HEPATITIS-C VIRUS HETERODUPLEX TRACKING ASSAY FOR GENOTYPE DETERMINATION REVEALS DIVERGING GENOTYPE-2 ISOLATES IN ITALIAN HEMODIALYSIS-PATIENTS

A heteroduplex tracking assay (HTA) was developed for genetic analyses of the hepatitis C virus (HCV) using single-stranded probes from the core (C)/E1 region. Nucleotide sequencing of reverse transcriptase (RT )-PCR products from 15 Italian dialysis patients confirmed the specifi city and accuracy of the HTA genotyping method, which identified 5 of 15 (33.3%) Ib, 7 of 15 (46.7%) 3a, and 3 of 15 (20%) type 2 infections . The genotypes of an additional 12 HCV antibody-positive blood donors from different geographical locations were also in agreement with the genotypes determined by the Inno-LiPA HCV II kit (Innogenetics) and/o r restriction fragment length polymorphism (RFLP). Isolates which had between 35 to 40% nucleotide divergence from control subtype la, Ib, 2 a, 2b, or 3a standards could be typed. Surprisingly, HTA detected one 1b-2 coinfection which was missed by DNA sequencing. Three samples tha t were designated non-2a of 2b type 2 by HTA were found to be type 2a by both RFLP and direct nucleotide sequencing of the 5' untranslated r egion. The genetic distance between patient type 2 and control 2a, 2b, and 2c isolates indicated that a new subtype was present in the popul ation being studied. Serotyping (RIBA serotyping strip immunoblot assa y kit) of 23 dialysis patients showed that the genotype could be deter mined in 6 of 8 (75%) C/E1 RT-PCR-negative and 15 of 23 (65.2%) RT-PCR -positive samples, indicating that the two tests complement each other .