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

Satoshi Yoshimi,Eisuke Murakami,Takuro Uchida,Nobuhiko Hiraga,C Nelson Hayes,Kazuaki Chayama,Hidenori Ochi,Michio Imamura,Sakura Akamatsu,Daiki Miki,Hiroshi Aikata,Hiromi Kan,Hiromi Abe,Ranjit Ray

doi:10.1371/journal.pone.0130022

Satoshi Yoshimi, Eisuke Murakami + Show 12 more

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https://doi.org/10.1371/journal.pone.0130022

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Journal: PloS one	Publication Date: Jun 17, 2015
Citations: 29	License type: CC BY 4.0

Affiliation: Hiroshima University

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.

Highlights

Hepatitis C virus (HCV) is a major cause of chronic liver disease, liver cirrhosis, and hepatocellular carcinoma, affecting up to 180 million people worldwide [1,2]
We developed a rapid NS5A-Y93H strain detection system based on the Invader assay to evaluate the proportion of HCV genotype 1b patients with pre-existing Y93H mutations
HCV genotype was determined at the central laboratory by polymerase chain reaction (PCR) amplification and direct sequencing

Summary

Introduction

Hepatitis C virus (HCV) is a major cause of chronic liver disease, liver cirrhosis, and hepatocellular carcinoma, affecting up to 180 million people worldwide [1,2]. The pre-existing Y93H mutation has been estimated by direct sequencing to be present in 8.3%–19% of Japanese patients [8,9]. Direct sequencing is commonly used to detect viral mutations. It is only capable of detecting viral subpopulations with frequencies no lower than 10% to 20% [10,11,12,13]. Generation sequencing (NGS), which has recently been applied to analyze viral mutations, can detect relatively low frequency variants ( 1%) [14,15], but it is still complex to perform and prohibitively expensive for widespread clinical use

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