Page 1

Infect Dis Clin N Am 20 (2006) 47–61

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 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 reading frames with four major genes designated pre-S/S, C, P, and X. The pre-S genes (S1 and S2) code for the hepatocyte receptor–binding 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 polymerase. The X gene encodes a protein that transactivates transcriptional promoters. Eight genotypes (A–H) of HBV have been identified based on nucleotide sequence divergences of at least 8%. HBV genotypes differ in their predominant geographic occurrence and response to therapy with interferon. 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 infection 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

A version of this article originally appeared in the 8:2 issue of Clinics in Liver Disease. * Corresponding author. Department of Medicine, Newton-Wellesley Hospital, 2014 Washington Street, Newton, MA 02462. E-mail address: (L.S. Friedman). 0891-5520/06/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.idc.2006.01.005



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 filled 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. Hepadnaviridae: the viruses and their replication. In: Fields BN, Knipe DM, Hawley PM, editors. Fundamental virology. 3rd edition. Philadelphia: Lippincott-Raven; 1996. p. 1199–234; with permission.)

[Fig. 2]). Patients then exhibit increased serum aminotransferase levels, usually 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 nonspecific symptoms such as fatigue and malaise as well as right upper quadrant pain and jaundice. 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, serum aminotransferase levels return to normal, HBeAg disappears and antibody 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;11:73–83; 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 equivalents/mL) of HBV DNA have been found in serum and peripheral blood mononuclear cells of patients up to 21 years after clinical and serologic recovery 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 ultimately enter a nonreplicative state characterized by seroconversion from HBeAg to anti-HBe, serum HBV DNA levels ! 105 copies/mL, and normalization 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 patients in the inactive carrier state may experience a reactivation to the replicative state, or flare, and may even cycle between the nonreplicative and replicative state [3]. Such flares 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 HBeAgnegative chronic hepatitis B characterized by circulating HBV DNA, fluctuating serum aminotransferase levels, and, in some cases, severe hepatic necroinflammatory 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 fulminant 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-methionineaspar-tate-aspartate) motif of the DNA polymerase gene and include substitutions 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 lamivudine-resistant mutants may be accompanied by HBeAg to anti-HBe seroconversion, a flare of serum aminotransferase levels, or hepatic



decompensation. Mutations that lead to resistance occur less frequently during 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 nonvirion associated spheres and tubules [7]. HBsAg is the first 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 exposure 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 different stages of infection Stage of HBV infection Acute HBV infection Early Window period Recovery Chronic HBV infection Replicative Nonreplicative/inactive carrier state Reactivation HBV HBeAg(-) chronic HBV (precore or core mutant)

HBsAg Anti-HBs HBeAg Anti-HBe Anti-HBc HBV DNA þ  





þ þ/ þ/a

þ þ




IgG, IgMb O105 copies/mL IgG !105 copies/mL

þ þ





O105 copies/mL O105 copies/mL

Abbreviations: anti-HBc, antibody to hepatitis B core antigen; anti-HBe, antibody to hepatitis B e antigen; anti-HBs, antibody to hepatitis B surface antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; IgG, IgG class of antibody; IgM, IgM class of antibody. a Low levels (101–102 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 associated 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 period 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 flares 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 signifies either resolved HBV infection (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 absence of HBsAg or anti-HBs. For example, up to 5% of healthy blood donors have isolated anti-HBc in the serum. Among human immunodeficiency 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 window period of acute HBV infection (see earlier discussion); (2) during chronic HBV infection as levels of HBsAg become undetectable; (3) after resolved 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 [11–13]. 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 infection (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 signifies active 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 detectable between 6 and 12 weeks after exposure to HBV. For patients who successfully 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 first 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 intermittent 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, reflecting active viral replication, infectivity, and hepatic inflammation. Three epidemiologic patterns of HBeAg-positive chronic HBV infection, defined by mode of transmission, have been identified. Pattern 1 is typical of chronic HBV infection resulting from perinatal transmission, in which HBeAg seroconversion usually does not occur until adulthood [3], This pattern is seen predominantly in Asia and Oceania, where patients present with HBeAg positivity, high serum HBV DNA levels, and normal serum aminotransferase levels, reflecting an ‘‘immune tolerant’’ state [3]. People with Pattern 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, seroconversion 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, affected people tend to have HBeAg in serum, elevated serum HBV DNA levels, and elevated serum AST or ALT levels (Fig. 2). These findings indicate active hepatic necroinflammation. 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-HBepositive still have active chronic HBV infection. Despite the of circulating HBeAg. suchjeopjejiave detectable HBV DNA (O105 copies/mL) and fluctuating 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 infection, the clinical course of people with HBeAg-negative chronic HBV infection typically is characterized by lower serum HBV DNA levels and intermittent, rather than sustained, periods of necroinflammatory 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, specifically mutations in the YMDD (tyrosine-methionine-aspartate-aspartate) motif of the HBV DNA polymerase 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, California) is available to identify the most common mutations in the DNA polymerase 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 quantitative tests in serum for HBV DNA is useful in several settings: (1) to diagnose some cases of acute HBV infection; (2) to distinguish replicative from non-replicative chronic HBV infection; and (3) to monitor a patient’s response to antiviral therapy. Determination of the HBV genotype will likely be used in the future to help predict response to antiviral therapy and can be performed with a commercially available line probe assay (INNO-LiPA HBV Genotyping Assay, Immunogenetics N.V., Ghent, Belgium) [16]. In most instances, the diagnosis of acute hepatitis B can be made with serologic testing for HBsAg. Coincident with the appearance of HBsAg in serum, markers of active HBV replication and infectivity, specifically HBeAg and HBV DNA, appear. If the results of tests for HBsAg are equivocal, assays 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 highrisk exposures, such as needlestick accidents in health care workers.



