Infection with multiple hepatitis C virus genotypes detected using commercial tests should be confirmed using next generation sequencing

Belén Fernández-Caso,Natalia Chueca,Jose Ángel Fernández-Caballero,Luisa García Buey,Eukene Rojo,Laura Cardeñoso,Federico García,Adolfo De Salazar

doi:10.1038/s41598-019-42605-z

Belén Fernández-Caso, Natalia Chueca + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-42605-z

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Abstract

Current HCV genotyping methods may have some limitations in detecting mixed infections. We aimed to determine the accuracy of genotyping and the detection of mixed-genotype infections using the Abbott-RealTime HCV Genotype II assay (Abbott-RT-PCR) in comparison with a Roche-Next Generation Sequencing assay (Roche-NGS). Plasma samples collected from 139 HCV-infected patients tested with Abbott-RT-PCR, 114 with single genotype (GT) and 25 with mixed GTs were genotyped using Roche-NGS. Roche-NGS confirmed all single GTs obtained with Abbott-RT-PCR. One case of Abbott GT 4 was found as GT 1a using Roche-NGS. Genotype 5 was confirmed using Roche-NGS in 75% cases (3 out of 4 cases). Twenty-five patients were identified as having mixed HCVinfections using Abbott-RT-PCR. The concordance between Abbott-RT-PCR and Roche-NGS was 76% (19 out of 25 cases). Three mixed-GT infections identified with the Abbott assay (two (1b + 4); one (1a + 3)) were reported as pure 1b using Roche-NGS. Very divergent results were found for the other three samples. When compared to Roche-NGS, Abbott-RT-PCR has performed excellently for the determination of patients infected with single GTs. For patients that are categorized as having a mixed infection using Abbott-RT-PCR, we recommend an NGS assay as a confirmation test.

Highlights

Hepatitis C virus (HCV) has been classified into seven major genotypes (GTs) (GT 1 – GT 7), with whole-genome nucleotide sequences differing between them by 30% approximately
HCV genotyping is still used in clinical practice and remains vital to determine the most appropriate antiviral regimen and treatment duration for each patient as well as for epidemiological studies[6,24]
HCV infection by mixed GTs has been frequently reported[3,10,27,28,29,30], the true incidence of HCV mixed infections is not yet clear because commercial genotyping assays are not intended to shed light on this issue and because the detection rate of mixed infections is highly variable depending on the methodology used and the geographic area

Summary

Introduction

Hepatitis C virus (HCV) has been classified into seven major genotypes (GTs) (GT 1 – GT 7), with whole-genome nucleotide sequences differing between them by 30% approximately. Several non-sequencing-based HCV genotyping assays, mainly based on reverse hybridization or real-time PCR (RT-PCR), such as the Abbott RealTime HCV Genotype II assay (Abbott RT-PCR), are commercially available and used for routine determination of the HCV GT and selected subtypes. Www.nature.com/scientificreports some limitations in detecting mixed infections, as they are designed to identify only the dominant HCV GT in the population and may not detect minor variants[7,19] For this reason, they may provide unambiguous results failing to assign the HCV GT/subtype. Next-generation sequencing (NGS) allows to investigate viral heterogeneity at much larger detail: its high throughput generates millions of reads in a single sequencing run, leading to a higher sensitivity compared to the detection of minor strains, which would go undetected by standard sequencing methods[21,22]

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