Detection of transrenal Mycobacterium tuberculosis DNA in urine

项目摘要 结核病(TB)是全球主要的健康负担，在#年导致160万人死亡。 2017年。这一高死亡率的一个主要驱动因素是延误及时诊断。常规结核病 诊断试验，如涂片显微镜或培养，用于确认结核病依赖于 痰，但许多结核病患者患有培养阴性疾病或不能产生痰(例如， 儿童或艾滋病毒携带者)，这阻碍了病例检测和治疗启动。 因此，迫切需要使用其他样本类型的诊断工具来确认结核病 疾病。 无细胞结核分枝杆菌(Mtb)DNA片段可以通过肾脏并被 在尿液中检测到经肾(TR)DNA，有望成为一种诊断工具。然而，测试 敏感性一直是可变的，影响细菌trDNA存在的因素尚不清楚。 作为秘鲁利马一项大型队列研究的一部分，我们收集了结核病患者的尿样。 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的水平 由于细菌杀灭增加导致释放，在开始治疗后不久增加 血液中的核酸。在本研究中，我们将(1)进行短片段实时 用聚合酶链式反应检测200例结核病患者提取液中结核分枝杆菌trDNA随时间的变化 三个系列样本，在治疗开始前、治疗后4天和7天收集；以及(2)联合 尿中结核分枝杆菌trDNA水平与宿主因素(如涂片或合并症)和尿液的关系 各种因素。这项工作将为开发基于trDNA的工具提供重要的信息 确认结核病，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.