Diagnosis of Hepatitis B Virus Infection Through Serological and Virological Markers

Jia-Horng Kao

Expert Rev Gastroenterol Hepatol. 2008;2(4):553-562.

Abstract and Introduction

Abstract

Hepatitis B virus (HBV) infection is an important health problem and the major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) worldwide. The natural history of chronic HBV infection can be divided into four dynamic phases in HBV carriers who acquire the virus early in life. Diagnosis of HBV infection is usually through serological and virological markers. Hepatitis B surface antigen (HBsAg) is the hallmark of HBV infection and is the first serological marker to appear in acute hepatitis B, and persistence of HBsAg for more than 6 months suggests chronic HBV infection. Hepatitis B e antigen (HBeAg) usually indicates active HBV replication and risk of transmission of infection. Recently, occult HBV infection is recognized as the absence of circulating HBsAg in individuals positive for serum or tissue HBV DNA, irrespective of other HBV serological markers. Meanwhile, monitoring the serum HBV DNA level is valuable for assessing liver disease activity, differentiating other etiologies of hepatitis activity in HBV carriers, predicting risk of HCC development or liver-related mortality, deciding to administer antiviral therapy, determination of the response to antiviral treatment, predicting the risk of developing drug resistance, and detecting the emergence of drug-resistant mutants. On the other hand, HBV genotype C, basal core promoter mutant and pre-S deletion mutant are reported to be associated with increased risk of HCC development. The roles of quantitative HBV serology and intrahepatic HBV covalently closed circular (ccc)DNA deserve further studies. In conclusion, it is particularly important for physicians to screen for HBV infection in HBV-endemic areas and to monitor liver disease progression in HBV carriers by using both serological and virological markers, so that effective treatment can be initiated early before the development of advanced liver disease.

Introduction

Hepatitis B virus (HBV) is an important public health problem and the major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) worldwide.^[1] HBV is the smallest human DNA virus, with a genome of 3200 base pairs, which belongs to the family Hepadnaviridae.^[2] The partially double-stranded circular DNA encodes four overlapping open reading frames including surface (S), core (C), polymerase (P) and X genes.^[2] These viral genes can be translated to different viral proteins from four major transcripts, such as surface antigen (HBsAg), e antigen (HBeAg) and core antigen (HBcAg). The host immune system therefore produces corresponding antibodies against these viral proteins, including anti-HBs, anti-HBe and anti-HBc. These antigens and antibodies lay the basis for serological diagnosis of HBV infection. Due to the high error rate of the viral reverse transcriptase, the HBV genome evolves and the estimated rate of nucleotide substitution is approximately 1.4-3.2 × 10^-5/site/year.^[3] This unique replication strategy accounts for the majority of point mutations and deletions or insertions observed in the HBV genome. A long period of time of evolution of HBV therefore leads to the occurrence of various genotypes, subgenotypes, variants or mutants, recombinants, and even quasispecies.^[1,2,3]

Hepatitis B virus infection is prevalent in Asia, Africa, Southern Europe and South America, where the prevalence of HBsAg in the general population ranges from 2 to 20%.^[1] HBV causes both acute and chronic infection in humans. Acute infection may result in classical acute hepatitis or fulminant hepatitis. In chronic infection, HBV replication persists throughout the course of chronic infection, and host immune response plays a pivotal role in HBV-related liver damage and control of HBV replication.^[2] In this article, the natural history of hepatitis B and the diagnosis of HBV infection through serological and virological markers are reviewed.

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Table 1. Four Dynamic Phases of Chronic Hepatitis B Virus Infection
	Immune tolerance	Immune clearance	Low replication	Reactivation^*
HBeAg	Positive	Positive	Negative	Negative
HBV DNA level (IU/ml)^‡	Very high (>2 × 10⁷)	High (>2 × 10⁴)	Low (<2 × 10³)	Moderate (>2 × 10³)
ALT level	Normal	Elevated	Normal	Elevated
Liver pathology	Normal or minimal change	Chronic inflammation and fibrosis	Normal or minimal change	Chronic inflammation and fibrosis

^*Reactivation phase may be recognized as a variant of immune clearance phase.
^Dagger;Approximately five copies = 1 IU.
ALT: Alanine aminotransferase; HBeAg: Hepatitis B e antigen; HBV: Hepatitis B virus.

