Viral infections are among the commonest causes of morbidity and mortality worldwide. In particular, HIV, HBV, HCV, and HCMV are some of the commonest agents responsible for human diseases. Despite the remarkable progresses in anti-viral therapy achieved, the emergence of drug-resistance is still a major clinical issue. Therefore, the prompt identification of resistance allows a quick therapeutic switch with both clinical benefits and economical advantages. Since somatic mutations occurring in specific gene sequences have been recognized as the major contributor to drug resistance emergence, molecular virology and gene sequencing during patient follow up has become a standard of care for virus-infected patients during treatment. Nonetheless, classical (Sanger) sequencing displays a relatively low sensitivity. As a consequence, mutations with low frequency or slowly emerging under the selective pressure of treatment may be missed until a definite clinical relapse. In this project, we aim to develop new assays for mutation detection in viral sequences by next generation sequencing (NGS) with special regard to HIV-1, HIV-2, HCV, HBV, and HCMV. To address this issue, collaboration between the Microbiology of Verona University and the Genomic Lab at the Euro-Mediterranean Institute of Science and Technology has been established. PCR-based amplification of targeted viral sequencing will be performed with original primer design in a selected panel of samples. Library preparation for Thermofisher and Illumina platforms will be then performed. NGS will be performed to ensure high target sequence coverage (>99%) and remarkable deepness (1000x, with and expected mean value of 500-600x). This is expected to allow the detection of mutation occurring in >5% of alleles and, consequently, an earlier detection of resistance occurrence.
Clinical validation will be performed by phase 3 and 4 diagnostic accuracy studies, the latter being planned in an independent panel of samples.
