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Next Generation Sequencing (NGS) for HCV Genotyping and optional Identification of Resistance-Associated Variants
Poster Title: Next Generation Sequencing (NGS) for HCV Genotyping and optional Identification of Resistance-Associated Variants
Submitted on 26 Oct 2016
Author(s): Elian Rakhmanaliev1, Zhang Rui1, Wen Huang1, Kok Siong Poon2, Cui Wen Chua2, Mui Joo Khoo2, Evelyn S. Koay2, Ekawat Passomsub3, Wasun Chantratita3, Gerd Michel1
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 The International Liver Congress (EASL), Barcelona, Spain, Apr, 2016
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Poster Information
Abstract: Despite the advent of a number of direct acting antiviral drugs interferon treatment is still a valid therapy option, requiring HCV genotype (GT) determination before initiation of therapy. Moreover, preliminary data from several studies indicate that genotype testing, in particular GT3, might be useful for decision making for some novel drug regimens. Therefore accurate detection of HCV GTs is critical and can have significant impact on outcome of therapy. The objective of this study was to compare a widely used line probe-based test (VERSANT HCV Genotype 2.0 LiPA) and a newly developed automated NGS-based integrated workflow, comprised of an upfront robotic liquid handling system and kits for RNA extraction and library preparation (Sentosa SQ HCV Genotyping Assay), instruments and kits for template preparation and Ion Torrent based deep sequencing, as well as bioinformatics analysis and reporting software. The latter gives full data reports on GTs 1a and 1b including known Resistance Associated Variants (RAVs). A total of 136 RAVs are included in the analysis across GTs and can be retrieved by the user. However, the system does not make direct treatment recommendations, which are left to the respective investigator. This study included a cohort of 346 patients with chronic HCV, eligible for therapy. Archived serum and plasma samples across all 6 HCV GTs 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. All discordant and indeterminate samples were subjected to Sanger sequencing. 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 SQ HCV Genotyping Assay. Among the 19 discordant samples, 10 GT6 were incorrectly classified as GT1b by line probing, 6 GT3 as GT4, 2 GT3 as GT6, and 1 GT1c as GT1a. GT distribution among the 47 samples indeterminate by VERSANT was: 5 GT1a, 1 GT2, 19 GT3, 1 GT4, 20 GT6 and 1 mixed infection (GTs 2 and 3). Clinical sensitivity aggregated was 86.4% (95%CI: 82.4–89.6%) for VERSANT and 100% (95%CI: 98.9–100%) for Sentosa SQ HCV Genotyping Assay. Our data indicate that NGS in combination with thorough bioinformatics data analysis is highly accurate for determination of HCV genotypes and identification of RAVs, both representing essential (GTs) and potentially important (RAVs) information for HCV treatment approaches.Summary: Next Generation Sequencing (NGS) for HCV Genotyping and optional Identification of Resistance-Associated Variants.Report abuse »
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