Routine detection and quantification of hepatitis B virus DNA in clinical laboratories: performance of three commercial assays

Pawlotsky, Jean‐Michel; Bastie, Anne; Hézode, Christophe; Lonjon, I.; Darthuy, Françoise; Rémiré, Jocelyne; Dhumeaux, Daniel

doi:10.1016/s0166-0934(99)00149-4

Cited by 82 publications

(50 citation statements)

References 20 publications

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“…These results compare favorably with sensitivities of 10 5 to 10 6 genomes/mL with some non-PCR assays and 10 to 10 3 genomes/mL with commercially available PCR-based assays. 31,32 Given that many HBV mutants tend to develop insidiously into the dominant form of virus, 33 it is of interest that real-time PCR with fluorescent hybridization probes can detect mutants representing as little as 5% of the wild-type population. In addition, the ratio of mutants among wild-type virus can be estimated by comparing the melting curves with those obtained by using a mixture of standard DNA.…”

Section: Discussionmentioning

confidence: 99%

Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis

2002

Hepatology

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Current methods of detecting hepatitis B virus (HBV) mutations are time consuming, labor intensive, and not suitable for screening large numbers of samples. In the present study, we documented the advantages of a system that exploits differences in thermal stability between perfect match and mismatch hybrids, and thereby distinguishes between wild-type and mutants. Hybridization probes were designed complementary to specific wild-type HBV sequences in surface (S), precore, and basal core promoter (BCP) regions of the HBV genome (nt 587, 1896, and 1762/1764, respectively). Two probes were designed for each mutation: anchor probes were 3 labeled with fluorescein and sensor probes, 5 labeled with LC-Red 640, and 3 phosphorylated. Temperatures for each probe melted from amplification products were then determined in a melting program. Sera from 12 patients, each containing identified HBV mutants (6 S-escape, 1 precore, 1 BCP, and 4 mixed precore and BCP), and 5 control sera from patients with wild-type virus were analyzed. Genomic sequences of mutant and wild-type viruses were confirmed by direct sequencing. Real-time polymerase chain reaction (PCR) with fluorescent hybridization probes accurately identified each mutant and wild-type genome. Melting temperatures obtained from probe-product duplexes for the 3 mutants were distinguished from wild-type (>4.0°C, minimal) within 45 minutes. The sensitivity of the system was 100 copies/mL and as few as 5% of mutant among wild-type virus were detected. In conclusion, real-time PCR with fluorescent hybridization probes is a specific, sensitive, quantitative, and rapid means of detecting clinically relevant HBV mutants. (HEPATOLOGY 2002;36:723-728.) T he hepatitis B virus (HBV) is one of the most common infectious agents in humans and a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. [1][2][3] It is a partially double-stranded DNA molecule virus that replicates through an RNA intermediate by reverse transcription. 4,5 The reverse transcriptase enzyme responsible for this aspect of the replication cycle lacks proofreading function and, therefore, mutations to the genome are common. Indeed, it is now recognized that the majority of chronic HBV infections are associated with emergence of mutations throughout the viral genome. Some of these mutations result in the generation of diverse viral populations and different clinical outcomes. Those in the surface, core, precore, and basal core promoter (BCP) regions appear to have the greatest clinical relevance. 6,7 Additional mutations have also been described after therapeutic interventions such as antiviral therapy and vaccination. 8,9 Thus, the ability to detect these mutants is essential to further our understanding of the natural history of HBV and its response to antiviral therapy and immunoprophylaxis.Presently, the detection of HBV mutants is largely performed by restriction fragment length polymorphism analysis, 10,11 gap ligase chain reaction assay and/or direct sequencing. 12,13 These methods ...

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Section: Discussionmentioning

confidence: 99%

Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis

2002

Hepatology

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“…In addition, a quantitative evaluation of HBV DNA concentrations can provide valuable information on the levels of viral replication and may be useful as a prognostic indicator of liver disease (4,21). A number of commercial assays are currently available for the quantification of HBV DNA in patient serum or EDTA-plasma, including hybridization-, signal-, and target-amplification-based technologies (5,11,(16)(17)(18)22). Selection of the optimal assay is dependent on the intrinsic performance characteristics of the methodology as well as the necessity to make appropriate clinical decisions in the context of HBV-associated disease (4,15,21).…”

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Multicenter Evaluation of the VERSANT Hepatitis B Virus DNA 3.0 Assay

