Characterization of a new sensitive PCR assay for quantification of viral DNA isolated from patients with hepatitis B virus infections

Abstract

Sensitive and accurate quantification of hepatitis B virus (HBV) DNA is necessary for monitoring patients with chronic hepatitis receiving antiviral therapy in order to determine treatment response and to adapt therapy in case of inadequate virologic control. The development of quantitative PCR assays has been crucial in meeting these needs. The objective of this study was to compare the performance of a new real-time PCR assay (Abbott RealTime) for HBV DNA with that of three other commercial assays for the detection of HBV DNA. These were the Versant 3.0 branched-chain DNA assay, the Cobas Amplicor HBV Monitor test, and the Cobas AmpliPrep-Cobas TaqMan hepatitis B virus assay (CAP-CTM). HBV DNA was measured in blood samples taken from two cohorts of patients with chronic hepatitis. HBV DNA levels measured with the Abbott RealTime assay were highly correlated with those measured with the other three tests over their respective dynamic ranges (r, 0.88 to 0.96). The sensitivity (detection limit, 10 IU/ml) and dynamic range of the Abbott RealTime assay (10(1) to 10(9) IU/ml) was superior to that of the Versant assay. The RealTime assay recognized both HBV strains belonging to genotypes A to G and those bearing polymerase gene mutations equivalently. In conclusion, this study demonstrates the utility of the Abbott RealTime assay for monitoring HBV DNA levels in patients with chronic hepatitis B. Its sensitivity and wide dynamic range should allow optimal monitoring of antiviral therapy and timely treatment adaptation.

FIG. 1.

Correlation between HBV DNA determinations within dynamic ranges using the Abbott RealTime and Versant assays for samples from 59 HBV-infected, HBeAg-negative patients for which a quantitative result was obtained with both assays. The identity line (solid line) as well as the best-fit correlation (broken line) is shown (y = 0.9863x − 0.154, R² = 0.8646).

FIG. 2.

Correlation between HBV DNA determinations within dynamic ranges using the RealTime and Versant assays for samples from 59 HBV-infected, HBeAg-negative patients according to surface gene variants (genotypes are indicated).

FIG. 3.

Correlation between HBV DNA determinations within dynamic ranges using the RealTime and Versant assays for samples from 59 HBV-infected, HBeAg-negative patients according to the number of polymerase gene mutations.

FIG. 4.

Correlation between HBV DNA determinations using the RealTime and other quantitative PCR assays in HBV DNA samples isolated from patients in cohort B for which a quantitative result was obtained with both assays. (Top) CAM assay (n = 66). (Bottom) CAP-CTM assay (n = 101). The best-fit correlation (solid line) is shown (for the CAM assay, y = 0.8653x + 0.3629 and R² = 0.7668; for the CAP-CTM assay, y = 0.9586x − 0.0543 and R² = 0.9276).