Nucleic acid and antigen detection tests for leptospirosis.

BACKGROUND: Early diagnosis of leptospirosis may contribute to the effectiveness of antimicrobial therapy and early outbreak recognition. Nucleic acid and antigen detection tests have the potential for early diagnosis of leptospirosis. With this systematic review, we assessed the sensitivity and specificity of nucleic acid and antigen detection tests. OBJECTIVES: To determine the diagnostic test accuracy of nucleic acid and antigen detection tests for the diagnosis of human symptomatic leptospirosis. SEARCH METHODS: We searched electronic databases including MEDLINE, Embase, the Cochrane Library, and regional databases from inception to 6 July 2018. We did not apply restrictions to language or time of publication. SELECTION CRITERIA: We included diagnostic cross-sectional studies and case-control studies of tests that made use of nucleic acid and antigen detection methods in people suspected of systemic leptospirosis. As reference standards, we considered the microscopic agglutination test alone (which detects antibodies against leptospirosis) or in a composite reference standard with culturing or other serological tests. Studies were excluded when the controls were healthy individuals or when there were insufficient data to calculate sensitivity and specificity. DATA COLLECTION AND ANALYSIS: At least two review authors independently extracted data from each study. We used the revised Quality Assessment of Diagnostic Accuracy Studies tool (QUADAS-2) to assess risk of bias. We calculated study-specific values for sensitivity and specificity with 95% confidence intervals (CI) and pooled the results in a meta-analysis when appropriate. We used the bivariate model for index tests with one positivity threshold, and we used the hierarchical summary receiver operating characteristic model for index tests with multiple positivity thresholds. As possible sources of heterogeneity, we explored: timing of index test, disease prevalence, blood sample type, primers or target genes, and the real-time polymerase chain reaction (PCR) visualisation method. These were added as covariates to the meta-regression models. MAIN RESULTS: We included 41 studies evaluating nine index tests (conventional PCR (in short: PCR), real-time PCR, nested PCR, PCR performed twice, loop-mediated isothermal amplification, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, immunochromatography-based lateral flow assay, and dipstick assay) with 5981 participants (1834 with and 4147 without leptospirosis). Methodological quality criteria were often not reported, and the risk of bias of the reference standard was generally considered high. The applicability of findings was limited by the frequent use of frozen samples. We conducted meta-analyses for the PCR and the real-time PCR on blood products.The pooled sensitivity of the PCR was 70% (95% CI 37% to 90%) and the pooled specificity was 95% (95% CI 75% to 99%). When studies with a high risk of bias in the reference standard domain were excluded, the pooled sensitivity was 87% (95% CI 44% to 98%) and the pooled specificity was 97% (95% CI 60% to 100%). For the real-time PCR, we estimated a summary receiver operating characteristic curve. To illustrate, a point on the curve with 85% specificity had a sensitivity of 49% (95% CI 30% to 68%). Likewise, at 90% specificity, sensitivity was 40% (95% CI 24% to 59%) and at 95% specificity, sensitivity was 29% (95% CI 15% to 49%). The median specificity of real-time PCR on blood products was 92%. We did not formally compare the diagnostic test accuracy of PCR and real-time PCR, as direct comparison studies were lacking. Three of 15 studies analysing PCR on blood products reported the timing of sample collection in the studies included in the meta-analyses (range 1 to 7 days postonset of symptoms), and nine out of 16 studies analysing real-time PCR on blood products (range 1 to 19 days postonset of symptoms). In PCR studies, specificity was lower in settings with high leptospirosis prevalence. Other investigations of heterogeneity did not identify statistically significant associations. Two studies suggested that PCR and real-time PCR may be more sensitive on blood samples collected early in the disease stage. Results of other index tests were described narratively. AUTHORS' CONCLUSIONS: The validity of review findings are limited and should be interpreted with caution. There is a substantial between-study variability in the accuracy of PCR and real-time PCR, as well as a substantial variability in the prevalence of leptospirosis. Consequently, the position of PCR and real-time PCR in the clinical pathway depends on regional considerations such as disease prevalence, factors that are likely to influence accuracy, and downstream consequences of test results. There is insufficient evidence to conclude which of the nucleic acid and antigen detection tests is the most accurate. There is preliminary evidence that PCR and real-time PCR are more sensitive on blood samples collected early in the disease stage, but this needs to be confirmed in future studies.

