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HCV Genotyping and Resistance-Associated Variants Detection Using Next-Generation Sequencing
Poster Title: HCV Genotyping and Resistance-Associated Variants Detection Using Next-Generation Sequencing
Submitted on 03 Nov 2016
Author(s): Zhang Rui1, Pramila Ariyaratne1, Kok Siong Poon2, Cui Wen Chua2, Mui Joo Khoo2, Evelyn S. Koay2, Ekawat Passomsub3, Wasun Chantratita3, Wen Huang1, Gerd Michel1, Elian Rakhmanaliev1
Affiliations: 1Vela Diagnostics Pte. Ltd., Singapore; 2Molecular Diagnosis Centre, Department of Laboratory Medicine, National University Hospital, Singapore; 3Department of Pathology, Faculty of Medicine, Ramathibodi Hospital Mahidol University, Bangkok, Thailand.
This poster was presented at 68th AACC Annual Scientific Meeting & Clinical Lab Expo, Philadelphia, PA, USA, 31 Jul. - 4 Aug. 2016
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Poster Information
Abstract: Both conventional interferon based regimens as well as the increasingly used direct acting antivirals (DAAs) provide current treatment options for HCV. Accurate genotyping remains a prerequisite for IFN based treatment. However, with regard to DAAs one of the most important considerations is the potential development of Resistance-Associated Variants (RAVs) that may negatively affect sustained virological response. Timely detection and reporting of RAVs and HCV genotypes (GTs) is critical for drug regiment and can minimize the development of resistance to antiviral drugs. In this study we investigated the frequency of RAVs across HCV GT1, which is highly prevalent across geographic regions. 346 prospective and retrospective EDTA-plasma and clinical serum samples from patents with chronic HCV infection across all 6 HCV GTs were tested in this study. We used a line probe-based test (VERSANT HCV Genotype 2.0 LiPA) in conjunction with the AutoBlot 3000H platform (SIEMENS) and a novel automated Next Generation Sequencing (NGS)-based integrated workflow, comprised of 1) a customized version of the epMotion 5075 (Eppendorf) robotic liquid handling system for RNA extraction and NGS library preparation (Sentosa SX101); 2) Ion Torrent technology for deep sequencing; 3) kits for nucleic acid extraction and NGS library preparation (Sentosa SQ HCV Genotyping Assay) and deep sequencing, respectively, and 4) data analysis and reporting software. The data reports on GTs 1a and 1b include 136 known RAVs in the NS3, NS5A and NS5B genes. Sanger sequencing was used as a reference method for all discordant and indeterminate samples. All 346 samples were tested on both platforms. For 47/346 (13.6%) samples GT results by VERSANT were “indeterminate”. In 19/299 (6.4%) of the samples, discordant results between the two methods were obtained. The ability to correctly determine HCV genotypes was 93.7% (95%CI: 90.3-95.9%) for VERSANT and 100% (95%CI: 98.7-100%) for Sentosa HCV Assay. Sanger sequencing confirmed that all 19 discordant samples were incorrectly classified by line probing. GT distribution among the 47 samples indeterminate by VERSANT was: 5 GT1a, 1 GT2, 19 GT3, 1 GT4, 20 GT6 and 1 mixed infection (GT2 and GT3). Clinical sensitivity aggregated was 86.4% (95%CI: 82.4-89.6%) for VERSANT and 100% (95%CI: 98.9-100%) for Sentosa HCV. 56 GT1a and 54 GT1b samples were used for further analysis of RAVs distribution among the GT1 population. 52.7%(58/110) of HCV strains were carrying 1 or multiple RAVs in 23 positions across all target genes. An unequal distribution of 4 mutations across the GT1 subtypes was observed. Frequency of the Q80K mutation (NS3) was 25%(14/56) in GT1a and 1.9%(1/56) in GT1b. While mutations Q54H and Y93H (NS5A) were prevalent in GT1b: 42.6%(23/56) and 18.5%(10/56) respectively. No Q54H mutation was present the GT1a population studies; Y93H in this group reached 1.8%(1/56). Mutation V499A in the NS5B gene was present in the GT1b population at 25.9%(14/54) and absent in the GT1a population. Simultaneous determination of HCV genotypes and detection of RAVs in single NGS runs provides comprehensive, clinically relevant information for optimal selection of HCV treatment regimens.Summary: HCV Genotyping and Resistance-Associated Variants Detection Using Next-Generation Sequencing.Report abuse »
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