Rapid quantitation of lamivudine-resistant mutants in lamivudine treated and untreated patients with chronic hepatitis B virus infection.

摘要

BACKGROUND: Long-term lamivudine treatment induces emergence of lamivudine-resistant hepatitis B virus (HBV) in a significant number of patients with chronic HBV infection. Rapid and quantitative methods to determine the percentage of lamivudine-resistant mutants in total HBV are important during lamivudine therapy. METHODS: We established a quantitative real-time PCR method with selective primers and TaqMan probe to detect the percentage of lamivudine-resistant mutants in total HBV without the need of external DNA standards. This percentage was calculated as the PCR efficiency raised to the differences between threshold cycle number (DeltaCt) of mutant and control reactions. Clones of the HBV polymerase gene containing the different YMDD variants were diluted in series and tested. Serum samples from 145 lamivudine-treated and 98 untreated patients with chronic hepatitis B virus infection were analyzed using this method and compared with DNA sequencing. RESULTS: As little as 0.1% mutant plasmids in 10(6)-10(9) copies/ml of wild-type plasmids were detected. Among the 145 patients treated with lamivudine, 42 of them had mutants with percentages of 5-100%. In six discordant results between real-time PCR and DNA sequencing, real-time PCR detected mutants with percentages of 5-20%, which were concordant with subclone sequencing. Five of 98 lamividine-untreated patients had mutants of 10-20% in wild-type virus populations. Compared to DNA sequencing, real-time PCR was fast and cost-effective. CONCLUSION: This real-time PCR is a rapid, sensitive and cost-effective method for relative quantitation of YMDD mutants of HBV.