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First CE-IVD Marked Next-Generation Sequencing (NGS)-based Automated Workflow for HCV Genotyping and Optional RAVs Detection.
Poster Title: First CE-IVD Marked Next-Generation Sequencing (NGS)-based Automated Workflow for HCV Genotyping and Optional RAVs Detection.
Submitted on 22 May 2017
Author(s): Rakhmanaliev Elian, Rui Zhang, Yeo Alex, Ariyaratne Pramila, Huang Wen, Lee Charlie
Affiliations: Vela Research Singapore Pte Ltd., Singapore
This poster was presented at 26th Conference of the Asian Pacific Association for the Study of the Liver (APASL), Shanghai, China, 15-19 Feb, 2017
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Poster Information
Abstract: Detection of resistance-associated mutations (RAVs) is increasingly used in HCV patients selected for treatment with direct acting antiviral agents (DAAs). Accurate determination of HCV genotypes (GTs) is essential for DAA treatment and conventional interferon-based therapy. Sanger sequencing has been the standard method in clinical diagnostics for several decades but it has recognized limitations in sensitivity and turn around time (TAT). NGS provides excellent accuracy, speed and sensitivity enabling detection of rare mutants, HCV subtypes as well as mixed infections. The objective was to develop and evaluate an automated NGS-based workflow for HCV genotyping for in vitro diagnostics.
The novel Sentosa® HCV genotyping workflow comprised of 1) a robotic liquid handling system for RNA extraction and NGS library preparation (Sentosa® SX101); 2) Ion Torrent NGS platform; 3) kits for RNA extraction, HCV NGS library preparation (Sentosa® SQ HCV Genotyping Assay) and deep sequencing, 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. However, the system does not make direct treatment recommendations, which are left to the investigator. Clinical sensitivity and clinical genotyping correctness was evaluated on 346 clinical samples across all 6 HCV GTs.
The Sentosa® HCV NGS workflow is highly automated and required <3 hrs. hands-on time with total TAT about 26 hrs. The assay is able to process up to 15 clinical samples simultaneously. The limit of detection was determined to be 1000 IU/mL for GTs 1-4, and 2000 IU/mL for GTs 5 and 6; reproducibility was 98.9%. For clinical evaluation we used a line probe-based test (VERSANT HCV Genotype 2.0 LiPA) in conjunction with the AutoBlot 3000H platform (SIEMENS). Sanger sequencing was used as a reference method for all discordant and indeterminate samples. Clinical sensitivity and genotyping correctness aggregated were 86.4% (95%CI: 82.4-89.6%) and 93.7% (95%CI: 90.3-95.9%) for VERSANT and 100% (95%CI: 98.9-100%) and 100% (95%CI: 98.7-100%) for Sentosa HCV, respectively. Also, 56 GT1a and 54 GT1b samples were used for further analysis of RAVs distribution. 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 in 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.
Given the crucial role of accurate sequencing analysis in HCV treatment management, the Sentosa® HCV NGS workflow appears as a highly reliable tool for differentiating HCV GTs and RAVs, which can help to prevent diagnostic errors potentially leading to suboptimal treatment.
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