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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.

Uchida Y, Kouyama J, Naiki K, Mochida S - PLoS ONE (2014)

Bottom Line: The percentage was 0% in 237 patients (80.3%) and 100% in 23 patients (7.8%), identical to the results of direct sequencing.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology & Hepatology, Saitama Medical University, Saitama, Japan.

ABSTRACT

Aim: 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%.

Conclusion: 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.

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Related in: MedlinePlus

Nucleotide (nt265–285) and Amino Acid (aa89–95) Sequences in the NS5A Region of 647 Genotype 1b HCV Strains in the Database.The figures in the lower panel denote the number of HCV strains showing each nucleotide (nt273, 276, 277, 279) among the 647 genotype 1b HCV strains.
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pone-0112647-g004: Nucleotide (nt265–285) and Amino Acid (aa89–95) Sequences in the NS5A Region of 647 Genotype 1b HCV Strains in the Database.The figures in the lower panel denote the number of HCV strains showing each nucleotide (nt273, 276, 277, 279) among the 647 genotype 1b HCV strains.

Mentions: In this study, we established a real-time PCR method using cycling probes to detect and quantify HCV stains showing Y93H mutation in the NS5A region. The percent RNA of Y93H HCV strains relative to the total HCV-RNA level in the serum can be calculated by this method. In this system, both the set of primers and the 2 types of cycling probe mixtures were designed based on nucleotide sequences of genotype 1b HCV strains reported in the database [21], since dual oral therapy with daclatasvir and asunaprevir, which is expected to be approved in the near feature in Japan, has been shown to be effective exclusively in patients with genotype 1b HCV infection [18]–[19]. As shown in Figure 4, a number of nucleotide polymorphisms (N at nt267, nt276, nt282, nt285, H at nt270 and T at nt283, nt279), which do not alter the amino acid sequences exist around the target nucleotide Y at nt277 in the NS5A region of HCV. Also, a nucleotide polymorphism that provokes an amino acid mutation, but with no alteration of the susceptibility to NS5A inhibitors, exists at nt282 showing N. Such abundant polymorphisms prevent detection of the Y93 wild and Y93H mutant strains by the usual PCR procedure; estimated maximal detection rates after such procedure were about 42% (614×311×609×634/6474 = 0.42). However, we overcame this problem through adoption of cycling probe mixtures (Ycnc and Hcnc), in which a nucleoside corresponding to nt278 was substituted for a nucleotide of the RNA. In general, cycling probes are designed to hybridize the target portion (nt277) at the RNA portion. In our preliminary experiments, however, amplification failure occurred frequently when the real-time PCR was done using such ordinary probes. The devise to shift the RNA portion enabled us to detect and quantify Y93 wild strains and Y93H mutant HCV strains separately in almost all patients with genotype 1b HCV infection. Considering that amplification failure occurred in 29 of the 332 patients (8.7%) even in the analysis by direct sequencing, in which a set of primers similar to that in the real-time PCR analysis is used, the efficacy rate of the cycling probe mixtures (Ycnc and Hcnc) for the detection of genotype 1b HCV strains was calculated as 92.5% (273/295).


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.

Uchida Y, Kouyama J, Naiki K, Mochida S - PLoS ONE (2014)

Nucleotide (nt265–285) and Amino Acid (aa89–95) Sequences in the NS5A Region of 647 Genotype 1b HCV Strains in the Database.The figures in the lower panel denote the number of HCV strains showing each nucleotide (nt273, 276, 277, 279) among the 647 genotype 1b HCV strains.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232421&req=5

pone-0112647-g004: Nucleotide (nt265–285) and Amino Acid (aa89–95) Sequences in the NS5A Region of 647 Genotype 1b HCV Strains in the Database.The figures in the lower panel denote the number of HCV strains showing each nucleotide (nt273, 276, 277, 279) among the 647 genotype 1b HCV strains.
Mentions: In this study, we established a real-time PCR method using cycling probes to detect and quantify HCV stains showing Y93H mutation in the NS5A region. The percent RNA of Y93H HCV strains relative to the total HCV-RNA level in the serum can be calculated by this method. In this system, both the set of primers and the 2 types of cycling probe mixtures were designed based on nucleotide sequences of genotype 1b HCV strains reported in the database [21], since dual oral therapy with daclatasvir and asunaprevir, which is expected to be approved in the near feature in Japan, has been shown to be effective exclusively in patients with genotype 1b HCV infection [18]–[19]. As shown in Figure 4, a number of nucleotide polymorphisms (N at nt267, nt276, nt282, nt285, H at nt270 and T at nt283, nt279), which do not alter the amino acid sequences exist around the target nucleotide Y at nt277 in the NS5A region of HCV. Also, a nucleotide polymorphism that provokes an amino acid mutation, but with no alteration of the susceptibility to NS5A inhibitors, exists at nt282 showing N. Such abundant polymorphisms prevent detection of the Y93 wild and Y93H mutant strains by the usual PCR procedure; estimated maximal detection rates after such procedure were about 42% (614×311×609×634/6474 = 0.42). However, we overcame this problem through adoption of cycling probe mixtures (Ycnc and Hcnc), in which a nucleoside corresponding to nt278 was substituted for a nucleotide of the RNA. In general, cycling probes are designed to hybridize the target portion (nt277) at the RNA portion. In our preliminary experiments, however, amplification failure occurred frequently when the real-time PCR was done using such ordinary probes. The devise to shift the RNA portion enabled us to detect and quantify Y93 wild strains and Y93H mutant HCV strains separately in almost all patients with genotype 1b HCV infection. Considering that amplification failure occurred in 29 of the 332 patients (8.7%) even in the analysis by direct sequencing, in which a set of primers similar to that in the real-time PCR analysis is used, the efficacy rate of the cycling probe mixtures (Ycnc and Hcnc) for the detection of genotype 1b HCV strains was calculated as 92.5% (273/295).

Bottom Line: The percentage was 0% in 237 patients (80.3%) and 100% in 23 patients (7.8%), identical to the results of direct sequencing.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology & Hepatology, Saitama Medical University, Saitama, Japan.

ABSTRACT

Aim: 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%.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus