RNA Detection as an Improved Diagnostic Assay for Human Leptospirosis

描述(申请人提供)：钩端螺旋体病是一种人畜共患病，是世界范围内一个重要的公共卫生问题。它是由钩端螺旋体属9个种200多个血清型的螺旋体细菌引起的。在美国，越来越多的人意识到钩端螺旋体病是市中心居民、军人和从事游泳和水上运动的个人的重要致病原因。在世界范围内，钩端螺旋体病给自给农民和城市贫民窟居民带来了最大的负担。如果在疾病过程的早期开始使用抗菌剂，似乎会带来最大的好处。然而，目前的钩端螺旋体病实验室诊断试验依赖过时的方法，灵敏度低，特别是在患病的头几天。现有的检测钩端螺旋体的PCR方法 DNA的灵敏度只有60%左右。我们建议实施一种新的检测方法来检测人体血液和尿样中的钩端螺旋体细菌。我们的初步结果表明，通过对来自16S RNA的cDNA分子进行PCR扩增，可以获得更高的灵敏度。由于16S RNA的丰度高于基因组DNA，预计这种方法将明显更加灵敏。我们将设计能成功检测到所有钩端螺旋体致病物种的16S RNA的引物对。然后，我们将使用添加了已知浓度的钩端螺旋体细菌的人类血液和尿液样本来比较三种不同扩增方法的性能：(1)实时PCR扩增，使用或不使用荧光TaqMan探针；(2)水滴数字PCR，这是一种非常新颖的扩增技术，具有比实时PCR更高的灵敏度和特异度；以及(3)重组酶聚合酶扩增，这是一种低成本技术，可能特别适合在资源匮乏的情况下实施护理点实施。我们将为每种扩增方法确定一个较低的检测阈值，并估计每个分析样本的成本。然后，我们将使用从钩端螺旋体病患者那里收集的160个现有样本和从健康对照那里收集的100个样本来确定我们的测试有多好。在第一阶段结束时，我们将选择引物对和平台技术进行进一步的开发。在第二阶段实验中，我们将在全球多个地点进行前瞻性研究，以比较我们的检测方法与现有测试的性能。

DESCRIPTION (provided by applicant): Leptospirosis, a zoonotic disease, is an important public health problem worldwide. It is caused by spirochete bacteria belonging to nine species and more than 200 serovars of the genus Leptospira. In the US, there is increasing awareness of the importance of leptospirosis as the cause of disease among inner-city populations, military personnel, and individuals engaged in swimming and water sports. Worldwide, leptospirosis imparts its greatest burden on subsistence farmers and urban slum dwellers. Antimicrobial agents appear to be of greatest benefit when initiated early in the disease process. However, current laboratory diagnostic tests for leptospirosis rely on antiquated methods and suffer from low sensitivity, especially in the first days of illness. Existing PCR methods to detect Leptospira DNA only have a sensitivity of approximately 60%. We propose to implement a novel testing modality to detect Leptospira bacteria in human blood and urine samples. Our preliminary results demonstrate that a greatly higher sensitivity can be achieved through the PCR amplification of cDNA molecules derived from 16S RNA. Such a method is expected to be markedly more sensitive because of the abundance of 16S RNA over that of genomic DNA. We will design primer pairs that successfully detect 16S RNA from all pathogenic species of Leptospira. We will then use human blood and urine samples spiked with known concentrations of Leptospira bacteria to compare the performance of three different amplification methods: (1) real-time PCR amplification, with or without a fluorescent TaqMan probe; (2) droplet digital PCR, a very novel amplification technique with improved sensitivity and specificity over real- time PCR; and (3) recombinase polymerase assay amplification, a low-cost technology which may be particularly suitable as a point-of-care implementation in resource-poor settings. We will determine a lower threshold of detection for each amplification method and estimate the cost per sample analyzed. We will then determine how well our test performs using 160 existing samples collected from patients with leptospirosis and 100 samples collected from healthy controls. At the conclusion of Phase I, we will select primer pairs and a platform technology for further development. In Phase II experiments, we will conduct a prospective study in multiple sites worldwide in order to compare the performance of our assay with that of existing tests.