title: Specific detection of naturally occurring hepatitis C virus mutants with resistance to telaprevir and boceprevir (protease inhibitors) among treatment-naïve infected individuals.
abstract: The use of telaprevir and boceprevir, both protease inhibitors (PI), as part of the specifically targeted antiviral therapy for hepatitis C (STAT-C) has significantly improved sustained virologic response (SVR) rates. However, different clinical studies have also identified several mutations associated with viral resistance to both PIs. In the absence of selective pressure, drug-resistant hepatitis C virus (HCV) mutants are generally present at low frequency, making mutation detection challenging. Here, we describe a mismatch amplification mutation assay (MAMA) PCR method for the specific detection of naturally occurring drug-resistant HCV mutants. MAMA PCR successfully identified the corresponding HCV variants, while conventional methods such as direct sequencing, endpoint limiting dilution (EPLD), and bacterial cloning were not sensitive enough to detect circulating drug-resistant mutants in clinical specimens. Ultradeep pyrosequencing was used to confirm the presence of the corresponding HCV mutants. In treatment-naïve patients, the frequency of all resistant variants was below 1%. Deep amplicon sequencing allowed a detailed analysis of the structure of the viral population among these patients, showing that the evolution of the NS3 is limited to a rather small sequence space. Monitoring of HCV drug resistance before and during treatment is likely to provide important information for management of patients undergoing anti-HCV therapy.
authors: Universidad Nacional Autónoma de México, Mexico City, Mexico.
authors: Salvador
authors: S
authors: Fonseca-Coronado
authors: Alejandro
authors: A
authors: Escobar-Gutiérrez
authors: Karina
authors: K
authors: Ruiz-Tovar
authors: Mayra Yolanda
authors: MY
authors: Cruz-Rivera
authors: Pilar
authors: P
authors: Rivera-Osorio
authors: Mauricio
authors: M
authors: Vazquez-Pichardo
authors: Juan Carlos
authors: JC
authors: Carpio-Pedroza
authors: Juan Alberto
authors: JA
authors: Ruíz-Pacheco
authors: Fernando
authors: F
authors: Cazares
authors: Gilberto
authors: G
authors: Vaughan
mesh_terms: D000328:Adult; D000998:Antiviral Agents / Q000494:pharmacology*; D058448:Biota; D024882:Drug Resistance, Viral; D005260:Female; D016174:Hepacivirus / Q000145:classification / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D006526:Hepatitis C / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009842:Oligopeptides / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods; D011392:Proline / Q000031:analogs & derivatives* / Q000494:pharmacology; D014773:Virology / Q000379:methods
chemical_list: D000998:Antiviral Agents; D009842:Oligopeptides; C486464:telaprevir; C512204:N-(3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl)-3-(2-((((1,1-dimethylethyl)amino)carbonyl)amino)-3,3-dimethyl-1-oxobutyl)-6,6-dimethyl-3-azabicyclo(3.1.0)hexan-2-carboxamide; D011392:Proline