Book on hepatitis from page 118 to 134

Book on hepatitis from page 118 to 134

Prophylaxis and Vaccination  117
Keeffe EB, Iwarson S, McMahon BJ, et al. Safety and immunogenicity of hepatitis A vaccine in
patients with chronic liver disease. Hepatology 1998;27:881-6. (Abstract)
Klade CS, Wedemeyer H, Berg T, et al. Therapeutic vaccination of chronic hepatitis C
nonresponder patients with the peptide vaccine IC41. Gastroenterology
2008;134:1385-95. (Abstract)
Kubitschke A, Bader.C Tillmann, H. L Manns, M. P Kuhn, S, Wedemeyer H. Injury with HCV-contaminated needles: What is the true rate of serconversion? Internist (Berl) 2007;
48: 1165-72. (Abstract)
Lalazar G, Rund D, Shouval D. Screening, prevention and treatment of viral hepatitis B
reactivation in patients with haematological malignancies. Br J Haematol
2007;136:699-712. (Abstract)
Loomba R, Rowley A, Wesley R, Liang TJ, et al. Systematic review: The effect of preventive
lamivudine on hepatitis B reactivation during chemotherapy. Ann Intern Med 2008;
148:519-28. (Abstract)
Low E, Vogel M, Rockstroh J, Nelson M. Acute Hepatitis C in HIV-Positive Individuals. AIDS
Rev 2008;10:245-53.
Martinez-Bauer E, Forns X, Armelles M, et al. Hospital admission is a relevant source of
hepatitis C virus acquisition in Spain. J Hepatol 2008;48:20-7. (Abstract)
Monteyne P, Andre FE. Is there a causal link between hepatitis B vaccination and multiple
sclerosis? Vaccine 2000;18:1994-2001. (Abstract)
Nothdurft H.D. Hepatitis A vaccines. Expert Rev Vaccines 2008;7:535-45. (Abstract)
Pestka JM, Zeisel MB, Blaser E, et al. Rapid induction of virus-neutralizing antibodies and viral
clearance in a single-source outbreak of hepatitis C. Proc Natl Acad Sci U S A
2007;104:6025-30. (Abstract)
Petersen J. HBV treatment and pregnancy. J Hepatol 2011;55:1171-3. (Abstract)
Pischke S, Suneetha PV, Baechlein C, et al. Hepatitis E Virus Infection as a Cause of Graft
Hepatitis in Liver Transplant Recipients. Liver Transplantation 2010;16:74-82.
(Abstract)
Pockros P, Jacobson I, Boyer TD, et al. Gi-5005 Therapeutic Vaccine Plus Peg-Ifn/Ribavirin
Improves Sustained Virologic Response Versus Peg-Ifn/Ribavirin in Prior Non-Responders with Genotype 1 Chronic Hcv Infection. Hepatology 2010;52:404a-5a.
Ranger-Rogez S, Denis F. Hepatitis B mother--to--child transmission. Expert Rev Anti Infect
Ther 2004;2:133-45. (Abstract)
Rehermann B. Hepatitis C virus versus innate and adaptive immune responses: a tale of
coevolution and coexistence. Journal of Clinical Investigation 2009;119:1745-54.
(Abstract)
Rein DB, Weinbaum CM. The cost-effectiveness of using hepatitis A/B combined vaccine
versus hepatitis B vaccine alone for high-risk heterosexuals. Vaccine 2008;26:5331-3. (Abstract)
Sadovnick AD, Scheifele DW. School-based hepatitis B vaccination programme and adolescent
multiple sclerosis. Lancet 2000;355:549-50. (Abstract)
Sallberg MM, Frelin L, Diepolder H, et al. A First Clinical Trial of Therapeutic Vaccination Using
Naked DNA Delivered by in Vivo Electroporation Shows Antiviral Effects in Patients
with Chronic Hepatitis C. Journal of Hepatology 2009;50:S18-S19.
Santantonio T, Medda E, Ferrari C, et al. Risk factors and outcome among a large patient
cohort with community-acquired acute hepatitis C in Italy. Clin Infect Dis
2006;43:1154-9. (Abstract)
Schattner A. Consequence or coincidence? The occurrence, pathogenesis and significance of
autoimmune manifestations after viral vaccines. Vaccine 2005;23:3876-86. (Abstract)
Shrestha M.P, Scott R.M, Joshi D.M, et al. Safety and efficacy of a recombinant hepatitis E
vaccine. N Engl J Med 2007;356:895-903. (Abstract)
Torresi J, Johnson D, Wedemeyer H. Progress in the development of preventative and
therapeutic vaccines for hepatitis C virus. J Hepatology 2011. (Abstract)
Turner J.M, Rider A.T, Imrie J, Copas A.J, Edwards S.G, Dodds J.P, Stephenson J.M.
Behavioural predictors of subsequent hepatitis C diagnosis in a UK clinic sample of
HIV positive men who have sex with men. Sex Transm Infect 2006;82:298-300.
(Abstract)
van de Laar T, Pybus O, Bruisten S, et al. Evidence of a large, international network of HCV
transmission in HIV-positive men who have sex with men. Gastroenterology
2009;136:1609-17. (Abstract)
118  Hepatology 2012
van der Sande MA, Waight PA, Mendy M, et al. Long-term protection against HBV chronic
carriage of Gambian adolescents vaccinated in infancy and immune response in HBV
booster trial in adolescence. PLoS ONE 2007;2: e753. (Abstract)
Van Herck K, Jacquet JM, Van Damme P. Antibody Persistence and Immune Memory in
Healthy Adults Following Vaccination With a Two-Dose Inactivated Hepatitis A
Vaccine: Long-Term Follow-Up at 15 Years. J Med Virol 2011;83:1885-91. (Abstract)
Vento S, Garofano T, Renzini C, Cainelli F, Casali F, Ghironzi G, et al. Fulminant hepatitis
associated with hepatitis A virus superinfection in patients with chronic hepatitis C. N
Engl J Med 1998;338:286-90. (Abstract)
Wedemeyer H, Cornberg M, Protzer U, Berg T, Dollinger MM. [German guidelines on diagnosis
and therapy of hepatitis B]. Dtsch Med Wochenschr 2007;132:1775-82.
