title: HCV genotyping by three methods: analysis of discordant results based on sequencing.
abstract: BACKGROUND
  Correct genotyping of hepatitis C virus (HCV) RNA-positive serum samples may have important clinical and therapeutic implications.
  
  
  OBJECTIVE
  Three methods were compared to improve accuracy of HCV genotyping.
  
  
  METHODS
  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) (Okamoto H, Tokita H, Sakamoto M, Kojima M, Iizuka H, Mishiro S. J Gen Virol 1993; 74: 2385-2390) 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 (Pohjanpelto P, Lappalainen M, Widell A, Asikainen K, Paunio M. Clin Diagn Virol 1996; 7: 7-16); and (ii) a type-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 1997; 35: 201-207). 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 > or = 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.
authors.affiliation: Servizio di Virologia, IRCCS Policlinico San Matteo, Pavia, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Furione
authors.forename: L
authors.initials: L
authors.lastname: Simoncini
authors.forename: M
authors.initials: M
authors.lastname: Gatti
authors.forename: F
authors.initials: F
authors.lastname: Baldanti
authors.forename: M
authors.initials: M
authors.lastname: Grazia Revello
authors.forename: G
authors.initials: G
authors.lastname: Gerna
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005838:Genotype; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D012150:Polymorphism, Restriction Fragment Length; D012367:RNA, Viral / Q000097:blood / Q000302:isolation & purification; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral