title: Multiclass HCV resistance to direct-acting antiviral failure in real-life patients advocates for tailored second-line therapies.
abstract: 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.
  
  
  
  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).
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Velia C
authors.initials: VC
authors.lastname: Di Maio
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Cento
authors.affiliation: Hepatology Unit, University Hospital of Rome Tor Vergata, Rome, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Lenci
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Marianna
authors.initials: M
authors.lastname: Aragri
authors.affiliation: Hepatology Unit, University Hospital of Rome Tor Vergata, Rome, Italy.
authors.forename: Piera
authors.initials: P
authors.lastname: Rossi
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Barbaliscia
authors.affiliation: Infectious Diseases Unit, University of Sassari, Sassari, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Melis
authors.affiliation: Infection Disease Unit, Policlinico S. Orsola-Malpighi, Bologna, Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Verucchi
authors.affiliation: Division of Infectious Disease, Hospital Sacco of Milan, Milan, Italy.
authors.forename: Carlo F
authors.initials: CF
authors.lastname: Magni
authors.affiliation: Infectious Diseases, University Hospital of Rome Tor Vergata, Rome, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Teti
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Ada
authors.initials: A
authors.lastname: Bertoli
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy.
authors.forename: FrancescoPaolo
authors.initials: F
authors.lastname: Antonucci
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Bellocchi
authors.affiliation: Unit of Microbiology, Hospital Sacco of Milan, Milan, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Micheli
authors.affiliation: Hepatology Unit, University Hospital of Rome Tor Vergata, Rome, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Masetti
authors.affiliation: Division of Infectious Disease, Hospital Sacco of Milan, Milan, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Landonio
authors.affiliation: Hepatology Unit, University Hospital of Rome Tor Vergata, Rome, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Francioso
authors.affiliation: Hepatology Unit, University Hospital of Rome Tor Vergata, Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Santopaolo
authors.affiliation: Liver Disease Unit, Department of Liver Transplantation, San Camillo Forlanini Hospital, Rome, Italy.
authors.forename: Adriano M
authors.initials: AM
authors.lastname: Pellicelli
authors.affiliation: Gastroenterology, "P. Giaccone" University Hospital, Palermo, Italy.
authors.forename: Vincenza
authors.initials: V
authors.lastname: Calvaruso
authors.affiliation: Infectious Diseases, Sant'Andrea Hospital - Sapienza University of Rome, Rome, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Gianserra
authors.affiliation: Gastroenterology, Catholic University of Rome, Rome, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Siciliano
authors.affiliation: Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Dante
authors.initials: D
authors.lastname: Romagnoli
authors.affiliation: Department of Gastroenterology, Scientific Institute for Digestive Disease "Saverio de Bellis" Hospital, Castellana Grotte, Bari, Italy.
authors.forename: Raffaele
authors.initials: R
authors.lastname: Cozzolongo
authors.affiliation: Gastroenterology, Catholic University of Rome, Rome, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Grieco
authors.affiliation: Infectious Disease Clinic, Hospital of Chieti, Chieti, Italy.
authors.forename: Jacopo
authors.initials: J
authors.lastname: Vecchiet
authors.affiliation: Gastroenterology, Department of Clinical Medicine and Surgery, University of Naples "Federico II", Naples, Italy.
authors.forename: Filomena
authors.initials: F
authors.lastname: Morisco
authors.affiliation: Gastroenterology, Department of Clinical Medicine, Sapienza University of Rome, Rome, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Merli
authors.affiliation: Infectious Diseases and Viral Hepatitis Unit, Second University, Naples, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Brancaccio
authors.affiliation: Infectious Diseases Unit, Department of Social Health (DISSAL) of the University of Genoa, IRCCS S. Martino-IST, Genoa, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Di Biagio
authors.affiliation: Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Loggi
authors.affiliation: Department of Public Health and Infectious Diseases, Sapienza University, Polo Pontino, Latina, Italy.
authors.forename: Claudio M
authors.initials: CM
authors.lastname: Mastroianni
authors.affiliation: Hepatology Unit, Pescara General Hospital, Pescara, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Pace Palitti
authors.affiliation: Infectious Disease, Hospital "G. Mazzini", Teramo, Italy.
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Tarquini
authors.affiliation: Department of Infectious Diseases, Hospital Niguarda Ca'Granda, Milan, Italy.
authors.forename: Massimo
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authors.lastname: Puoti
authors.affiliation: Infectious and Tropical Diseases Unit, Department of Clinical Medicine, Sapienza University of Rome, Rome, Italy.
authors.forename: Gloria
authors.initials: G
authors.lastname: Taliani
authors.affiliation: Infectious Diseases, University Hospital of Rome Tor Vergata, Rome, Italy.
authors.forename: Loredana
authors.initials: L
authors.lastname: Sarmati
authors.affiliation: Department of Internal Medicine, Gastroenterology Unit, University of Genova, Genoa, Italy.
authors.forename: Antonino
authors.initials: A
authors.lastname: Picciotto
authors.affiliation: Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Vullo
authors.affiliation: Gastroenterology, Department of Clinical Medicine and Surgery, University of Naples "Federico II", Naples, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Caporaso
authors.affiliation: Infectious Disease Unit, Avezzano General Hospital, Avezzano, Italy.
authors.forename: Maurizio
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authors.affiliation: Infectious Diseases, Sant'Andrea Hospital - Sapienza University of Rome, Rome, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Pasquazzi
authors.affiliation: Division of Infectious Disease, Hospital Sacco of Milan, Milan, Italy.|School of Clinical Medicine, Faculty of Health Science University of the Witwatersrand, Johannesburg, South Africa.
authors.forename: Giuliano
authors.initials: G
authors.lastname: Rizzardini
authors.affiliation: Infectious Disease Unit, Pescara General Hospital, Pescara, Italy.
authors.forename: Giustino
authors.initials: G
authors.lastname: Parruti
authors.affiliation: Gastroenterology, "P. Giaccone" University Hospital, Palermo, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Craxì
authors.affiliation: Infectious Diseases Unit, University of Sassari, Sassari, Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Babudieri
authors.affiliation: Infectious Diseases, University Hospital of Rome Tor Vergata, Rome, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Andreoni
authors.affiliation: Hepatology Unit, University Hospital of Rome Tor Vergata, Rome, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Angelico
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Carlo F
authors.initials: CF
authors.lastname: Perno
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Ceccherini-Silberstein
mesh_terms: D000368:Aged; D000998:Antiviral Agents / Q000627:therapeutic use*; D024882:Drug Resistance, Viral / Q000235:genetics*; D004359:Drug Therapy, Combination; D005260:Female; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics*; D019698:Hepatitis C, Chronic / Q000188:drug therapy*; D006801:Humans; D007372:Interferons / Q000627:therapeutic use; D007558:Italy; D008297:Male; D008875:Middle Aged; D009154:Mutation; D012008:Recurrence; D012254:Ribavirin / Q000627:therapeutic use; D017422:Sequence Analysis, DNA; D000069474:Sofosbuvir / Q000627:therapeutic use; D000072230:Sustained Virologic Response; D017211:Treatment Failure; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D007372:Interferons; D012254:Ribavirin; D000069474:Sofosbuvir; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: antiviral therapy; direct-acting antivirals; hepatitis C virus; resistance test; resistance-associated substitutions