Multiclass HCV Resistance to Direct-acting Antiviral Failure in Real-life Patients Advocates for Tailored Second-line Therapies

Velia C. Di Maio; Valeria Cento; Ilaria Lenci; Marianna Aragri; Piera Rossi; Silvia Barbaliscia; Michela Melis; Gabriella Verucchi; Carlo F. Magni; Elisabetta Teti; Ada Bertoli; FrancescoPaolo Antonucci; Maria C. Bellocchi; Valeria Micheli; Chiara Masetti; Simona Landonio; Simona Francioso; Francesco Santopaolo; Adriano M. Pellicelli; Vincenza Calvaruso; Laura Gianserra; Massimo Siciliano; Dante Romagnoli; Raffaele Cozzolongo; Antonio Grieco; Jacopo Vecchiet; Filomena Morisco; Manuela Merli; Giuseppina Brancaccio; Antonio Di Biagio; Elisabetta Loggi; Claudio M. Mastroianni; Valeria Pace Palitti; Pierluigi Tarquini; Massimo Puoti; Gloria Taliani; Loredana Sarmati; Antonino Picciotto; Vincenzo Vullo; Nicola Caporaso; Maurizio Paoloni; Caterina Pasquazzi; Giuliano Rizzardini; Giustino Parruti; Antonio Craxì; Sergio Babudieri; Massimo Andreoni; Mario Angelico; Carlo F. Perno; Francesca Ceccherini-Silberstein


Liver International. 2017;37(4):514-528. 

In This Article

Material and Methods


One hundred and ninety-seven chronically HCV-infected patients who experienced a virological failure to a DAA-containing regimen, between 2011 and 2016, in different Italian clinical centres were included in the analysis. All of them had a resistance test at failure, performed at least on the target gene(s) of DAAs used (overall NS3/NS5A/NS5B sequences at failure were N=192/129/133 respectively). Furthermore, 70 patients had also a baseline NS3 and/or NS5A and/or NS5B resistance test. Approval by ethics committee was deemed unnecessary for all patients evaluated for diagnostic purpose according to Italian law (art. 6 and art. 9, leg. decree 211/2003). In the few cases evaluated for research purpose, approval by the local Ethics Committees of Fondazione PTV Policlinico Tor Vergata and patient written informed consent were obtained. This study was conducted on anonymous samples (leg. decree 196/2003) and in accordance with the principles of the Declaration of Helsinki. All information, including virological, clinical and therapy data, were recorded in an anonymous database.

Sanger HCV Sequencing

Hepatitis C virus-RNA was extracted with NucliSENS easyMAG system (bioMérieux, Boxtel, The Netherlands), according to the manufacturer's instructions. Then, synthesis and amplification of cDNA were performed in a single step using the commercial SuperScript III One-Step RT-PCR System with Platinum (Invitrogen Corp, Carlsbad, CA, USA) and specific primers for each HCV genotype/subtype (Table S1). If necessary, Nested PCR was also performed with specific HCV genotype/subtype primers (Table S1). Finally, NS3-protease, NS5A- and NS5B-amplified products were purified and sequenced by an automated sequencer (ABI-3130XL) in sense and antisense orientation using the BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems, Foster City, CA, USA). The sensitivity for detection of RASs using Sanger sequencing is approximately 10%-20%.[18]

HCV Resistance Mutation Analysis

Prevalence of drug class-specific RASs was evaluated at baseline (when available) and at failure.

The list of RASs analysed, known to confer reduced susceptibility (>2-fold increased 50% effective drug concentrations [EC50]) of different HCV genotypes/subtypes to currently available HCV DAAs,[1–3] is the following:

  • NS3-RASs (asunaprevir [ASU]; boceprevir [BOC]; paritaprevir [PTV]); simeprevir [SMV]; telaprevir [TVR]: V36A/G/M, F43I/L/S, T54S, V55A, Y56H, Q80K/R, S122R, R155G/K/Q/T/W, A156S/G/T/V, any168A/C/E/G/H/I/N/T/V/Y, any170A/T;

  • NS5A-RASs (daclatasvir [DCV]; ledipasvir [LDV]; ombitasvir [OMV]): any24G/N/R, any28A/G/M/S/T/V, any30D/E/G/H/L/Q/K/R/S/T, any31I/F/M/V, any32L/F, S38F, any58D/S, any92K/R/T, Y93C/F/H/N/R/S/T/W;

  • NS5B Nucleotide (NI)-RASs (sofosbuvir [SOF]): L159F*, S282T, C316H/N*, L320F*, V321A*;

  • NS5B Non-nucleotide (NNI)-RASs (dasabuvir [DSV]): C316H/N/Y, S368T, M414I/T, E446K/Q, Y448C/H, A553T/V, G554S*, S556G/N/R, G558R*, D559G/N*, Y561H.

*These mutations have been reported as treatment-emergent variants at failure, but exhibited no EC50 increase in vitro >2 or EC50 was not determined in vitro.

Statistical Analysis

Results are expressed as median values and interquartile range (IQR) for continuous data and number (percentage) for categorical data. Categorical variables were compared using the Chi-squared test and Fisher's exact test when was appropriate. All analyses were performed using the SPSS software package (version 19.0) for Windows (SPSS Inc., Chicago, IL).