Human Leptospirosis Cases Research Articles

Comparison of two PCR methods for detection of Leptospira interrogans in formalin-fixed and paraffin-embedded tissues

In this study we compared two polymerase chain reaction (PCR) methods using either 16S ribosomal RNA (rRNA) or 23S rRNA gene primers for the detection of different Leptospira interrogans serovars. The performance of these two methods was assessed using DNA extracted from bovine tissues previously inoculated with several bacterial suspensions. PCR was performed on the same tissues before and after the formalin-fixed, paraffin-embedding procedure (FFPE tissues). The 23S rDNA PCR detected all fresh and FFPE positive tissues while the 16S rDNA-based protocol detected primarily the positive fresh tissues. Both methods are specific for pathogenic L. interrogans. The 23S-based PCR method successfully detected Leptospira in four dubious cases of human leptospirosis from archival tissue specimens and one leptospirosis-positive canine specimen. A sensitive method for leptospirosis identification in FFPE tissues would be a useful tool to screen histological specimen archives and gain a better assessment of human leptospirosis prevalence, especially in tropical countries, where large outbreaks can occur following the rainy season.

Atypical lymphocytosis in leptospirosis: a cohort of hospitalized cases between 1996 and 2009 in State of Rio de Janeiro, Brazil

Leptospirosis is a zoonotic disease found in tropical and temperate countries, and its clinical diagnostic confusion with arboviruses (dengue fever, oropouche fever and yellow fever), Brazilian spotted fever, viral hepatitis and hantaviruses has been an ongoing public health concern. The aim of this observational study was to demonstrate an association between findings of atypical lymphocytosis and the progression of endemic leptospirosis. A retrospective analysis was performed on the demographic, epidemiological, clinical and laboratory aspects of 27 human leptospirosis cases that occurred over a period of 13 years (1996-2009) with no reported epidemic outbreaks in Rio de Janeiro, Brazil. The overall mortality rate was 11.1% in our cohort of hospitalized cases. However, there was no mortality among patients with atypical lymphocytosis (OR = 11.1; 95% CI = 1.12-110.9; p = 0.04). Two patients who were in the septicemic phase showed signs of expansion of γδ T cell responses in peripheral blood. Atypical lymphocytosis may be observed in patients with leptospirosis. Our observations suggest that these atypical leukocyte subsets are associated with partial protection during the disease course of leptospirosis.

Highly Virulent Leptospira borgpetersenii Strain Characterized in the Hamster Model

Abstract. A recent study by our group reported the isolation and partial serological and molecular characterization of four Leptospira borgpetersenii serogroup Ballum strains. Here, we reproduced experimental leptospirosis in golden Syrian hamsters (Mesocricetus auratus) and carried out standardization of lethal dose 50% (LD50) of one of these strains (4E). Clinical disease features and histopathologic analyses of tissue lesions were also observed. As results, strain 4E induced lethality in the hamster model with inocula lower than 10 leptospires, and histopathological examination of animals showed typical lesions found in severe leptospirosis. Gross pathological findings were peculiar; animals that died early had more chance of presenting severe jaundice and less chance of presenting pulmonary hemorrhages (P < 0.01). L. borgpetersenii serogroup Ballum has had a considerable growth in human leptospirosis cases in recent years. This strain has now been thoroughly characterized and can be used in more studies, especially evaluations of vaccine candidates.

Strikes, flooding, rats, and leptospirosis in Marseille, France

The aim of this study was to examine the mechanisms by which weather conditions, the incidence of Leptospira in reservoir populations, and various socio-ecological factors are driving the emergence of leptospirosis in Marseille, France. Over the last 30 years our laboratory has surveyed all human cases of leptospirosis in Marseille using clinical, epidemiological, serological, and molecular tools. Data for the weather conditions in Marseille were collected from the official meteorological station, and garbage management strikes were monitored through the local press. Rats were trapped in alleys near to where the patients may have acquired leptospirosis. Three new cases of autochthonous leptospirosis are reported. The global tendency for rainfall showed a slight increase over the period under analysis. The index case of leptospirosis occurred after a heavy rainfall with flooding. Over the last 10 years Marseille has undergone 82 days of garbage management strikes. Leptospira DNA was detected in two of 11 rats. The emergence of leptospirosis has become a public health problem in Marseille, and is associated with a combination of heavy rainfall and garbage collection strikes in which garbage is left on the street and thereby contributes to the expansion of the rat population on the surface.

