Identification of Different States of Hepatitis B Virus Infection with a Quantitative PCR Assay

Kessler, Harald H.; Preininger, Sabine; Stelzl, Evelyn; Daghofer, Elisabeth; Santner, Brigitte I.; Marth, Egon; Lackner, Herwig; Stauber, Rudolf

doi:10.1128/cdli.7.2.298-300.2000

Cited by 58 publications

(41 citation statements)

References 21 publications

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“…That 98% of HBsAg-containing samples also contain HBV DNA allows us to equate the presence of HBsAg and infectivity. This percentage of concordance is higher than reported by other investigators using QPCR 10,11 or other quantitative methods, 12 who found no more than 84% DNA positivity in HBsAg-containing samples in asymptomatic carriers. The percentage of concordant results decreased to 77% or less in anti-HBe-positive samples.…”

Section: Discussioncontrasting

confidence: 52%

The risk of hepatitis B virus infection by transfusion in Kumasi, Ghana

Allain

Candotti

Soldan

et al. 2003

Blood

162

160

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The risk of hepatitis B virus (HBV) transmission by transfusion in sub-Saharan Africa is considered to be relatively low, and testing of blood donors is often not done or is done relatively poorly. To reexamine this attitude, we identified HBV chronically infected blood donors from a major hospital in Ghana with a range of hepatitis B surface antigen (HBsAg) assays. Test efficacy was estimated using HBV DNA as a gold standard, and the risk of HBV infection in blood recipients was estimated for different testing strategies.

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Section: Discussioncontrasting

confidence: 52%

The risk of hepatitis B virus infection by transfusion in Kumasi, Ghana

Allain

Candotti

Soldan

et al. 2003

Blood

162

160

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“…In chronic infection, viremia in general persists lifelong but at highly variable levels, from more than 10 9 copies/ml to below 100 copies/ml. Quantitation of HBV DNA is important for staging and pretreatment evaluation (5,6,11,15). Moreover, antiviral therapy requires highly sensitive detection of viremia, both for monitoring the initial response and later for identifying increasing HBV DNA levels indicating drug resistance (10,12,14).…”

mentioning

confidence: 99%

Dynamic Range and Reproducibility of Hepatitis B Virus (HBV) DNA Detection and Quantification by Cobas Taqman HBV, a Real-Time Semiautomated Assay

Lindh

Hannoun

2005

J Clin Microbiol

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The Cobas Taqman assay for hepatitis B virus (HBV) DNA showed linear detection over 7 logs for genotypes A to D. The coefficient of variation was 1.2% at >1,000 IU/ml and 22.0% at 10 IU/ml. In 97 clinical samples, the log HBV DNA/ml differed by 0.11 between Cobas Amplicor and Cobas Taqman (r 2 ‫؍‬ 0.97).Hepatitis B virus (HBV) infection is a major cause of chronic liver disease and may result in liver cirrhosis or hepatocellular carcinoma. In chronic infection, viremia in general persists lifelong but at highly variable levels, from more than 10 9 copies/ml to below 100 copies/ml. Quantitation of HBV DNA is important for staging and pretreatment evaluation (5,6,11,15). Moreover, antiviral therapy requires highly sensitive detection of viremia, both for monitoring the initial response and later for identifying increasing HBV DNA levels indicating drug resistance (10,12,14).Methods for analyzing HBV DNA by quantitative PCR have had limitations, mainly concerning the detection range (7,9,18). In the last few years, real-time PCR methods with wider detection ranges have been reported (1-4, 8, 10, 12, 13, 16, 17, 19-24). Here we evaluate the detection range, reproducibility, and clinical applicability of the Cobas Taqman HBV assay (Roche Molecular Systems, Branchburg, NJ), a real-time PCR method which includes an internal quantitation standard (23).The analyses were performed with a Cobas Taqman 48 instrument according to the manufacturer's instructions. First, HBV DNA was manually isolated from 500 l serum. A known number of quantitation standard (QS) molecules were introduced into each specimen and were carried through the specimen preparation, amplification, and detection steps, serving as both a quantitation standard and an inhibition control. The DNA was eluted in a volume of about 80 l, of which 50 l was used for PCR in a reaction mixture of 100 l, amplifying a 105-bp segment of the precore-core region. The C T values, i.e., the cycles in which the fluorescence becomes detectable for target HBV and QS, are used to calculate the target HBV concentration, which essentially equals to [QS](2 ⌬C T ), where [QS] represents the concentration of added QS and ⌬C T is the difference in C T for HBV and QS.Linearity panels consisting of samples (genotypes A, B, C, and D) with high HBV DNA levels (Ͼ10 9 copies/ml as measured by Cobas Amplicor [Roche Diagnostics, Branchburg, NJ]) were prediluted to approximate levels of 10 8 copies/ml and serially diluted in 1:10 steps to around 10 2 copies/ml. At each level, two replicates were analyzed by Cobas Taqman (DNA extraction and real-time PCR). As shown in Fig. 1, there was a good linearity over 7 logs for all genotypes (A to D). The r 2 values were 0.997, 0.997, 0.997, and 0.991 for genotypes A, B, C, and D.Reproducibility was evaluated by analyzing a genotype D sample that was diluted to six replicates at four different levels (5 ϫ 10 7 , 5 ϫ 10 5 , 5 ϫ 10 3 , and 50 copies/ml). Each of the 24 replicates was analyzed by Cobas Taqman (DNA extraction and real-time PCR) on 4 subs...