Wedemeyer H, Pethig K, Wagner D, et al. Long-term outcome of chronic hepatitis B in heart
transplant recipients. Transplantation 1998;66:1347-53. (Abstract)
Wedemeyer H, Schuller E, Schlaphoff V, et al. Therapeutic vaccine IC41 as late add-on to
standard treatment in patients with chronic hepatitis C. Vaccine 2009;27:5142-51.
(Abstract)
Wedemeyer H, Janczewska-Kazek E, Mazur WW, et al. HCVac study: Antiviral activity of
TG4040 therapeutic vaccine in genotype-1 chronic HCV patients. Hepatology 2011.
Wedemeyer H, Pischke S. Hepatitis: Hepatitis E vaccination--is HEV 239 the breakthrough?
Nat Rev Gastroenterol Hepatol 2011;8:8-10. (Abstract)
WHO, Revised WHO position paper on hepatitis B vaccine, Oct 2009,
http://www.who.int/immunization/documents/positionpapers/en/index.html#hepb,
accessed 2011. (Abstract)
Wiegand J, Cornberg M, Aslan N, et al. Fate and function of hepatitis-C-virus-specific T-cells
during peginterferon-alpha 2b therapy for acute hepatitis C. Antivir Ther 2007;12:303-16. (Abstract)
Wichmann O, Schimanski S, Koch J, Kohler M, Rothe C, Plentz A, et al. Phylogenetic and
case-control study on hepatitis E virus infection in Germany. J Infect Dis
2008;198:1732-41. (Abstract)
Wolters B, Junge U, Dziuba S, Roggendorf M. Immunogenicity of combined hepatitis A and B
vaccine in elderly persons. Vaccine 2003;21:3623-28. (Abstract)
Zanetti AR, Mariano A, Romano L, et al. Long-term immunogenicity of hepatitis B vaccination
and policy for booster: an Italian multicentre study. Lancet 2005;366:1379-84.
(Abstract)
Zhu FC, Zhang J, Zhang XF, et al. Efficacy and safety of a recombinant hepatitis E vaccine in
healthy adults: a large-scale, randomised, double-blind placebo-controlled, phase 3
trial. Lancet 2010;376:895-902. (Abstract)
Zuckerman J, van Hattum J, Cafferkey M, Gjorup I, Hoel T, Rummukainen M.L, Weiland O.
Should hepatitis B vaccination be introduced into childhood immunisation
programmes in northern Europe? Lancet Infect Dis 2007;7:410-19. (Abstract)
Hepatitis B: Diagnostic Tests  119
8.  Hepatitis B: Diagnostic Tests
Jörg Petersen
Introduction
The diagnosis of hepatitis B virus (HBV) infection was initiated by the discovery of
Australia antigen (hepatitis B surface antigen, HBsAg). During the following
decades, serologic assays were established for HBsAg and other HBV antigens and
antibodies. Advances in molecular biology techniques led to the development of
polymerase chain reaction (PCR) assays for direct determination of hepatitis B virus
DNA (HBV DNA).
Diagnosis of Hepatitis B Virus (HBV) infection tests for a series of serological
markers of HBV and excludes alternative etiological agents such as hepatitis A, C,
and D viruses. Serological tests are used to distinguish acute, self-limited infections
from chronic HBV infections and to monitor vaccine-induced immunity. These tests
are also performed to determine if the patient should be considered for antiviral
therapy. Nucleic acid testing for HBV DNA is used as the standard to quantify HBV
viral load and measure the effectiveness of therapeutic agents.
Other causes of chronic liver disease should be systematically looked for
including coinfection with HCV, HDV or HIV.  Cytomegalovirus, Epstein-Barr
virus, enteroviruses, other hepatotoxic drugs, and even herbal medicines should be
considered when appropriate. Moreover, co-morbidities, including alcoholic,
autoimmune, metabolic liver disease with steatosis or steatohepatitis should be
assessed. Finally, vaccination status and previous tests results should be used to
guide appropriate testing.
Serological tests for HBV
Collection and transport
Serological tests for viral antigens can be performed on either serum or plasma
(Yang 2002). The World Health Organization (WHO) has defined an international
standard for normalisation of expression of HBV DNA concentrations (Quint 1990).
Serum HBV DNA levels should be expressed in IU/ml to ensure comparability; the
same assay should be used in the same patient to evaluate antiviral efficacy. Both
HBV antigens and antibody are stable at room temperature for days, at 4°C for
months, and frozen at -20°C to -70°C for many years. Because current testing
120  Hepatology 2012
involves automated enzyme immunoassays that depend on colourimetric or
chemiluminescence signal measurement, care should be taken to avoid hemolysis of
the sample because it may interfere with the ability of the assay to accurately detect
these markers. Care must be taken to avoid the degradation of the viral nucleic acid
in the specimen, which can result in falsely low or no measurable viral load. Serum
should therefore be removed from clotted blood within 4 hrs of collection and stored
at -20°C to -70°C (Krayden 1998). Alternatively, the presence of EDTA in plasma
is known to stabilize viral nucleic acids. EDTA blood can be stored for up to five
days at 4°C without affecting the viral load. Polymerase chain reaction-based tests
that are routinely used as standard can use either serum or plasma. The diagnosis of
HBV infection can also be made by the detection of HBsAg or hepatitis B core
antigen (HBcAg) in liver tissues by immunohistochemical staining and of HBV
DNA by Southern hybridization, in situ hybridisation, or PCR.
