A Novel Diagnostic Target in the Hepatitis C Virus Genome

Abstract

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 (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.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.

Differential Infection Patterns and Recent Evolutionary Origins of Equine Hepaciviruses in Donkeys

“…The primer sequences were as follows: HCV-F150, GSWSCYYCYAGGICCMCCCC; HCV-R371, CTCRTGIISYAIGGTCTACRAGRCC; and HCV-R342, GGIGCICTCGCAAGCRYGCCYATCA (I ϭ inosine, S ϭ C/G, W ϭ A/T, Y ϭ C/T, M ϭ A/C, and R ϭ A/G). The limits of detection were determined as the number in probit analyses conducted with SPSS V23 (IBM, Ehningen, Germany) using eight replicates per RNA concentration as described previously (26). The 95% lower limit of detection of the EqHV 5=-UTR assay was 5.7 ϫ 10 2 RNA copies per reaction (range, 3.8 ϫ 10 2 to 1.2 ϫ 10 3 ), which was well below the commonly observed viral loads in EqHV-infected horses (25).…”

“…The first assay targeted specifically the EqHV 5=-untranslated region (5= UTR) commonly used for HV detection (26) and a second assay targeted the NS3 domain that is more conserved among diverse HVs than the 5= UTR (11). One donkey from France (sampled in 1979, age and gender unknown), one donkey from Bulgaria (sampled in 2015, a 10-year-old male), and one mule from Bulgaria (sampled in 2015, a 16-year-old female) tested positive for EqHV RNA using the 5=-UTR-based assay (0.3% of all 882 donkey and mule sera).…”

Highly Divergent Hepaciviruses from African Cattle

“…RNA secondary structures in viral genome ends were inferred manually based on covariant base pairing and thermodynamic predictions from mfold (16). The quantification of viral loads relied on photometrically quantified cRNA controls in vitro transcribed from cloned PCR amplicons containing the target sites within the NS3 gene as described previously (17). For quantification, a 25-l real-time RT-PCR was set up using a SuperScript III (SSIII) one-step RT-PCR kit (Invitrogen, Karlsruhe, Germany) with 5 l of template RNA, 400 nM both primers cattleHV-FWD (CTYAAATTGGCNTCCTAYAAAACWGG) and cattleHV-REV (GCICGRATGCCCCTAGAACG), 200 nM the probe cattleHV-P (6-carboxyfluorescein [FAM]-AYTGTGARGCKCTCGCT-GCTGACCT-black hole quencher 1), 1 g bovine serum albumin, 0.2 mM each deoxynucleoside triphosphate (dNTP), and 2.4 mM MgSO 4 .…”

Performance of the Novel QiagenartusQS-RGQ Viral Load Assays Compared to That of the Abbott RealTime System with Genetically Diversified HIV and Hepatitis C Virus Plasma Specimens

“…Variation between the results determined by different assays bears the risk of misguiding treatment decisions (19). Reagent costs for all viral load assays are high (in the range of $50 to $100 per test), highlighting the need for robust systems that avoid the necessity to retest patients (9,10,22). However, even widely used proprietary systems have shown shortcomings in their subtype/genotype coverage.…”

“…For HIV, 111 clinical plasma specimens representing 15 different subtypes/circulating recombinant forms (CRF) were available. For HCV, 91 samples of all six genotypes were collected from routine diagnostics in 2002 through 2011 and a previous study (9). Clinical specimens were genotyped by sequencing of an approximate 1,600-bp fragment in the HIV pol gene (PCR primers available upon request) and a 389-bp fragment in the HCV NS5b gene (14), followed by comparison with online databases (2,3).…”