HCV genotyping by three methods

Analysis of discordant results based on sequencing

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)121-130
Number of pages10
JournalJournal of Clinical Virology
Volume13
Issue number3
DOIs
Publication statusPublished - Aug 1999

Fingerprint

Hepacivirus
Restriction Fragment Length Polymorphisms
Genotype
Reverse Transcription
Polymerase Chain Reaction
Serum
RNA
Viral Genome
5' Untranslated Regions
Italy

Keywords

  • Genotyping
  • Hepatitis C virus
  • Restriction fragment length polymorphism analysis
  • RT-nPCR

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Virology
  • Immunology and Allergy
  • Infectious Diseases

Cite this

HCV genotyping by three methods : Analysis of discordant results based on sequencing. / Furione, Milena; Simoncini, Lavinia; Gatti, Marta; Baldanti, Fausto; Grazia Revello, M.; Gerna, Giuseppe.

In: Journal of Clinical Virology, Vol. 13, No. 3, 08.1999, p. 121-130.

Research output: Contribution to journalArticle

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abstract = "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.",
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T2 - Analysis of discordant results based on sequencing

AU - Furione, Milena

AU - Simoncini, Lavinia

AU - Gatti, Marta

AU - Baldanti, Fausto

AU - Grazia Revello, M.

AU - Gerna, Giuseppe

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N2 - 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.

AB - 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.

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