Clinical significance of periodic detection of hepatitis B virus YVDD mutation by ultrasensitive real-time amplification refractory mutation system quantitative PCR during lamivudine treatment in patients with chronic hepatitis B.

Abstract

Monitoring hepatitis B virus (HBV) mutants periodically during nucleoside analogue treatment is of great clinical significance, particularly in persistently HBV DNA-positive patients. However, few studies have investigated the dynamic changes of HBV YMDD (Tyr-Met-Asp-Asp) and YVDD (Tyr-Val-Asp-Asp) populations in chronic hepatitis B (CHB) patients whilst undergoing lamivudine (LMV) treatment. In this study, we sought to investigate the dynamic changes of HBV YMDD and YVDD variants by ultrasensitive real-time amplification refractory mutation system quantitative PCR (RT-ARMS-qPCR) and evaluate its significance for changes in the treatment of CHB patients. RT-ARMS-qPCR was established and evaluated with standard recombinant plasmids. Fifteen CHB patients receiving LMV (100 mg daily) were consecutively recruited and followed up for 60 weeks. Serum samples were obtained from each patient at baseline and every 12 weeks. The total HBV DNA, HBV YMDD DNA and YVDD DNA levels were measured using RT-ARMS-qPCR at all given time points after treatment. Routine liver biochemistry parameters, including aspartate aminotransferase and alanine aminotransferase, were also measured every 12 weeks. The linear range of the assay was between 1×10(12) and 1×10(5) copies ml(-1). The low detection limit was 1×10(4) copies ml(-1). After 60 weeks of LMV treatment, nine patients experienced virological breakthrough. The YVDD variant could be detected 12-48 weeks before virological breakthrough. The YVDD variant was detected as the predominant population (range 69.4-100 %) in patients by the time virological breakthrough appeared. We concluded that RT-ARMS-qPCR was sensitive for the detection and quantification of low levels of HBV mutation. Periodic detection of HBV YM(V)DD every 12 weeks during LMV treatment is helpful for therapeutic decision making.