**4. Discussion**

In order to explore the spread of HCV1b in the Calabria Region, we analyzed NS5B population sequences, obtained from two cohorts of positive individuals, enrolled in different time spans, using phylogenetic analysis. In addition, viral isolates collected between 2015 and 2016 from naïve and IFN/pegIFNα/RBV treated patients were analyzed in the NS5B and NS5A regions to assess the presence of RASs with the potential to impact on DAA therapy.

Molecular analysis was carried out on 53 sequences of HCV subtype 1b, previously characterized by Inno-Lipa and confirmed by sequencing analysis. As reported in previous studies, subtype 1b, together with subtype 2c, are the most prevalent genotypes in Italy followed by genotypes 3 and 4 [3,6]. HCV1b diffusion worldwide is related to several risk factors, such as blood transfusions, dental treatment, unsafe reuse of nondisposable syringes [27,28]. In previous studies, transmission of two subtypes was already correlated to specific risk factors in the Calabria Region. HCV4d was found related to intravenous drug use and blood transfusion, while HCV2c infection was maintained by unsafe use of glass syringes followed by surgery and unsafe blood transfusion [29,30].

In this work, we investigated possible transmission patterns in a regionally representative sample from a small village (Sersale), where a seminal HCV prevalence study was conducted, and a metropolitan area of the Calabria region [13]. The ML phylogenetic tree shows that the HCV1b Calabria sequences were distributed in 16 statistically supported clusters. Twelve clusters (75%), contained Italian sequences mixed with foreign HCV1b references while four statistically significant monophyletic clusters included only sequences from Calabria (clusters: 1, 3, 4, 12). In particular, cluster 4 contained only seven closely related Italian sequences collected from Sersale village.

In this study, the majority (58.5%) of the enrolled individuals reported multiple risk factors, most of which were surgical intervention and dental treatment (*n* = 8) or surgical intervention and glass syringes (*n* = 7). Individually, we observed that the most frequent risk factors were dental treatment (16.9%) and surgical intervention (13.2%). Interestingly, the risk factors for HCV acquisition in cluster 4 were medical interventions and multiple use of glass syringes in a family setting as reported in 71% (no. 5/7) of patients (ISS 695, 1003, 1805, 1821, 1836).

Our analysis indicates that in the past, subtype 1b was maintained, by sporadic infections, mainly acquired through unsafe use of glass syringes especially in some limited areas of southern Italy, such as Sersale, a small town located 30 miles from Catanzaro. Conversely, in the metropolitan area, other transmission routes, such as dental treatment and surgical intervention had a significant influence on the dissemination of HCV subtype 1b throughout the Calabria Region. Interestingly, a community-based survey in the Calabria Region, revealed a high percentage of possible risk factors for HCV acquisition, such as dental treatment (69.5%) and glass syringes injections (25.8%) [31].

On the other hand, DAA treatment of hepatitis C could be influenced by baseline RASs naturally occurring in the viral genome [25,32]. It has been reported that 3% of HCV positive patients have no virological response, due to the presence of comorbidities and/or RASs in viral isolates, especially in the NS5A viral region [33]. We detected NS5B L159F alone in 15/41 (36.5%) and in association with C316N in 8/41 (19.5%) patients, respectively. This last substitution, showing a global frequency of 31.4% in HCV1b, is now defined as a

fitness-associated substitution when combined with the L159F [34]. Therefore, both amino acid variants were associated with a lower response to SOF [35]. Interestingly, NS5B S282T conferring high-level resistance to SOF-containing regimens, was not detected among our isolates, despite being present in 99.1% of worldwide strains [25]. In three patients, NS5A sequences carried the Y93H substitution, currently the major clinically relevant RAS contributing to failure of many approved IFN-free regimens [36]. Additionally, all three isolates showed the Y93H + K108R profile, which is associated with a minor affinity to OMV drug with respect to the Y93H + R108K combination as previously reported [37]. However, the 97% of treated patients with DAAs achieved sustained virological response (SVR). According to our experience about a single-center cohort in Southern Italy, the SVR rate was 97% for the older age group, 96% for people under 65 years old, finally 94% and 100% for cirrhotic and non-cirrhotic patients, respectively [38].