Hepatitis B surface antigen and antibody
Hepatitis B surface antigen (HBsAg) is the serologic hallmark of HBV infection. It
can be detected by radioimmunoassays (RIA) or enzyme immunoassays (EIA).
HBsAg appears in serum 1 to 10 weeks after acute exposure to HBV, prior to the
onset of hepatitis and elevation of serum alanine aminotransferase. HBsAg usually
becomes undetectable after four to six months in patients who recover from
hepatitis B. Persistence of HBsAg for more than six months implies chronic
infection. It is estimated that about 5 percent of immunocompetent adult patients
with genuine acute hepatitis B progress to chronic infection (Chu 1989). Among
patients with chronic HBV infection, the rate of clearance of HBsAg is
approximately 0.5 to 1 percent per year (Liaw 1991). The disappearance of HBsAg
is followed by the appearance of hepatitis B surface antibody (anti-HBs). In most
patients, anti-HBs persists for life, thereby conferring long-term immunity.
Coexistence of HBsAg and anti-HBs has been reported in HBsAg positive
individuals (Tsang 1986, Dufour 2000). In most instances, the antibodies are unable
to neutralize the circulating virions. These individuals should therefore be regarded
as carriers of the hepatitis B virus.
In recent years the quantification of HBsAg levels (qHBsAg) has been used to
determine threshold levels to distinguish between patients with active hepatitis B
and inactive carrier state (Brunetto 2010). Furthermore, a continuous decline of
qHBsAg during IFN α therapy has been used as a response marker of therapy
(Marcellin 2009). In contrast, in patients with nucleos(t)ide therapy the
measurement of qHBsAg levels over time have not yielded definite answers yet in
helping to distinguish patients that will clinically resolve chronic hepatitis B
infection with HBsAg loss or seroconversion.
Hepatitis B core antigen and antibody
Hepatitis B core antigen (HBcAg) is an intracellular antigen that is expressed in
infected hepatocytes. It is  not  detectable in serum. Anti-HBc can be detected
throughout the course of HBV infection in the serum.
During acute infection, anti-HBc is predominantly of IgM class. IgM anti-HBc is
the important marker of HBV infection during the window period between the
disappearance of HBsAg and the appearance of anti-HBs. IgM anti-HBc may
remain detectable up to two years after acute infection. Furthermore, the titer of IgM
Hepatitis B: Diagnostic Tests  121
anti-HBc may increase to detectable levels during exacerbations of chronic hepatitis
B (Maruyama 1994). This can present a diagnostic problem, incorrectly suggesting
acute hepatitis B. Other common causes of acute exacerbation of chronic hepatitis B
are superinfection with hepatitis D virus (delta virus) or hepatitis C virus. IgG anti-HBc persists along with anti-HBs in patients who recover from acute hepatitis B. It
also persists in association with HBsAg in those who progress to chronic HBV
infection.
Isolated detection of anti-HBc can occur in three settings: during the window
period of acute hepatitis B when the anti-HBc is predominantly IgM; many years
after recovery from acute hepatitis B when anti-HBs has fallen to undetectable
levels; and after many years of chronic HBV infection when the HBsAg titer has
decreased to below the level of detection. HBV DNA can be detected in the liver of
most persons with isolated anti-HBc. Transmission of HBV infection has been
reported from blood and organ donors with isolated anti-HBc. There are, in a small
percentage of cases, false-positive isolated anti-HBc test results.
The evaluation of individuals with isolated anti-HBc should include repeat testing
for anti-HBc, HBsAg, anti-HBe, and anti-HBs. Those who remain isolated anti-HBc
positive should be tested for the presence of IgM anti-HBc to rule out recent HBV
infection. Individuals with evidence of chronic liver disease should be tested for
HBV DNA to exclude low-level chronic HBV infection.
Hepatitis B e antigen and antibody
Hepatitis B e antigen (HBeAg) is a secretory protein processed from the precore
protein. It is generally considered to be a marker of HBV replication and infectivity.
The presence of HBeAg is usually associated with high levels of HBV DNA in
serum and higher rates of transmission of HBV infection. HBeAg to anti-HBe
seroconversion occurs early in patients with acute infection, prior to HBsAg to anti-HBs seroconversion. However, HBeAg seroconversion may be delayed for years to
decades in patients with chronic HBV infection. In such patients, the presence of
HBeAg is usually associated with the detection of high levels of HBV DNA in
serum and active liver disease. However, HBeAg-positive patients with perinatally
acquired HBV infection may have normal serum ALT concentrations and minimal
inflammation in the liver (Chang 1988).
Seroconversion from HBeAg to anti-HBe is usually associated with a decrease in
serum HBV DNA and remission of liver disease. However, some patients continue
to have active liver disease after HBeAg seroconversion. Such individuals may have
low levels of wild type HBV or HBV variants with a stop codon in the precore or
dual nucleotide substitutions in the core promoter region that prevent or decrease
the production of HBeAg (Carman 1989).
Serum HBV DNA assays
Qualitative and quantitative tests for HBV DNA in serum have been developed to
assess HBV replication. Currently, most HBV DNA assays use real-time PCR
techniques, report results in IU/mL, have a lower limit of detection of around 20
IU/mL and a range of linearity up to 8 log10
IU/mL.
Recovery from acute hepatitis B is usually accompanied by the disappearance of
HBV DNA in serum. However, HBV DNA may remain detectable in serum for
122  Hepatology 2012
many years if tested by PCR assays (Cornberg 2011) suggesting that the virus
persists but is controlled by the immune system.
In patients with spontaneous or treatment-induced HBeAg seroconversion in
chronic hepatitis B, PCR assays usually remain positive except in patients with
HBsAg seroconversion. By contrast, most patients who develop HBeAg
seroconversion during nucleos(t)ide analog therapy have undetectable serum HBV
DNA. In fact, many patients receiving nucleos(t)ide analog therapy remain HBeAg
positive despite having undetectable serum HBV DNA for months or years. The
explanation for this phenomenon is unclear but is likely related to the lack of direct
effect of nucleos(t)ide analogs on covalently closed circular HBV DNA (ccc DNA)
and viral RNA transcription and viral protein expression.
