Development of a PCR method for rapid detection of Leptospira from one microliter of whole blood

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. Its diagnosis is usually difficult, often resulting in delayed antimicrobial therapy and worse outcomes. Nucleic acid amplification tests of blood provide reliable Leptospira detection in acute infections, but the requirement for skilled personnel and expensive equipment still limits their widespread use, especially in low-resource settings. We, therefore, developed a microfluidic-based PCR (mbPCR) assay to detect pathogenic Leptospira, aiming to simplify sample preparation. The PicoGene (R) PCR1100 device was integrated with primers and probes targeting the 16S rDNA of pathogenic Leptospira. The assay was evaluated for its ability to detect the spirochete directly in specimens, omitting the DNA purification step. Direct detection from blood samples was assessed using cultured live Leptospira interrogans strain L495 spiked into whole blood of hamsters and humans to determine the lower limit of detection. In addition, whole blood collected from the infected hamsters was examined by mbPCR. Using the mbPCR assay, live leptospires were detected at a concentration of 7.78 (95 % CI, 3.83-15.8) leptospires/mu L in phosphate-buffered saline (PBS) and 1 x 102 leptospires/mu L in whole blood. The detection sensitivity was unaffected by the host animal species and the type of anticoagulant. Furthermore, mbPCR successfully identified the bacteria in as little as 1 mu L of whole blood obtained from a hamster model of leptospirosis. Although further validation is required, this method has the potential to provide timely and straightforward point-of-care diagnostics, and is anticipated to see expanded use in developing regions.