Performance of version 2.0 of the Cobas AmpliPrep/Cobas TaqMan real-time PCR assay for hepatitis B virus DNA quantification

Abstract

The detection and quantification of hepatitis B virus (HBV) DNA are essential for the diagnosis and treatment of chronic HBV infection. The use of real-time PCR assays for HBV DNA quantification is strongly recommended. The goal of this study was to evaluate the intrinsic characteristics and clinical performance of version 2.0 (v2.0) of the Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) assay, a fully automated platform for HBV DNA quantification in serum or in plasma with a claimed lower limit of detection of 20 IU/ml and a claimed upper limit of quantification of 1.7 × 10(8) IU/ml. The specificity of the assay was 99% (95% confidence interval, 94.7 to 100%). Intra-assay and interassay coefficients of variation ranged from 0.21% to 2.67% and from 0.65% to 2.25%, respectively. The calibration of the assay was found to be satisfactory. Study of blood specimens from patients infected with HBV genotypes A to F showed good correspondence between HBV DNA levels measured by the CAP/CTM v2.0 assay, version 1.0 of the same assay, and the third-generation "branched DNA" assay. The CAP/CTM v2.0 assay quantified HBV DNA levels in serum or plasma from the same patients equally. In conclusion, the new version of the CAP/CTM assay is sensitive, specific, and reproducible. It accurately quantifies HBV DNA levels in patients chronically infected with HBV genotypes A to F. Improvements made to ensure equal quantification of HBV DNA in serum and plasma have been successful. Overall, the CAP/CTM assay, version 2.0, is well suited to monitoring clinical HBV DNA levels according to current clinical practice guidelines.

FIG. 1.

Quantification by the CAP/CTM v2.0 assay of HBV DNA levels in 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 (the actual HBV DNA contents of the panel members). The dashed line is the equality line.

FIG. 2.

Correlation between the HBV DNA levels determined by the CAP/CTM v2.0 and bDNA assays (A) or by the CAP/CTM v2.0 and v1.0 assays (B) in 51 clinical samples (group C) containing HBV genotypes A (n = 12), B (n = 9), C (n = 8), D (n = 9), E (n = 10), and F (n = 3).

FIG. 3.

(A) Bland-Altman plot of HBV DNA levels measured by the CAP/CTM v2.0 and bDNA assays in the 51 group B samples. The difference between the HBV DNA levels obtained by the CAP/CTM v2.0 assay and those obtained by the bDNA assay is plotted as a function of the mean of the two values. Different genotypes are represented by different colors. The shaded area corresponds to the mean difference ± 1.96 standard deviation. (B) Distribution of the differences between the HBV DNA levels obtained by the CAP/CTM v2.0 assay and those obtained by the bDNA assay for the same samples, according to the HBV genotype (genotypes A to E). The difference was not significant.

FIG. 4.

Kinetics of HBV DNA levels measured in individual patients' plasma specimens with the CAP/CTM v1.0 assay (solid lines and open circles), the CAP/CTM v2.0 assay (solid lines and filled circles), and the bDNA assay (dashed lines). The top and bottom shaded areas correspond to the lower limits of detection of the bDNA (2.55 log₁₀ IU/ml) and CAP/CTM v2.0 (1.30 log₁₀ IU/ml) assays, respectively. HBV DNA levels (log₁₀ IU/ml) are given on the y axis.