Source Universidad Nacional Autónoma de México, Mexico.

The use of telaprevir and boceprevir, both protease inhibitors (PI), as part of the specifically targeted antiviral therapy for hepatitis C (STAT-C) has significantly improved sustained virologic response (SVR) rates. However, different clinical studies have also identified several mutations associated with viral resistance to both PIs. Under no selective pressure, HCV resistant mutants are generally present at low frequency, making mutation detection challenging. Here, we describe a mismatch amplification mutation assay (MAMA) PCR method for the specific detection of naturally occurring HCV resistant mutants. MAMA PCR successfully identified the corresponding HCV variants while conventional methods such as direct sequencing, end-point limiting-dilution (EPLD) and bacterial cloning were not sensitive enough to detect circulating resistant mutants in clinical specimens. Ultra-deep pyrosequencing was used to confirm the presence of the corresponding HCV mutants. In treatment naïve patients, frequency of all resistant variants was below 1%. Deep amplicon sequencing allowed a detailed analysis of the structure of the viral population among these patients showing that the evolution of the NS3 is limited to a rather small sequence space. Monitoring of HCV resistance before and during treatment is likely to provide important information for management of patients undergoing anti-HCV therapy.