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“…The HBcrAg assay demonstrated higher sensitivity than HBV-DNA transcription-mediated amplification ( Many hepatitis B virus (HBV) markers are used for diagnosing and monitoring hepatitis B patients. HBV-DNA tests, such as the branched-chain DNA (b-DNA) signal amplification assay (7, 31), and transcription-mediated amplification (TMA)-based (11) or PCR-based (12,14,20) assays are used to diagnose and monitor the efficacy of treatment. However, these methods require cumbersome procedures and expensive equipment, thus requiring considerable skill and high costs.…”

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confidence: 99%

Sensitive Enzyme Immunoassay for Hepatitis B Virus Core-Related Antigens and Their Correlation to Virus Load

et al. 2002

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A sensitive enzyme immunoassay (EIA) specific for hepatitis B virus core antigen (HBcAg) and hepatitis B e antigen (HBeAg) was developed. We designated the precore/core gene products as hepatitis B virus (HBV) core-related antigens (HBcrAg). In order to detect HBcrAg even in anti-HBc/e antibody-positive specimens, the specimens were pretreated in detergents. The antibodies are inactivated by this pretreatment and, simultaneously, the antigens are released and the epitopes are exposed. The assay demonstrated 71 to 112% recovery using HBcrAg-positive sera. We observed no interference from the tested anticoagulants or blood components. When the cutoff value was tentatively set at 10 3 U/ml, all healthy control (HBsAg/HBV-DNA negative; n ‫؍‬ 108) and anti-HCV antibody-positive (n ‫؍‬ 59) sera were identified as negative. The assay showed a detection limit of 4 ؋ 10 2 U/ml using recombinant antigen. Detection limits were compared in four serially diluted HBV high-titer sera. The HBcrAg assay demonstrated higher sensitivity than HBV-DNA transcription-mediated amplification ( Many hepatitis B virus (HBV) markers are used for diagnosing and monitoring hepatitis B patients. HBV-DNA tests, such as the branched-chain DNA (b-DNA) signal amplification assay (7, 31), and transcription-mediated amplification (TMA)-based (11) or PCR-based (12,14,20) assays are used to diagnose and monitor the efficacy of treatment. However, these methods require cumbersome procedures and expensive equipment, thus requiring considerable skill and high costs. These gene amplification assays also present some limitations (22,23,35). The b-DNA assay provides quantitative results but requires a long incubation time and lacks adequate sensitivity. Amplification assays have adequate sensitivity but are less quantitative.Immunoassays are generally easy and inexpensive. There have been a few reports of serum HBcAg assays with specimen pretreatment (4, 32). 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 is limited because of relatively low sensitivity and complex procedures.Serum HBeAg concentration reflects virus replication and hepatitis activity and is closely correlated with virus load in anti-HBe antibody-negative patients (8). Seroconversion of HBeAg to anti-HBe antibody reveals the inactive phase of infection (17,25). However, after seroconversion, many patients may exhibit reactivation and high viral load (3,10,18). In these cases, HBeAg is usually negative due to masking by anti-HBe antibody (24), although the HBeAg/anti-HBe immune complex can be indirectly detected according to the levels of alanine aminotransferase (ALT) and HBV-DNA (6). Therefore, HBcAg and HBeAg could be expected to be efficient markers of virus load if antibodies were inactivated and the antigens released.In the present study, for the purpose of developing a simple, sensitive, and inexpensive assay for determining HBV virus load, we targete...

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Identification of Different States of Hepatitis B Virus Infection with a Quantitative PCR Assay

Cited by 58 publications

References 21 publications

The risk of hepatitis B virus infection by transfusion in Kumasi, Ghana

The risk of hepatitis B virus infection by transfusion in Kumasi, Ghana

Dynamic Range and Reproducibility of Hepatitis B Virus (HBV) DNA Detection and Quantification by Cobas Taqman HBV, a Real-Time Semiautomated Assay

Sensitive Enzyme Immunoassay for Hepatitis B Virus Core-Related Antigens and Their Correlation to Virus Load

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