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Leptospirosis, caused by a pathogenic Leptospira bacteria, is the most prevalent zoonosis worldwide and in this context has been extensively investigated through a One Health framework. Diagnosis of human leptospirosis includes molecular and serological tools, with serological Microscopic Agglutination Test (MAT) still being considered as a gold standard. Mammals considered as biological reservoirs include species or populations that are able to maintain chronic infection and shed the bacteria via their urine in the environment. Leptospira bacteria are often investigated using the same diagnosis tool, serological MAT. However, MAT testing of putative animal reservoirs can lead to mis-interpretations as it can signal previous infection and not necessarily bring in robust information regarding the capacity of such sero-positive animals to maintain chronic infection. We use previously published data and present new results on introduced and endemic small mammals to show that MAT should not be used for the identification of reservoirs. By contrast, serological data are informative on the level of exposure of animals occupying a specific environment. Finally, we present a sequential methodology to investigate human leptospirosis in a One Health framework that associates molecular detection in humans and animals, together with MAT of human samples using Leptospira isolates obtained from reservoir animals occurring in the same environment.

BACKGROUND:Leptospirosis is a neglected zoonosis affecting mainly tropical and subtropical regions worldwide, particularly South America and the Caribbean. As in many other countries, under-reporting of cases was suspected in the French West Indies because of inadequate access to diagnostic tests for the general population. METHODOLOGY/PRINCIPAL FINDINGS:In order to estimate the real incidence of leptospirosis in Guadeloupe and Martinique, a study was performed in 2011 using the three prevailing available biological tests for diagnosis: Microscopic Agglutination Test (MAT), IgM ELISA and PCR. The study investigated inpatients and outpatients and used active case ascertainment from data provided by a general practitioners' sentinel network. The epidemiology of the disease was also described in terms of severity and demographic characteristics. Leptospirosis incidence was estimated at 69.4 (95%CI 47.6-91.1) and 60.6 (95%CI 36.3-85.0) annual cases per 100,000 inhabitants in Guadeloupe and Martinique, respectively, which was 3 and 4 times higher than previous estimations. CONCLUSION/SIGNIFICANCE:Inclusion of PCR and IgM ELISA tests for diagnosis of leptospirosis resulted in improved sensitivity in comparison with MAT alone. Our results highlighted the substantial health burden of the disease in these two territories and the importance of access to appropriate laboratory tests. Based on our results, PCR and IgM ELISA tests have now been included in the list of tests reimbursed by the national system of social security insurance in France. Our results also underline the relevance of implementing an integrated strategy for the surveillance, prevention and control of leptospirosis in the French West Indies.

NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden., Instituto de Biotecnologia y Biologia Molecular, Facultad de Ciencias Exactas, Facultad de Ciencias Médicas

Background Leptospirosis is a worldwide zoonosis that is endemic in tropical areas, such as Reunion Island. The species Leptospira interrogans is the primary agent in human infections, but other pathogenic species, such as L. kirschner and L. borgpetersenii, are also associated with human leptospirosis. Methods and Findings In this study, a melting curve analysis of the products that were amplified with the primer pairs lfb1 F/R and G1/G2 facilitated an accurate species classification of Leptospira reference strains. Next, we combined an unsupervised high resolution melting (HRM) method with a new statistical approach using primers to amplify a two variable-number tandem-repeat (VNTR) for typing at the subspecies level. The HRM analysis, which was performed with ScreenClust Software, enabled the identification of genotypes at the serovar level with high resolution power (Hunter-Gaston index 0.984). This method was also applied to Leptospira DNA from blood samples that were obtained from Reunion Island after 1998. We were able to identify a unique genotype that is identical to that of the L. interrogans serovars Copenhageni and Icterohaemorrhagiae, suggesting that this genotype is the major cause of leptospirosis on Reunion Island. Conclusions Our simple, rapid, and robust genotyping method enables the identification of Leptospira strains at the species and subspecies levels and supports the direct genotyping of Leptospira in biological samples without requiring cultures.