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

Abstract and Introduction


Background & Aims: Despite the excellent efficacy of direct-acting antivirals (DAA) reported in clinical trials, virological failures can occur, often associated with the development of resistance-associated substitutions (RASs). This study aimed to characterize the presence of clinically relevant RASs to all classes in real-life DAA failures.

Methods: Of the 200 virological failures that were analyzed in 197 DAA-treated patients, 89 with pegylated-interferon+ribavirin (PegIFN+RBV) and 111 without (HCV-1a/1b/1g/2/3/4=58/83/1/6/24/25; 56.8% treatment experienced; 65.5% cirrhotic) were observed. Sanger sequencing of NS3/NS5A/NS5B was performed by home-made protocols, at failure (N=200) and whenever possible at baseline (N=70).

Results: The majority of the virological failures were relapsers (57.0%), 22.5% breakthroughs, 20.5% non-responders. RAS prevalence varied according to IFN/RBV use, DAA class, failure type and HCV genotype/subtype. It was 73.0% in IFN group vs 49.5% in IFN free, with the highest prevalence of NS5A-RASs (96.1%), compared to NS3-RASs (75.9% with IFN, 70.5% without) and NS5B-RASs (66.6% with IFN, 20.4% without, in sofosbuvir failures). In the IFN-free group, RASs were higher in breakthrough/non-responders than in relapsers (90.5% vs 40.0%, P<.001). Interestingly, 57.1% of DAA IFN-free non-responders had a misclassified genotype, and 3/4 sofosbuvir breakthroughs showed the major-RAS-S282T, while RAS-L159F was frequently found in sofosbuvir relapsers (18.2%). Notably, 9.0% of patients showed also extra target RASs, and 47.4% of patients treated with ≥2 DAA classes showed multiclass resistance, including 11/11 NS3+NS5A failures. Furthermore, 20.0% of patients had baseline-RASs, which were always confirmed at failure.

Conclusions: In our failure setting, RAS prevalence was remarkably high in all genes, with a partial exception for NS5B, whose limited resistance is still higher than previously reported. This multiclass resistance advocates for HCV resistance testing at failure, in all three genes for the best second-line therapeutic tailoring.


Remarkable changes in hepatitis C virus (HCV) treatment were observed in the latest years with the introduction of direct-acting antivirals (DAAs). In 2014/2015, several new DAAs have been approved and new interferon (IFN)-free regimen combinations are currently available in many countries.

Interferon-free treatment strategies are based generally on the combination of two or more drugs of different HCV classes: NS3, NS5A, NS5B non-nucleotide (NI) or non-nucleoside (NNI), with or without ribavirin (RBV).[1,2]

Despite the excellent efficacy of DAA-containing regimens reported in clinical trials (sustained virologic response, SVR >90%), virological failures can occur, often associated with the development of resistance-associated substitutions (RASs) related to the drugs used in the therapeutic regimen.[3–8] Furthermore, as a result of the highly variable nature of HCV and the potential presence of natural resistance,[9–11] RASs can be present at failure also in an extra-target out of the DAAs used, as natural occurring variants.

The presence of drug resistance could represent an important issue for retreatment options, especially when RASs are present in NS5A and/or in multiple DAA targets. Indeed, NS5A resistance-associated variants (RAVs) are very fit and tend to persist longer compared to NS3 and NS5B ones.[12–15] Furthermore, the presence of specific NS5A RAVs before a first- and, even more, a second-line NS5A-including regimen can significantly affect the achievement of a SVR in cirrhotic patients.[16–19] At present, however, few real-life data are available on DAA failures, and an exhaustive description of clinical characteristics of failing patients, along with their resistance profile, is still largely missing.[17,20–22]

A simple approach for retreatment after a virological failure even in the presence of resistance is to switch the DAA class (because of the absence of cross-resistance among different DAA classes).[23,24] However, in patients harbouring viruses with RASs in multiple DAA targets, the retreatment could represent a big challenge. In this context, HCV resistance test could represent a helpful tool to characterize the presence of clinically relevant RASs for the different DAA classes. Nevertheless, indications to perform a resistance test, after virological failure, to a DAA regimen are not clearly recommended in all currently available clinical guidelines. Indeed, the last published version of European guidelines do not yet presently endorse the resistance testing at virological failure,[1] because its utility prior to retreatment in patients who failed on any of the DAA-containing treatment regimens is unknown, while the last version of American Association for the Study of the Liver (AASLD)[2] recommend to perform a resistance test for RASs only in patients who failed a NS5A inhibitor and are in urgent need to be retreated. In these patients, it is recommended to test for NS5A and NS3 RASs.[2] Interestingly, the resistance testing for all three genes (NS3, NS5A and NS5B), at virological failure after a DAA-based regimen, was proposed, by a panel of Italian experts on HCV treatment in a recent paper, in order to better guide retreatment decisions.[25]

Therefore, more real-life data are required to improve knowledge regarding the types of failures, the characteristics of patients who fail and the role of HCV drug-resistance testing in this setting. Starting from all these observations, the aim of this study was to characterize the presence of clinically relevant RASs in all the three genes (including NS5B) in a real-life setting of patients who failed a DAA-containing regimen.