Determining hepatitis C genotype by analyzing the sequence of the NS5b region

Sandres-Sauné, Karine; Dény, Paul; Pasquier, Christophe; Thibaut, V; Duverlie, Gilles; Izopet, Jacques

doi:10.1016/s0166-0934(03)00070-3

Cited by 147 publications

(129 citation statements)

References 38 publications

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“…Also, in accordance with previous studies 6,8,12,14,17,18,21 , discrepant results at the subtype level, mainly subtypes 1a and 1b, were found here (two samples identified as 1a by NS5B sequencing were subtyped as 1b by 5' NC sequencing). Although both regions provide similar results at the genotype level, which is adequate for clinical practice, especially in the management of antiviral therapy, the NS5B sequencing appears to be more useful for epidemiological investigations.…”

Section: Resultssupporting

confidence: 85%

“…Of these, 9 (17.7%) could not be genotyped by NS5B sequence analysis due to failure of the amplification procedure despite successive attempts to amplify cDNA with the NS5B primers. Although these primers are thought to bind highly conserved sequences 21 , primer-target mismatching within the NS5B region is still likely because the relative variability of its sequence that may not always be recognized by the used primers. Despite this inability to amplify all HCV RNA positive samples, NS5B sequence analysis was used here as a reference method for accurate genotyping 8,21 .…”

Section: Resultsmentioning

confidence: 99%

“…cDNA was amplified using the primers complementary to the conserved area of the 5' NC and NS5B regions and PCR conditions described by GINABREDA et al 11 and SANDRES-SAUNÉ et al 21 , respectively.…”

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confidence: 99%

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Hepatitis C virus genotypes in hemodialysis patients in the Federal District, Brazil

Amorim

Raiol

Trevizoli

et al. 2010

Rev. Inst. Med. trop. S. Paulo

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SUMMARYHepatitis C virus (HCV) genotypes and subtypes were determined in hemodialysis patients in the Federal District, Brazil, by sequencing of the 5' noncoding (NC) and nonstructural 5B (NS5B) regions. From 761 patients, 66 anti-HCV-positive samples were tested for HCV RNA. All 51 HCV RNA-positive samples by PCR of the 5' NC region were genotyped as genotypes 1 (90.2%) and 3 (9.8%). Subtype 1a (82.3%) was the most prevalent, followed by subtypes 3a (9.8%), 1b (5.9%) and 1a/1b (2.0%). Forty-two samples could be amplified and genotyped in the NS5B region: 38 (90.5%) as genotype 1, subtypes 1a, and 8 (9.5%) as genotype 3, subtype 3a. For the 42 samples sequenced in both regions, the genotypes and subtypes determined were concordant in 100% and 95.2% of cases, respectively. Two samples presented discrepant results, with the 5' NC region not distinguishing correctly the subtypes 1a and 1b. These findings indicate that the HCV genotype 1, subtype 1a, is the most prevalent among hemodialysis patients in the Federal District, Brazil.

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Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

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Hepatitis C virus genotypes in hemodialysis patients in the Federal District, Brazil

Amorim

Raiol

Trevizoli

et al. 2010

Rev. Inst. Med. trop. S. Paulo

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“…However, the genotyping methods employed in those studies, which were based on 5' noncoding (5'NC) region analyses, did not permit the correct identification of HCV subtypes (10)(11)(12)(13)(14)(15)(16)(17). To our knowledge, the present study is the first to analyze Brazilian HCV samples using two genotyping methods: line probe assay (LiPA, based on the 5'NC region) and the nucleotide sequencing analysis of the nonstructural 5B (NS5B) region, which has been used as a reference method for accurate genotyping (10)(11)(12)(13)(14)(15)(16)(17). Thus, our objective was to determine which HCV genotypes and subtypes occur in hemodialysis patients in Central Brazil and also to compare the genotyping efficiency of LiPA and direct sequencing of the NS5B region.…”

mentioning

confidence: 99%

“…Genotyping by sequence analysis was performed after nested RT-PCR for the NS5B region using the primers and PCR conditions described by Sandres-Sauné et al (17). The nested RT-PCR products were purified using the QIAquick gel extraction kit (Qiagen -GmbH, Hilden, Germany) and submitted to a direct nucleotide sequencing reaction in both directions using a Big Dye Terminator kit (version 3.1, Applied Biosystems, Foster…”

