Limits...
Rapid, Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H) Strain Frequency in Genotype 1b HCV by Invader Assay.

Yoshimi S, Ochi H, Murakami E, Uchida T, Kan H, Akamatsu S, Hayes CN, Abe H, Miki D, Hiraga N, Imamura M, Aikata H, Chayama K - PLoS ONE (2015)

Bottom Line: Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Histogram of Y93H frequency by deep sequencing according to detectability by the Invader assay.Deep-sequencing was performed using 55 sera of HCV 1b patients with various Y93H frequencies by the Invader assay. White and black bars represent successful detection cases and detection failure cases, respectively. Closed circles represent the success rate.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4470996&req=5

pone.0130022.g006: Histogram of Y93H frequency by deep sequencing according to detectability by the Invader assay.Deep-sequencing was performed using 55 sera of HCV 1b patients with various Y93H frequencies by the Invader assay. White and black bars represent successful detection cases and detection failure cases, respectively. Closed circles represent the success rate.

Mentions: Sensitivity for NS5A-Y93H strain detection was evaluated in comparison with the deep-sequencing data of 55 sera of HCV 1b patients. These were selected to include various proportions of NS5A-Y93H strain by this assay. As shown in Fig 6, among samples containing more than 2.8% mutant, detection of Y93H mutant variant by the Invader assay was consistently successful, and the lower detection limit of the proportion of Y93H strains could be estimated at 1 to 2%.


Rapid, Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H) Strain Frequency in Genotype 1b HCV by Invader Assay.

Yoshimi S, Ochi H, Murakami E, Uchida T, Kan H, Akamatsu S, Hayes CN, Abe H, Miki D, Hiraga N, Imamura M, Aikata H, Chayama K - PLoS ONE (2015)

Histogram of Y93H frequency by deep sequencing according to detectability by the Invader assay.Deep-sequencing was performed using 55 sera of HCV 1b patients with various Y93H frequencies by the Invader assay. White and black bars represent successful detection cases and detection failure cases, respectively. Closed circles represent the success rate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4470996&req=5

pone.0130022.g006: Histogram of Y93H frequency by deep sequencing according to detectability by the Invader assay.Deep-sequencing was performed using 55 sera of HCV 1b patients with various Y93H frequencies by the Invader assay. White and black bars represent successful detection cases and detection failure cases, respectively. Closed circles represent the success rate.
Mentions: Sensitivity for NS5A-Y93H strain detection was evaluated in comparison with the deep-sequencing data of 55 sera of HCV 1b patients. These were selected to include various proportions of NS5A-Y93H strain by this assay. As shown in Fig 6, among samples containing more than 2.8% mutant, detection of Y93H mutant variant by the Invader assay was consistently successful, and the lower detection limit of the proportion of Y93H strains could be estimated at 1 to 2%.

Bottom Line: Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus