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Int. J. Mol. Sci. 2016, 17(10), 1679; doi:10.3390/ijms17101679

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

Laboratory of Virology, National Institute for Infectious Diseases “Lazzaro Spallanzani”—IRCCS, Via Portuense 292, Rome 00149, Italy
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Academic Editor: Tatsuo Kanda
Received: 22 July 2016 / Revised: 13 September 2016 / Accepted: 23 September 2016 / Published: 7 October 2016
(This article belongs to the Special Issue Hepatitis Virus Infection and Research)
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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. View Full-Text
Keywords: HCV genotyping; HCV subtype; ultra-deep pyrosequencing; mixed HCV infection; indeterminate genotype HCV genotyping; HCV subtype; ultra-deep pyrosequencing; mixed HCV infection; indeterminate genotype
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MDPI and ACS Style

Minosse, C.; Giombini, E.; Bartolini, B.; Capobianchi, M.R.; Garbuglia, A.R. Ultra-Deep Sequencing Characterization of HCV Samples with Equivocal Typing Results Determined with a Commercial Assay. Int. J. Mol. Sci. 2016, 17, 1679.

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