HBV DNA levels are also detectable in patients with HBeAg negative chronic
hepatitis, although levels are generally lower than in patients with HBeAg positive
chronic hepatitis. Because of the fluctuations in HBV DNA levels there is no
absolute cutoff level that is reliable for differentiating patients in the inactive carrier
state from those with HBeAg negative chronic hepatitis B (Chu 2002).
HBV genotypes
HBV can be classified into eight genotypes and four major serotypes. There have
been reports about differing therapeutic responses with nucleos(t)ide analogs and
interferon α with respect to different genotypes. Furthermore, some genotypes, such
as B and C, may have a greater risk for the development of hepatocellular
carcinomas. Nevertheless, in the clinical setting in contrast to hepatitis C, the
diagnosis of HBV genotypes is not part of clinical routine (Thursz 2011).
Antiviral resistance testing
Drug-resistant hepatitis B virus (HBV) mutants frequently arise, leading sometimes
to treatment failure and progression to liver disease. There has been much research
time invested into the mechanisms of resistance to nucleos(t)ides and the selection
of mutants. The genes that encode the polymerase and envelope proteins of HBV
overlap, so resistance mutations in the polymerase usually affect the hepatitis B
surface antigen; these alterations affect infectivity, vaccine efficacy, pathogenesis of
liver disease, and transmission throughout the population (see  Chapter 2).
Associations between HBV genotype and resistance phenotype have allowed cross-resistance  profiles to be determined for many commonly detected mutants, so
genotyping assays can be used to adapt therapy. In vitro phenotyping procedures are
established in a rather small number of HBV laboratories and are not commercially
available. Known mutations can be detected by commercially available tests with a
threshold of about 5% (line probe assays, Inno-Lipa
®
) whereas determination of
novel mutations remain to be detected by research oriented laboratories with full
length sequencing methods. Novel ultra-deep pyrosequencing techniques are much
more sensitive in order to detect many more viral variants but are a tool only for
specialised research laboratories and not part of clinical routine (Zoulim 2009,
Margeridon-Thermet 2009).
Hepatitis B: Diagnostic Tests  123
Assessment of liver disease
As a first step, the causal relationship between HBV infection and liver disease has
to be established and an assessment of the severity of liver disease needs to be
performed. Not all patients with chronic hepatitis B virus infection have persistently
elevated aminotransferases. Patients in the immune tolerant phase have persistently
normal ALT levels and a proportion of patients with HBeAg-negative chronic
hepatitis B may have intermittently normal ALT levels. Therefore appropriate,
longitudinal long-term follow-up is crucial.
The assessment of the severity of liver disease should include: biochemical
markers, including aspartate aminotransferase (AST) and ALT, gammaglutamyl
transpeptidase (GGT), alkaline phosphatase, prothrombin time and serum albumin;
blood counts; and hepatic ultrasound. Usually, ALT levels are higher than AST.
However, when the disease progresses to cirrhosis, the ratio may be reversed. A
progressive decline in serum albumin concentrations and prolongation of the
prothrombin time, often accompanied by a drop in  platelet counts, are
characteristically observed once cirrhosis has developed (EASL 2009).
Acute HBV infection
The diagnosis of acute hepatitis B is based upon the detection of HBsAg and IgM
anti-HBc. During the initial phase of infection, markers of HBV replication, HBeAg
and HBV DNA, are also present. Recovery is accompanied by the disappearance of
HBV DNA, HBeAg to anti-HBe seroconversion, and subsequently HBsAg to anti-HBs seroconversion.
The differential diagnosis of HBsAg-positive acute hepatitis includes acute
hepatitis B, exacerbations of chronic hepatitis B, reactivation of chronic hepatitis B,
superinfection of a hepatitis B carrier with hepatitis C or D virus (Tassopoulos
1987), and acute hepatitis due to drugs or other toxins in a hepatitis B carrier.
Past HBV infection
Previous HBV infection is characterized by the presence of anti-HBs and IgG anti-HBc. Immunity to HBV infection after vaccination is indicated by the presence of
anti-HBs only.
HBsAg
−  If negative, acute HBV infection is ruled out (Dufour 2000).
−  If positive, the patient is infected with HBV. A repeat test six months later will
determine if the infection has resolved or is chronic.
Anti-HBs
−  If negative, the patient has no apparent immunity to HBV
−  If positive, the patient is considered immune to HBV (either because of
resolved infection or vaccination).
Anti-HBc-immunoglobulin M
In rare cases, anti-HBc immunoglobulin (Ig) M may be the only HBV marker
detected during the early convalescence or 'window period' when the HBsAg and
anti-HBs tests are negative. Because current tests for HBsAg are very sensitive, an
anti-HBc IgM that is typically positive with acute HBV infection is not generally
124  Hepatology 2012
required to diagnose active infection. Because some chronic HBV carriers remain
anti-HBc IgM positive for years, epidemiological information is necessary to
confirm that the infection is indeed acute. A negative anti-HBc IgM in the presence
of a positive HBsAg suggests that the infection is likely chronic. For these reasons,
routine testing for anti-HBc IgM is not generally recommended to screen for acutely
infected patients.
Chronic HBV infection
Chronic HBV infection is defined by the continued presence of HBsAg in the blood
for longer than six months. Additional tests for HBV replication, HBeAg and serum
HBV DNA, should be performed to determine if the patient should be considered
for antiviral therapy. All patients with chronic HBV infection should be regularly
monitored because HBV DNA and ALT levels vary during the course of infection
to monitor for progression of liver disease. In addition, patients who are not
candidates for treatment at the time of presentation may become candidates for
treatment during follow-up.
