Comparison of real-time PCR assays for monitoring serum HBV DNA levels during antiviral therapy

Abstract

INTRODUCTION & AIM: Monitoring of serum HBV DNA levels is established as a gold standard to assess antiviral potency. However, currently used commercial assays have limitations with respect to sensitivity and dynamic range of quantification. The objective of the present study was to compare two real-time PCR based assays, the RealARTTM and Molecular Beacons assays, with the Digene Hybrid Capture II assay (ultrasensitive) in patients undergoing antiviral therapy. METHODS: A total of 224 serum samples from 14 hepatitis B e antigen-positive chronic hepatitis B patients; 9 on adefovir dipivoxil plus emtricitabine combination therapy and 5 on adefovir dipivoxil monotherapy, were tested for HBV DNA using the three assays. RESULTS: The RealARTTM and Digene Hybrid assays were significantly related (r = 0.94, p < 0.001). The Molecular Beacons and Digene assays were also correlated (r = 0.79, p < 0.001). There was a good agreement between the RealARTTM and Digene assays. The mean difference was −1.05, as expected for good agreement, and 95% of the data were between +0.12 and −2.19 after logarithmic transformation. The RealARTTM assay diverged from the Digene assay in a range of 2 orders of magnitude. On the other hand, the mean difference was −1.73 when Molecular Beacons was compared with the Digene assay and the limits of agreement were +0.33 and −3.79 respectively. Molecular Beacons differed from the Digene assay by 4 orders of magnitude.The RealARTTM Digene and Molecular Beacons assays revealed coefficients of variation of 5.33%, 6.03% and 6.47%, respectively. CONCLUSION: The RealARTTM assay has a better correlation with the Digene assay and can be used effectively to monitor patients with high serum HBV DNA levels. It is also effective in the monitoring of patients whose serum HBV DNA levels have been suppressed to undetectable level by other commercial assays after antiviral therapy.