Use of flow cytometry for rapid and accurate enumeration of live pathogenic Leptospira strains.

Enumeration of Leptospira, the causative agent of leptospirosis, is arduous mainly because of its slow growth rate. Rapid and reliable tools for numbering leptospires are still lacking. The current standard for Leptospira cultures is the count on Petroff-Hausser chamber under dark-field microscopy, but this method remains time-consuming, requires well-trained operators and lacks reproducibility. Here we present the development of a flow-cytometry technique for counting leptospires. We showed that upon addition of fluorescent dyes, necessary to discriminate the bacterial population from debris, several live Leptospira strains could be enumerated at different physiologic states. Flow cytometry titers were highly correlated to counts with Petroff-Hausser chambers (R2 >0.99). Advantages of flow cytometry lie in its rapidity, its reproducibility significantly higher than Petroff-Hausser method and its wide linearity range, from 104 to 108 leptospires/ml. Therefore, flow cytometry is a fast, reproducible and sensitive tool representing a promising technology to replace current enumeration techniques of Leptospira in culture. We were also able to enumerate Leptospira in artificially infected urine and blood with a sensitivity limit of 105 leptospires/ml and 106 leptospires/ml, respectively, demonstrating the feasibility to use flow cytometry as first-line tool for diagnosis or bacterial dissemination studies.