Table 2. Common Serological Markers of HBV Infection
Marker	Definition and diagnostic use
HBsAg	General marker of HBV infection First serologic marker to appear Persistence for more than 6 months suggests chronic HBV infection
Anti-HBs	Neutralizing antibody Recovery and/or immunity to HBV The only marker detectable after immunity conferred by HBVimmunization
HBeAg	Active replication of HBV and high risk of transmission
Anti-HBe	Less active HBV replication Decrease of HBV infectivity and remission of disease Precore/core promoter mutations in HBV genome
IgM anti-HBc	Presence with high index value during acute HBV infection and usually disappears within 6 months 10-20% of chronic hepatitis B patients with hepatitis flares also positive for IgM anti-HBc with low index value
IgG anti-HBc	Not a neutralizing antibody and presence indicates an exposure to HBV Isolated IgG anti-HBc may indicate occult HBV infection

anti-HBs: HBsAg antibody; HBc: Hepatitis B core antigen; HBe: Hepatitis B e antigen; HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus;
Ig: Immunoglobulin.

Table 3. Comparison of Commercial Quantitative *HBV* DNA Assays
Assay (manufacture)	Sensitivity^* (copies/ml)	Dynamic range (copies/ml)	Genotype independent
Branched DNA 3.0 (Bayer)	2 × 10³	2 × 10³–1 × 10⁸	A, B, C, D, E, F
PCR (Roche)	1.7^-4 × 10²		(A), B, C, D, E
Amplicor monitor		400-1 × 10⁷	(A), B, C, D, E
Amplicor Cobas^®		200-1 × 10⁵
Taqman (RT-PCR)		170-1 × 10⁸
RealTime HBV (Abbott)	34	34-1 × 10⁹	A, B, C, D, E, F
Molecular beacons	<50	50-1 × 10⁹	A, B, C, D, E, F

^*Approximately five copies = 1 IU.
HBV: Hepatitis B virus; RT: Real-time.

Table 4. Methods for *HBV* Genotyping
Method	Advantage	Limitation
Direct sequencing of PCR products and homology comparison or phylogenetic tree analysis	Reference method; suitable for analysis of new genotypes or recombination between genotypes	Labor intensive and time consuming
PCR followed by restriction fragment length polymorphism	Easy to perform	Only for known genotypes
PCR with type-specific primers	Easy to perform; can identify mixed genotypes	Only for known genotypes
Serological genotyping	Easy to perform; can identify mixed genotypes; suitable for subjects negative for viremia	Only for known genotypes; cannot confirm viremiastatus
Hybridization technologies
Reverse hybridization (INNO-LIPA)	Commercial kit; easy to perform; could identify mixed genotype	Costly; only for known genotypes
Real-time PCR formats with specific probes and melting curve analysis	Easy to perform; could identify mixed genotypes	Only for known genotypes; can quantify viremia level simultaneously

HBV: Hepatitis B virus; INNO-LIPA: Innogenetics line probe assay.

Table 5. Methods for Identifying HBV Mutants
Method	Advantage	Limitation
Direct sequencing of PCR products	Gold standard; all mutations can be detected	Labor intensive and time consuming; detect 20-30% of total population
PCR followed by multiple cloning sequence analysis	More sensitive and quantitative; evaluate the proportion of mixed mutant andwild-type populations	Costly; labor intensive and time consuming
PCR followed by restriction fragment length polymorphism	Easy to perform; detect 5-10% of total viral population	Only for known mutations
Real time PCR formats with specific probes and melting curveanalysis	Easy to perform; detect 5-10% of total viral population	Only for known mutations
Reverse hybridization (INNO-LIPA)	Commercial kit; easy to perform; detect 5-10% of total viralpopulation	Only for known mutations
MALDI-TOF MS-based restriction fragment mass polymorphism	Easy to perform; detect 0.1% of total viral population	Costly; only for known mutations

HBV: Hepatitis B virus; INNO-LIPA: Innogenetics line probe assay.

Table 6. Serological and Virological Profiles in Patients With HBV Infection
Marker	Acute hepatitis B	Recovery from HBV	Chronic hepatitis B^*	Inactive carrier^‡	Occult hepatitis B
HBsAg	+	-	+	+	-
Anti-HBs	-	+	-	-	-/+
Anti-HBc	+	+	+	+	-/+
HBeAg	+	-	±	-	-/+
Anti-HBe	-	+	-	+	-/+
HBV DNA	+	-	+; >2000 IU/ml	+; <2000 IU/ml	+; <200 IU/ml

^*Chronic hepatitis B: HBV carriers with abnormal ALT level for more than 6 months;
^‡Inactive carrier: HBV carriers with persistently normal ALT level.
ALT: Alanine aminotransferase; Anti-HBc: Hepatitis B core antibody; Anti-HBe: Hepatitis B e antibody; Anti-HBs: Hepatitis B surface antibody; HBeAg: Hepatitis B e antigen; HBsAg: Hepatitis B surface antigen.

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