Seasonality of Human Leptospirosis in Reunion Island (Indian Ocean) and Its Association with Meteorological Data

BackgroundLeptospirosis is a disease which occurs worldwide but particularly affects tropical areas. Transmission of the disease is dependent on its excretion by reservoir animals and the presence of moist environment which allows the survival of the bacteria.Methods and FindingsA retrospective study was undertaken to describe seasonal patterns of human leptospirosis cases reported by the Centre National de Références des Leptospiroses (CNRL, Pasteur Institute, Paris) between 1998 and 2008, to determine if there was an association between the occurrence of diagnosed cases and rainfall, temperature and global solar radiation (GSR). Meteorological data were recorded in the town of Saint-Benoît (Météo France “Beaufonds-Miria” station), located on the windward (East) coast. Time-series analysis was used to identify the variables that best described and predicted the occurrence of cases of leptospirosis on the island. Six hundred and thirteen cases were reported during the 11-year study period, and 359 cases (58.56%) were diagnosed between February and May. A significant correlation was identified between the number of cases in a given month and the associated cumulated rainfall as well as the mean monthly temperature recorded 2 months prior to diagnosis (r = 0.28 and r = 0.23 respectively). The predictive model includes the number of cases of leptospirosis recorded 1 month prior to diagnosis (b = 0.193), the cumulated monthly rainfall recorded 2 months prior to diagnosis (b = 0.145), the average monthly temperature recorded 0 month prior to diagnosis (b = 3.836), and the average monthly GSR recorded 0 month prior to diagnosis (b = −1.293).ConclusionsLeptospirosis has a seasonal distribution in Reunion Island. Meteorological data can be used to predict the occurrence of the disease and our statistical model can help to implement seasonal prevention measures.

Leptospiroses em suínos no Brasil

Leptospirosis is a zoonotic disease of worldwide distribution, reported in humans,pigs, cattle, horses, goats, dogs, rodents, and several species of wild animals. The swineleptospirosis is a disease of obligatory notification classified in the List B by the OfficeInternational des Epizooties (OIE), as a disease common to many species. Accordingto the standards for certification of breeding swine farms, the whole farm must becertified free of classical swine fever, Aujeszky’s disease, brucellosis, tuberculosis,scabies, and free or controlled for leptospirosis. Thus, this review aimed to describeaspects of Leptospirosis in swine in Brazil, especially in Piauí and Maranhão State,where the registry of animal and human leptospirosis cases are scarce or inexistent.

Sorovares de Leptospira spp. predominantes em exames sorológicos de caninos e humanos no município de Uberlândia, Estado de Minas Gerais

This study aimed to verify the occurrence of the principal Leptospira spp. serovars in domestic dogs and humans, notified in 2008, and the main risk factors in a geographic approach to the disease in the City of Uberlândia, State of Minas Gerais, Brazil. Canine blood serum samples (n=268) from different districts, belonging to the Northern, Southern, Eastern, Western and Central Sanitary Districts of Uberlandia, were collected during an animal vaccination campaign against rabies, in August 2008. A geographic approach to the city was conducted, including evaluation of locations in peripheral areas, waste landfills, garbage collection, rodent notification, human leptospirosis cases and overflow flood areas, during 2008. Leptospirosis was diagnosed using the microscopic agglutination test (MAT), the gold standard for animal and human leptospirosis diagnosis. Dogs mainly reacted to Autumnalis (34.2%) and Tarassovi (23.7%) serovars, while in humans, predominance of Tarassovi serovars occurred in the cases registered. The occurrence of these serovars could be related to an infection source common to both species, or dogs could be the infection source for humans. The Eastern Sanitary District showed a greater number of reactive dogs. Leptospirosis occurred in dogs and humans of the City of Uberlandia in 2008. This often neglected disease must be prevented because represents a public health risk and resembles other endemic illness like dengue.

The impact of climate on Leptospirosis in São Paulo, Brazil

In this work, we correlate the daily number of human leptospirosis cases with several climatic factors. We used a negative binomial model that considers hospital daily admissions due to leptospirosis as the dependent variable, and the climatic variables of daily precipitation pattern, and maximum and minimum temperature as independent variables. We calculated the monthly leptospirosis admission probabilities from the precipitation and maximum temperature variables. The month of February showed the highest probability, although values were also high during the spring months. The month of February also showed the highest number of hospital admissions. Another interesting result is that, for every 20 mm precipitation, there was an average increase of 31.5% in hospital admissions. Additionally, the relative risk of leptospirosis varied from 1.1 to 2.0 when the precipitation varied from 20 to 140 mm.

