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

Abstract

Persons with hepatitis C virus (HCV) genotype 1a (GT1a) infections harboring a baseline Q80K polymorphism in nonstructural protein 3 (NS3) have a reduced virologic response to simeprevir in combination with pegylated interferon-alfa and ribavirin. We aimed to develop, validate, and freely disseminate an NS3 clinical sequencing assay to detect the Q80K polymorphism and potentially other HCV NS3 drug resistance mutations. HCV RNA was extracted from frozen plasma using a NucliSENS easyMAG automated nucleic acid extractor, amplified by nested reverse transcription-PCR, and sequenced using 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 genome, and consensus NS3 sequences were generated with nucleotides present at >20% called as mixtures. The accuracy, precision, and sensitivity for detecting the Q80K polymorphism were assessed in 70 samples previously sequenced by an external laboratory. A comparison of the sequences generated by the Sanger and MiSeq methods with those determined by an external lab revealed >98.5% nucleotide sequence concordance and zero discordant calls of the Q80K polymorphism. The results were both highly repeatable and reproducible (>99.7% nucleotide concordance and 100% Q80K concordance). The limits of detection (>2 and ϳ5 log 10 IU/ml for the Sanger and MiSeq assays, respectively) are sufficiently low to allow genotyping in nearly all chronically infected treatment-naive persons. No systematic bias in the under-or overamplification of minority variants was observed. Coinfection with other viruses (e.g., HIV and hepatitis B virus [HBV]) did not affect the assay results. The two independent HCV NS3 sequencing assays with the automated analysis procedures described here are useful tools to screen for the Q80K polymorphism and other HCV protease inhibitor drug resistance mutations. Until recently, the standard of care for treating hepatitis C virus (HCV) infection has been combination antiviral therapy with pegylated interferon-alfa and ribavirin (Peg-IFN/RBV). In 2011, the nonstructural protein 3 (NS3) protease inhibitors (PI) telaprevir and boceprevir, in combination with Peg-IFN/RBV, were the first direct-acting antiviral (DAA) agents approved for treatment of chronic HCV genotype 1 infection (1-3). The success of HCV therapy with DAA, however, is complicated by the incredible genetic diversity of the virus and its capacity to mutate in response to drug selection pressure (4, 5). Treatment failure is often accompanied by the emergence of resistance mutations in the genes targeted by these drugs (6, 7). Furthermore, certain drug resistance mutations exist as naturally occurring polymorphisms in a small proportion of treatment-naive patients and can compromise PI treatment in these individuals (8)(9)(10)(11).In combination with Peg-IFN/RBV, the second-generation PI simeprevir was approved in Canada in 2013 for the treatment...

Performance comparison of next generation sequencing analysis pipelines for HIV-1 drug resistance testing

“…This additional information strengthens our ability to assess the clinical impact of a given DRM and to determine and track its overall frequency within a population, which may significantly impact drug regimens and public health approaches to control and reduce HIV transmission 10,14,[49][50][51][52] . Notably, while many NGS HIVDR data analysis pipelines exist 25,[31][32][33][34][35][36][37][38][39] , their design and implementation was conducted independently by different research groups with little coordination among the developers. Given the complexity of the analysis and the varied approaches adopted by the different development teams, this historical lack of coordination results in uncertainties in the reliability of the data.…”

“…The standardization of NGS-based HIVDR assays is more complex and it includes three main steps: (1) wet-lab steps to generate PCR amplicons that cover the pol region and prepare libraries; (2) NGS platforms; and (3) bioinformatics pipelines which convert NGS data into user-interpretable HIVDR results 13,15,30 . Several bioinformatical pipelines have been independently developed to address the needs for automated NGS-based HIVDR genotyping 25,[31][32][33][34][35][36][37][38][39] . We recently published guidelines on the standards for bioinformatics analysis and reporting conventions for HIVDR research and clinical purposes in the "Winnipeg Consensus".…”

Hepatitis C Virus Core Antigen: A Simplified Treatment Monitoring Tool, including for Post-Treatment Relapse

“…the "grey-zone") were retested per manufacturer recommendations where sample was available. For participants with quantifiable HCV RNA, and yet consistently negative HCVcAg results, the HCV Core gene was amplified and sequenced using previously published methods where sample was available [15,17] to identify potential mutations in the antibody binding region of the ARCHITECT HCV Ag assay [18][19][20].…”

HCV Genotyping with Concurrent Profiling of Resistance-Associated Variants by NGS Analysis

“…The Vela NGS offers information on RAVs in HCV 1a or 1b positive samples, where such profiling will be useful when prescribing DAA regimes, and detecting of baseline or emerging RAVs. Targeted assays had been previously developed to identify a specific RAV [27,28]. RAVs which are found at levels with at least 15% variant frequency, at baseline, are known to confer resistance to certain DAAs [29], and therefore may impact on the effectiveness of DAA treatment [30].…”