Are Pathogenic Leptospira Species Agents of Community-Acquired Pneumonia? Case Reports of Leptospirosis Presenting as Pneumonia.

We report four Indonesian cases meeting the clinical and radiological criteria for community-acquired pneumonia and other findings suggestive of leptospirosis. Quantitative PCR (qPCR) analyses of serum and urine samples and serology confirmed the diagnosis of leptospirosis in each. Results of qPCR analysis of throat swabs were concordant with those obtained with acute-phase serum samples, which suggests its potential for use as a noninvasive diagnostic tool for leptospirosis.

The role of leptospirosis reference laboratories.

The general goal of reference centres is to support the community, from diagnostic laboratories to research institutions, in the execution of their work by providing reference strains and reagents and giving instructions and recommendations to individual colleagues and national and international organisations on a wide variety of issues. There are different levels of reference centres, from local to international, with an increasing package of tasks and responsibilities. Local reference centres might limit activities to diagnostic confirmation by applying standard testing, while international reference centres cover a wider range of activities from design, validation and harmonisation of diagnostic and reference technologies to international monitoring associated with recommendations on the global burden and distribution of leptospirosis and its prevention and control to national and international health decision makers. This chapter focusses on four major pillars constituting reference tasks in addition to the obvious provision of reference substances, i.e. Research and training, Diagnosis, Identification of Leptospira and Surveillance. Due to financial and organisational constraints, reference centres are restricted in their capacity for basic research and consequently focus on applied research into various aspects of leptospirosis. They offer training, either individually or groupwise, that might vary from standard technologies to novel sophisticated methodologies, depending on the need and requests of the trainee. Most reference centres are involved in the confirmation of preliminary diagnosis obtained at peripheral levels, such as local hospitals and health centres, while other major activities involve the design and validation of diagnostics, their international harmonisation and quality assurance. Identification of causative Leptospira strains (or serovars) is key to the identification of infection sources and is critical for surveillance. Hence, reference centres also focus on the development, application and provision of methods that are required for unambiguous characterisation of new and recognised Leptospira strains and the maintenance of the integrity of strain collections. In line with their central role, reference centres are frequently associated with local, national and/or international surveillance activities linked to an advisory role and the production of guidelines. Such surveillance activities usually comprise collation of morbidity and mortality data, signalling of outbreaks and the investigation of infection sources and risks.

Marked increase in leptospirosis infections in humans and dogs in the Netherlands, 2014.

In the Netherlands, 97 human leptospirosis cases were notified in 2014. This represents a 4.6-fold increase in autochthonous cases (n = 60) compared with the annual average between 2010 and 2013. Most cases had symptom onset between June and November. This marked increase in humans coincided with an increase of leptospirosis in dogs. In 2014, 13 dogs with leptospirosis were reported, compared with two to six dogs annually from 2010 to 2013. The majority of the autochthonous cases (n = 20) were linked to recreational exposure, e.g. swimming or fishing, followed by occupational exposure (n = 15). About sixty per cent (n = 37) of the autochthonous cases were most likely attributable to surface water contact, and 13 cases to direct contact with animals, mainly rats. A possible explanation for this increase is the preceding mild winter of 2013-2014 followed by the warmest year in three centuries, possibly enabling rodents and Leptospira spp. to survive better. A slight increase in imported leptospirosis was also observed in Dutch tourists (n = 33) most of whom acquired their infection in Thailand (n = 18). More awareness and early recognition of this mainly rodent-borne zoonosis by medical and veterinary specialists is warranted.

Human leptospirosis trends, the Netherlands, 1925-2008.

To increase knowledge of leptospirosis in the Netherlands and identify changing trends of this disease over time, we analyzed historical passive surveillance reports for an 84-year period (1925-2008). We found that 2,553 mainly severe leptospirosis cases were diagnosed (average annual incidence rate 0.25 cases/100,000 population). The overall case-fatality rate for patients with reported leptospirosis was 6.5% but decreased over the period, probably because of improved treatment. Ninety percent of reported leptospirosis cases were in male patients. Most autochthonous leptospirosis infections were associated with recreational exposures, but 15.5% of the cases were attributed to accidents that resulted in injury and to concomitant water contact. Since the end of the 1950s, the proportion of imported infections gradually increased, reaching 53.1% of the total during 2005-2008. Most (80.1%) imported infections were associated with sporting and adventurous vacation activities.