Epidemiological Aspects of Human Brucellosis and Leptospirosis Outbreaks in Korea

In order to compare the epidemiological aspects of human brucellosis (HB) and human leptospirosis (HL) outbreaks in Korea, we have analyzed the current state both of the disease incidence and related risk factors. A total 651 cases of HB occurred between 2001 and 2010 in Korea, and the average incidence rate per 100,000 populations was 0.15. A total 1,153 of HL cases occurred during the same period, and its rate was 0.24. While most of the HB prevalence occurred in the whole year round, prevalence in summer was more prominent for HB (p < 0.01), while outbreaks in autumn was more frequent for HL (p < 0.01). Geographical distribution HB cases were eastern and western regions of the rural (88.6% of total) in the Korean peninsula, showing higher outbreaks than other areas, while HL occurred in easterly regions (64.5%). Significantly more males were infected in both HB (86.8%) and HL (59.5%) than those of females in both HB (13.1%) and HL (41.1%), respectively (p < 0.01). The distribution by age groups were different between HB and HL, while the outbreaks over 62.1% of the cases of HB occurred in 40- to 59-year-old age group, and that of HL was clearly showing a high incidence in the elderly age over 60-year-old (59.6%) (p < 0.01). In both diseases, elderly people especially in famers showed a very high incidence rate (57.9% of HB and 52.2% of HL), which is possible due to increased outdoor activities and a decreased number of young people in those areas. The occupational distribution of HB cases was famers, veterinarians, dairyman and others, and those of HL cases were broad. In conclusion, the difference between HB and HL risk factors reflects the different influence of host/vector, climate, and geographical and environmental characteristics in the epidemiological patterns.KeywordsHuman brucellosis and leptospirosis; Epidemic aspects; Risk factors

Acute kidney injury in human leptospirosis: an immunohistochemical study with pathophysiological correlation

Tubulointerstitial nephritis is a common clinicopathological finding in leptospirosis. Clinically, nonoliguric acute kidney injury (AKI), hypokalemia, sodium, and magnesium wasting frequently occur in leptospirosis. The exact mechanisms of renal involvement remain largely unclear. Immunohistochemistry to detect expression of the endogenous sodium/hydrogen exchanger isoform 3 (NHE 3), aquaporin 1 and 2, alpha-Na(+)K(+)ATPase, and sodium-potassium-chloride cotransporter in its NKCC2 isoform was performed on kidneys removed during autopsy of human leptospirosis cases and kidneys removed during autopsy of human non-leptospirosis cases with and without evidence of acute tubular necrosis (ATN). A decrease in NHE 3, aquaporin 1, and alpha-Na(+)K(+)ATPase expression occurred in proximal convoluted tubule cells. Expression of aquaporin 1 was preserved along the descending thin limb of the loop of Henle in the outer medulla. alpha-Na(+)K(+)ATpase expression was essentially preserved in the distal tubules, i.e., the thick ascending limb of the loop of Henle, macula densa, and distal convoluted tubule. Aquaporin 2 expression in the collecting tubules was enhanced compared to those of non-leptospirotic kidneys. NKCC2 cotransport isoform was expressed in the thick ascending limb of the loop of Henle and was essentially preserved in leptospirotic kidneys. Primary injury of the proximal convoluted tubules is regarded as the hallmark of the kidney in leptospirosis. Sodium and water transport are particularly affected with increased distal potassium excretion, hypokalemia, and polyuria. Enhanced expression of aquaporin 2 in medullary collecting tubules is probably an attempt to retain water during the nonoliguric phase of renal failure.

Incidence of Leptospirosis in Mazandaran Province, North of Iran: A One Year Survey

The aim of this study was to provide the first report of incidence of leptospirosis and to determine the epidemiology of this zoonotic disease in Mazandaran Province, North of Iran. In the period of one year, from April 2007 to April 2008, forty seven confirmed reports of human cases of leptospirosis was received by Mazandaran Health Centre from local hospitals and leptospirosis laboratory. The annual incidence rate for the total population was 1.6 per 100,000 person-year. The majority of cases were males (84.1%). The maximum number of cases was seen to occur between 40 and 59 years of age. Seasonal outbreak of leptospirosis was seen in summer (70.3%). Farmers (57.4%) more frequently affected by disease than other occupations. In conclusion, it is necessary for medical practitioner to pay attention to leptospirosis in farmers during summer season.

Human leptospirosis cases and the prevalence of rats harbouring Leptospira interrogans in urban areas of Tokyo, Japan

Thirteen patients with leptospirosis were identified, as confirmed by laboratory analysis during the last 5 years in our laboratory, who came from urban areas of Tokyo, Japan. All of the patients came into contact with rats before the onset of illness. Seventeen per cent of Norway rats captured in the inner cities of Tokyo carried leptospires in their kidneys. Most of these rat isolates were Leptospira interrogans serovar Copenhageni/Icterohaemorrhagiae. Antibodies against these serovars and their DNA were detected in the patients. This suggests that rats are important reservoirs of leptospirosis, and that rat-borne leptospires occur in urban areas of Tokyo.

Leptospirosis in Taiwan, 2001–2006

To the Editor: Leptospirosis is a zoonotic disease that has now been identified as an emerging infectious disease (1,2). It is caused by pathogenic spirochetes of the genus Leptospira. The natural hosts for Leptospira spp. come from a variety of species (2–4), of which the rodent is the most important reservoir (4,5). The incubation period range for leptospirosis is usually 5–14 days, with a range of 2–30 days (4). Leptospirosis is a disease of humid tropical and subtropical countries. According to the World Health Organization (4), probable leptospirosis incidence ranges from ≈0.1–1 case/100,000 population/year in temperate climates to 10–100 cases/100,000 population/year in humid tropical climates. Leptospirosis epidemics are often related to heavy rainfall and flooding (1,6,7). Because of its climate, Taiwan may be at high risk for leptospirosis. We therefore investigated human leptospirosis in Taiwan and the relationship between leptospirosis incidence and rainfall pattern. Taiwan is a medium-sized archipelago in East Asia; the Tropic of Cancer runs through its center. The northern part of Taiwan is subtropical; the southern part is tropical. Taiwan lies in the path of many tropical storms and typhoons that bring extremely heavy rainfall usually during July–September. The annual “plum rain” season in May and June also brings a lot of precipitation. Because of its tropical and subtropical marine climate, Taiwan enjoys rich agricultural productivity throughout the year, which is favorable for rodent infestations (8,9). In Taiwan, reported cases of leptospirosis have been investigated by the Centers for Disease Control since 2001. Leptospirosis should be suspected in patients who have fever; headache; myalgia; abdominal pain; prostration; conjunctival suffusion; meningeal irritation and aseptic meningitis; anuria, oliguria, or proteinuria; jaundice; acute renal insufficiency; or gastrointestinal or lung hemorrhage. Patients with suspected leptospirosis are reported by physicians to Taiwan’s Centers for Disease Control through the Notifiable Disease Surveillance System, after which local health bureaus collect urine and blood samples for confirmation by serologic testing. Urine and blood samples from patients with clinically suspected leptospirosis are inoculated into Ellinghausen-McCullough-Johnson-Harris culture medium plus 5-fluorouracil and incubated at 30°C for 8–12 weeks. Cultures are examined by dark-field microscopy every week. Alternatively, latex agglutination assay may be used for rapid serologic diagnosis of serum from patients with clinically suspected leptospirosis (10). Samples with positive latex agglutination assay results should be confirmed by microscopic agglutination test (MAT). An antibody titer >100 as determined by MAT is regarded as a probable case of leptospirosis. The local health bureau again collects patients’ serum during the convalescent phase of illness for confirmation by MAT. A laboratory-confirmed case is defined as the isolation of leptospires from urine and blood or a 4-fold increase in antibody titer between acute- and convalescent-phase samples. During 2001–2006, of 7,733 suspected human cases of leptospirosis, 291 cases were confirmed. The major serotype identified was L. santarosai serovar Shermani. The mean annual incidence was 0.21 cases/100,000 population. The laboratory-confirmed cases were observed in Taiwan, mostly in male patients (83.5%) (Figure, panel A). Cases occurred in all age groups but were more common (90%) in those 25–74 years of age. Age-specific incidence was highest for persons 55–64 years of age; mean annual incidence was 0.57/100,000 population. Figure Leptospirosis cases (N = 291) in Taiwan, 2001–2006. A) Distribution by age and sex. B) Relationship between rainfall and leptospirosis incidence. Data represent averages for each month during the 6 years. Rainfall data from the Central Weather Bureau of the Republic of China showed typically high rainfall (252–433 mm/month) in Taiwan during May–September. Heavy rains were followed by an increase in laboratory-confirmed cases of leptospirosis (Figure, panel B); June–October accounted for 60% of cases, with a higher incidence of 0.022–0.028 cases/100,000 population. In October–December, monthly rainfall was below average (201.1 mm/month), but leptospirosis incidence was above average (0.018/100,000). Specifically, 25 (74%) of leptospirosis cases in October, 10 (42%) in November, and 4 (19%) in December were likely associated with several days of heavy rainfall from typhoons. Therefore, the typhoons may be the reason for high incidence in October–December. The annual incidence of leptospirosis in Taiwan is relatively lower than that in other countries with tropical or subtropical climates. Our study does not conclusively document the reason for lower incidence, although it does suggest an association between amount of rainfall and incidence. An understanding of the relationship between leptospirosis incidence and rainfall is indispensable for implementing appropriate preventive measures.

