A novel simple assay system to quantify the percent HCV-RNA levels of NS5A Y93H mutant strains and Y93 wild-type strains relative to the total HCV-RNA levels to determine the indication for antiviral therapy with NS5A inhibitors.
OBJECTIVE
Oral treatment with asunaprevir and daclatasvir has been reported to yield a SVR ratio of 80% in patients with genotype 1b HCV infection, however, treatment failure has been reported, especially in patients with HCV strains showing the NS5A-Y93H mutation at baseline. An assay system to detect such strains was established to facilitate selection of appropriate candidates for this antiviral therapy.


METHODS
Primer sets and 2 types of cycling probe mixtures were designed, and real-time PCR was performed with HCV-RNA purified from 332 patients with genotype 1b HCV infection, and the results were compared with those obtained by direct sequencing.


RESULTS
Both the wild-type and mutant strains were quantified, with a threshold of 4.0 Log copies/mL, in 295 of the 332 patients (88.9%), and the percentage of the mutant strains relative to the total HCV-RNA level in the serum was calculated. The percentage was 0% in 237 patients (80.3%) and 100% in 23 patients (7.8%), identical to the results of direct sequencing. Both wild-type and mutant strains were detected in the remaining 35 patients (11.9%), at levels between 1% and 99%, despite the mutant strains having been undetectable by direct sequencing in 11 patients with percentages of these strains of less than 25%.


CONCLUSIONS
A novel assay system to quantify the percent RNA of Y93H mutant strains relative to the total HCV-RNA level was established. This system may be useful to determine the indication for treatment with NA5A inhibitors in patients with HCV.
Department of Gastroenterology & Hepatology, Saitama Medical University, Saitama, Japan.
Yoshihito
Y
Uchida
Department of Gastroenterology & Hepatology, Saitama Medical University, Saitama, Japan.
Jun-ichi
J
Kouyama
Department of Gastroenterology & Hepatology, Saitama Medical University, Saitama, Japan.
Kayoko
K
Naiki
Department of Gastroenterology & Hepatology, Saitama Medical University, Saitama, Japan.
Satoshi
S
Mochida
D000998:Antiviral Agents / Q000008:administration & dosage; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D006526:Hepatitis C / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis*; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
D000998:Antiviral Agents; D017931:DNA Primers; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase