Ultra-Deep Sequencing Characterization of HCV Samples with Equivocal Typing Results Determined with a Commercial Assay.

Claudia Minosse,Anna R Garbuglia,Maria R Capobianchi,Emanuela Giombini,Barbara Bartolini

doi:10.3390/ijms17101679

Abstract

Hepatitis C virus (HCV) is classified into seven phylogenetically distinct genotypes, which are further subdivided into related subtypes. Accurate assignment of genotype/subtype is mandatory in the era of directly acting antivirals. Several molecular methods are available for HCV genotyping; however, a relevant number of samples with indeterminate, mixed, or unspecified subtype results, or even with misclassified genotypes, may occur. Using NS5B direct (DS) and ultra-deep pyrosequencing (UDPS), we have tested 43 samples, which resulted in genotype 1 unsubtyped (n = 17), mixed infection (n = 17), or indeterminate (n = 9) with the Abbott RealTime HCV Genotype II assay. Genotype 1 was confirmed in 14/17 samples (82%): eight resulted in subtype 1b, and five resulted in subtype 1a with both DS and UDPS, while one was classified as subtype 1e by DS and mixed infection (1e + 1a) by UDPS. Three of seventeen genotype 1 samples resulted in genotype 3h with both sequencing approaches. Only one mixed infection was confirmed by UDPS (4d + 1a), while in 88% of cases a single component of the mixture was detected (five genotype 1a, four genotype 1b, two genotype 3a, two genotype 4m, and two genotype 4d); 44% of indeterminate samples resulted genotype 2c by both DS and UDPS, 22% resulted genotype 3a; one indeterminate sample by Abbott resulted in genotype 4d, one resulted in genotype 6n, and one was classified as subtype 3a by DS, and resulted mixed infection (3a + 3h) by UDPS. The concordance between DS and UDPS was 94%, 88%, and 89% for genotype 1, co-infection, and indeterminate results, respectively. UDPS should be considered very useful to resolve ambiguous HCV genotyping results.

Highlights

Hepatitis C virus (HCV) is the unique recognized member of the genus Hepacivirus in the Flaviviridae family, including positive-strand RNA viruses
The NS5B amplicons obtained from 43 plasma samples (genotype 1, n = 17; indeterminate HCV genotype, n = 9; HCV multiple infections, n = 17; median of HCVRNA viral load (IU/mL): 6.26 × 105;: 1.04 × 105 − 3.11 × 106) and successfully amplified with the protocol described by Quer et al [8] were analyzed by direct sequencing (DS) and ultra-deep pyrosequencing (UDPS)
Among genotype 1 samples, eight resulted in subtype 1b, five resulted in subtype 1a when analyzed with both methods, DS and UDPS; while one sample, classified as subtype 1e by DS, was found to be a mixed infection of HCV subtype 1e (2320 reads, 98.85%) and 1a (27 reads, 1.15%) by UDPS

Summary

Introduction

Hepatitis C virus (HCV) is the unique recognized member of the genus Hepacivirus in the Flaviviridae family, including positive-strand RNA viruses. HCV has been classified into seven different genotypes and 67 confirmed subtypes; divergence of the whole genome sequence is over 30% for genotypes and between 15% and 30% for subtypes [1]. Sequencing of conserved HCV genome regions (core/E1 or the NS5B) is the reference method for assigning HCV genotype/subtype [2], several molecular methods have been established for HCV genotyping in diagnostic routine. The most used commercial assays in clinical practice are Versant HCV Genotype 2.0 assay and the Abbott RealTime HCV Genotype v 2.0. Both assays use the 5 untranslated region (5 UTR) to define HCV 1–6 genotypes [3], and additional targets to define the subtype. While HCV subtyping is not clinically relevant for Peg-interferon-α (PegIFN-α) and Ribavirin (RBV)

Methods

Results

Discussion

Conclusion