Evaluation of the COBAS AmpliPrep-Total Nucleic Acid Isolation-COBAS TaqMan Hepatitis B Virus (HBV) Quantitative Test and Comparison to the VERSANT HBV DNA 3.0 Assay

Abstract

Quantitative detection of hepatitis B virus (HBV) in serum or plasma has become the most direct and reliable method for monitoring chronic hepatitis B. Here, we report the performance characteristics of a real-time PCR hepatitis B DNA quantitative assay, the COBAS TaqMan (CTM) HBV test (Roche Diagnostics, Meylan, France), in combination with an automated DNA extraction on the COBAS AmpliPrep (CAP) instrument using the total nucleic acid isolation kit (TNAI kit), a generic reagent for nucleic acid isolation (both from Roche Diagnostics). The linearity, accuracy, and specificity of the CAP-TNAI-CTM HBV test were evaluated using various reference panels and standards (HBV panel 2004 from Quality Control for Molecular Diagnostics, OptiQuant HBV panel from AcroMetrix, WHO International Standard for HBV, and Teragenix hepatitis B genotype panel). Quantitative results show that the CAP-TNAI-CTM HBV test performed well with respect to linearity, accuracy, and reproducibility from at least 100 to 500,000 HBV DNA IU/ml. Based on the log 10 IU of HBV DNA/ml measured, the intra-assay variation ranged from 2.49% to 8.46% and the interassay variation ranged from 1.88% to 7.83%. The test was extremely sensitive and could detect samples containing HBV DNA below the reported quantification threshold (<30 IU/ml). All HBV genotypes were correctly amplified, and no cross-contamination occurred during the automated sample preparation. In addition, 402 human serum samples were tested comparatively to the VERSANT HBV DNA 3.0 assay (bDNA; Bayer Diagnostics, Puteaux, France). The viral load results of the CAP-TNAI-CTM test and bDNA were significantly correlated, but the agreement between the two tests was poor, with large differences between results for individual samples. The hands-on time was estimated to be reduced from 2.30 h with bDNA to 45 min with the CAP-TNAI-CTM test, and up to 84 samples were completely processed within a working day. Overall, the performance characteristics of the CAP-TNAI-CTM test demonstrated that it provides a high-throughput sensitive and reliable method for quantitation of HBV DNA levels in the routine molecular laboratory.Hepatitis B virus (HBV) is a major causative agent of chronic hepatitis. Approximately 350 million to 400 million people worldwide are chronically infected. Chronic HBV infection can lead to liver cirrhosis and hepatocellular carcinoma (13). Detection and quantification of HBV DNA in serum or plasma are now considered important indicators for managing disease in HBVinfected patients and predicting and monitoring the efficiency of antiviral treatment as well as identifying the emergence of drug resistance by detecting HBV DNA breakthrough (4, 24). Several commercial molecular tests for the quantitation of HBV DNA in serum or plasma have been developed and are routinely used in diagnostic virology laboratories (10, 29). These molecular tests are based on target amplification (HBV Monitor test; Roche Diagnostics, Meylan, France), branched-DNA signal amplification (VERSANT HBV DNA 3.0 assa...

Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

“…Comparison of the two assays for quantitative detection of HBV DNA levels in the 118 serum samples showed that the two sets of results were highly correlated (r = 0.948, P < 0.001). This was in accordance with the results of previous studies that have evaluated other realtime PCR assays for quantitation of HBV DNA [13,14,[22][23][24][25][26] .…”

“…Many studies have shown that real-time PCR is accurate, reproducible, sensitive, and rapid for quantitative detection of HBV levels in clinical samples [13,14,[17][18][19][20][21] . Realtime PCR also has the widest dynamic range among all the assays available commercially [14] .…”

“…The Ct value is smaller when the initial DNA target level is higher. Several evaluation studies have shown that real-time PCR has a higher sensitivity, a broader dynamic range, and an accurate quantitation of HBV DNA compared to those of the existing commercial methods [12][13][14][15] .…”

Outcomes of Infants Born to Women Infected With Hepatitis B

“…A viral load of 2000 IU/mL was considered equivalent to 10 000 copies/mL and 3.3 log IU/mL. 10 Viral load was dichotomized as $2000 IU/mL and ,2000 IU/mL in accordance with previous studies defining viral load thresholds. 11,12 Viral load was recoded as missing when reported in unknown units or in units other than IU/mL, copies/mL, or log IU/mL.…”

LNA real-time PCR probe quantification of hepatitis B virus DNA

“…According to the related reference standard (15)(16)(17)(18), the LNA real-time PCR viral load in 35 HBV DNA-positive samples compared well with the TaqMan real-time PCR assay viral load (r=0.9636, p<0.05) (Fig. 5).…”