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Pyrosequencing method for sensitive detection of HBV drug resistance mutations.

Guan Huei LeeMasafumi InoueRoxanne Hui-Heng ChongJimmy TohSue Yuen WeeKah-Sin LohSeng-Gee Lim
Published in: Journal of medical virology (2018)
Hepatitis B (HBV) drug resistance assay is important for guiding therapy after the development of virologic breakthrough for patients receiving nucleoside/-tide analog therapy. However, the existing genotyping tools are either costly or lack sensitivity to detect mixed genotypes, and an improved method of resistant mutation detection is needed. An assay protocol for clinical application using pyrosequencing method was developed, capable of detecting all known validated HBV polymerase gene mutations that impart resistance to lamivudine, adefovir, tenofovir, and entecavir. Sixty-eight serum samples with known HBV resistance genotypes, previously tested with either Sanger sequencing assay or commercial line probe assay, were used for validation. Where there were discrepancies between the two methods, clonal sequencing by Sanger's method was used for confirmation. The modified pyrosequencing method accurately identified all the cloned polymerase genotypes and was able to distinguish as little as 5% of the mutant populations. This assay can be performed on serum sample with HBV DNA as low as 13.5 IU/mL. The cost per test was less than existing commercial assay. HBV drug resistance pyrosequencing assay was accurate, more sensitive and cheaper compared with the existing methods. It can detect minor populations of drug-resistant clones earlier, before the drug resistant clones become dominant, allowing the opportunity for an earlier change of therapy.
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