HBeAg-negative patients who have normal serum ALT and low (<2000 IU/mL)
or undetectable HBV DNA are considered to be in an inactive carrier state. These
patients generally have a good prognosis and antiviral treatment is not indicated.
However, serial tests are necessary to accurately differentiate them from patients
with HBeAg-negative chronic hepatitis who have fluctuating ALT and/or HBV
DNA levels (Lok 2007). Patients who are truly inactive carriers should continue to
be monitored but at less frequent intervals. HBeAg-negative patients with elevated
serum ALT concentrations should be tested for serum HBV DNA to determine if
the liver disease is related to persistent HBV replication.
HBsAg
−  If negative, chronic HBV infection is typically ruled out.
−  If positive,  the patient is considered HBV-infected. Chronic infection is
diagnosed when the HBsAg remains detectable for longer than six months.
Antibody to hepatitis B core protein
−  If negative, past infection with HBV is typically ruled out.
−  If positive, the patient has been infected with HBV. Infection may be resolved
(HBsAg-negative) or ongoing (HBsAg-positive). If the infection is resolved,
the person is considered naturally immune to HBV infection.
Antibody to hepatitis B surface protein
−  If negative, the patient has no apparent immunity to HBV
−  If positive, the patient is considered immune to HBV (either because of
resolved infection or as the result of prior vaccination). Very rarely (less than
1%) chronic carriers can be positive for HBsAg and antibody to hepatitis B
surface protein (anti-HBs) at the same time (Tsang 1986, Dufour 2000). In
such cases, the patient is considered infectious.
Serum transaminases
Once an individual has been diagnosed with chronic HBV infection, follow-up
testing must be performed for alanine aminotransferase (ALT), a marker of liver cell
Hepatitis B: Diagnostic Tests  125
inflammation. Repeat periodic testing is indicated because the ALT levels can
fluctuate (e.g., from less than the upper limit of normal to intermittently or
consistently elevated). Sustained and intermittent elevations in ALT beyond the
upper limit of normal are indicative of hepatic inflammation and correlate with an
increased risk of progressive liver disease. It must be noted that the normal ALT
ranges are both age and sex dependent and, occasionally, individuals with severe
liver disease may not manifest elevated ALT (Cornberg 2011, EASL 2009).
Occult HBV infection
This is defined as the presence of detectable HBV DNA by PCR in patients who are
negative for HBsAg. Most of these patients have very low or undetectable serum
HBV DNA levels accounting for the failure to detect HBsAg. Infections with HBV
variants that decrease HBsAg production or have mutations in the S gene with
altered S epitopes that evade detection in serology assays for HBsAg are
uncommon. HBV DNA is often detected in the liver and transplantation of livers
from these persons can result in de novo HBV infection (Margeridon-Thermet
2009).
Assessment of HBV immunity
Immunity to HBV is acquired from a resolved infection or from vaccination. The
HBV vaccine has been shown to induce protective immunity in 90% to 95% of
vaccinees. Most vaccinees will have protective levels of anti-HBs for 5-10 years
after vaccination, although the exact duration of immunity remains undefined.
Anti-HBs
−  If the anti-HBs level is less than 10 mIU/mL, this implies that the person is
nonimmune to HBV. In individuals who have received a complete course of
HBV vaccine, the level of anti-HBs may drop to less than 10 mIU/mL after
five to 10 years, but these individuals are generally considered to be immune
based on their vaccination history (Maruyama 1994).
−  If the anti-HBs result is greater than 10 mIU/mL, the person is considered to be
immune. Immunity may be due to immunization or resolved natural infection.
These two states can be distinguished by testing for antibody to hepatitis B
core protein (anti-HBc), which is present in subjects that have had HBV
infection but absent in vaccinees (see below).
Anti-HBc
−  If the anti-HBc total test is positive, this is compatible with current or resolved
HBV infection. A negative HBsAg confirms a resolved infection. HBV
vaccination does not induce anti-HBc total.
Liver biopsy and noninvasive liver transient elastography
A liver biopsy is recommended for determining the degree of necroinflammation
and fibrosis in patients with either increased ALT or HBV DNA levels >2000 IU/ml
(or both) since hepatic morphology can assist the decision to start treatment. Biopsy
is also useful for evaluating other possible causes of liver disease such as steatosis
or steatohepatitis. Although liver biopsy is an invasive procedure, the risk of severe
complications is low. It is important that the size of the needle biopsy specimen be
126  Hepatology 2012
large enough to precisely analyse the degree of liver injury and fibrosis. A liver
biopsy is usually not required in patients with clinical evidence of cirrhosis or in
those in whom treatment is indicated irrespective of the grade of activity or the
stage of fibrosis.
There is growing interest in the use of noninvasive methods, including serum
markers and transient elastography, to assess hepatic fibrosis to complement or
avoid a liver biopsy (Cornberg 2011, EASL 2009).
References
Brunetto MR, Oliveri F, Colombatto P, et al. Hepatitis B surface antigen serum levels help to
distinguish active from inactive hepatitis B virus genotype D carriers.
Gastroenterology 2010;139:483-90. (Abstract)
Carman WF, Jacyna MR, Hadziyannis S, et al. Mutation preventing formation of hepatitis B e
antigen in patients with chronic hepatitis B infection. Lancet 1989;2:588. (Abstract)
Chang MH, Hwang LY, Hsu HC, et al. Prospective study of asymptomatic HBsAg carrier
children infected in the perinatal period: clinical and liver histologic studies.
Hepatology 1988;8:374 (Abstract)
Chu CJ, Hussain M, Lok AS. Quantitative serum HBV DNA levels during different stages of
chronic hepatitis B infection. Hepatology 2002;36:1408. (Abstract)
Chu CM, Liaw YF, Pao CC, Huang MJ. The etiology of acute hepatitissuperimposed upon
previously unrecognized asymptomatic HBsAg carriers. Hepatology 1989;9:452.