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confidence: 99%

Genotyping hepatitis C virus from hemodialysis patients in Central Brazil by line probe assay and sequence analysis

Espírito-Santo

Carneiro

Reis

et al. 2007

Braz J Med Biol Res

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The present study examined the distribution of hepatitis C virus (HCV) genotypes and subtypes in a hemodialysis population in Goiás State, Central Brazil, and evaluated the efficiency of two genotyping methods: line probe assay (LiPA) based on the 5' noncoding region and nucleotide sequencing of the nonstructural 5B (NS5B) region of the genome. A total of 1095 sera were tested for HCV RNA by RTnested PCR of the 5' noncoding region. The LiPA assay was able to genotype all 131 HCV RNA-positive samples. Genotypes 1 (92.4%) and 3 (7.6%) were found. Subtype 1a (65.7%) was the most prevalent, followed by subtypes 1b (26.7%) and 3a (7.6%). Direct nucleotide sequencing of 340 bp from the NS5B region was performed in 106 samples. The phylogenetic tree showed that 98 sequences (92.4%) were classified as genotype 1, subtypes 1a (72.6%) and 1b (19.8%), and 8 sequences (7.6%) as subtype 3a. The two genotyping methods gave concordant results within HCV genotypes and subtypes in 100 and 96.2% of cases, respectively. Only four samples presented discrepant results, with LiPA not distinguishing subtypes 1a and 1b. Therefore, HCV genotype 1 (subtype 1a) is predominant in hemodialysis patients in Central Brazil. By using sequence analysis of the NS5B region as a reference standard method for HCV genotyping, we found that LiPA was efficient at the genotype level, although some discrepant results were observed at the subtype level (sensitivity of 96.1% for subtype 1a and 95.2% for subtype 1b). Thus, analysis of the NS5B region permitted better discrimination between HCV subtypes, as required in epidemiological investigations.

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Transcription‐mediated amplification linked to line probe assay as a routine tool for HCV typing in clinical laboratories

Roß

Viazov

Kpakiwa

et al. 2007

Clinical Laboratory Analysis

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Typing of hepatitis C virus (HCV) isolates is currently a prerequisite for adequate tailoring of antiviral combination therapy. In many diagnostic laboratories, there seems to be a tendency toward convenient and time-saving procedures utilizing amplification products, which are already available from preceding qualitative or quantitative HCV ribonucleic acid (RNA) assays. In this context, we evaluated the performance characteristics of a combination of techniques, i.e., transcription-mediated amplification-line probe assay (TMA-LiPA), which links highly sensitive TMA of HCV RNA to the VERSANT HCV Genotype Assay (version 1). A total of 100 clinical samples were genotyped by TMA-LiPA. The obtained results were compared to those recorded by the original, nested reverse transcription (RT)-polymerase chain reaction (PCR)-based VERSANT assay, the core-related GEN-ETI-K DEIA, and phylogenetic analyses of partial sequences from the HCV core and NS5B regions. TMA-LiPA assigned the correct genotype to all 100 HCV isolates. For subtyping of genotype 1 and 2 isolates, TMA-LiPA only showed discriminatory powers of 82% and 53%, respectively. Thus, TMA-LiPA in our hands turned out as a convenient and time-saving routine procedure for HCV typing which currently provides sufficient information for clinical purposes. Like all 5'untranslated region (UTR)-based assays, the technique is limited, however, in its potentials to resolve the complexity of existing HCV subtypes.

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Determining hepatitis C genotype by analyzing the sequence of the NS5b region

Cited by 147 publications

References 38 publications

Hepatitis C virus genotypes in hemodialysis patients in the Federal District, Brazil

Hepatitis C virus genotypes in hemodialysis patients in the Federal District, Brazil

Genotyping hepatitis C virus from hemodialysis patients in Central Brazil by line probe assay and sequence analysis

Transcription‐mediated amplification linked to line probe assay as a routine tool for HCV typing in clinical laboratories

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