Assays for HBV DNA in serum are also used to characterize the replicative state of chronic HBV infection. Patients with chronic HBV infection may continue to display markers of active viral replication or may cycle between 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 increasing sensitivity of molecular assays that allows quantification of HBV DNA levels, the threshold that distinguishes the replicative from the nonreplicative state has been defined as 105 copies of HBV DNA/mL. These assays allow patients to be classified as having replicative, nonreplicative, or reactivation HBV infection. Furthermore, in patients with precore or core mutations 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 confirmation 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 Gastroenterological Association proposed criteria to define 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 compared with findings on a pretreatment liver biopsy. A critical component of VR is undetectable HBV DNA levels (!105 copies/mL) with the use of an unamplified assay (see later discussion) and loss of HBeAg in serum in patients 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 quantification of hepatitis B virus DNA There are both qualitative and quantitative assays for HBV DNA. However, the qualitative, PCR-based, assays are not necessary to assess the success 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. Quantification of HBV DNA in serum is performed using either signal or target amplification techniques. Signal amplification techniques require the use of a specific ‘‘capture’’ oligonucleotide probe that hybridizes to denatured DNA [17]. Then, the signal (radioisotope, chemi luminescence) from the probe-DNA hybrid is amplified for



detection and quantification [17], Target amplification requires amplification of the viral genome (amplicons); the amplicons are then detected and quantified. Assays using signal amplification 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, California). The liquid hybridization assay uses iodine 125-labeled nucleic acid probes that hybridize to soluble, denatured HBV DNA [9]. After hybridization, the radiolabeled probes are detected and quantified using a gamma scintillation counter [18]. The sensitivity of liquid hybridization is 6  105 copies/mL or l–2 pg/mL [9]. In the Hybrid Capture System [17,19,20], specific RNA probes are hybridized to the target HBV DNA to create RNA–DNA hybrids (Fig. 3). Then, multiple RNA–DNA hybrids are captured onto microplate wells, using

Fig. 3. Principle of hybrid capture signal amplification assay. The target sequence is doublestranded HBV DNA. Hybridization to specific RNA probes creates RNA–DNA hybrids, which are captured on a solid phase (a tube in the first-generation assay, a microplate in the secondgeneration assay) by means of universal capture antibodies specific for RNA–DNA hybrids. Detection is performed after signal amplification with multiple antibodies conjugated to a revelation system based on chemiluminescence. Light emission is measured and compared with a standard curve generated simultaneously with known standards. (From Pawlotsky J. Molecular diagnosis of viral hepatitis. Gastroenterology 2002;122:1554–68; with permission from the American Gastroenterological Association.)



universal capture antibodies specific for the hybrids. The captured RNA– DNA hybrids are then detected using multiple antibodies (creating signal amplification) conjugated to alkaline phosphatase. The bound alkaline phosphatase is detected with a chemiluminescent dioxetane substrate that produces light, which is then measured. The signal can be amplified 3000 fold. The sensitivity of the Hybrid Capture System is 4700 copies/mL [17]. The bDNA technique [17,21] involves the use of specific oligonucleotide probes to hybridize HBV DNA to plastic microwells (Fig. 4). Signal

Fig. 4. Principle of branched DNA (bDNA) signal amplification assay. The target sequences are captured on the wells of a microtiter plate by means of specific ‘‘capture probes.’’ ‘‘Extender probes’’ are used to hybridize synthetic bDNA amplifier molecules (in first-generation HBV DNA and hepatitis C virus (HCV) RNA assays, and in second-generation HCV RNA assays) or, as shown in the figure, preamplifier molecules that in turn hybridize bDNA molecules (thirdgeneration HCV and HBV assays). The multiple repeat sequences within each bDNA molecule serve as sites for hybridization to alkaline phosphatase–conjugated oligonucleotide probes. Alkaline 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;122:1554–68; with permission from the American Gastroenterological Association.)



