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

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.

HCV Genotype-3h, a Difficult-to-Diagnose Sub-Genotype in the DAA Era

“…Interestingly, three of the four patients with subtype-3h had been erroneously identified as harbouring genotype-1 or mixed genotypes. Similarly, of 17 subjects with non-subtyped genotype-1 by Abbott Realtime HCV Genotype II, Minosse et al [20] identified 3 with genotype-3h by NS5B sequencing and ultra-deep pyrosequencing. Moreover, two of our four patients with genotype-3h had Y93H in NS5A, which may affect the second-line DAA regimen.…”

Mixed Infections Unravel Novel HCV Inter-Genotypic Recombinant Forms within the Conserved IRES Region

“…However, the true prevalence of this phenomenon remains uncertain and is likely influenced by methodological bias. Although many strategies have been developed for HCV genotyping over time, current HCV genotyping methods may have some limitations in detecting mixed infections [ 41 , 67 , 68 , 69 , 70 ]. Next-generation sequencing techniques provide an alternative to traditional sequencing methods for studying viral genetic diversity.…”