(Abstract)
Cornberg, M, Protzer U, Petersen J, et al. Aktualisierung der S3 Leitliniezur Prophylaxe,
Diagnostik und Therapie der Hepatitis-B-Virusinfektion. Z Gastroenterol 2011;49:871-930
Dufour DR, Lott JA, Nolte FS, Gretch DR, Koff RS, Seeff LB. Diagnosis and monitoring of
hepatic injury. I. Performance characteristics of laboratory tests. Clin Chem
2000;46:2027-49. (Abstract)
EASL Clinical Practice Guidelines: Management of chronic Hepatitis B. J Hepatology
2009;50:227-42
Hepatitis B virus resistance to nucleos(t)ide analogues. Zoulim F, Locarnini S. Gastroenterology
2009;137:1593-608 (Abstract)
Krajden M, Comanor L, Rifkin O, Grigoriew A, Minor JM, Kapke GF. Assessment of hepatitis B
virus DNA stability in serum . J Clin Microbiol 1998;36:382-6.
Liaw YF, Sheen IS, Chen TJ, et al. Incidence, determinants and significance of delayed
clearance of serum HBsAg in chronic hepatitis B virus infection: a prospective study.
Hepatology 1991;13:627. (Abstract)
Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 2007;45:507. (Abstract)
Marcellin P, Bonino F, Lau GK, et al. Sustained response of hepatitis B e antigen-negative
patients 3 years after treatment with peginterferon alpha-2a. Gastroenterology
2009;136:2169-79. (Abstract)
Margeridon-Thermet S, Shulman NS, Ahmed A, et al. Ultra-deep pyrosequencing of hepatitis B
virus quasispecies from nucleoside and nucleotide reverse-transcriptase inhibitor
(NRTI)-treated patients and NRTI-naive patients. J Infect Dis 2009;199:1275-85
(Abstract)
Maruyama T, Schödel F, Iino S, et al. Distinguishing between acute and symptomatic chronic
hepatitis B virus infection. Gastroenterology 1994;106:1006. (Abstract)
Quint WG, de Bruijn I, Kruining H, Heijtink RA. HBV-DNA detection by gene amplification in
acute hepatitis B. Hepatology 1990;12:653-6. (Abstract)
Raimondo G, Allain JP, Brunetto MR, et al. Statements from the Taormina expert meeting on
occult hepatitis B virus infection. J Hepatol 2008;49:652. (Abstract)
Tassopoulos NC, Papaevangelou GJ, Sjogren MH, et al. Natural history of acute hepatitis B
surface antigen-positive hepatitis in Greek adults. Gastroenterology 1987;92:1844.
(Abstract)
Thursz M, Yee L, Khakoo S Understanding the host genetics of chronic hepatitis B and C.
Semin Liver Dis 2011;31:115-127. (Abstract)
Hepatitis B: Diagnostic Tests  127
Tsang TK, Blei AT, O'Reilly DJ, Decker R. Clinical significance of concurrent hepatitis B surface
antigen and antibody positivity. Dig Dis Sci 1986;31:620. (Abstract)
Yang HI, Lu SN, Liaw YF, et al. Hepatitis B e antigen and the risk of hepatocellular carcinoma.
N Engl J Med 2002;347:168-74. (Abstract)
128  Hepatology 2012
9.  Hepatitis B Treatment
Florian van Bömmel, Johannes Wiegand, Thomas Berg
Introduction
Individuals with HBV infection carry a significantly increased risk of life-threatening complications such as hepatic decompensation, liver cirrhosis and hepa-tocellular carcinoma (HCC) (Beasley 1988). Recent studies have shown that the
level of serum HBV DNA correlates higher with the risk of developing cirrhosis and
HCC as compared to other baseline or virologic parameters (Chen 2006, Iloeje
2006) (Figure 1). Thus, suppressing the replication of HBV to undetectable levels is
now a major goal in HBV treatment (Liaw 2008, Lok 2007, EASL 2009, Cornberg
2011). Moreover, it has now become clear that continuous suppression of HBV
replication can revert liver fibrosis or even cirrhosis in most patients (Marcellin
2011, Schiff 2011). HBeAg seroconversion is another endpoint, provided that HBV
replication remains durably suppressed to low levels. The ultimate treatment goal,
however, the loss of HBsAg or HBsAg seroconversion, remains difficult to achieve.
The level of hepatitis B surface antigen (HBsAg) before and during interferon-based
treatment is becoming a marker for response to interferon based treatment.
There are two drug classes available for the treatment of chronic HBV infection:
the immune modulator interferon  α  (standard or pegylated (PEG)-INF α) and
nucleoside or nucleotide analogs, which act as reverse transcriptase inhibitors of the
HBV polymerase. Currently, the nucleoside analogs lamivudine (LAM), telbivudine
(LdT), entecavir (ETV) and the acyclic nucleotide analogs adefovir dipivoxil
(ADV) and tenofovir disoproxil fumarate (TDF) are available. Due to this broad
spectrum of therapeutic options disease progression and complications can be
prevented if the infection is diagnosed early and treated effectively. The early
diagnosis of chronic hepatitis B by HBsAg screening in high-risk groups and in
patients with elevated transaminases plays a crucial role in the management of HBV
infection.
