A novel diagnostic target in the hepatitis C virus genome.
<h4>Background</h4>Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5'-noncoding region (5'-NCR), and all show genotype-dependent variation of sensitivities and viral load resul...
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Public Library of Science (PLoS)
2009
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oai:doaj.org-article:38f6f63165004e9a85c200b78d3062882021-11-25T05:37:21ZA novel diagnostic target in the hepatitis C virus genome.1549-12771549-167610.1371/journal.pmed.1000031https://doaj.org/article/38f6f63165004e9a85c200b78d3062882009-02-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19209955/?tool=EBIhttps://doaj.org/toc/1549-1277https://doaj.org/toc/1549-1676<h4>Background</h4>Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5'-noncoding region (5'-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays.<h4>Methods and findings</h4>In this study we determined by de novo sequencing that the 3'-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1-6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3-24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10(-9) IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1-6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5'-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties.<h4>Conclusion</h4>This study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.Jan Felix DrexlerBernd KupferNadine PetersenRejane Maria Tommasini GrottoSilvia Maria Corvino RodriguesKlaus GrywnaMarcus PanningAugustina AnnanGiovanni Faria SilvaJill DouglasEvelyn S C KoayHeidi SmutsEduardo M NettoPeter SimmondsMaria Inês de Moura Campos PardiniW Kurt RothChristian DrostenPublic Library of Science (PLoS)articleMedicineRENPLoS Medicine, Vol 6, Iss 2, p e31 (2009) |
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Medicine R |
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Medicine R Jan Felix Drexler Bernd Kupfer Nadine Petersen Rejane Maria Tommasini Grotto Silvia Maria Corvino Rodrigues Klaus Grywna Marcus Panning Augustina Annan Giovanni Faria Silva Jill Douglas Evelyn S C Koay Heidi Smuts Eduardo M Netto Peter Simmonds Maria Inês de Moura Campos Pardini W Kurt Roth Christian Drosten A novel diagnostic target in the hepatitis C virus genome. |
| description |
<h4>Background</h4>Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5'-noncoding region (5'-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays.<h4>Methods and findings</h4>In this study we determined by de novo sequencing that the 3'-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1-6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3-24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10(-9) IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1-6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5'-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties.<h4>Conclusion</h4>This study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings. |
| format |
article |
| author |
Jan Felix Drexler Bernd Kupfer Nadine Petersen Rejane Maria Tommasini Grotto Silvia Maria Corvino Rodrigues Klaus Grywna Marcus Panning Augustina Annan Giovanni Faria Silva Jill Douglas Evelyn S C Koay Heidi Smuts Eduardo M Netto Peter Simmonds Maria Inês de Moura Campos Pardini W Kurt Roth Christian Drosten |
| author_facet |
Jan Felix Drexler Bernd Kupfer Nadine Petersen Rejane Maria Tommasini Grotto Silvia Maria Corvino Rodrigues Klaus Grywna Marcus Panning Augustina Annan Giovanni Faria Silva Jill Douglas Evelyn S C Koay Heidi Smuts Eduardo M Netto Peter Simmonds Maria Inês de Moura Campos Pardini W Kurt Roth Christian Drosten |
| author_sort |
Jan Felix Drexler |
| title |
A novel diagnostic target in the hepatitis C virus genome. |
| title_short |
A novel diagnostic target in the hepatitis C virus genome. |
| title_full |
A novel diagnostic target in the hepatitis C virus genome. |
| title_fullStr |
A novel diagnostic target in the hepatitis C virus genome. |
| title_full_unstemmed |
A novel diagnostic target in the hepatitis C virus genome. |
| title_sort |
novel diagnostic target in the hepatitis c virus genome. |
| publisher |
Public Library of Science (PLoS) |
| publishDate |
2009 |
| url |
https://doaj.org/article/38f6f63165004e9a85c200b78d306288 |
| work_keys_str_mv |
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