Evaluation of a New Assay in Comparison with Reverse Hybridization and Sequencing Methods for Hepatitis C Virus Genotyping Targeting Both 5′ Noncoding and Nonstructural 5b Genomic Regions

Microbiology Department, Hospital Universitari Germans Trias i Pujol, Ctra de Canyet, s/n. 08916 Badalona, Spain.
Journal of clinical microbiology (Impact Factor: 3.99). 02/2008; 46(1):192-7. DOI: 10.1128/JCM.01623-07
Source: PubMed


We report the evaluation of a new real-time PCR assay for hepatitis C virus (HCV) genotyping. The assay design is such that genotype 1 isolates are typed by amplification targeting the nonstructural 5b (NS5b) subgenomic region. Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5' noncoding region (5'NC) (method 1; HCV genotyping analyte-specific reagent assay). This method was compared with 5'NC reverse hybridization (method 2; InnoLiPA HCV II) and 5'NC sequencing (method 3; Trugene HCV 5'NC). Two hundred ninety-five sera were tested by method 1; 223 of them were also typed by method 2 and 89 by method 3. Sequencing and phylogenetic analysis of an NS5b fragment were used to resolve discrepant results. Suspected multiple-genotype infections were confirmed by PCR cloning and pyrosequencing. Even though a 2% rate of indeterminates was obtained with method 1, concordance at the genotype level with results with methods 2 and 3 was high. Among eight discordant results, five mixed infections were confirmed. Genotype 1 subtyping efficiencies were 100%, 77%, and 74% for methods 1, 2, and 3, respectively; there were 11/101 discordants between methods 1 and 2 (method 1 was predominantly correct) and 2/34 between methods 2 and 3. Regarding genotype 2, subtyping efficiencies were 100%, 45%, and 92% by methods 1, 2, and 3, respectively; NS5b sequencing of discordants (16/17) revealed a putative new subtype within genotype 2 and that most subtype calls were not correct. Although only sequencing-based methods provide the possibility of identifying new variants, the real-time PCR method is rapid, straightforward, and simple to interpret, thus providing a good single-step alternative to more-time-consuming assays.