Indication for antiviral therapy
Acute hepatitis B
Acute hepatitis resolves spontaneously in 95-99% of cases (McMahon 1985, Liaw
2009). Therefore, treatment with the currently available drugs is generally not
Hepatitis B Treatment  129
indicated. However, in a recent trial comparing treatment with LAM 100 mg/day
versus no treatment in 80 Chinese patients with fulminant hepatitis B, a reduced
mortality of 7.5% was found in patients receiving LAM treatment compared to 25%
in the control group (p=0.03) (Yu 2010). These observations are supported by a
placebo-controlled trial investigating the use of LAM in 71 patients with fulminant
hepatitis B in India (Kumar 2007). Several case reports from Europe also revealed
that patients with severe and fulminate hepatitis B may benefit from early antiviral
therapy with LAM or other nucleos(t)ide analogs by reducing the need for high-urgency liver transplantation (Tillmann 2006). As a result, treatment for fulminant
hepatitis B with LAM is recommended by EASL and with LAM or LdT by AASLD
(EASL 2009, Lok 2009). Interferon therapy is contraindicated in patients with acute
HBV infection because of risk of increasing hepatitis. The endpoint of treatment of
acute HBV infections is HBsAg clearance (EASL 2009, Lok 2007).
Figure 1. Cumulative incidence of liver cirrhosis in untreated HBV-infected individuals
within a mean observation period of 11.4 years (REVEAL Study). The incidence of liver
cirrhosis increases over time depending on baseline HBV DNA levels (Iloeje 2006). The relative
risk for developing HCC was 1.4 in patients with HBV DNA levels of 300 to 1,000 and increased
to 2.4 in patients with 1,000-10,000 to 5.4 in patients with 10,000 to 100,000 and to 6.7 in
patients with HBV DNA levels >1 million copies/ml. A similar association between HBV DNA
levels and the risk of HCC development was shown (Chen 2006).
Chronic hepatitis B
All patients with HBsAg positive chronic hepatitis should be considered as possible
candidates for antiviral therapy especially in situations when there is a significant
level of HBV replication (Chen 2006, Iloeje 2006). Differentiation between
HBeAg-positive and HBeAg-negative chronic hepatitis B is not necessary anymore
for treatment indication, although with respect to the choice of the appropriate
antiviral drug these criteria may be still useful.
130  Hepatology 2012
Table 1. Key guideline recommendations for indication for antiviral treatment of
HBV infection.
AASLD
(Lok 2007, Lok
2009)
Consider treatment:
•  HBeAg(+): HBV DNA >20,000 IU/ml + ALT ≤2x ULN + biopsy shows
moderate/severe inflammation or significant fibrosis
•  HBeAg(+): HBV DNA >20,000 IU/ml + ALT >2x ULN. Observe for 3-6
months and treat if no spontaneous HBeAg loss
•  HBeAg(-): HBV DNA >20,000 IU/ml + ALT >2x ULN
Consider biopsy:
•  HBeAg(+): HBV DNA >20,000 IU/ml + ALT >2x ULN + compensated
•  HBeAg(+): HBV DNA >20,000 IU/ml + ALT 1-2x ULN + age >40 years
or family history of HCC
•  HBeAg(-): HBV DNA >2,000-20,000 IU/ml + ALT 1-2x ULN
APASL
(Liaw 2008)
Consider treatment:
•  All patients: HBV DNA detectable + advanced fibrosis/cirrhosis
•  HBeAg(+): HBV DNA >20,000 IU/ml + ALT >2x ULN + impending/
overt decompensation
•  HBeAg(-): HBV DNA > 2,000 + ALT >2x ULN + impending/ overt
decompensation
EASL
(EASL 2009)
Consider treatment:
•  HBV DNA >20,000 IU/ml + ALT >2x ULN + moderate to severe
necroinflammation
Belgian
(Colle 2007)
Consider treatment:
•  HBeAg(+): HBV DNA >20,000 IU/ml + ALT >2x ULN (or
moderate/severe hepatitis on biopsy)
•  HBeAg(-): HBV DNA ≥2,000 IU/ml and elevated ALT
Consider biopsy:
•  Fluctuating or minimally elevated ALT (especially in those older than
35-40 years)
Dutch
(Buster 2008)
Consider treatment:
•  HBeAg(+) and HBeAg(-): HBV DNA ≥20,000 IU/ml and ALT ≥2x ULN
or active necrotic inflammation
•  HBeAg(-): HBV DNA ≥2,000–20,000 IU/ml and ALT ≥2x ULN (and
absence of any other cause of hepatitis)
German
(Cornberg 2011)
Consider treatment:
•  HBV DNA >2,000 IU/ml + minimal inflammation/low fibrosis or ALT
elevation
Italian
(Carosi 2008)
Consider treatment:
•  HBeAg(+): HBV DNA >20,000 IU/ml + ALT >2x ULN
•  HBeAg(-): HBV DNA >2,000 IU/ml + abnormal ALT and or fibrosis
(Ishak ≥S2)
Consider biopsy:
•  HBeAg(-): HBV DNA >2,000 IU/ml + borderline ALT, or if DNA 2,000–
20,000 IU/ml + high ALT
Turkish TASL
(Akarca 2008)
Consider treatment:
•  HBV DNA >2,000 IU/ml + histological fibrosis >2
•  HBV DNA >20,000 IU/ml + any histological finding + ALT >2x ULN
Current recommendations of the different national and international societies are
shown in Table 1 (Akarca 2008, Carosi 2008, Colle 2007, Cornberg 2011, EASL
2009, Janssen 2008, Juszczyk 2008, Keeffe 2007, Liaw 2008, Lok 2009, Waked
2008). In most of these guidelines, the most relevant factor for a decision to initiate
treatment has shifted from histological proven disease activity to the level of HBV
Hepatitis B Treatment  131
DNA. Thus, most of the recently published guidelines now recommend antiviral
treatment for patients with HBV DNA levels >2,000 IU/mL (corresponding to
>10,000 copies/mL) in association with a sign of ongoing hepatitis which can either
be ALT levels greater than 2 times the upper limit of normal or significant fibrosis
demonstrated by liver histology greater than A1/F1. The recently updated German
treatment guidelines emphasise the importance of suppression of HBV replication
by recommending treatment in patients with HBV DNA levels >2,000 IU/mL and
any elevation of ALT levels or signs of fibrosis (Cornberg 2011).