Molecular and serological characterization of Leptospira interrogans serovar Canicola isolated from dogs, swine, and bovine in Brazil.

The identification of Leptospira clinical isolates through genotyping and serotyping, besides the recognition of its reservoirs, are important tools for understanding the epidemiology of leptospirosis, and they are also keys for identifying new species and serovars. Fourteen clinical isolates from animals were characterized by means of single enzyme amplified length polymorphism, variable number of tandem repeat analysis, pulsed field gel electrophoresis, and serotyping. All isolates were identified as Leptospira interrogans, serovar Canicola. Infections by this serovar occur in urban regions, where dogs represent the main maintenance hosts, whereas bovine and swine may act as reservoirs of serovar Canicola in rural areas. Both urban and rural aspects of leptospirosis, and the role of domestic animals as maintenance hosts, cannot be neglected in developing and developed countries.

Surveillance for leptospirosis in the Americas, 1996-2005: a review of data from ministries of health.

OBJECTIVE: To characterize current leptospirosis reporting practices in the Americas. METHODS: Information was collected from the official websites of national ministries of health from the Americas region and two international organizations; personal communications; and three international morbidity databases. For all sources other than the morbidity databases, the review was limited to official reports citing clinically suspected and laboratory confirmed leptospirosis cases or deaths during the period 1996-2005. RESULTS: A total of 73 out of 1 644 reports met the selection criteria and were included in the analysis. Published leptospirosis data were available from half of the countries/sovereign territories (24 out of 48), and 18 of them had mandatory notification policies for leptospirosis. The sum of the median number of leptospirosis cases notified annually by the 24 countries/territories was 4 713.5, but just three countries (Brazil, Costa Rica, and Cuba) accounted for 83.1% (3 9cas20 es) of the notifications. Eight (16.7%) countries reported deaths due to leptospirosis. The sum of the median number of deaths reported annually for the eight countries was 380, but 349 (91.8%) were reported by Brazil. CONCLUSIONS: Notification practices in the Americas for leptospirosis are limited. Therefore, the numbers of cases and deaths reported are not representative for the region. The lack of leptospirosis data for many countries/territories may reflect weaknesses in certain aspects of national surveillance systems, including mandatory reporting policies, clinical laboratory infrastructure for performing case confirmation, and capacity to collect reported cases. Improved surveillance of leptospirosis cases and deaths in the Americas is needed to allow monitoring of regional epidemiological patterns and to estimate the burden of this important disease.

Mutations affecting Leptospira interrogans lipopolysaccharide attenuate virulence.

Leptospira interrogans is the causative agent of leptospirosis. Lipopolysaccharide (LPS) is the major outer membrane component of L. interrogans. It is the dominant antigen recognized during infection and the basis for serological classification. The structure of LPS and its role in pathogenesis are unknown. We describe two defined mutants of L. interrogans serovar Manilae with transposon insertions in the LPS locus. Mutant M895 was disrupted in gene la1641 encoding a protein with no known homologues. M1352 was disrupted in a gene unique to serovar Manilae also encoding a protein of unknown function. M895 produced truncated LPS while M1352 showed little or no change in LPS molecular mass. Both mutants showed altered agglutination titres against rabbit antiserum and against a panel of LPS-specific monoclonal antibodies. The mutants were severely attenuated in virulence via the intraperitoneal route of infection, and were cleared from the host animal by 3 days after infection. M895 was also highly attenuated via the mucosal infection route. Resistance to complement in human serum was unaltered for both mutants. While complementation of mutants was not possible, the attenuation of two independently derived LPS mutants demonstrates for the first time that LPS plays an essential role leptospiral virulence.

Evaluation of the use of selective PCR amplification of LPS biosynthesis genes for molecular typing of leptospira at the serovar level.

Leptospirosis is an important epidemic zoonosis worldwide. Currently, there are more than 250 Leptospira pathogenic serovars known that can potentially infect humans. Conventional classification of leptospires with the serovar as the basic taxon, based on serological recognition of lipopolysaccharide (LPS) composition does not correlate well with species determination, based on general genomic features. Here, we investigate the selective amplification of polymorphic regions from the LPS biosynthesis loci (rfb) as a potential tool for serovar typing of Leptospira interrogans species. Eight pairs of primers were designed to target six ORFs from the rfb operon with varying levels of sequence polymorphism. They were tested both separately and multiplexed. Half of these primer pairs produced serovar-specific amplicons, allowing the identification of some specific serovars and also groups of serovars. It was shown that the serovar classification of Leptospira can be accessed by selective amplification of rfb operons in some cases, which may permit a parallel between the serological and the genomic classifications of Leptospira. As a conclusion, the selective amplification of rfb generated promising and already useful results, but it appears necessary to characterize a larger variety of Leptospira genomes or rfb operons to fully develop this method.

Interactions of leptospira with environmental bacteria from surface water.

Leptospires can persist for months in nutrient-poor aqueous environments prior to transmission to a mammalian host. Interactions with environmental bacteria and biofilm formation are possible mechanisms of persistence of leptospires in the environment. Bacteria isolated from rivers in the Ecuadorian rainforest were tested for their ability to support leptospiral viability. We found that co-culture with Sphingomonas spp., but not Flavobacterium spp. or Delftia spp., enabled survival of L. biflexa and L. meyeri for up to a year in distilled water. We also found that L. interrogans biofilms formed in distilled water contained viable organisms that rapidly dispersed into the planktonic phase in the presence of nutrients in serum or EMJH medium. These data inform our understanding of leptospiral survival strategies that enable long-term persistence in nutrient-poor conditions yet allow rapid mobilization when nutrients become available.

Are Pathogenic Leptospira a Possible Cause of Aseptic Meningitis in Suspected Children in Sudan?

INTRODUCTION: Clinical presentations of leptospirosis are diverse, with meningitis easily confused with other microbial causes. We aimed to investigate the involvement of pathogenic leptospira in the cerebrospinal fluid (CSF) of meningitis-suspected children in Sudan. METHODS: A total of 153 CSF specimens were collected over 5 months from patients attending a reference pediatric hospital in Omdurman, Sudan. All patients had provisionally been diagnosed with meningitis on admission. Demographic, clinical, and conventional laboratory findings were obtained. DNA was extracted using a QIAamp mini kit, and the secY gene investigated using real-time PCR. RESULTS: Nine of 153 (6%) CSF specimens were positive for pathogenic leptospiral DNA. All these patients were male (seven infants and two toddlers aged ˂4 years). Typical conventional laboratory findings for aseptic meningitis (ie, CSF turbidity/pleocytosis, normal or reduced CSF glucose, normal or elevated proteins) were seen in five (56%). All patients presented with fever and seizures, 56% vomiting and stiff neck, and 29% bulging fontanel. Most (67%) patients presented in summer (March to May). Polymicrobial infections were identified in three patients (33%). CONCLUSION: We conclude that pathogenic leptospira are probably a common cause of meningitis in children in Sudan; therefore, we recommend including leptospirosis in the differential diagnosis of CNS infections and other undifferentiated febrile illnesses in this country.

Draft Genome Sequence of a Leptospira kirschneri Serovar Mozdok Type 2 Strain Isolated from a Horse in Portugal.

Leptospira kirschneri is an agent causing leptospirosis in animals and humans. We report the draft genome sequence of Leptospira kirschneri serovar Mozdok type 2 strain Horse 112, comprising 485 contigs and having a genome size of 4,301,784 bp. This genome will facilitate studying important mechanisms for clinical outcomes.

Genetic diversity of pathogenic leptospires from wild, domestic and captive host species in Portugal.

