Leptospirosis is endemic in the French West Indies. The first human cases were first documented in 1932 in Guadeloupe 
and 1938 in Martinique 
. The annual incidence of leptospirosis in the French West Indies was estimated to be approximately 10 cases per 100,000 inhabitants in the 1990s. The incidence of leptospirosis during 2002–2004 was affected by the El Nino phenomenon, which resulted in increases in rainfall and the number of cases in Guadeloupe 
. A prospective study of patients with acute febrile illness in Martinique and Guadeloupe (InVS, CIRE Antilles-Guyane) in 2011 improved the surveillance of leptospirosis. This increased awareness could explain the record incidence in 2011, peaking at <39 cases per 100,000 inhabitants in both Guadeloupe and Martinique.
Although the use of PCR diagnostic testing is becoming more common in the French West Indies, the diagnosis of leptospirosis is mostly dependent on MAT, which can identify the presumptive serogroup of the infecting bacterium. MAT has been used to show that the most frequent serogroups in Guadeloupe are Icterohaemorrhagiae and Ballum, followed by Sejroe and Canicola 
(data from the NRC for Leptospirosis). Serogroups Cynopteri, Tarassovi, Panama, Grippotyphosa and Autumnalis are less common. The sensitivity of MAT is low during the acute stage of disease 
and, because of paradoxical reactions and cross-reactions between serogroups, the accuracy of MAT in identifying the infecting serovar or serogroup can also be poor 
, limiting its epidemiological value. In this study, MAT serological data from culture-positive patients were reviewed retrospectively, allowing the identification of a total of 36 patients with MAT data for serum samples (data not shown). It was possible to infer the serogroup identity of infecting leptospires from the MAT results for 26 of these 36 patients (72%). Similarly, only a small proportion of PCR-positive samples were correctly identified by MAT (). This further confirms that only the isolation of Leptospira
from patients allowed definitive identification of the infecting serovar and is therefore essential for the study of the epidemiology of the disease.
We determined 16S rRNA sequences to identify the isolates to the species level, and then used serogrouping, PFGE, secY sequences, and MLVA to sub-type the species.
For most of the isolates (101/110), the PFGE patterns were mostly consistent with those of known serovars: i.e. serovars Bogvere, Tabaquite, Bajan, and Icterohaemorrhagiae or Copenhageni. For the serogroup Ballum, the PFGE patterns of the reference isolates for serovars Ballum, Castellonis, Guangdong, Arborea, and Soccoestomes were all similar, with fewer than three band differences 
. Serovar Arborea was previously identified by CAAT as the major serovar from the serogroup Ballum in the Caribbean island Barbados 
, suggesting that our strains may belong to serovar Arborea. For the remaining five isolates which were serogrouped (Tarassovi and Celledoni), comparison of PFGE patterns with reference strains was inconclusive for the serovar. Finally, for four isolates the rabbit antisera used did not lead to agglutination such that comparison with the reference strains was not possible. Surprisingly, none of the isolates in the last ten years from the French West Indies were identified as belonging to serogroups Canicola or Sejroe, although up to14% of MAT-positive sera correspond to these two serogroups. Similar findings were reported in Barbados 
. This may be due to cross-reactions between serogroups in MAT and/or difficulties in isolating these strains from patients (for example patients not hospitalized because of less severe symptoms or the strains fail to grown in EMJH medium).
Typing by PCR-based methods for amplification of 16S rRNA, secY
, and VNTR loci can be used directly on biological samples, thus avoiding culturing of the pathogen. The bacterial load in blood during the acute phase ranges from 102
/ml. The Leptospira
count decreases with time, and can be detected for up to 15 days 
. Thus, if the bacterial load is low, it may be necessary to use nested-PCR for amplification of the target sequences. The classification according to secY
sequences was in good agreement with the groupings determined by PFGE and MLVA, further confirming our previous data on clinical isolates from Mayotte 
Sequencing of secY
in DNA extracted from the clinical isolates and blood samples allowed a simple and rapid first-line screening and the identification of the presumptive serovar. A total of thirteen genotypes were found in our study, a large proportion of strains (75%) being of only three genotypes associated with serovars Icterohaemorrhagiae/Copenhageni, Bogvere, and Arborea. The secY
sequences from the L. santarosai
isolates showed the highest nucleotide diversity. Six genotypes were not found in the MLST database and may therefore be specific to the French West Indies. Further characterization of these isolates should include the use of the CAAT, which requires the preparation of antibodies against the strain of interest, for definitive identification of the serovar. We also detected the appearance of strains related to the pathogenic species L. kmetyi
in Martinique in at least two patients, one of which was probably exposed during canyoneering activities in the tropical forest (Hochedez et al.
