Development of diagnostic assays for Hepatitis C Virus genotyping and identification of drug-resistance mutations

Hepatitis C Virus (HCV) is one of the most widespread viruses in the world. World Health Organisation (WHO) planned to eliminate the disease in 2030. In order to do so, many efforts have to be made, especially in the countries where HCV is still endemic. HCV is an extremely variable virus and it is divided into genetic groups called genotypes. In 2011 Direct Antiviral Agents (DAAs) have been introduced for the HCV therapy. In comparison with the previous therapy, these drugs have a higher Sustained Virological Response (SVR). However, they are genotypedependent, and the identification of the viral genotype was considered the priority for the choice of a correct therapeutic regimen. Since genotyping analysis was considered mandatory during the care pathway, many in vitro diagnostic assays were developed by different companies to fill this clinical need. Biofield Innovation decided to redesign an HCV genotyping assay based on Reverse Line Blot technology to improve the performance of it. In this study, development and validation process of the new assay are discussed. In 2016 pangenotypic antiviral drugs were commercialized. Their introduction makes the choice of the therapy regimen less and less influenced by the viral genotype. However, even with angenotypic drugs, a low percentage of therapeutic failure is still registered. One of the main reasons of the treatment failure is the presence of Resistance Associated Substitutions (RASs) that are point mutations that confer drug-resistance to the virus. To avoid the viral relapse and to prevent the therapeutic failure, the choose of the most suitable treatment for the therapy course must involve the consideration of the presence of RASs. Nowadays, the most powerfull technology that can be used to investigate the presence of RASs is Next Generation Sequencing (NGS). In this study the development of an in vitro diagnostic device for the identification of the genotype and the presence of RASs based on the NGS is described. In particular, prototype pre-validation and validation processes results of the prototype will be reported and analysed.