Naturally occurring resistance-associated substitutions (RASs) can negatively impact the response to direct-acting antivirals (DAAs) agents-based therapies for hepatitis C virus (HCV) infection. Herein, we set out to characterize the RASs in the HCV1b genome from serum samples of DAA-naïve patients in the context of the SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology, 2014) project. We deep-sequenced the NS3/4A protease region of the viral population using the Ion Torrent Personal Genome Machine, and patient-specific majority rule consensus sequence summaries were constructed with a combination of freely available next generation sequencing data analysis software. We detected NS3/4A protease major and minor variants associated with resistance to boceprevir (V36L), telaprevir (V36L, I132V), simeprevir (V36L), and grazoprevir (V36L, V170I). Furthermore, we sequenced part of HCV NS5B polymerase using Sanger-sequencing and detected a natural RAS for dasabuvir (C316N). This mutation could be important for treatment strategies in cases of previous therapy failure.

Direct-acting antiviral drugs to cure infections with Hepatitis C virus (HCV) achieve a sustained virological response (SVR) in more than 90% of adult patients. At present, clinical trials are ongoing and real-life data are still limited in children. Herein, we report two cases of pediatric patients treated with fixed-dose combination of sofosbuvir/ledipasvir, already approved to treat HCV4 genotype. Both young girls achieved SVR even though HCV4 isolates carried L28M and M31L NS5A resistance-associated substitutions (RASs). Therefore, possible effects of these RASs merit further study, especially in children.