Lsa21, a novel leptospiral protein binding adhesive matrix molecules and present during human infection

BackgroundIt has been well documented over past decades that interaction of pathogens with the extracellular matrix (ECM) plays a primary role in host cell attachment and invasion. Adherence to host tissues is mediated by surface-exposed proteins expressed by the microorganisms during infection. The mechanisms by which pathogenic leptospires invade and colonize the host remain poorly understood since few virulence factors contributing to the pathogenesis of the disease have been identified. Whole-genome sequencing analysis of L. interrogans allowed identification of a repertoire of putative leptospiral surface proteins.ResultsHere, we report the identification and characterization of a new leptospiral protein that exhibits extracellular matrix-binding properties, called as Lsa21 (leptospiral surface adhesin, 21 kDa). Compatible with its role in adhesion, the protein was shown to be surface-exposed by indirect immunofluorescence. Attachment of Lsa21 to laminin, collagen IV, and plasma fibronectin was specific and dose dependent. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. The gene coding for Lsa21 is present in pathogenic strains belonging to the L. interrogans species but was not found in the saprophytic L. biflexa serovar Patoc strain Patoc 1. Loss of gene expression occurs upon culture attenuation of pathogenic strains. Environmental factors such as osmolarity and temperature affect Lsa21 expression at the transcriptional level. Moreover, anti-Lsa21 serum labeled liver and kidney tissues of human fatal cases of leptospirosis.ConclusionOur data suggest a role of Lsa21 in the pathogenesis of leptospirosis.

Anticorpos anti-Leptospira em pacientes de Mato Grosso do Sul com suspeita clínica de dengue ou hepatite viral

In view of the lack of clinical and epidemiological data on human leptospirosis in the State of Mato Grosso do Sul, and the possibility of confounding it with other diseases, sera from patients with a preliminary clinical suspicion of dengue or viral hepatitis but without laboratory confirmation were examined by means of microscopic seroagglutination techniques for leptospirosis. The seroreactivity rates among the samples with clinically suspected dengue or viral hepatitis were 15.9% and 9%. The most frequent serovar was Hurstbridge (70.4%) and the serovar with the highest titer was Canicola (1:51,200). No association was found between seropositivity and the patients sex, age or occupation. This study demonstrated that, although the present notifications of leptospirosis cases in Mato Grosso do Sul are negligible, the prevalence of antibodies was high in the groups investigated. Therefore, the hypothesis that there is undernotification of human leptospirosis cases in this State and difficulties in the differential diagnosis between dengue and viral hepatitis should be considered.

The incidence and clinical characteristics of the immune phase eye disease in treated cases of human leptospirosis

Uveitis is increasingly being reported from south India following epidemics of leptospirosis. The incidence of eye involvement in treated patients has not been investigated properly in prospective studies. To determine the incidence, clinical spectrum and risk factors for Leptospiral uveitis in antibiotic-treated patients. A prospective cohort study conducted among the patients treated for leptospirosis at Calicut Medical College between July and November 2002 and a seroprevalence study among sewage workers. As many as 360 patients admitted with suspected leptospirosis were studied by clinical examination, baseline ophthalmic survey, laboratory investigations and Leptospira serology (Microagglutination test - MAT; and IgM and IgG using ELISA) during the epidemic. Of the 282 seropositive and antibiotic-treated cases, 174 patients who completed 30 months of regular follow-up were analyzed. A cross-sectional serosurveillance also was performed among 50 sewage workers to determine the baseline MAT titer. Univariate analysis and logistic regression. Thirty-two patients (18.4%) developed the eye disease during follow-up. The mean age was 43.9 years and the sex ratio was equal. Twenty-one patients (65.6%) had anterior uveitis. Only six patients (18.8%) had visual symptoms. Median duration for developing anterior uveitis was 4 weeks. Recurrent uveitis was not seen following treatment. None had vision-threatening eye disease. Clinical and laboratory abnormalities during the acute phase did not pose risk for development of the eye disease later. Forty-six sewage workers (92%) showed a MAT titer of 1/25. Uveitis is common following acute leptospirosis. Antibiotic-treated patients during the acute phase of illness developed only mild uveitis.

