Development and Validation of Two Screening Assays for the Hepatitis C Virus NS3 Q80K Polymorphism Associated with Reduced Response to Combination Treatment Regimens Containing Simeprevir

摘要: Persons with hepatitis C virus (HCV) genotype 1a (GT1a) infections harboring a baseline Q80K polymorphism in nonstructural protein 3 (NS3) have reduced virologic response to simeprevir combination pegylated interferon-alfa and ribavirin. We aimed develop, validate, freely disseminate an NS3 clinical sequencing assay detect the potentially other HCV drug resistance mutations. RNA was extracted from frozen plasma using NucliSENS easyMAG automated nucleic acid extractor, amplified by nested reverse transcription-PCR, sequenced Sanger and/or next-generation (MiSeq) methods. chromatograms were analyzed in-house software (RECall), nucleotide mixtures called automatically. MiSeq reads iteratively mapped H77 reference genome, consensus sequences generated nucleotides present at >20% as mixtures. The accuracy, precision, sensitivity for detecting assessed 70 samples previously external laboratory. A comparison of methods those determined lab revealed >98.5% sequence concordance zero discordant calls polymorphism. results both highly repeatable reproducible (>99.7% 100% concordance). limits detection (>2 ∼5 log10 IU/ml assays, respectively) are sufficiently low allow genotyping nearly all chronically infected treatment-naive persons. No systematic bias under- or overamplification minority variants observed. Coinfection viruses (e.g., HIV B [HBV]) did not affect results. two independent assays analysis procedures described here useful tools screen protease inhibitor