title: Rapid, Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H) Strain Frequency in Genotype 1b HCV by Invader Assay.
abstract: Daclatasvir and asunaprevir dual oral therapy is expected to achieve high sustained virological response (SVR) rates in patients with HCV genotype 1b infection. However, presence of the NS5A-Y93H substitution at baseline has been shown to be an independent predictor of treatment failure for this regimen. By using the Invader assay, we developed a system to rapidly and accurately detect the presence of mutant strains and evaluate the proportion of patients harboring a pre-treatment Y93H mutation. This assay system, consisting of nested PCR followed by Invader reaction with well-designed primers and probes, attained a high overall assay success rate of 98.9% among a total of 702 Japanese HCV genotype 1b patients. Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed. Our assay system showed a better lower detection limit of Y93H proportion than using direct sequencing, and Y93H frequencies obtained by this method correlated well with those of deep-sequencing analysis (r = 0.85, P <0.001). The proportion of the patients with the mutant strain estimated by this assay was 23.6% (164/694). Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10-4), higher serum HCV RNA levels (p=4.3 x 10-7), and lower HCC risk (p=6.9 x 10-3) than those with the wild type strain. Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients.
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yoshimi
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Laboratory for Digestive Diseases, Center for Genomic Medicine, RIKEN, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Ochi
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors.forename: Eisuke
authors.initials: E
authors.lastname: Murakami
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors.forename: Takuro
authors.initials: T
authors.lastname: Uchida
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Kan
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors.forename: Sakura
authors.initials: S
authors.lastname: Akamatsu
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Laboratory for Digestive Diseases, Center for Genomic Medicine, RIKEN, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors.forename: C Nelson
authors.initials: CN
authors.lastname: Hayes
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Laboratory for Digestive Diseases, Center for Genomic Medicine, RIKEN, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Abe
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Laboratory for Digestive Diseases, Center for Genomic Medicine, RIKEN, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors.forename: Daiki
authors.initials: D
authors.lastname: Miki
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Hiraga
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors.forename: Michio
authors.initials: M
authors.lastname: Imamura
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Aikata
authors.affiliation: Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan; Laboratory for Digestive Diseases, Center for Genomic Medicine, RIKEN, Hiroshima, Japan; Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Chayama
mesh_terms: D000368:Aged; D001483:Base Sequence; D004252:DNA Mutational Analysis; D024882:Drug Resistance, Viral / Q000235:genetics*; D005260:Female; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics
chemical_list: D009841:Oligonucleotides; D012367:RNA, Viral