et al. 2004

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Infection with hepatitis B virus (HBV) remains a difficult worldwide challenge to public health. The World Health Organization estimates that more than one-third of the world's population has been infected with HBV (25). Epidemiological trends suggest there are currently 400 million HBV chronic carriers worldwide, with over 1 million deaths annually due to HBV-associated liver disease (13,23). HBV is the leading cause of cirrhosis and hepatocellular carcinoma globally (25; Centers for Disease Control and Prevention hepatitis fact sheet [www.cdc.gov/hepatitis]). The development and utilization of molecular diagnostic assays for the detection and quantification of HBV genomes have provided insight into the natural history of HBV and the pathogenesis of HBV infection as well as facilitated the monitoring of viral response to treatment (15,17). In addition, a quantitative evaluation of HBV DNA concentrations can provide valuable information on the levels of viral replication and may be useful as a prognostic indicator of liver disease (4, 21). A number of commercial assays are currently available for the quantification of HBV DNA in patient serum or EDTA-plasma, including hybridization-, signal-, and target-amplification-based technologies (5,11,(16)(17)(18)22). Selection of the optimal assay is dependent on the intrinsic performance characteristics of the methodology as well as the necessity to make appropriate clinical decisions in the context of HBV-associated disease (4, 15, 21).The VERSANT HBV 3.0 Assay (referred to herein as VER-SANT 3.0) is a third-generation branched-DNA (bDNA) assay for the direct quantification of HBV DNA in human serum and plasma. After HBV genomic DNA is released from the virions, the viral DNA is captured by a set of specific, synthetic oligonucleotide capture probes fixed in a microtiter well. A set of target probes (or label extender probes) then hybridizes to both the captured viral DNA and unique preamplifier probes. The capture probes and the target probes bind to conserved DNA regions throughout the entire HBV genome. The amplifier probes subsequently hybridize to the preamplifier probes, forming a bDNA complex. Multiple copies of an alkaline phosphatase-labeled probe are then hybridized to this immobilized complex. Detection is achieved by incubating the alkaline phosphatase-bound complex with a chemiluminescent substrate. The intensity of light emission is directly related to the amount of HBV DNA present in each sample, and results are recorded as relative light units by the luminometer. A standard curve is defined by light emission from quantitative standards containing known concentrations of recombinant DNA. Concentrations of HBV DNA in specimens are determined from this standard curve. This third-generation sandwich nucleic acid hybridization procedure differs from earlier bDNA assays by using the unique preamplifier probes to increase the number of labeled probes that can bind to the target, thereby

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“…Several assays for the quantitative measurement of HBV DNA have been developed, such as the branched-chain DNA signal amplification assay (5,7,28) and transcriptionmediated amplification (TMA)-based (10) or PCR-based (6,11,13,17) nucleic acid amplification assays. However, these methods tend to generate highly divergent results (20,21,22,31) and require cumbersome procedures and expensive equipment, in turn requiring considerable skill and high costs.…”

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New Enzyme Immunoassay for Detection of Hepatitis B Virus Core Antigen (HBcAg) and Relation between Levels of HBcAg and HBV DNA

et al. 2003

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A new enzyme immunoassay specific for hepatitis B virus (HBV) core antigen (HBcAg) was developed. In order to detect HBcAg, specimens were pretreated with detergents to release HBcAg from the HBV virion and disassemble it to dimers, and simultaneously, the treatment inactivated anti-HBc antibodies. HBcAg detected by the assay peaked with HBV DNA in density gradient fractions of HBV-positive sera. The assay showed a wide detection range from 2 to 100,000 pg/ml. We observed no interference from anti-HBc antibody or blood components, but the assay was inhibited by very high concentrations (>1 g/ml; corresponding to 80 signal/cutoff) of HBeAg. When the cutoff value was tentatively set at 4 pg/ml, all healthy control (HBsAg and HBV DNA negative, n ‫؍‬ 160) and anti-hepatitis C virus-positive (n ‫؍‬ 55) sera were identified as negative. HBcAg concentrations correlated very closely with HBV DNA (r ‫؍‬ 0.946, n ‫؍‬ 145) in 216 samples from 72 hepatitis B patients. In seroconversion panels, HBcAg concentrations changed in parallel with HBV DNA levels. The assay, therefore, offers a simple method for monitoring hepatitis B patients. With a series of sera during lamivudine therapy, HBV DNA levels fell sharply and the HBcAg concentration also decreased, but the change in HBcAg was smaller and more gradual. The supposed mechanism of these changes and their clinical significance are discussed.Diagnosis of chronic hepatitis B virus (HBV) infection has long been based on HBV serology and measurement of liver enzymes. With the development of therapies for chronic HBV infection including interferon and lamivudine (9, 16), quantitative detection of HBV DNA has been used increasingly as the most important marker for monitoring HBV replication activity and disease progression as well as for assessing responses to antiviral treatment of patients with chronic hepatitis B (7,8). Several assays for the quantitative measurement of HBV DNA have been developed, such as the branched-chain DNA signal amplification assay (5, 7, 28) and transcriptionmediated amplification (TMA)-based (10) or PCR-based (6, 11, 13, 17) nucleic acid amplification assays. However, these methods tend to generate highly divergent results (20,21,22,31) and require cumbersome procedures and expensive equipment, in turn requiring considerable skill and high costs.On the other hand, immunoassays are generally easy and inexpensive. The nucleocapsid of HBV is composed of either 90 or 120 dimers of HBV core antigen (HBcAg) (3), released into circulation after envelopment. Hence, the quantity of HBcAg in serum would demonstrate virus load as well as HBV DNA. Serum HBcAg assays with specimen pretreatment have been reported previously (4, 29), and the concentration of HBcAg in these assays correlated with levels of HBV-associated DNA polymerase (4). Thus, HBcAg could be a marker for virus load.However, the use of these assays was limited because of relatively low sensitivity and complexity in the procedures.We have developed an enzyme immunoassay (EIA) for hepatitis B virus c...

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Routine detection and quantification of hepatitis B virus DNA in clinical laboratories: performance of three commercial assays

Cited by 82 publications

References 20 publications

Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis

Rapid detection of hepatitis B virus mutations using real-time PCR and melting curve analysis

Multicenter Evaluation of the VERSANT Hepatitis B Virus DNA 3.0 Assay

New Enzyme Immunoassay for Detection of Hepatitis B Virus Core Antigen (HBcAg) and Relation between Levels of HBcAg and HBV DNA

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