title: Development of rare resistance-associated variants that are extremely tolerant against NS5A inhibitors during daclatasvir/asunaprevir therapy by a two-hit mechanism.
abstract: OBJECTIVE
  The virologic characteristics of resistance-associated variants (RAVs) developing in patients receiving dual oral therapy with daclatasvir/asunaprevir, including those with previous triple therapy with simeprevir, were evaluated.
  
  
  METHODS
  A total of 206 patients with genotype-1b HCV infection, including 5 patients with previous simeprevir therapy, were treated with daclatasvir/asunaprevir for 24 weeks. Resistance-associated variants in the NS5A regions at baseline and during/after therapy were evaluated using cycling-probe real-time polymerase chain reaction combined with direct sequencing. The dynamics of rare RAVs were also assessed using ultra-deep sequencing.
  
  
  RESULTS
  A sustained virologic response (SVR12) was achieved in 180 patients (87%); the rates were 95% in patients without baseline NS5A-RAVs and 83%, 59%, and 77% in those with hepatitis C virus (HCV) strains carrying NS5A-L31M, NS5A-Y93H/C, and NS5A-R30Q/H/L mutations, respectively. A multivariate analysis revealed baseline NS5A-R30Q/H/L mutation and NS5A-Y93H mutations as significant factors associated with SVR12. Virologic failure developed in all 5 patients with previous simeprevir treatment, and rare RAVs (HCV strains with NS5A-R30H, NS5A-A92K, NS5A-P29del, and NS5A-P32del) developed at virologic failure. Ultra-deep sequencing revealed that HCV strains with NS5A-P29del or NS5A-P32del were absent at baseline and emerged within 4 weeks of dual oral therapy among the strains appearing after simeprevir administration.
  
  
  CONCLUSIONS
  NS5A-R30Q/H/L and NS5A-Y93H mutations at baseline determined the therapeutic efficacy of dual oral therapy with daclatasvir/asunaprevir, but rare NS5A-RAVs developed frequently in patients with previous simeprevir treatment. Such RAVs may develop in a two-hit manner, with simeprevir altering the quasispecies of HCV strains in the NS5A regions, leading to the emergence of HCV strains with NS5A-P29del and NS5A-P32del during exposure to daclatasvir/asunaprevir.
authors.affiliation: Department of Gastroenterology and Hepatology, Faculty of Medicine, Saitama Medical University, Moroyama, Japan.
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Uchida
authors.affiliation: Department of Gastroenterology and Hepatology, Faculty of Medicine, Saitama Medical University, Moroyama, Japan.
authors.forename: Jun-Ichi
authors.initials: JI
authors.lastname: Kouyama
authors.affiliation: Department of Gastroenterology and Hepatology, Faculty of Medicine, Saitama Medical University, Moroyama, Japan.
authors.forename: Kayoko
authors.initials: K
authors.lastname: Naiki
authors.affiliation: Department of Gastroenterology and Hepatology, Faculty of Medicine, Saitama Medical University, Moroyama, Japan.
authors.forename: Kayoko
authors.initials: K
authors.lastname: Sugawara
authors.affiliation: Department of Gastroenterology and Hepatology, Faculty of Medicine, Saitama Medical University, Moroyama, Japan.
authors.forename: Mie
authors.initials: M
authors.lastname: Inao
authors.affiliation: Department of Gastroenterology and Hepatology, Faculty of Medicine, Saitama Medical University, Moroyama, Japan.
authors.forename: Yukinori
authors.initials: Y
authors.lastname: Imai
authors.affiliation: Department of Gastroenterology and Hepatology, Faculty of Medicine, Saitama Medical University, Moroyama, Japan.
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Nakayama
authors.affiliation: Department of Gastroenterology and Hepatology, Faculty of Medicine, Saitama Medical University, Moroyama, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Mochida
keywords: HCV; NS3/4A protease inhibitor; NS5A inhibitor; resistance-associated variants