A novel diagnostic target in the hepatitis C virus genome.
A novel diagnostic target in the hepatitis C virus genome.
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DOI：
10.1371/journal.pmed.1000031
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发表时间：
2009-02-10
期刊：
影响因子：
15.8
通讯作者：
Drosten, Christian
中科院分区：
文献类型：
--
作者：
Drexler, Jan Felix;Kupfer, Bernd;Petersen, Nadine;Tommasini Grotto, Rejane Maria;Corvino Rodrigues, Silvia Maria;Grywna, Klaus;Panning, Marcus;Annan, Augustina;Silva, Giovanni Faria;Douglas, Jill;Koay, Evelyn S. C.;Smuts, Heidi;Netto, Eduardo M.;Simmonds, Peter;de Moura Campos Pardini, Maria Ines;Roth, W. Kurt;Drosten, Christian
丙型肝炎病毒（HCV） RNA的检测和定量是诊断和治疗方案不可或缺的一部分。所有的分子检测都以病毒5′-非编码区（5′-NCR）为靶点，并且都显示出敏感性和病毒载量结果的基因型依赖性差异。非西方HCV基因型在评价研究中代表性不足。HCV基因组内的另一种诊断靶区可能促进新一代检测。在这项研究中，我们通过从头测序确定了3 ' -X-tail元件，其特征明显晚于基因组的其他部分，在基因型中高度保守。为了证明其作为分子诊断靶点的临床实用性，开发了一种原型定性和定量检测方法，并对来自四大洲（德国、英国、巴西、南非、新加坡）的725份临床血浆样本进行了多中心评估，涵盖了HCV基因型1-6。据我们所知，这是迄今为止用于丙型肝炎病毒核酸检测（NAT）验证的最多样化和最全面的临床和基因型标本面板。检测下限（LOD）为18.4 IU/ml(95%可信区间15.3 ~ 24.1 IU/ml)，适用于供血筛查。最高LOD超过10−9 IU/ml，便于在大动态范围内监测病毒载量。在598份基因型样本中，采用Bayer VERSANT 3.0分支DNA （bDNA）定量，所有基因型中基于x尾的病毒载量与bDNA高度一致。基因型1 ~ 6的bDNA与x尾NAT的相关系数分别为：0.92、0.85、0.95、0.91、0.95和0.96；仅在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）。在巴西的一个实验室中，X-tail NAT的成功引入证实了基于X-tail协议的实际稳定性和鲁棒性。该检测方法的反应成本低（每个样品8.70美元），周转时间短（最多96个样品2.5小时），并且没有技术困难。本研究为从根本上改善HCV病毒载量监测和感染筛查提供了一条途径。我们的原型分析可以作为新一代病毒载量分析的模板。此外，据我们所知，这项研究提供了第一个开放协议，允许在资源有限的情况下进行工业级HCV检测和量化。Christian Drosten及其同事开发、验证并公开提供了一种基于保守的3' x尾元素的丙型肝炎病毒检测原型，具有在发展中国家临床使用的潜力。世界上约3%的人口（1.7亿人）长期（慢性）感染丙型肝炎病毒（HCV），每年约有300万至400万人新感染这种病毒。hcv是慢性肝炎（肝脏炎症）的主要原因，通过接触感染者的血液传播。在全球范围内，主要传播途径是使用未经筛选的血液进行输血和重复使用消毒不充分的医疗器械，包括针头。在富裕国家，捐献的血液被常规筛查是否存在丙型肝炎病毒，大多数传播是通过吸毒者之间共用针头。性传播和母婴传播丙型肝炎病毒的风险很低。虽然丙型肝炎病毒感染偶尔会引起以疲劳和黄疸（眼睛和皮肤发黄）为特征的急性（短期）疾病，但大多数新感染者会发展为无症状的慢性感染，最终可能导致肝硬化（瘢痕形成）和肝癌。丙型肝炎病毒感染可以用干扰素和利巴韦林两种药物联合治疗，但这些药物价格昂贵，而且对许多患者无效。限制丙型肝炎病毒全球传播的一种有效方法可能是对发展中国家用于输血的血液进行常规筛查。在发达国家，对献血者的丙型肝炎病毒筛查使用昂贵的、商业化的“RT-PCR”检测方法来检测少量的丙型肝炎病毒核糖核酸（RNA；丙型肝炎病毒储存了复制自身所需的信息——它的基因组——作为“核糖核苷酸”序列）。目前所有的HCV检测，也可以在治疗期间定量血液中病毒RNA的数量（病毒载量），检测病毒基因组中称为5 ' -非编码区（5 ' -NCR）的靶序列。然而，有几种不同的HCV“基因型”（株）。这些基因型在地理分布上各不相同，尽管常见基因型（HCV基因型1-6）的5′-NCR序列非常相似（高度保守），但现有的检测方法并不能同样有效地检测所有变异。这一缺点，再加上它们的高成本，意味着5 ' -NCR RT-PCR测定法不适合在许多发展中国家使用。在这项研究中，研究人员在HCV基因组中确定了另一种诊断靶标序列——3 ' -X-tail元素，并询问该序列是否可以用于开发新一代的HCV感染检测，这种检测可能更适合在发展中国家使用。研究人员在主要HCV基因型的参考样本中测定了3 ' - x尾元件的RNA序列，并表明HCV基因组的这一区域与5 ' -NCR一样高度保守。然后，他们开发了一种原型x尾RT-PCR分析方法，并测试了其在基因型参考样本和在德国、英国、巴西、南非和新加坡收集的大量HCV感染血液样本中检测少量HCV和测量病毒载量的能力。新的检测方法在所有基因型参考样本中检测到低水平的HCV RNA，并且也能够量化高RNA浓度。它为临床样本提供的病毒载量估计值与使用商业测定法获得的结果非常吻合，而不考虑样本的HCV基因型。最后，X-tail RT-PCR测定法在巴西实验室中用于测量在巴西收集的127例患者样本的独立组中的病毒载量时，其结果与标准测定法相似，而成本只是标准测定法的一小部分。这些发现表明，无论HCV基因型如何，HCV 3 ' -X-tail元件都可以为筛查HCV感染的血液样本和监测治疗期间的病毒载量提供替代靶标。此外，他们认为X-tail RT-PCR检测方法可能足够稳定和可靠，可用于新兴国家的实验室。总的来说，这些发现应该刺激新一代临床丙型肝炎病毒检测方法的发展，因为X-tail检测中使用的方案是免费的，可以通过提供一种廉价和有效的替代现有专有丙型肝炎病毒检测方法来改善发展中国家的血液安全性。请通过本摘要的在线版本http://dx.doi.org/10.1371/journal.pmed.1000031访问这些网站。世界卫生组织有一份关于丙型肝炎的情况说明书（英文和法文），美国疾病控制和预防中心为公众和卫生专业人员提供丙型肝炎的信息（也有西班牙文），美国国家糖尿病、消化和肾脏疾病研究所提供丙型肝炎的基本信息（英文和西班牙文），MedlinePlus百科全书有一个关于丙型肝炎的页面；MedlinePlus还提供有关丙型肝炎的更多信息的链接（英文和西班牙文）。
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. Christian Drosten and colleagues develop, validate, and make openly available a prototype hepatitis C virus assay based on the conserved 3' X-tail element, with potential for clinical use in developing countries. About 3% of the world's population (170 million people) harbor long-term (chronic) infections with the hepatitis C virus (HCV) and about 3–4 million people are newly infected with this virus every year. HCV—a leading cause of chronic hepatitis (inflammation of the liver)—is spread through contact with the blood of an infected person. Globally, the main routes of transmission are the use of unscreened blood for transfusions and the reuse of inadequately sterilized medical instruments, including needles. In affluent countries, where donated blood is routinely screened for the presence of HCV, most transmission is through needle sharing among drug users. The risk of sexual and mother-to-child transmission of HCV is low. Although HCV infection occasionally causes an acute (short-lived) illness characterized by tiredness and jaundice (yellow eyes and skin), most newly infected people progress to a symptom-free, chronic infection that can eventually cause liver cirrhosis (scarring) and liver cancer. HCV infections can be treated with a combination of two drugs called interferon and ribavirin, but these drugs are expensive and are ineffective in many patients. An effective way to limit the global spread of HCV might be to introduce routine screening of the blood that is used for transfusions in developing countries. In developed countries, HCV screening of blood donors use expensive, commercial “RT-PCR” assays to detect small amounts of HCV ribonucleic acid (RNA; HCV stores the information it needs to replicate itself—its genome—as a sequence of “ribonucleotides”). All the current HCV assays, which can also quantify the amount of viral RNA in the blood (the viral load) during treatment, detect a target sequence in the viral genome called the 5′-noncoding region (5′-NCR). However, there are several different HCV “genotypes” (strains). These genotypes vary in their geographical distribution and, even though the 5′-NCR sequence is very similar (highly conserved) in the common genotypes (HCV genotypes 1–6), the existing assays do not detect all the variants equally well. This shortcoming, together with their high cost, means that 5′-NCR RT-PCR assays are not ideal for use in many developing countries. In this study, the researchers identify an alternative diagnostic target sequence in the HCV genome—the 3′-X-tail element—and ask whether this sequence can be used to develop a new generation of tests for HCV infection that might be more appropriate for use in developing countries. The researchers determined the RNA sequence of the 3′-X-tail element in reference samples of the major HCV genotypes and showed that this region of the HCV genome is as highly conserved as the 5′-NCR. They then developed a prototype X-tail RT-PCR assay and tested its ability to detect small amounts of HCV and to measure viral load in genotype reference samples and in a large panel of HCV-infected blood samples collected in Germany, the UK, Brazil, South Africa, and Singapore. The new assay detected low levels of HCV RNA in all of the genotype reference samples and was also able to quantify high RNA concentrations. The viral load estimates it provided for the clinical samples agreed well with those obtained using a commercial assay irrespective of the sample's HCV genotype. Finally, the X-tail RT-PCR assay gave similar results to a standard assay at a fraction of the cost when used to measure viral loads in a Brazilian laboratory in an independent group of 127 patient samples collected in Brazil. These findings suggest that the HCV 3′-X-tail element could provide an alternative target for screening blood samples for HCV infection and for monitoring viral loads during treatment, irrespective of HCV genotype. In addition, they suggest that X-tail RT-PCR assays may be stable and robust enough for use in laboratories in emerging countries. Overall, these findings should stimulate the development of a new generation of clinical HCV assays that, because the protocol used in the X-tail assay is freely available, could improve blood safety in developing countries by providing a cheap and effective alternative to existing proprietary HCV assays. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000031. The World Health Organization has a fact sheet about hepatitis C (in English and French) The US Centers for Disease Control and Prevention provides information on hepatitis C for the public and for health professionals (information is also available in Spanish) The US National Institute of Diabetes and Digestive and Kidney Diseases provides basic information on hepatitis C (in English and Spanish) The MedlinePlus Encyclopedia has a page on hepatitis C; MedlinePlus also provides links to further information on hepatitis C (in English and Spanish)
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影响因子：
9.4
作者：
Caliendo, A. M.;Valsamakis, A.;Lurain, N. S.
通讯作者：
Lurain, N. S.
影响因子：
9.4
作者：
Colucci, G.;Ferguson, J.;Sarrazin, C.
通讯作者：
Sarrazin, C.
影响因子：
9.4
作者：
Elbeik, T;Surtihadi, J;Terrault, NA
通讯作者：
Terrault, NA
影响因子：
9.4
作者：
Highbarger, Helene C.;Hu, Zonghui;Dewar, Robin L.
通讯作者：
Dewar, Robin L.
影响因子：
9.3
作者：
Drosten, Christian;Panning, Marcus;Preiser, Wolfgang
通讯作者：
Preiser, Wolfgang