Performance of the Cobas AmpliPrep/Cobas TaqMan real-time PCR assay for hepatitis B virus DNA quantification

Abstract

Hepatitis B virus (HBV) DNA quantification is used to establish the prognosis of chronic HBV-related liver disease, to identify those patients who need to be treated, and to monitor the virologic response and resistance to antiviral therapies. Real-time PCR-based assays are gradually replacing other technologies for routine quantification of HBV DNA in clinical practice. The goal of this study was to evaluate the intrinsic characteristics and clinical performance of the real-time PCR Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) platform for HBV DNA quantification. Specificity was satisfactory (95% confidence interval, 98.1 to 100%). Intra-assay coefficients of variation ranged from 0.22% to 2.68%, and interassay coefficients of variation ranged from 1.31% to 4.13%. Quantification was linear over the full dynamic range of quantification of the assay (1.7 to 8.0 log(10) IU/ml) and was not affected by dilution. The assay was accurate regardless of the HBV genotype. Samples containing HBV DNA levels above 4.5 log(10) IU/ml were slightly underestimated relative to another accurate assay based on branched-DNA technology, but this is unlikely to have noteworthy clinical implications. Thus, the CAP/CTM HBV DNA assay is sensitive, specific, and reproducible, and it accurately quantifies HBV DNA levels in patients chronically infected by HBV genotypes A to F. Samples with HBV DNA concentrations above the upper limit of quantification need to be diluted and then retested. Broad use of fully automated real-time PCR assays should improve the management of patients with chronic HBV infection.

FIG. 1.

CAP/CTM (A) and bDNA (B) assay quantification of a commercial standard panel containing 2 × 10² (2.3 log₁₀) to 2 × 10⁷ (7.3 log₁₀) IU of HBV DNA/ml (OptiQuant HBV DNA; AcroMetrix, Benicia, CA). The average measured values are shown as a function of the expected values (actual HBV DNA content of the panel sample). The dashed line is the equality line.

FIG. 2.

Correlation between measurements by the CAP/CTM and bDNA assays of HBV DNA levels in 52 clinical samples (group B) containing HBV genotypes A, B, C, D, E, and F.

FIG. 3.

(A) Bland-Altman plot of HBV DNA levels in the 52 group B samples measured by the CAP/CTM and bDNA assays. The difference between the CAP/CTM and bDNA measurements is represented as a function of the mean of the two values. Different genotypes are represented by different colors. The gray area corresponds to the mean difference ± 1.96 SD. (B) Distribution of the differences between HBV DNA levels in the same samples measured by the CAP/CTM and bDNA assays according to the HBV genotype (A to E). The differences were not significant.

FIG. 4.

Individual differences between HBV DNA levels measured by the CAP/CTM and bDNA assays, ranked in increasing order of bDNA values, according to the HBV genotype. Different HBV genotypes are represented by different colors. Bars above the zero line correspond to samples for which the CAP/CTM result was higher than the bDNA result, whereas bars below that line correspond to samples for which the CAP/CTM result was lower than the bDNA result.

FIG. 5.

HBV DNA levels in serial 1/5 dilutions of patients' clinical samples containing HBV genotypes A through F (panels A through F, respectively), measured by the CAP/CTM assay. Two examples are shown for each genotype.