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Available from: Elisa Martró
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    • "By contrast, the GT II kit was unable to distinguish between genotypes in several mixed-genotype plasma samples. A previous study has also demonstrated that the GT II kit was unable to detect some genotypes in a mixed infection in which the ratio of the viral load of the minority genotype to that of the dominant genotype was less than 1:100 (Martro et al., 2008). Furthermore, because of the similar routes of viral transmission, HCV co-infection with HBV and/or HIV is commonly observed, especially in hemophiliac patients and IDUs (Soriano et al., 2006; Koziel and Peters, 2007). "
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    ABSTRACT: The genotyping of the hepatitis C virus (HCV) plays an important role in the treatment of HCV because genotype determination has recently been incorporated into the treatment guidelines for HCV infections. Most current genotyping methods are unable to detect mixed genotypes from two or more HCV infections. We therefore developed a multiplex genotyping assay to determine HCV genotypes using a bead array. Synthetic plasmids, genotype panels and standards were used to verify the target-specific primer (TSP) design in the assay, and the results indicated that discrimination efforts using 10 TSPs in a single reaction were extremely successful. Thirty-five specimens were then tested to evaluate the assay performance, and the results were highly consistent with those of direct sequencing, supporting the reliability of the assay. Moreover, the results from samples with mixed HCV genotypes revealed that the method is capable of detecting two different genotypes within a sample. Furthermore, the specificity evaluation results suggested that the assay could correctly identify HCV in HCV/human immunodeficiency virus (HIV) co-infected patients. This genotyping platform enables the simultaneous detection and identification of more than one genotype in a same sample and is able to test 96 samples simultaneously. It could therefore provide a rapid, efficient and reliable method of determining HCV genotypes in the future.
    Full-text · Article · Aug 2014 · Microbial Biotechnology
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    • "The detection limit was 12 IU/mL. HCV genotypes were determined by VERSANT HCV Genotype 2.0 Assay (LiPa) (Innogenetics, Ghent, Belgium; distributed by Siemens Healthcare Diagnostics) [22] or by Abbott RealTime HCV Genotype II Assay (Abbott) [23]. "
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    ABSTRACT: A good correlation between HCV core antigen (HCVAg) and different HCV-RNA assays has been described, but little data are available in HCV/HIV co-infection. We aimed to evaluate HCVAg in comparison with HCV-RNA and to determine their kinetics during antiviral treatment in selected HCV/HIV co-infected patients. 355 samples from 286 HCV/HIV co-infected subjects for whom HCV-RNA (Abbott RealTime) was requested were analysed also for HCVAg (Abbott ARCHITECT) in order to evaluate the correlation between the two parameters both in patients treated or untreated for chronic hepatitis C and according to different HCV genotypes. The differences between percentages were evaluated by chi square or Fisher's exact test, while mean and median values were compared by Student's t test or the Mann-Whitney test, respectively. All differences were considered significant for a p value <0.05. HCVAg was detectable on 288/315 sera (91.4%) positive for HCV-RNA and in 5 out of40(12.5%) sera with undetectable HCV-RNA for a total concordance of 90.1%. The correlation was fair both in untreated (r = 0.742) and in treated (r = 0.881) patients and stronger for genotypes 1 and 4 than for genotype 3. Both HCV-RNA and HCVAg levels were significantly higher (p = 0.028 and p = 0.0098, respectively) in patients infected by genotype 1 than by genotype 3. The mean ratio of Log values between HCV-RNA (IU/mL) and HCVAg (fmol/liter) was 2.27 + 1.09 in untreated and 2.20 + 0.82 in treated patients (p = n.s.),consistent with a sensitivity of HCVAg corresponding to about 1,000 IU/mL of HCV-RNA, and ranged from 2.21 to 2.32 among HCV genotypes with no significant differences; five samples (1.4%; 2 genotype 1a or 1c, 3 genotype 3a) showed highly divergent values. The analysis of 18 monitoring profiles from patients treated with PEG-IFN and Ribavirin showed similar trends, except in one case in which relapse could be predicted by HCVAg and not by HCV-RNA. These results suggest that HCVAg represents an adequate tool for determining an ongoing HCV infection also in HIV co-infected patients, with lower costs and faster turnaround time that HCV-RNA.
    Full-text · Article · Apr 2014 · BMC Infectious Diseases
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    • "For these latter drugs, the distinction between subtypes 1a and 1b is also of clinical relevance due to their varying propensity for the rapid development of drug resistance [2]. Other commercially available HCV genotyping assays include the Trugene HCV 5 NC genotyping kit (Bayer Healthcare, Berkeley, CA, USA), a semi-automated sequencing assay targeting the highly conserved 5 NCR (non-coding region), and VERSANT ® HCV Genotype Assay (LiPA) 2.0 (Siemens, Tarrytown, NY, USA), an automated reverse hybridization line-probe assay targeting the 5 NCR and core regions [3] [4] [5] [6]. The automated Abbott m2000 Real- Time HCV Genotype II assay (Abbott Molecular Inc., Des Plaines, IL, USA) is a relatively new addition to these commercially available assays and is a PCR-based assay targeting specific regions of the 5 NCR and the NS5b genes, and reports the genotypes as 1, 1a, 1b, 2, 3, 4, 5 or 6 [7]. "
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    ABSTRACT: Hepatitis C (HCV) genotyping is important for treatment planning. The Abbott m2000 RealTime HCV Genotype II assay is a PCR-based assay targeting specific regions of the 5'NCR gene for genotypes 1-6, and the NS5b gene for subgenotypes 1a/1b. However, not all genotypes can be resolved, with results being reported as: 'indeterminate', 'mixed', 'genotype X reactivity with Y', or just the major genotype 1 alone. To assess the supplementary testing required for these unresolved HCV genotypes, these samples were tested further using an in-house core/E1 sequencing assay. The resulting genotypes/subgenotypes were assigned using phylogenetic analysis with reference HCV genotype sequences. Additional testing was conducted using the INNO-LiPA HCV II assay for truly mixed genotypes. Out of 1052 samples tested, 89 (8.5%) underwent further sequencing to determine the HCV genotype: 16 that were 'indeterminate' on the m2000, were mostly genotype 2s and 3s by sequencing; 12 that were 'mixed', were mostly one of the genotypes reported in the mixture; 7 that were 'X reactivity with Y', were usually genotype X; 54 that gave just a major genotype 1 result were mostly 1a, with some 6 and 1b, and a few 1c. For three truly mixed genotypes, additional testing using the VERSANT(®) HCV Genotype Assay (LiPA) 2.0, showed two mixed 1 and 3, and one indistinguishable 6c-6l genotypes. The Abbott m2000 RealTime HCV Genotype II assay can resolve most (∼90%) HCV genotypes. However in 9-10% of cases, to fully resolve the genotype, additional testing is required.
    Full-text · Article · Apr 2014 · Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology
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