Abstract
Affordable next generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting anti-viral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV viral sequence datasets and evaluated their utility for diagnostics and clinical assessment.NGS methods using (1) unselected HCV RNA (metagenomic); (2) pre-enrichment of HCV RNA by probe capture and (3) HCV pre-amplification by PCR implemented in four UK centres were compared. Metrics of sequence coverage and depth, quasispecies diversity and detection of DAA-resistance associated variants (RAVs), mixed HCV genotype and other co-infections were compared using a panel of samples of varying viral load, genotype and mixed HCV geno(sub)types. Each NGS method generated near complete genome sequences from over 90% of samples. Enrichment methods and PCR pre-amplification generated greater sequence depth and were more effective for low viral load samples. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus co-infections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing genotype and information on potential DAA resistance.
Highlights
Hepatitis C virus (HCV) chronically infects more than 150 million people globally and is associated with the development of liver fibrosis, cirrhosis, hepatic failure, and hepatocellular cancer [1]
Samples were used with informed consent conforming to the ethical guidelines of the 1975 Declaration of Helsinki, and study protocols were approved by the National Research Ethics Service (NRES) Committee East Midlands
Each method was effective at detecting hepatitis C virus (HCV) sequences in most or all panel samples with a wide range of viral loads, including those as low as 2,000 international units (IU)/ml
Summary
Hepatitis C virus (HCV) chronically infects more than 150 million people globally and is associated with the development of liver fibrosis, cirrhosis, hepatic failure, and hepatocellular cancer [1]. New all-oral directly acting antivirals (DAAs) with high efficacy rates and an improved safety profile have recently revolutionized the treatment of HCV. Oral therapies that target NS3, NS5A, and NS5B HCV proteins have been approved by the Food and Drug Administration and European Medicines Agency regulatory bodies [2, 3] and, used in combination, these DAAs achieve high sustained virological response (SVR) rates with minimal side effects [4]. The accurate assignment of viral genotype and subtype remains an important stratification parameter both in clinical trials of DAA therapy and in clinical practice. A minority of patients fail to achieve SVR with all-oral combination therapy, failure more commonly occurs in patients with advanced liver disease, and optimal retreatment strategies in all patients who fail DAA therapies are currently unclear.
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