All patients with liver cirrhosis or high-grade liver fibrosis and any measurable
HBV DNA should be considered for antiviral therapy (EASL 2009, Lok 2007,
Cornberg  2011). The indication for antiviral treatment according to the recent
German guidelines is depicted in Figure 2 (Cornberg 2011). In patients with
decompensated cirrhosis Child-Pugh score B or C, INF α is contraindicated.
Figure 2. Indication for antiviral treatment according to the German guidelines for the
treatment of chronic HBV infection. Treatment should be considered if HBV DNA levels
exceed 10
4
copies/ml and if ALT are elevated or if liver histology is abnormal. Of note,
asymptomatic carriers with family history of HCC should receive treatment even if signs of
hepatitis are absent (Cornberg 2011).
Inactive chronic HBsAg carriers, characterised by negative HBeAg and positive
anti-HBeAg, low HBV DNA levels (<2,000 IU/ml) and serum aminotransferases
within normal levels do not have an indication for antiviral therapy (Cornberg 2011,
Brunettto 2011). However, differentiation between inactive HBsAg carriers and
patients with chronic HBeAg-negative hepatitis may be difficult in some cases.
Elevated transaminases are no reliable parameter for assessing the stage of liver
fibrosis and long-term prognosis of HBV-infected patients. Even in patients with
normal or slightly elevated aminotransferases there can be a significant risk for the
development of HBV-associated complications (Chen 2006, Iloeje 2006, Kumar
2008). It is reasonable to perform a liver biopsy in these individuals and to control
132  Hepatology 2012
the level of HBV DNA at three-month intervals. However, a liver biopsy is not
mandatory to initiate treatment for the majority of patients (Table 1).
HBV immunotolerant patients are mostly under 30 years old and can be recog-nised by their high HBV DNA levels, positive HBeAg, normal ALT levels and
minimal or absence of significant histological changes. According to most practice
guidelines immediate therapy is not required (Akarca 2007, Balik 2008, Carosi
2008, Colle 2007, Cornberg 2011, EASL 2009, Buster 2008, Juszczyk 2008, Keeffe
2007, Liaw 2008, Lok 2009, Waked 2008). However, patients with elevated risk for
HCC development, such as those with a positive family history, and patients from
high endemic areas like Asia or Africa may perhaps benefit from early antiviral
therapy (Cornberg 2011). Studies are under way  to further clarify this issue,
especially to answer the question whether early intervention with antiviral therapy
will positively influence the long-term risk for HCC.
Summary of treatment indications in the German Guidelines of 2011
–  All patients with chronic hepatitis B should be evaluated for treatment.
Indication for treatment initiation depends on the level of viral replication
(HBV DNA ≥2,000IU/mL, corresponding to ml ≥10,000 copies/mL), any
elevation of serum aminotransferases and the histological  grading and
staging.
–  Patients with advanced fibrosis or cirrhosis and detectable viremia need
consistent antiviral therapy.
–  Reactivation of HBV replication due to immunosuppression should be
avoided by preventive therapy.
–  Alcohol and drug consumption are not a contraindication for treatment with
nucleos(t)ide analogs.
–  Therapy with nucleos(t)ide analogs during pregnancy may be considered if
the benefit outweighs the risk. A running treatment with LAM or TDF can be
continued during pregnancy.
–  Occupational and social aspects and extrahepatic complications may justify
therapy in individual cases.
Endpoints of antiviral treatment
Due to persistence of episomal covalently closed circular DNA (cccDNA), a
template of the HBV genome located in the nucleus of infected hepatocytes, a
complete eradication of HBV infection is currently impossible (Rehermann 1996).
Reactivation of an HBV infection can occur in certain circumstances from these
nuclear reservoirs even decades after HBsAg loss, for instance during
immunosuppressive therapy. The aim of treatment of chronic hepatitis B is to
reduce complications such as liver failure and HCC and to increase survival (EASL
2009, Lok 2009, Cornberg 2011). To determine the success of antiviral therapy
surrogate markers are used during and after treatment. These parameters include
virologic (HBeAg and HBsAg status, HBsAg levels, HBV DNA level) and patient-related parameters (aminotransferases, liver histology).
Suppression of HBV replication. In two recent studies a close correlation be-tween baseline HBV DNA levels and progression of the disease was demonstrated.
In the REVEAL study, 3774 untreated HBV-infected individuals were followed
Hepatitis B Treatment  133
over a mean time period of 11.4 years in Taiwan (Chen 2006, Iloeje 2006). HBV
DNA levels at baseline were the strongest predictors of cirrhosis and HCC devel-opment (Figure 1). In multivariate models, the relative risk of cirrhosis increased
when HBV DNA reached levels greater than 300 copies/mL, independent of
whether patients were negative or positive for HBeAg. In addition, individuals with
HBV DNA levels ≥10
4
copies/mL (or ≥2,000 IU/mL) were found to have a 3-15
fold greater incidence of HCC as compared to those with a viral load <10
4
copies/mL. According to these results, a meta-analysis covering 26 prospective
studies revealed a statistically significant and consistent correlation between viral
load levels and histologic, biochemical, or serologic surrogate markers (Mommeja-Marin 2003). It can therefore be concluded that the complete and persistent
suppression of HBV replication is a reliable endpoint for the treatment of chronic
HBV infection.
Induction of HBeAg seroconversion.  In HBeAg-positive patients,
seroconversion from HBeAg to anti-HBe was found to be a reliable surrogate
marker for prognosis of chronic HBV infection leading in many cases to an inactive
HBsAg carrier state (Figure 3). In these patients, HBsAg remains detectable but
HBV replication continues at low or even undetectable levels and transaminases are
generally within normal ranges.
Figure 3. Possible endpoints of treatment of chronic HBV infection. After achieving
HBeAg or HBsAg seroconversion, antiviral treatment can be stopped. However, it is
recommended to maintain treatment for a period of 6-12 months after HBeAg or HBsAg
seroconversion.