HCV genotyping by three methods: analysis of discordant results based on sequencing

Background: Correct genotyping of hepatitis C virus (HCV) RNA-positive seru m samples may have important clinical and therapeutic impIications. Objectives: Three methods were compared to improve accuracy of HCV genotypi ng. Study design: PI panel of 144 HCV RNA-positive sera prospectively tested by a modified Okamoto's type-specific reverse transcription-nested polymerase chain reaction (RT-nPCR) (Okamoto H, Tokita H, Sakamoto M, Kojima M, Iizuk a H, Mishiro S. J Gen Virol 1993; 74: 2385-2390) was retrospectively analyz ed by two recently described methods which were reported to identify all HC V types and the majority of HCV subtypes: (i) a restriction fragment length polymorphism (RFLP) analysis of PCR products amplified from the 5' untrans lated region (5'UTR) of the viral genome (Pohjanpelto P, Lappalainen M, Wid ell A, Asikainen K, Paunio M. Clin Diagn Virol 1996; 7. 7-16); and (ii) a t ype-specific RT-nPCR relevant to the core region (Ohno T, Mizokami M, Wu R, Saleh M, Ohba K, Orito E, Mukaide M, Williams R, Lau J. J Clin Microbiol 1 997; 35: 201-207). The panel (according to results given by the modified Ok amoto's method) consisted of: (i) 105 sera belonging to five different HCV subtypes; (ii) 20 specimens containing a mixture of greater than or equal t o 2 genotypes; and (iii) 19 untypeable clinical samples. Results: There was agreement of the three methods for 78/144 (54.2%) blood samples, whereas discordant results were obtained for the remaining 66 samp les, 56 of which could be typed by sequencing. Of these, 51 (91.7%) were co rrectly typed by RFLP, 37 (66.0%) by Ohno's; and 27 (48.2%) by the modified Okamoto's procedure. The overall genotyping sensitivity of each method ove r the total number of 134 samples whose genotype was ascertained, was 96.2% for RFLP, 85.8% for Ohno's and 78.3% for the modified Okamoto's procedure. Conclusions: RFLP analysis, notwithstanding some limitations in subtyping e fficiency of genotype 1 samples, appears superior to the two RT-nPCR method s because: (i) it is able to type a larger number of samples; (ii) it is mo re efficient in identifying genotypes 2a/c, which are widespread in Italy; (iii) it is highly sensitive (together with Ohno's method) in recognizing g enotypes 3 and 4. (C) 1999 Published by Elsevier Science B.V. All rights re served.