Development and validation of two screening assays for the HCV NS3 Q80K polymorphism associated with reduced response to combination treatment regimens containing simeprevir

2015 | J Clin Microbiol

Program(s): Laboratory |

Abstract:

BACKGROUND:

Persons with Hepatitis C Virus (HCV) genotype 1a (GT1a) infections harbouring a baseline Q80K polymorphism in NS3 have a reduced virologic response to simeprevir in combination with pegylated interferon-alfa and ribavirin. We aimed to develop, validate, and freely disseminate a NS3 clinical sequencing assay to detect Q80K and potentially other HCV NS3 drug resistance mutations.

METHODS:

HCV RNA was extracted from frozen plasma using a NucliSENS easyMAG, amplified by nested RT-PCR and sequenced by Sanger and/or next-generation (MiSeq) methods. Sanger chromatograms were analyzed using in-house software (RECall) and nucleotide mixtures were called automatically. MiSeq reads were iteratively mapped to the H77 reference and consensus NS3 sequences were generated with nucleotides present at >20% called as mixtures. The accuracy, precision and sensitivity in detecting Q80K were assessed in 70 samples previously sequenced by an external laboratory.

RESULTS:

Comparison of sequences generated by the Sanger and MiSeq methods to those determined by an external lab revealed >98.5% nucleotide sequence concordance and zero discordant calls of Q80K. Results were both highly repeatable and reproducible (>99.7% nucleotide concordance; 100% Q80K concordance). The limits of detection (>2 and ∼5 log₁₀ IU/mL for the Sanger and MiSeq assays, respectively) are sufficiently low to allow genotyping in nearly all chronically-infected, treatment-naïve persons. No systematic bias in the under- or over-amplification of minority variants was observed. Co-infection with other viruses (e.g. HIV, HBV) did not affect assay results.

CONCLUSION:

The two independent HCV NS3 sequencing assays with automated analysis procedures described here are useful tools to screen for the Q80K polymorphism and other HCV protease inhibitor drug resistance mutations.

Text or Node

Chui CK

BC Centre for Excellence in HIV/AIDS

Search form

Development and validation of two screening assays for the HCV NS3 Q80K polymorphism associated with reduced response to combination treatment regimens containing simeprevir

Search form