RNA Detection as an Improved Diagnostic Assay for Human Leptospirosis

RNA 检测作为人类钩端螺旋体病诊断方法的改进

基本信息

批准号：
8782404
负责人：
Albert Icksang Ko
金额：
$ 30万
依托单位：
L2 DIAGNOSTICS, LLC
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8782404
关键词：
Acute Acute Kidney Failure Affect Antibiotics Awareness Bacteria Biological Assay Blood Brazil Centers for Disease Control and Prevention (U.S.)Characteristics Clinical Collection Complementary DNA Country DNA Dengue Destinations Detection Developing Countries Development Diagnosis Diagnostic Diagnostic tests Disasters Disease Disease Outbreaks Emerging Communicable Diseases Ensure Evaluation Event Exhibits Fever Genomic DNA Goals Hawaii Health Hemorrhage Human Illness Days Individual Infection Laboratories Leptospira Leptospirosis Life Lung Malaria Methods Military Personnel Modality National Institute of Allergy and Infectious Disease Order Spirochaetales Patients Performance Phase Polymerase Polymerase Chain Reaction Population Process Prospective Studies Protocols documentation Public Health Puerto Rico RNA Recruitment Activity Resources Ribosomal RNA Sampling Sensitivity and Specificity Serum Site Slum Specimen Sports Swimming Syndrome Techniques Technology Testing Time United States National Institutes of Health Universities Urine Variant Water Whole Blood antimicrobial drug base burden of illness cost cost effective design digital farmer field study improved inner city man neglect novel pathogen point of care prospective prototype public health relevance recombinase research study response sample collection

项目摘要

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.

描述（由申请人提供）：钩端螺旋体病是一种人畜共患疾病，是全球重要的公共卫生问题。它是由属于钩端螺旋体属 9 个物种、200 多个血清型的螺旋体细菌引起的。在美国，人们越来越认识到钩端螺旋体病作为市中心人口、军事人员以及从事游泳和水上运动的个人疾病病因的重要性。在世界范围内，钩端螺旋体病给自给自足的农民和城市贫民窟居民带来了最大的负担。在疾病过程的早期开始使用抗菌药物似乎具有最大的益处。然而，目前钩端螺旋体病的实验室诊断测试依赖于过时的方法，并且灵敏度较低，尤其是在患病的最初几天。现有的PCR方法检测钩端螺旋体 DNA 的灵敏度仅为 60% 左右。我们建议实施一种新的检测方式来检测人体血液和尿液样本中的钩端螺旋体细菌。我们的初步结果表明，通过 PCR 扩增 16S RNA 衍生的 cDNA 分子可以实现更高的灵敏度。由于 16S RNA 的丰度高于基因组 DNA，因此这种方法预计会更加灵敏。我们将设计引物对，成功检测钩端螺旋体所有致病物种的 16S RNA。然后，我们将使用掺有已知浓度钩端螺旋体细菌的人体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) 液滴数字PCR，一种非常新颖的扩增技术，其灵敏度和特异性比实时PCR有所提高； (3) 重组酶聚合酶检测扩增，这是一种低成本技术，特别适合在资源匮乏的环境中作为现场护理实施。我们将为每种扩增方法确定较低的检测阈值，并估算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收集的 160 个现有样本和从健康对照收集的 100 个样本来确定我们的测试效果如何。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，以便将我们的检测方法与现有测试的性能进行比较。