ABSTRACT
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.
Subject(s)
Drug Resistance, Viral , Genotyping Techniques/methods , Hepatitis B virus/enzymology , Hepatitis B virus/genetics , High-Throughput Nucleotide Sequencing/methods , Mutation , RNA-Directed DNA Polymerase/genetics , Adult , Costs and Cost Analysis , Female , Hepatitis B/virology , Hepatitis B virus/isolation & purification , Humans , Male , Microbial Sensitivity Tests/methods , Middle Aged , Sensitivity and SpecificityABSTRACT
AIM: Few cases of primary entecavir resistance in chronic hepatitis B patients have been reported to date. The serial profiling of the HBV polymerase gene mutations from a treatment-naive patient who developed drug resistance after 32 months of entecavir therapy is presented here. DESIGN: Serum samples were collected at multiple time points from before the start of therapy to virological and biochemical breakthrough. The evolution of the hepatitis B virus polymerase gene mutations was analysed with commercial line probe assay and pyrosequencing. RESULTS: Drug resistance mutation analysis by pyrosequencing revealed a two-step process in the selection of drug resistance. The patient had a good initial response to entecavir 0.5 mg/day. A partially resistant HBV strain first emerged as the predominant species from as early as 2 weeks. After a period of non-compliance to therapy, there was virological breakthrough, which resolved on restarting entecavir. Shortly after, there was secondary failure of entecavir therapy, caused by a new resistant strain carrying all three mutations required. CONCLUSION: In this patient, pre-existence of minor population of partially resistant viral strains and treatment non-compliance probably contributed to his development of primary entecavir resistance.
