Ruolo del sequenziamento diretto nella tipizzazione di HCV-RNA a fini clinico-epidemiologici

Abstract

Background/Aims: Genotyping and subtyping of hepatitis C virus (HCV) is epidemiologically and clinically relevant to prognosis and therapeutical management of HCV infection.Aim was to study the feasibility of an “in house” direct sequencing method for the genotyping and subtyping of HCV strains compared with a commercially available genotyping assay (Inno-LiPA,VERSANT® HCV Genotype 2.0 Assay, Bayer). Methods: 74 clinical plasma samples cross-representing HCV genotypes 1 to 5 typed with the line probe assay Inno-LiPA (LiPA) were subjected to a laboratory-developed 5’UTR direct sequencing protocol (5’UTR Seq).A NS5B direct sequencing protocol (NS5B Seq) was apply to 23/74 samples (17 negative by the 5’UTR).Two libraries of 5’UTR and NS5B HCV prototypes were constructed; BioEdit 7.0.0 and ClustalW were used for alignments and phylogenetic analysis. Results: 5’UTR Seq typed 47/74 LiPA positive samples (64%). Concordance between the two assays was 91% (43/47) and 72% (31/43) for HCV genotyping and subtyping, respectively. LiPA and 5’UTR Seq discordant samples were typed by NS5B Seq. NS5B Seq typed 13/17 samples negative with 5’UTR Seq.All genotypes and subtypes detected with NS5B Seq were concordant with LiPA. 5’UTR and NS5B direct sequencing pointed out a wider HCV subtype distribution than LiPA for genotype 1, 2 and 4. By the combination of 5’UTR and NS5B direct sequencing, 81% (60/74) of LiPA positive samples were typed. Conclusion: HCV typing and subtyping with the line probe assay Inno-LiPA is highly recommended for clinical purposes.A more detailed HCV typing for epidemiologic purposes can by achieved by the direct sequencing of a region with a moderate degree of genetic variability such as NS5B. HCV NS5B analysis is more efficient in resolving viral strain genetic variability than direct sequencing of a highly conserved region such as 5’UTR.