Detection of transrenal Mycobacterium tuberculosis DNA in urine

项目摘要 结核病（TB）是一个主要的全球健康负担，导致160万人死亡， 2017.这种高死亡率的一个主要驱动因素是及时诊断的延误。常规TB 用于确认结核病的涂片显微镜检查或培养等诊断测试依赖于 痰，但许多TB患者具有培养阴性疾病或不能产生痰（例如， 儿童或艾滋病毒感染者），这阻碍了病例发现和治疗的启动。 因此，迫切需要使用其他样本类型来确认结核病的诊断工具 疾病 无细胞结核分枝杆菌（Mtb）DNA片段可以通过肾脏， 在尿液中检测到的跨肾（tr）DNA，提供了作为诊断工具的希望。然而，测试 敏感性是可变的，影响细菌trDNA存在的因素是未知的。 作为在秘鲁利马进行的一项大型队列研究的一部分，我们收集了结核病患者的尿液样本 并从这些样本中提取了DNA我们的初步数据表明，结核分枝杆菌trDNA水平 由于细菌杀灭增加导致释放， 血液中的核酸。在本研究中，我们将（1）进行短片段实时 PCR检测200例结核病病例提取物中Mtb trDNA随时间的变化， 三个系列样品，在治疗开始前、治疗开始后四天和七天收集;和（2）相关 尿液中Mtb trDNA水平与宿主因素（如涂片状态或合并症）和尿液 因素这项工作将提供重要的信息，以制定战略的trDNA为基础的工具 确认结核病并监测结核病治疗反应。

Project Summary Tuberculosis (TB) is a major global health burden and led to the death of 1.6 million people in 2017. A major driver of this high mortality rate is delay in timely diagnosis. Conventional TB diagnostic tests such as smear microscopy or culture used to confirm TB disease rely on sputum, but many TB patient have culture-negative disease or cannot produce sputum (e.g., children or people living with HIV), which hampers case detection and treatment initiation. Therefore, there is an urgent need for diagnostic tools using other sample types to confirm TB disease. Fragments of cell-free Mycobacterium tuberculosis (Mtb) DNA can pass the kidneys and be detected in urine as transrenal (tr) DNA, offering promise as a diagnostic tool. However, test sensitivity has been variable and factors influencing presence of bacterial trDNA are unknown. As part of a large cohort study in Lima, Peru, we have collected urine samples from TB patients and extracted DNA from these samples. Our preliminary data suggest that levels of Mtb trDNA increase shortly after initiation of treatment due to increased bacterial killing leading to release of nucleic acids in the bloodstream. In this study, we will (1) conduct short-fragment real-time PCR to detect changes in Mtb trDNA over time in extracts from 200 TB cases, of whom we have three serial samples, collected pre-, four, and seven days post treatment start; and (2) associate levels of Mtb trDNA in urine with host factors (such as smear status or comorbidities) and urine factors. This work will provide important information to develop strategies for trDNA-based tools to confirm TB disease and monitor TB treatment response.