title: A novel diagnostic target in the hepatitis C virus genome.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Clinical Virology Group, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
authors.forename: Jan Felix
authors.initials: JF
authors.lastname: Drexler
authors.forename: Bernd
authors.initials: B
authors.lastname: Kupfer
authors.forename: Nadine
authors.initials: N
authors.lastname: Petersen
authors.forename: Rejane Maria Tommasini
authors.initials: RM
authors.lastname: Grotto
authors.forename: Silvia Maria Corvino
authors.initials: SM
authors.lastname: Rodrigues
authors.forename: Klaus
authors.initials: K
authors.lastname: Grywna
authors.forename: Marcus
authors.initials: M
authors.lastname: Panning
authors.forename: Augustina
authors.initials: A
authors.lastname: Annan
authors.forename: Giovanni Faria
authors.initials: GF
authors.lastname: Silva
authors.forename: Jill
authors.initials: J
authors.lastname: Douglas
authors.forename: Evelyn S C
authors.initials: ES
authors.lastname: Koay
authors.forename: Heidi
authors.initials: H
authors.lastname: Smuts
authors.forename: Eduardo M
authors.initials: EM
authors.lastname: Netto
authors.forename: Peter
authors.initials: P
authors.lastname: Simmonds
authors.forename: Maria Inês de Moura Campos
authors.initials: MI
authors.lastname: Pardini
authors.forename: W Kurt
authors.initials: WK
authors.lastname: Roth
authors.forename: Christian
authors.initials: C
authors.lastname: Drosten
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics*; D001483:Base Sequence; D016679:Genome, Viral / Q000235:genetics; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000097:blood* / Q000235:genetics; D019562:Viral Load / Q000379:methods*
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral