Author(s): Drexler, Jan Felix ; Kupfer, Bernd ; Petersen, Nadine ; Grotto, Rejane Maria Tommasini [UNESP] ; Corvino Rodrigues, Silvia Maria [UNESP] ; Grywna, Klaus ; Panning, Marcus ; Annan, Augustina ; Silva, Giovanni Faria [UNESP] ; Douglas, Jill ; Koay, Evelyn S. C. ; Smuts, Heidi ; Netto, Eduardo M. ; Simmonds, Peter ; Pardini, Maria Ines de Moura Campos [UNESP] ; Roth, W. Kurt ; Drosten, Christian
Date: 2014
Persistent ID: http://hdl.handle.net/11449/11468
Origin: Oasisbr
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Qiagen, Germany
German Ministry of Health
National Reference Centre for Tropical Infections at the Bernhard Nocht Institute
European Union
BackgroundDetection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5'-noncoding region (59-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.Methods and FindingsIn 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.ConclusionThis 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.
Univ Bonn, Inst Virol, D-5300 Bonn, Germany
Bernhard Nocht Inst Trop Med, Clin Virol Grp, Hamburg, Germany
Universidade Federal da Bahia (UFBA), Univ Hosp Prof Edgard Santos, Infect Dis Res Lab, Salvador, BA, Brazil
UNESP, Botucatu Med Sch, Ctr Blood Transfus, Mol Biol Lab, Botucatu, SP, Brazil
UNESP, Dept Internal Med, Botucatu, SP, Brazil
Univ Edinburgh, Ctr Infect Dis, Virus Evolut Grp, Edinburgh, Midlothian, Scotland
Natl Univ Singapore, Yong Loo Lin Sch Med, Dept Pathol, Singapore, Singapore
Natl Univ Singapore Hosp, Mol Diag Ctr, Singapore 119074, Singapore
Univ Cape Town, Fac Hlth Sci, Dept Clin Lab Sci, Div Med Virol,Natl Hlth Lab Serv, ZA-7925 Cape Town, South Africa
GFE Blut MbH, Frankfurt, Germany
UNESP, Botucatu Med Sch, Ctr Blood Transfus, Mol Biol Lab, Botucatu, SP, Brazil
UNESP, Dept Internal Med, Botucatu, SP, Brazil
EU: SSPE-CT-2005-022639
