It has recently been estimated that hepatitis C virus (HCV) affects between 64 and 103 million people worldwide, making it a major public health concern. With the introduction of direct-acting antiviral agents (DAA) and the use of DAA combinations, there have been better outcomes for this infected group yielding a success rate of approximately 90%.
NS5A inhibitors are one of four classes of DAAs approved by the US Food and Drug Administration (FDA) and the European Medecines Agency (EMA) to treat HCV infection.
French researchers noted that, "The combination of the high level of HCV replication and the lack of proofreading by the RNA-dependent RNA polymerase leads to a high degree of genetic variability resulting in different genotypes at the population scale and in the constitution of distinct viral quasi-species in each infected individual. Consequently, in this new context of DAA treatment, genetic variability must be taken into account."
In addition, they noted that the specific DAA eligibility, resistance prevalence, and efficacy depend on the genotypes and subtypes of the HCV.
"To improve the monitoring of HCV infection and determine the baseline, emergence, and persistence of Resistant Variants (RVs), the development of HCV gene target sequencing is required in clinical virology laboratories," they pointed out. "In our team, we already have a pangenotypic sequencing technique for NS3 and NS5B genes, and therefore we wanted to develop a similar technique for the NS5A gene."
The objective of the current study was to develop a method for HCV genome sequencing that allowed for simultaneous genotyping and NS5A inhibitor resistance profiling. They developed a tool (Geno2pheno [hcv] v0.92 tool) that allows them to determine the HCV genotypes, subtypes, and identification of NS5A mutations, according to a study published in the journal PLOS One.
"This single method can be used to detect pre-existing resistance mutations in NS5A before treatment and to check the emergence of resistant viruses while undergoing treatment in major HCV genotypes (G1-5) in the EU and the US," explained the researchers, led by Elisabeth Andre-Garnier of the Service de Virologie, CHU Nantes, and the Centre de Recherche en Transplantation et Immunologie UMR1064, INSERM, Universite´ de Nantes in Nantes, France.
In the study, investigators attempted to validate the use of a unique RT-PCR for HCV genotypes 1 to 5 by analyzing 142 plasma samples from patients infected with HCV. The NS4B-NS5A partial region was successfully amplified and sequenced in all samples. At the same time, partial NS3 sequences were analyzed and obtained for genotyping.
The phylogenetic analysis demonstrated concordance of genotypes and subtypes with a bootstrap >95% for each type cluster. NS5A resistance mutations were analyzed using the Geno2pheno [hcv] v0.92 tool and were compared with the recently published list of known Resistant Associated Substitutions (RAS).
The investigators noted that with the protocol developed in this study, virologists and hepatologists have a solution to homogenize the response of RVs.
"Our method will allow the investigation of pre-existing NS5A RASs, which will help to prospectively evaluate their effects," they stated. "To our knowledge, this is the first method using only one reaction mixture to amplify all main genotypes and subtypes HCV for NS5A. This transposable technique will allow a standardization of NS5A genotyping that could be used to develop a fully automated method for HCV genotype/resistance typing."