Acute and chronic hepatitis B

Introduction

Over the last three decades, laboratory diagnostics of viral infections have become
influenced more and more by molecular biology, the field of technology that has
grown the fastest in this same period of time. Classical serologic and virologic tests
have advanced and sometimes been replaced by novel detection methods that rely
on genome amplification procedures like PCR and NASBA.
Especially for the human hepatitis B virus this technological development has been
extremely important. As mentioned in Chapter 6, in contrast to other viruses, HBV
is extremely hard to cultivate as it does not replicate in any cell line used regularly
in diagnostic laboratories. Furthermore, earlier techniques were not sensitive
enough to detect small amounts of virus in blood and blood products, and consequently
failed to avoid unintentional transmission of virus from donors to blood
product recipients.

Aims of diagnostic tests in the management of HBV infection

The first test of an HBV infection is to diagnose whether it is acute or chronic. As a
standard procedure, the patient with an HBV infection diagnosed by clinical symptoms
or elevated alanine aminotransferase (ALT) levels needs to test positive for
anti-hepatitis B core antigen (HBcAg) antibodies. HBcAg is massively expressed in
both acute and chronic infections and is a clear sign of HBV infection. After a positive
result for anti-HBcAg antibodies, antibodies reactant to the surface antigen
(HbsAg) are looked for. If found, this indicates that the patient has a past, currently
inactive HBV infection. In case of negative anti-HBcAg antibodies and the presence
of anti-HBsAg antibodies the individual has been successfully vaccinated
against HBV.
Based on these initial serologic diagnostics, other efforts to define the status of the
infection are made. An anti-HBcAg positive but anti-HBsAg negative patient may
be chronically infected with a pre-core mutant HBV or may have low level replication
with a wild type HBV. In these cases a number of parameters should be investigated,
namely early antigen (HbeAg), anti-HBeAg, HBsAg, HBcAg, and finally,
the viral load, measured as genome equivalents per ml in serum. HBeAg is normally
only expressed in case of an acute and/or ongoing infection with active replication.
Unfortunately, so-called pre-core mutants exist that display active replication
without expressing HBeAg while bearing a high risk for progression to cirrhosis
and hepatocellular carcinoma (HCC). It is worth noting that HBeAg seroconversion
occurs in up to 98% of subjects, and this is not a marker for a cure as it would
be in wild type HBV, although it does act as a marker for healing.
After serologic screening is completed and a replicative HBV infection is assumed,
the more expensive molecular methods such as HBV DNA are performed. This is
generally to decide on whether to start treatment, to monitor treatment efficacy and
treatment adherence, to identify resistant strains, and to identify pre-core mutant
strains of HBV.