amplification occurs when bDNA amplifier molecules are hybridized to the target HBV DNA hybrids in the microwell. Multiple repeat sequences within the bDNA amplifier molecule are then conjugated with an alkaline phosphatase–catalyzed chemiluminescence probe similar to that used in the Hybrid Capture System. The lower limit of detection for the bDNA assay is 7  105 DNA equivalents/mL [17]. Although the signal amplification techniques offer highly specific assays to detect HBV DNA, they are unable to detect low levels of HBV DNA (!w5,000 copies/mL). Target amplification 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 specific primers that attach to each strand of target dsDNA, Then, new DNA strands are synthesized and amplified behind the primer. This cycle of DNA denaturing, primer annealing, and strand synthesis is repeated multiple times, thereby resulting in amplification 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 Molecular Systems, Pleasanton, California). The ranges of HBV DNA detection 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 assays and uses an Amplicor analyzer that automates the amplification and detection process. Recent advances in PCR technology include the development of ‘‘realtime’’ PCR techniques to increase the sensitivity of the assay. Real-time PCR refers to the simultaneous amplification and quantification of viral genomes, thereby obviating the need for post-PCR manipulations [23,24]. Real-time PCR can detect a wide range of HBV DNA levels and offers 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 LCPCR 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 fluorescent probe annealed to target DNA sequences for quantification 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 significance of such low serum 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 definition 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 quantification assays Assay

Versant HBV DNA 1.0 Assay

HBV Hybrid-Capture I HBV Hybrid-Capture II Ultra-sensitive HBV Hybrid-Capture II Target amplification Amplicor HBV Monitor Test v2.0 Cobas Amplicor HBV Monitor Test v2.0



Lower detection cutoff

Dynamic range of quantification

Abbott Labs, Chicago, IL Bayer Corp, Diagnostics Division, Tarrytown, NY Digene Corp., Gaithersburg, MD Digene Corp., Gaithersburg, MD Digene Corp., Gaithersburg, MD

Liquid hybridization

1–2 pg/mL (w600,000 copies/mL) 700,000 genome equivalents/mL

1–2 pg/mL –w 800 pg/mL (600,000–300,000,000 copies/mL) 700,000–5,000,000,000 genome equivalents/mL

Hybrid capture signal amplification in tubes Hybrid capture signal amplification in microplates Hybrid capture signal amplification in microplates after centrifugation

700,000 copies/mL

700,000–560,000,000 copies/mL

142,000 copies/mL

142,000–1,700,000,000 copies/mL

4700 copies/rnL

4700–57,000,000 copies/mL

Roche Molecular Systems, Pleasanton, CA Roche Molecular Systems, Pleasanton, CA

Manual quantitative RT-PCR

1000 copies/mL

1000–40,000,000 copies/mL

Semi-automated quantitative RT-PCR

200 copies/mL

200–200,000 copies/mL

Manual branched DNA (bDNA) signal amplification


Signal amplification Genostics Assay

Adapted from Pawlotsky J. Molecular diagnosis of viral hepatitis. Gastroenterology 2002;122:1554–68; with permission from the American Gastroenterological Association.)




fluctuate and intermittently fall below 105 copies/mL. Second, the threshold HBV DNA level associated with the development of hepatic fibrosis is unknown. Third, currently available HBV DNA assays have not been standardized 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 defining clinically appropriate treatment guidelines based on serum HBV DNA levels [17].

Summary Serologic assays for HBV are the mainstay diagnostic tools for HBV infection. However, the advent of molecular biology–based 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 redefinitions of chronic HBV infection, as well as thresholds for antiviral treatment. As the sensitivity of these molecular techniques continues to improve, the challenge will be to standardize these assays as well as define clinically significant levels of HBV replication.