, submitted). To our knowledge, L. kmetyi
which was first isolated from soil in Malaysia 
, has never been isolated from a patient with leptospirosis. Further studies are needed to determine the serological and molecular features of these strains and their distribution in the French West Indies.
The distribution of the predominant pathogenic leptospiral serovars differed between Guadeloupe and Martinique. Serovars Bogvere, Arborea, and Icterohaemorrhagiae/Copenhageni made up 35, 31, and 23% respectively of all Leptospira
isolates in Guadeloupe since 2004. In Martinique, serovar Icterohaemorrhagiae is the most frequent (35%), followed by Arborea (9%) and Bogvere (6%). In the Caribbean island of Barbados, 140 miles from Martinique, serovars Arborea (14%) and Icterohaemorrhagiae (26%) similarly cause many human infections, but the serovar Bogvere 
, which was first isolated in Jamaica 
does not. Serovar Tabaquite (serogroup Mini), which was found in Guadeloupe, was first isolated from a patient in Trinidad 
. Serovar Bim (serogroup Autumnalis) is the most frequently isolated serovar in Barbados (75% of all isolates) 
, was not isolated in the French West Indies. This suggests that some strains circulate throughout the Caribbean islands but others are highly prevalent only in restricted areas. This may be related to the distribution of the animal reservoirs for the different serovar in these islands.
can colonize or infect renal tubules of a wide variety of wild and domesticated mammals. In the Caribbean, numerous mammalian species including rodents, opossums, mongoose, bats, pigs, cattle, and dogs have been demonstrated to be hosts of pathogenic Leptospira
. Isolates from the serogroup Icterohaemorrhagiae, including serovars Icterohaemorrhagiae and Bogvere, have been isolated from the kidneys of rats, mice, and mongoose and isolates from the serogroup Ballum were isolated from rats and mice, suggesting predominantly rodent-borne transmission of the disease. Serovar Arborea was reported to be prevalent in both humans and animals in Barbados 
. Serovar Bajan was originally isolated from toads and frogs in Barbados 
. In our study, human and rat isolates from Guadeloupe and belonging to serovars Icterohaemorrhagiae/Copenhageni, Bogvere, and Arborea all showed identical genotypes, consistent with rats being responsible for the transmission of the disease.
The identification of the circulating etiological agents of leptospirosis in the French West Indies will help establish appropriate control and prevention measures in this area where the disease is endemic. For example, the reference technique, MAT, requires a panel of live antigens representing a broad range of serogroups. The use of local isolates in the panel of antigens may maximize the chances of detecting an immune response to the infecting bacterium. At the NRC for Leptospirosis (Institut Pasteur), the initial panel of 18 antigens, which already included strains representative of the serogroups Icterohaemorrhagiae, Ballum, Australis and Tarassovi, was thus expanded to include local isolates from serogroups Celledoni and Mini for serum samples originating from the French West Indies. Knowledge of leptospiral epidemiology may also be useful for the development of a whole bacterial vaccine against leptospirosis. Vaccines currently available for use in animals and, in a few countries, in humans generally consist of one, two or more locally prevalent serovars. In France, a human vaccine containing only serovar Icterohaemorrhagiae has been used since 1981 
. However, we report here that only one-third of the infections in the French West Indies are due to serovar Icterohaemorrhagiae (not including serovar Bogvere from the serogroup Icterohaemorrhagiae), and the corresponding figure for Barbabos is 22.5% 
. Immunity is restricted to antigenically related serovars, so the vaccine used in France may not be effective against the majority of strains circulating in the French West Indies.
Further studies should include the analysis of the influence of serovar and strain genetic background on the clinical presentation and outcome of the disease. It would also be valuable to investigate the reasons for differences in the distributions of Leptospira serovars in the Caribbean islands.