Prevalence of Single and Multiple Natural NS3, NS5A and NS5B Resistance-Associated Substitutions in Hepatitis C Virus Genotypes 1-4 in Italy

Abstract

Natural resistance-associated substitutions (RASs) are reported with highly variable prevalence across different HCV genotypes (GTs). Frequency of natural RASs in a large Italian real-life cohort of patients infected with the 4 main HCV-GTs was investigated. NS3, NS5A and NS5B sequences were analysed in 1445 HCV-infected DAA-naïve patients. Sanger-sequencing was performed by home-made protocols on 464 GT1a, 585 GT1b, 92 GT2c, 199 GT3a, 16 GT4a and 99 GT4d samples. Overall, 20.7% (301/1455) of patients showed natural RASs, and the prevalence of multiclass-resistance was 7.3% (29/372 patients analysed). NS3-RASs were particularly common in GT1a and GT1b (45.2-10.8%, respectively), mainly due to 80K presence in GT1a (17%). Almost all GTs showed high prevalence of NS5A-RASs (range: 10.2-45.4%), and especially of 93H (5.1%). NS5A-RASs with fold-change >100x were detected in 6.8% GT1a (30H/R-31M-93C/H), 10.3% GT1b (31V-93H), 28.4% GT2c (28C-31M-93H), 8.5% GT3a (30K-93H), 45.5% GT4a (28M-30R-93H) and 3.8% GT4d (28V-30S-93H). Sofosbuvir RAS 282T was never detected, while the 159F and 316N RASs were found in GT1b (13.4-19.1%, respectively). Natural RASs are common in Italian patients infected with HCV-GTs 1-4. High prevalence of clinically-relevant RASs (such as Y93H) supports the appropriateness of HCV resistance-test to properly guide DAA-based therapy.

Figure 1

Prevalence of natural NS3, NS5A and NS5B resistance-associated substitutions by HCV genotype and subtype. NS3 resistance-associated substitutions (RASs) in panel a were classified according to the in vitro fold-change reduction in protease inhibitors activity in the specific HCV genotype. Low-level RASs were defined by fold-changes between 2 and 100, while intermediate-level RASs were defined by fold-change between 100 and 1000. The overall prevalence also include RASs observed in vivo or proposed to be associated with resistance (no fold-change available). NS5A substitutions in panel b were divided both according to the in vitro fold-change reduction, and to their potential association with resistance in vivo. For 1st generation NS5A-inhibitors, RASs with fold-change 2.5–20× are reported as “likely susceptible”; RASs with fold-change 20–100 or only in vivo RAS (no fold-change available) are reported as “resistance possible”. Lastly, RASs with fold-change >100× are defined as “resistance likely”. For 2nd generation NS5A-inhibitors elbasvir, pibrentasvir and velpatasvir, RASs with fold-change <2.5× are reported as “likely susceptible”, RASs with fold-change 2.5–9 or only in vivo RAS (no fold-change available) are reported as “resistance possible”. Lastly, RASs with fold-change >10x are reported as “resistance likely”. Additional rule applied: 1 level up if found in virologic failure. NS5B substitutions in panel c were reported separately for dasabuvir or sofosbuvir. RAS, resistance-associated substitution.