Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology · 2019
A pan-genotypic Hepatitis C Virus NS5A amplification method for reliable genotyping and resistance testing.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод пока не подготовлен
Обработка статьи · этапы, ошибки и управление
Загрузка состояния…
Ошибки и ограничения
История · было → стало
Состояние обновляется каждые 5 секунд, пока блок открыт. Процент показывается только там, где известен объём работы. Пауза останавливает продолжение этапа; текущий запрос к источнику может завершиться.
Walker Andreas, Ennker Kim Sophie, Kaiser Rolf, Lübke Nadine, Timm Jörg
Аннотация
BACKGROUND: Chronic infection with the Hepatitis C Virus (HCV) is associated with the risk of progressive liver disease. Although, HCV treatment options and viral cure rates have tremendously increased over the last decade, all currently licensed combination therapies contain inhibitors of the replication complex NS5A. Resistance-associated substitutions (RAS) in NS5A can limit the efficacy of therapy; however, resistance testing is routinely not recommended for all patients. Notably, pan-genotypic combinations have been approved, however the correct identification of the HCV genotype is still required for treatment decisions and is a good predictor for treatment success.
OBJECTIVE: The aim of this study was the establishment of a pan-genotypic NS5A amplification method for reliable genotyping and simultaneous resistance testing in a fast and cheap routine diagnostic setup.
STUDY DESIGN: Pan-genotypic degenerated nested PCR primer were designed and tested in 262 HCV-patients. The collection included samples from genotypes 1-7 and the median viral load was 1.07 × 106 IU/ml (range 248-21 × 106 IU/ml).
RESULTS: Amplification of the expected 747bp fragment was successful in 257 of 262 (98.1%) samples including samples <1000 IU/ml. The direct comparison of the genotype information obtained with core sequencing to those obtained by NS5A prediction showed high concordance (97.3%) and discrepancies occurred only for relatively rare subtypes. Resistance analysis using Geno2Pheno[HCV] showed NS5A-RAS in 23 of 257 (8.9%) of samples.
CONCLUSIONS: We successfully developed a routine diagnostic method for pan-genotypic amplification of NS5A. This amplicon can be used for simultaneous genotyping and resistance testing for enhancing and improving routine HCV diagnostic.
OBJECTIVE: The aim of this study was the establishment of a pan-genotypic NS5A amplification method for reliable genotyping and simultaneous resistance testing in a fast and cheap routine diagnostic setup.
STUDY DESIGN: Pan-genotypic degenerated nested PCR primer were designed and tested in 262 HCV-patients. The collection included samples from genotypes 1-7 and the median viral load was 1.07 × 106 IU/ml (range 248-21 × 106 IU/ml).
RESULTS: Amplification of the expected 747bp fragment was successful in 257 of 262 (98.1%) samples including samples <1000 IU/ml. The direct comparison of the genotype information obtained with core sequencing to those obtained by NS5A prediction showed high concordance (97.3%) and discrepancies occurred only for relatively rare subtypes. Resistance analysis using Geno2Pheno[HCV] showed NS5A-RAS in 23 of 257 (8.9%) of samples.
CONCLUSIONS: We successfully developed a routine diagnostic method for pan-genotypic amplification of NS5A. This amplicon can be used for simultaneous genotyping and resistance testing for enhancing and improving routine HCV diagnostic.