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.