In:
Viruses, MDPI AG, Vol. 13, No. 9 ( 2021-08-30), p. 1721-
Kurzfassung:
Despite the effectiveness of direct-acting antiviral agents in treating hepatitis C virus (HCV), cases of treatment failure have been associated with the emergence of resistance-associated substitutions. To better guide clinical decision-making, we developed and validated a near-whole-genome HCV genotype-independent next-generation sequencing strategy. HCV genotype 1–6 samples from direct-acting antiviral agent treatment-naïve and -treated HCV-infected individuals were included. Viral RNA was extracted using a NucliSens easyMAG and amplified using nested reverse transcription-polymerase chain reaction. Libraries were prepared using Nextera XT and sequenced on the Illumina MiSeq sequencing platform. Data were processed by an in-house pipeline (MiCall). Nucleotide consensus sequences were aligned to reference strain sequences for resistance-associated substitution identification and compared to NS3, NS5a, and NS5b sequence data obtained from a validated in-house assay optimized for HCV genotype 1. Sequencing success rates (defined as achieving 〉 100-fold read coverage) approaching 90% were observed for most genotypes in samples with a viral load 〉 5 log10 IU/mL. This genotype-independent sequencing method resulted in 〉 99.8% nucleotide concordance with the genotype 1-optimized method, and 100% agreement in genotype assignment with paired line probe assay-based genotypes. The assay demonstrated high intra-run repeatability and inter-run reproducibility at detecting substitutions above 2% prevalence. This study highlights the performance of a freely available laboratory and bioinformatic approach for reliable HCV genotyping and resistance-associated substitution detection regardless of genotype.
Materialart:
Online-Ressource
ISSN:
1999-4915
Sprache:
Englisch
Verlag:
MDPI AG
Publikationsdatum:
2021
ZDB Id:
2516098-9