Molecular assays in the diagnosis and management of HBV

Utility of quantitative HBV DNA assays
Many scientific societies have published consensus papers and/or guidelines for the
management of chronically-infected HBV patients (Cornberg 2007; de Franchis
2003; Keeffe 2006; Liaw 2005; Lok 2001; Lok 2004a; Lok 2004b). All of them
recommend an initial quantification of viral load and continuous measurements
during follow-up monitoring. Follow-up is considered to be important for deciding
on initiation of treatment or changes to the drug regimen of the patient. Furthermore,
sensitive methods for quantification are needed for detection of low-level
viremia in patients infected with strains that are of high risk for the development of
hepatocellular carcinoma such as HBeAg negative strains with the precore mutation.
One agreed upon criterion for chronic HBV infection is a detectable viral load –
measured as viral DNA in serum or plasma - for a minimum of 6 months (de Franchis
2003; Keeffe 2006; Liaw 2005; Lok 2001; Lok 2004a; Lok 2004b). In this
case, replication is considered to be active if > 20,000 IU/ml or > 100,000 copies/ml
can be detected (Cornberg 2007). Also, in HbeAg-negative chronic hepatitis B virus
infections, HBV DNA is the only marker that needs to be monitored (Manesis
2003; Zacharakis 2005).
Furthermore, qualitative and quantitative measurement of viral DNA is important
for monitoring another condition, occult hepatitis. This is characterized as HBV
infection with measurable DNA levels in the absence of detectable HBsAg. Testing
for occult hepatitis B virus infection is recommended if (a) cryptogenic liver disease
is observed, (b) prior to immunosuppression, and (c) in solid organ transplant donors
with positive HBV serology (HBcAg antibodies) (Conjeevaram 2001; Torbenson
2004; Torbenson 2002). It is recommended that viral load should be measured
every 3-6 months while on HBV therapy or to monitor chronic HBV infection (de
Franchis 2003; Liaw 2005).
Furthermore, the measurement of viral load after the onset of therapy is a useful and
standard tool to identify non-responders (Schildgen 2004; Schildgen 2006; Sirma
2007; Volz 2007). Non-response to therapy can be induced by host factors, viral
resistance, or non-compliance (reviewed by Tillmann 2007). For quantification of
the HBV viral load, several assays are commercially available, each having advantages
and disadvantages (reviewed by Valsamakis 2007).
Utility of HBV genotyping
Genotyping of the HBV genome, while not a standard procedure in the clinic, can
be useful. First, viral genotype may influence success of the therapy, e.g., patients
with an HBV genotype A infection have a better chance of a more favorable outcome
than those infected with genotype non-A (Chen 2004; Colombo 2003; Enomoto
2006; Erhardt 2005; Flink 2006; Fung 2004; Guettouche 2005; Kao 2002;
Kao 2003; Kao 2000; Kobayashi 2002; Liu 2002; Peters 2004; Sanchez-Tapias
2002; Zhang 1996).
Second, genotyping is the simplest method for identification of resistance mutations
that are associated with non-response to nucleoside and nucleotide analogues. This
may guide the decision on how to switch therapy, because cross-resistance between
HBV polymerase inhibitors plays an important role (Schildgen 2004; Schildgen
2006; Sirma 2007).
Third, genotyping plays an important role in the identification of chains of infection
in a nosocomial setting or if transmission by blood donations or blood products has
occurred. Genotyping can be performed by in-house or commercial full genome
sequencing. PCR followed by INNO-LiPA hybridisation has recently been developed
and covers pre-identified mutations. Regular updates of the test are mandatory
as new mutations are identified; newly found mutations will not be always detectable,
in contrast to full genome sequencing (Hussain 2003; Hussain 2006; Osiowy
2003; Osiowy 2006). INNO-LiPA has the major advantage of being able to detect
mixed infections as well.
Utility of antiviral resistance testing
With the introduction of more and more antiviral compounds into clinical practice
in the last decade the option for new and combination treatments of HBV infections
has increased greatly, and will continue to grow. As a side effect of the number of
novel antivirals, the development of resistance mutations has also started to increase,
and it can be assumed that the problem of antiviral resistance observed in
HBV will become as complicated as what is happening today in HIV treatment.
After genotypic analysis, as mentioned above, mutations already known can be
identified and associated with resistance. Major evidence for resistance is if such
mutations, like the mutations in the polymerase YMDD motif, evolve during ongoing
therapy. The real question for the virologist is when none of the known mutations
is observed at failure. In such cases it has to be estimated how far other
novel mutations not yet associated with resistance contribute to therapeutic failure.
In that case, in vitro phenotyping procedures established in a rather small number of
HBV laboratories (see Chapter 6) need to be performed. Unfortunately, known
mutations can be detected and classified by commercial methods whereas novel
mutations remain speculative; in phenotyping, these possibly important mutations
will remain undetected or underestimated. However, it will probably be helpful to
make use of novel e-learning database solutions that can help in the interpretation
of sequencing results
Utility of core promotor and precore mutation detection
assays
Today, the diagnosis of HBeAg-negative chronic hepatitis B virus infection is based
on the assessment of a combination of infection markers, namely positive HBsAg,
negative HBeAg, and detectable viral DNA, together with anti-HBeAg antibodies
and the evidence for liver injury measured by elevated liver enzymes, non-invasive
fibrosis tests or histopathological findings. Assays are commercially available in
formats of PCR sequencing and hybridisation, INNO-LiPA hybridisation and sequencing,
as well as the Affigene HBV mutant VL19 test (Olivero 2006; Qutub
2006).
Conclusion and future aspects
The major challenge for HBV diagnostics in the future will be the increasing number
of resistance mutations and the immune escape mutants, occult hepatitis and
HBeAg-negative chronic hepatitis. Novel tools such as the virtual phenotype based
on database interpretation of genotype results as established for HIV resistance interpretation
may help in the interpretation of laboratory results. However, a downside
of the rapid increase in these sophisticated diagnostic tools are the costs for
these tests which may help guide treatment decisions and avoid suboptimal HBV
therapy.