Hepatitis B: Diagnostic Tests

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.

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Prophylaxis and Vaccination

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

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

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HCV Virology

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

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HBV Virology

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

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Hepatitis E: an underestimated problem?

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

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