title: Use of flow cytometry for rapid and accurate enumeration of live pathogenic Leptospira strains.
abstract: 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 104to 108leptospires/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 105leptospires/ml and 106leptospires/ml, respectively, demonstrating the feasibility to use flow cytometry as first-line tool for diagnosis or bacterial dissemination studies.
authors: Merial SAS, 254 rue Marcel Mérieux, 69007 Lyon, France; Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
authors: Célia
authors: C
authors: Fontana
authors: Merial SAS, 254 rue Marcel Mérieux, 69007 Lyon, France.
authors: Steve
authors: S
authors: Crussard
authors: Merial SAS, 254 rue Marcel Mérieux, 69007 Lyon, France.
authors: Nathalie
authors: N
authors: Simon-Dufay
authors: Merial SAS, 254 rue Marcel Mérieux, 69007 Lyon, France.
authors: Daniel
authors: D
authors: Pialot
authors: Merial SAS, 254 rue Marcel Mérieux, 69007 Lyon, France. Electronic address: natalia.bomchil@merial.com.
authors: Natalia
authors: N
authors: Bomchil
authors: Merial SAS, 254 rue Marcel Mérieux, 69007 Lyon, France. Electronic address: jean.reyes@merial.com.
authors: Jean
authors: J
authors: Reyes
mesh_terms: D000818:Animals; D015169:Colony Count, Microbial / Q000379:methods*; D004285:Dogs; D005240:Feasibility Studies; D005434:Flow Cytometry; D007921:Leptospira interrogans / Q000302:isolation & purification*; D007922:Leptospirosis / Q000097:blood / Q000175:diagnosis* / Q000652:urine; D016014:Linear Models; D008853:Microscopy; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
keywords: Enumeration; Flow cytometry; Leptospira