ethanol injection
for HCC, 1213 T
Pradefovir TACE. See Transarterial chemoembolization
for HBV infection, 75 (TACE)
Protease inhibitors Telbivudine
for nonresponders to chronic HCV for HBV infection, 74
management, 125
Tenofovir
Protein(s) for HBV infection, 72
core
implications for design of novel Transarterial chemoembolization (TACE)
anti-HCV strategies, 85 for HCC, 1516
Transplantation
R liver. See Liver transplantation
Radiofrequency ablation
for HCC, 1315 V
Ribavirin Vaccine(s)
in maintenance management of HCV HBV, 2745. See also Hepatitis B virus
infection in patients who failed to (HBV) infection, vaccines for

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HEPATITIS

The Progression of Hepatitis B and CInfections

VOLUME 20 NUMBER 1 MARCH 2006 v

Assessment and Management of Chronic Hepatitis B 63

Molecular Virology of the Hepatitis C Virus: Implication

Antiviral Therapy for Treatment Nave Patients

Approach to the Management of Patients with Chronic

Treatment of Relapsers After Combination Therapy

Management of Recurrent Hepatitis C in Liver Transplant

The Clinics are now available online!

Access your subscription at:

Several viruses are capable of causing hepatic inammation. These in-

make it increasingly dicult for the practicing physician to keep up with

Robert C. Moellering, Jr, MD

The Progression of Hepatitis

Patients with, or at risk for, hepatocellular carcinoma (HCC) present

expertise and preference. US is also quite useful, but a lesion detected on US

Box 1. European Association for the Study of Liver Disease

Sensitivity Specificity Sensitivity Specificity

P ! .0001). Resectability and survival were also signicantly better in those

imaging in 3 months to determine lesion persistence or growth; lesions 1 to

part of standard care. One placebo-controlled study was stopped on interim

of more than 14% of indocyanine green at 15 minutes indicates poor tolerabil-

accrued waiting time were ranked higher. As waiting times lengthened

Posttransplant hepatitis C has a highly variable course from rapid decline

Percutaneous ethanol injection

study from ve dierent Italian centers reported approximately six sessions

by intratumor and surrounding vasculature can create a signicant heat

occurred in completely excised specimens taken 4 weeks later. The foci of

of just the tumor-feeding artery and chemotherapy delivery without embo-

therapies. It is not surprising that chemotherapy clinical trials have shown

provides a detailed therapeutic algorithm (Fig. 4) [96]. Patients are stratied

Stage 0 Stage A-C Stage D

HBV & HCV: CHRONIC LIVER DISEASE & HCC

Single 3 nodules all <3 cm

Portal invasion, N1, M1?

Both normal No Yes No Yes

Resection Transplantation RFA/PEI TACE New agents Supportive

transplantation. Conversely, if recurrence presents at greater than stage T2,

Box 1. Indications for pre-exposure hepatitis B vaccination

professions or institutions for inmates or mentally retarded people are typ-

immunization should be tested for the presence of antiHBs. If antiHBs is

Prevaccination screening and isolated antibody to hepatitis B core antigen

chain reaction (PCR). Alternatively, an individual with isolated positive

Pennsylvania) and Engerix-B (Glaxo-SmithKline, Philadelphia, Pennsylva-

subjects who were younger (aged 20 to 39 years versus 40 to 59 years), sup-

Routes of vaccine administration

Box 2. Risk factors associated with poor anti-HBs response to

nonresponders mounted an appreciable antiHBs titer. When nine of the 12

Ecacy of hepatitis B vaccines

vaccine injection, probably attributable to immunologic protection against

other hand, the prevalence of S gene mutations among HBsAgpositive chil-

Vaccine nonresponse and its management

Adverse eects of hepatitis B vaccines

Immunotherapy using hepatitis B vaccine

Serologic and Molecular Diagnosis

Hepatitis B virus (HBV) is a hepadnavirus with a 3200-base-pair genome

decompensation. Mutations that lead to resistance occur less frequently dur-