Clinical Application of Pyrosequencing in Detection of YMDD Mutations

Abstract

It has been estimated that more than 300 million individuals worldwide are chronically infected with HBV and chronic hepatitis B (CHB). It is important to conduct antiviral therapy against CHB to minimize the amount of liver damage. Lamivudine has been shown to be effective in supressing virus replication; unfortunately, drug-resistant viral stains may everge after prolonged treatment. It is possible to detect the emergence of lamivudine-resistant HBV mutants by direct sequencing of HBV DNA; however, this is time consuming and laborious. Other molecular genetic techniques are available, but these, too, are time consuming. Pyrosequencing has been reported to be a potential tool to resolve the above problems in detection of YMDD mutaions. Serum samples were collected from 271 patients with chronic HBV infection. DNA was extracted from the serum samples and serum samples were prepared for pyrosequencing by sample extraction and PCR. Fifty samples were subjected to PCR and pyrosequencing and the sensitivity and specificity were calculated. The sensitivity of PCR was investigated, and the results indicated it should be possible to perform resistance tests with samples containing as little as 1,000 HBV DNA copies/mL. Detection by pyrosequencing had a 95% concordance in the samples categorized as wild type (WT), 91% for predominately mutant, and 85% in equal mixtures. The authors find that pyrosequencing for the identification of lamivudine-resistance in samples from HBV-infected patients is a rapid, high-throughput method and can finely detect minor sequence varients. However, one present limitation to pyrosequencing is that relatively few laboratories have access to pyrosequencing equipment.