A model of Leptospirosis infection in an African rodent to determine risk to humans: Seasonal fluctuations and the impact of rodent control

Human leptospirosis ( Leptospira spp. infection) is a worldwide public health problem that is of greatest concern for humid tropical and subtropical regions. The magnitude of the problem in these areas is larger because of the climatic and environmental conditions the bacterium face outside their hosts but also because of the frequency of contacts between people and sources of infection. Rodents are thought to play the most important role in the transmission of human leptospirosis. We here model the dynamics of infection in an African rodent ( Mastomys natalensis) that is thought to be the principal source of infection in parts of Tanzania. Our model, representing the climatic conditions in central Tanzania, suggests a strong seasonality in the force of infection on humans with a peak in the abundance of infectious mice between January and April in agricultural environments. In urban areas the dynamics are predicted to be more stable and the period of high numbers of infectious animals runs from February to July. Our results indicate that removal of animals by trapping rather than reducing the suitability of the environment for rodents will have the greater impact on reducing human cases of leptospirosis.

Immunohistochemical and in situ hybridization studies of the liver and kidney in human leptospirosis

An in situ hybridization (ISH) assay for the detection of leptospiral DNA in tissues was described and its diagnostic and pathogenetic usefulness in combination with immunohistochemistry (IHC) was evaluated in formalin-fixed, paraffin-embedded liver and kidney samples from human fatal cases of leptospirosis. IHC assays with anti-E-cadherin antibodies assessed the liver-plate disarray frequently observed in leptospirosis. Immunohistochemistry detected leptospiral antigen (LAg) in macrophages, both in human liver and kidney. In guinea pigs, in addition to these findings, staining on cell membranes of hepatocytes and, occasionally, in apical membrane of kidney tubular cells was demonstrated. Positive ISH signal was observed chiefly in the nuclei of human hepatocytes and in the cytoplasm and nuclei of liver cells of experimentally infected guinea pigs. Loss of E-cadherin membrane expression is associated with liver-plate disarray. These findings were discussed in the contention that, in leptospirosis, cell membrane damage might be important for the pathogenesis of the disease. Finally, it was suggested that both IHC and/or ISH might be used for both diagnostic and research purposes.

Leptospira broomii sp. nov., isolated from humans with leptospirosis

Isolates of Leptospira from two human cases of leptospirosis in Denmark and France were studied using DNA-DNA relatedness, G + C content, 16S rRNA gene sequence data and pulsed-field gel electrophoresis. These isolates differed from previously described species of Leptospira and are defined as Leptospira broomii sp. nov. The type strain is 5399T (= ATCC BAA-1107T = KIT 5399T).

Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species

BackgroundLeptospira are the parasitic bacterial organisms associated with a broad range of mammalian hosts and are responsible for severe cases of human Leptospirosis. The epidemiology of leptospirosis is complex and dynamic. Multiple serovars have been identified, each adapted to one or more animal hosts. Adaptation is a dynamic process that changes the spatial and temporal distribution of serovars and clinical manifestations in different hosts. Serotyping based on repertoire of surface antigens is an ambiguous and artificial system of classification of leptospiral agents. Molecular typing methods for the identification of pathogenic leptospires up to individual genome species level have been highly sought after since the decipherment of whole genome sequences. Only a few resources exist for microbial genotypic data based on individual techniques such as Multiple Locus Sequence Typing (MLST), but unfortunately no such databases are existent for leptospires.ResultsWe for the first time report development of a robust MLST method for genotyping of Leptospira. Genotyping based on DNA sequence identity of 4 housekeeping genes and 2 candidate genes was analyzed in a set of 120 strains including 41 reference strains representing different geographical areas and from different sources. Of the six selected genes, adk, icdA and secY were significantly more variable whereas the LipL32 and LipL41 coding genes and the rrs2 gene were moderately variable. The phylogenetic tree clustered the isolates according to the genome-based species.ConclusionThe main advantages of MLST over other typing methods for leptospires include reproducibility, robustness, consistency and portability. The genetic relatedness of the leptospires can be better studied by the MLST approach and can be used for molecular epidemiological and evolutionary studies and population genetics.