Background: Correct genotyping of hepatitis C virus (HCV) RNA-positive serum samples may have important clinical and therapeutic implications. Objectives: Three methods were compared to improve accuracy of HCV genotyping. Study design: A panel of 144 HCV RNA-positive sera prospectively tested by a modified Okamoto's type-specific reverse transcription-nested polymerase chain reaction (RT-nPCR) was retrospectively analyzed by two recently described methods which were reported to identify all HCV types and the majority of HCV subtypes: (i) a restriction fragment length polymorphism (RFLP) analysis of PCR products amplified from the 5' untranslated region (5'UTR) of the viral genome; and (ii) a type-specific RT-nPCR relevant to the core region. The panel (according to results given by the modified Okamoto's method) consisted of: (i) 105 sera belonging to five different HCV subtypes; (ii) 20 specimens containing a mixture of ≥ 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 samples, 56 of which could be typed by sequencing. Of these, 51 (91.7%) were correctly 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 over 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 efficiency of genotype 1 samples, appears superior to the two RT-nPCR methods because: (i) it is able to type a larger number of samples, (ii) it is more efficient in identifying genotypes 2a/c, which are widespread in Italy; (iii) it is highly sensitive (together with Ohno's method) in recognizing genotypes 3 and 4.
- Hepatitis C virus
- Restriction fragment length polymorphism analysis
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology
- Immunology and Allergy
- Infectious Diseases