Leptospirosis is a neglected zoonotic disease of worldwide distribution with a significant veterinary and public health impact. It is caused by pathogenic bacteria of the genus Leptospira. The availability of effective tools to accurately identify and type leptospires is of utmost importance for the diagnosis of the disease and for assessing its epidemiology. Several multi-locus sequence typing (MLST) approaches were described for the typing of worldwide isolates of Leptospira but an extensive agreement towards the adoption of a unique consensus scheme for this agent is still lacking. Most genotyped strains originate from Asian and South American countries, with a minority originating from Europe (being most countries represented only by one or a few isolates). The knowledge of the diversity of circulating leptospires is the key to understanding the disease transmission and its zoonotic implications. In this study, we revisited the taxonomy of several isolates of pathogenic Leptospira obtained from domestic, wild and captive animals in Portugal, between 1990 and 2012. A selection of these isolates was genotyped using two previously published MLST schemes. A total of seven distinct sequence types (STs) were detected among the Portuguese isolates with two STs representing L. borgpetersenii (ST149 and ST152), two STs representing L. kirschneri (ST117 and ST100) and three STs representing L. interrogans (ST17, ST24 and ST140). Global widespread (and maybe more virulent) Leptospira genotypes seem to circulate in Portugal, particularly the L. interrogans ST17 isolates which are associated with several outbreaks of leptospirosis among humans and animals in different regions of the world. This study contributes to the enrichment of the global MLST databases with a new set of allele and sequence type information also providing novel data on circulating Leptospira serovars in Portugal.

Murine typhus and leptospirosis as causes of acute undifferentiated fever, Indonesia.

To investigate rickettsioses and leptospirosis among urban residents of Semarang, Indonesia, we tested the blood of 137 patients with fever. Evidence of Rickettsia typhi, the agent of murine typhus, was found in 9 patients. Another 9 patients showed inconclusive serologic results. Thirteen patients received a diagnosis of leptospirosis. No dual infections were detected.

Arenal, a new Leptospira serovar of serogroup Javanica, isolated from a patient in Costa Rica.

Leptospirosis is a worldwide distributed zoonotic disease caused by pathogenic spirochetes of the genus Leptospira. The basic taxon of Leptospira is the serovar. Currently, nearly 300 serovars have been identified. Leptospirosis is particularly prevalent in warm and humid tropical regions where conditions for transmission and survival of pathogenic leptospires in the environment are optimal. Leptospirosis probably constitutes a serious veterinary and public health problem in Central America but solid figures are missing. To determine distribution of leptospirosis in Costa Rica and to identify locally circulating pathogenic serovars, we performed a sentinel-based study, isolating and characterizing leptospires from patients attending hospitals. Strain MAVJ 401 was isolated from a hospitalized patient in the Alajuela province. The isolate produced agglutination titers notably with reference rabbit antisera against serovars of serogroup Javanica but appeared serologically unique in the standard Cross Agglutinin Absorption Test. Therefore, MAVJ 401 was considered to represent a new serovar, designated Arenal, of the serogroup Javanica. Genotypic analysis revealed that strain MAVJ 401 belongs to Leptospira santarosai, a species that almost exclusively occurs in Latin America. This is not a unique finding of an exotic serovar. Recent isolates from severely ill patients in the same region appeared to be identical to Arenal. We have identified a novel highly virulent serovar from a patient in Costa Rica that is common in this area, thus posing a threat for the local public and veterinary health.

Seroepidemiology and serological follow-up of anti-leptospiral IgG in children in Southern Vietnam.

A follow-up study was conducted with 23 months interval to investigate the seroepidemiology and persistence of Leptospira IgG antibodies among healthy children in Binh Thuan province, Southern Vietnam. Sera from 262 children (7-13 years of age) were collected and analysed with a commercially available enzyme-linked immunosorbent assay (ELISA) for Leptospira IgG. Seroconversion was observed in 10.4% (22 of 211, 95% CI: 5.6-26.7) of the children, of whom 18 (8.5%) had probably and four (1.9%) had certainly been exposed to Leptospira. Based on the reduction of sero-negatives of 1.9% among children who have been certainly exposed, the annual seroconversion rate, a measure of the incidence rate of Leptospira infections, corresponds to 0.99% (95% CI: 0.39-2.52). In 61% (31 of 51, 95% CI: 47.1-73.0) of the children with past-infection, Leptospira IgG antibodies remain detectable after 2 years. Data from this study indicate that IgG antibody responses against Leptospira may persist at least for 2 years in children without manifestations of leptospirosis. Results of study uncover the true incidence of leptospirosis infection, the dynamics of waxing and waning antibody concentrations and points at a larger burden of clinically non-significant Leptospira infections in Southern Vietnam. This also indicates background reactivity for serological testing and thus serological result of a single serum sample must be carefully interpreted.

