Drexler Jan Felix, Kupfer Bernd, Petersen Nadine, Grotto Rejane Maria Tommasini, Rodrigues Silvia Maria Corvino, Grywna Klaus, Panning Marcus, Annan Augustina, Silva Giovanni Faria, Douglas Jill, Koay Evelyn S C, Smuts Heidi, Netto Eduardo M, Simmonds Peter, Pardini Maria Inês de Moura Campos, Roth W Kurt, Drosten Christian
Clinical Virology Group, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
PLoS Med. 2009 Feb 10;6(2):e31. doi: 10.1371/journal.pmed.1000031.
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.
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.
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.
丙型肝炎病毒（HCV）RNA的检测和定量对于诊断和治疗方案至关重要。所有分子检测均针对病毒5'-非编码区（5'-NCR），并且所有检测都显示出敏感性和病毒载量结果的基因型依赖性变异。非西方HCV基因型在评估研究中的代表性不足。HCV基因组内的另一个诊断靶区域可能有助于新一代检测方法的开发。
在本研究中，我们通过从头测序确定，3'-X尾元件的特征比基因组的其余部分确定得晚得多，在各基因型中高度保守。为了证明其作为分子诊断靶标的临床实用性，我们开发了一种定性和定量检测原型，并在来自四大洲（德国、英国、巴西、南非、新加坡）的725份完整临床血浆样本的大型综合样本组上进行了多中心评估，样本涵盖HCV基因型1-6。据我们所知，这是迄今为止用于HCV核酸检测（NAT）验证的最多样化和最全面的临床及基因型样本组。检测下限（LOD）为18.4 IU/ml（95%置信区间，15.3 - 24.1 IU/ml），表明适用于献血者筛查。检测上限超过10^(-9) IU/ml，便于在宽动态范围内进行病毒载量监测。在598份已进行基因分型的样本中，通过拜耳VERSANT 3.0分支DNA（bDNA）进行定量，基于X尾的病毒载量与所有基因型的bDNA高度一致。基因型1 - 6的bDNA与基于X尾的NAT之间的相关系数分别为：0.92、0.85、0.95、0.91、0.95和0.96；仅12%的样本中基于X尾的病毒载量与基于5'-NCR的病毒载量偏差超过0.5 log10（最大偏差为0.85 log10）。在巴西一家实验室成功引入基于X尾的NAT证实了基于X尾方案的实际稳定性和稳健性。该检测方法以低反应成本（每份样本8.70美元）、短周转时间（最多96份样本需2.5小时）实施，且无技术困难。
本研究指出了一种从根本上改善HCV病毒载量监测和感染筛查的方法。我们的检测原型可作为新一代病毒载量检测方法的模板。此外，据我们所知，本研究提供了首个开放方案，允许在资源有限的环境中进行工业级HCV检测和定量。
