Comparison of HCV genotype and subtype determination using Inno-LiPA HCV II and a laboratory-developed HCV 5'UTR sequencing procedure

Abstract number: p655

Gabella  S., Allice  T., Varetto  S., Pittaluga  F., Ghisetti  V.

Background: 

Genotyping and subtyping of hepatitis C virus (HCV) is clinically relevant to epidemiology, prognosis and therapeutical management of HCV infection.

Objectives: 

To compare HCV genotyping and subtyping for clinical samples using an established Line Probe assay (InnoLiPA HCV II, Innogenetics, Ghent, B) and a laboratory-developed HCV 5'UTR direct sequencing protocol (5'UTR Seq) with ABI Prism 310.

Methods: 

Twenty-five clinical samples cross-representing HCV genotypes 1–5 were analysed with InnoLiPA and a direct 5'UTR sequencing method. A library of 5'UTR HCV prototypes (n = 64) was constructed; BioEdit 7.0 and ClustalW were used for alignments and phylogenetic analysis of samples. Concordance for genotype and subtype determination was compared between the commercial test and HCV sequencing. HCV viral load was quantified in each sample using the bDNA technology (VERSANT HCV RNA 3.0 Assay).

Results: 

5'UTR provided genotype determination in all the samples. Concordance with InnoLiPA for genotype determination was 92% (22/25). Discrepant results occurred in 3 samples misclassified as genotype 2, 4 and 5 by InnoLiPA versus genotype 3 and 4 (2 samples) by 5'UTR Seq. Subtype concordance was much lower (44%, 11/25). 5'UTR Seq provided subtype determination in 96% of the clinical samples (24/25; one sample subtype 5a by InnoLiPA was classified as type 4r/4m without further subtyping), while InnoLiPA accounted for subtyping 13 of 25 samples (52% of all samples). 5'UTR Seq classified the 24 samples in 8 subtypes: 1a (n = 2), 1b (n = 5), 1c (n = 1), 2a (n = 2), 2c (n = 1), 2k (n = 1), 3a (n = 6) and 4a (n = 6), one sample misclassified as 4r/4m. InnoLiPA allowed the classification of the samples in only 5 subtypes: 1a (n = 2), 1b (n = 4), 3a (n = 4), 4e (n = 1) and 5a (n = 1) (13 not subtyped samples). Average (+SD) viral load for all genotypes was 5.9 + 0.5 log IU/ml and no significant differences were observed according to the genotype distribution.

Conclusion: 

5'UTR Sequencing protocol provided HCV genotype determination for all samples and therefore it is suitable for determining HCV genotypes from clade 1–5 in clinical samples from patients with HCV infection. 5'UTR Sequencing allowed a much wider subtype determination from all the clades but particularly for genotype 1 and 2 compared with the commercial established InnoLiPA HCV II assay. HCV subtype sequencing provides a relevant tool for epidemiological purposes and the surveillance of nosocomial transmission of HCV in high risk units.