References [1] Schaefer S. Hepatitis B virus: significance of genotypes. J Viral Hepat 2005;12:111–24. [2] Cabrerizo M, Bartolome J, Caramelo C, Barril G, Carreno V. Molecular analysis of hepatitis B virus DNA in serum and peripheral blood mononuclear cells from hepatitis B surface antigen-negative cases. Hepatology 2000;32:116–23. [3] Lok ASF, McMahon BJ. Chronic hepatitis B. Hepatology 2001;34:1225–41. [4] Okamoto H, Tsuda F, Akahane Y, Sugai Y, Toshiba M, Moriyama K, et al. Hepatitis B virus with mutations in the core promoter for an e antigen-negative phenotype in carriers with antibody to e antigen. J Virol 1994;68:8102–10. [5] Laskus T, Rakela J, Nowicki MJ, Pershing DH. Hepatitis B core promoter sequence analysis in fulminant and chronic hepatitis B. Gastroenterology 1995;109:1618–23. [6] Locarnini S. Molecular virology and the development of resistant mutants: implications for therapy. Semin Liver Dis 2005;25(Suppl 1):9–19. [7] Martin P, Friedman LS, Dienstag JL. Diagnostic approach. In: Zuckerman AJ, Thomas HC, editors. Viral hepatitis: scientific basis and clinical management. Edinburgh: Churchill Living-stone; 1993. p. 393–408. [8] CDC. Sensitivity of the test for antibody to hepatitis B surface antigendUnited States. MMWR 1993;42:707–10. [9] Berenguer M, Wright TL. Viral hepatitis. In: Feldman M, Friedman LS, Sleisenger MH, editors. Sleisenger and Fordtran’s gastrointestinal and liver disease: pathophysiology diagnosis management. 7th edition. Philadelphia: W.B. Saunders; 2002. p. 1278–341. [10] Gandhi RT, Wurcel A, Lee H, McGovern B, Boczanowski M, Gerwin R, et al. Isolated antibody to hepatitis B core antigen in human immunodeficiency virus type-1–infected individuals. Clin Infect Dis 2003;36:1602–5. [11] Douglas DD, Taswell HF, Rakela J, Rabe D. Absence of hepatitis B virus DNA detected by polymerase chain reaction in blood donors who are hepatitis B surface antigen negative and



[13] [14] [15] [16] [17] [18] [19]

[20] [21]



[24] [25]

[26] [27] [28]


antibody to hepatitis B core antigen positive from a United States population with a low prevalence of hepatitis B serologic markers. Transfusion 1993;33:212–6. Silva AE, McMahon BJ, Parkinson AJ, Sjogren MH, Hoofnagle JH, Di Bisceglie AM. Hepatitis B virus DNA in persons with isolated antibody to hepatitis B core antigen who subsequently received hepatitis B vaccine. Clin Infect Dis 1998;26:895–7. Chung HT, Lee STK, Lok ASF. Prevention of posttransfusion hepatitis B and C by screening for antibody to hepatitis C virus and antibody to HBcAg. Hepatology 1993;18:1045–9. Lok AS, Heathcote EJ, Hoofnagle JH. Management of hepatitis B 2000: summary of a workshop. Gastroenterology 2001;120:1828–53. Hadziyannis SJ. Hepatitis B e antigen negative chronic hepatitis B: from clinical recognition to pathogenesis and treatment. Viral Hepat Rev 1995;1:7–36. Osiowy C, Giles E. Evaluation of the INNO-LiPA HBV genotyping assay for the determination of hepatitis B virus genotype. J Clin Microbiol 2003;41:5473–7. Pawlotsky JM. Molecular diagnosis of viral hepatitis. Gastroenterology 2002;122:1554–68. Hu KQ, Vierling JM. Molecular diagnostic techniques for viral hepatitis. Gastroenterol Clin North Am 1994;23:479–98. Barlet V, Cohard M, Thelu MA, Chaix MJ, Baccard C, Zarski JP, et al. Quantitative detection of hepatitis B virus DNA in serum using chemiluminescence: comparison of radioactive solution hybridization assay. J Virol Methods 1994;49:141–51. Digene Corporation Website. Hybrid Capture Technology. Available at: http://www. Accessed December 18, 2005. Urdea MS, Horn T, Fultz TJ, Anderson M, Running JA, Hamren S, et al. Branched DNA amplification multimers for the sensitive, direct detection of human hepatitis viruses. Nucleic Acids Symp Ser 1991;24:197–200. Loeb KR, Jerome KR, Goddard J, Huang M, Cent A, Corey L. High-throughput quantitative analysis of hepatitis B virus DNA in serum using the TaqMan fluorogenic detection system. Hepatology 2000;32:626–9. Ho SKN, Yam W, Leung ETK, Wong L, Leung JKH, Lai K, et al. Rapid quantification of hepatitis B virus DNA by real-time PCR using fluorescent hybridization probes. J Med Micro-biol 2003;52:397–402. Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (NY) 1993;11:1026–30. Sum SS, Wong DK, Yuen JC, et al. Comparison of the COBAS Taq Man HBV test with the COBAS Amplicor monitor test for measurement of hepatitis B virus DNA in serum. J Med Virol 2005;77:486–90. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996;6:986–94. Gibson UE, Heid CA, Williams PM. A novel method for real time quantitative RT-PCR. Genome Res 1996;6:995–1001. Saldanha J, Gerlich W, Lelie N, Dawson P, Heermann K, Heath A, WHO Collaborative Study Group. An international collaborative study to establish a World Health Organization international standard for hepatitis B virus DNA nucleic acid amplification techniques. Vox Sang 2001;80:63–71.



Read more
Read more
Similar to
Popular now
Just for you