Assessment of animal hosts of pathogenic Leptospira in northern Tanzania.

Leptospirosis is a zoonotic bacterial disease that affects more than one million people worldwide each year. Human infection is acquired through direct or indirect contact with the urine of an infected animal. A wide range of animals including rodents and livestock may shed Leptospira bacteria and act as a source of infection for people. In the Kilimanjaro Region of northern Tanzania, leptospirosis is an important cause of acute febrile illness, yet relatively little is known about animal hosts of Leptospira infection in this area. The roles of rodents and ruminant livestock in the epidemiology of leptospirosis were evaluated through two linked studies. A cross-sectional study of peri-domestic rodents performed in two districts with a high reported incidence of human leptospirosis found no evidence of Leptospira infection among rodent species trapped in and around randomly selected households. In contrast, pathogenic Leptospira infection was detected in 7.08% cattle (n = 452 [5.1-9.8%]), 1.20% goats (n = 167 [0.3-4.3%]) and 1.12% sheep (n = 89 [0.1-60.0%]) sampled in local slaughterhouses. Four Leptospira genotypes were detected in livestock. Two distinct clades of L. borgpetersenii were identified in cattle as well as a clade of novel secY sequences that showed only 95% identity to known Leptospira sequences. Identical L. kirschneri sequences were obtained from qPCR-positive kidney samples from cattle, sheep and goats. These results indicate that ruminant livestock are important hosts of Leptospira in northern Tanzania. Infected livestock may act as a source of Leptospira infection for people. Additional work is needed to understand the role of livestock in the maintenance and transmission of Leptospira infection in this region and to examine linkages between human and livestock infections.

Genomic Comparison Among Global Isolates of L. interrogans Serovars Copenhageni and Icterohaemorrhagiae Identified Natural Genetic Variation Caused by an Indel.

Leptospirosis is a worldwide zoonosis, responsible for more than 1 million cases and 60,000 deaths every year. Among the 13 pathogenic species of the genus Leptospira, serovars belonging to L. interrogans serogroup Icterohaemorrhagiae are considered to be the most virulent strains, and responsible for majority of the reported severe cases. Serovars Copenhageni and Icterohaemorrhagiae are major representatives of this serogroup and despite their public health relevance, little is known regarding the genetic differences between these two serovars. In this study, we analyzed the genome sequences of 67 isolates belonging to L. interrogans serovars Copenhageni and Icterohaemorrhagiae to investigate the influence of spatial and temporal variations on DNA sequence diversity. Out of the 1072 SNPs identified, 276 were in non-coding regions and 796 in coding regions. Indel analyses identified 258 indels, out of which 191 were found in coding regions and 67 in non-coding regions. Our phylogenetic analyses based on SNP dataset revealed that both serovars are closely related but showed distinct spatial clustering. However, likelihood ratio test of the indel data statistically confirmed the presence of a frameshift mutation within a homopolymeric tract of lic12008 gene (related to LPS biosynthesis) in all the L. interrogans serovar Icterohaemorrhagiae strains but not in the Copenhageni strains. Therefore, this internal indel identified can genetically distinguish L. interrogans serovar Copenhageni from serovar Icterohaemorrhagiae with high discriminatory power. To our knowledge, this is the first study to identify global sequence variations (SNPs and Indels) in L. interrogans serovars Copenhageni and Icterohaemorrhagiae.

Environmental risk of leptospirosis infections in the Netherlands: Spatial modelling of environmental risk factors of leptospirosis in the Netherlands.

Leptospirosis is a globally emerging zoonotic disease, associated with various climatic, biotic and abiotic factors. Mapping and quantifying geographical variations in the occurrence of leptospirosis and the surrounding environment offer innovative methods to study disease transmission and to identify associations between the disease and the environment. This study aims to investigate geographic variations in leptospirosis incidence in the Netherlands and to identify associations with environmental factors driving the emergence of the disease. Individual case data derived over the period 1995-2012 in the Netherlands were geocoded and aggregated by municipality. Environmental covariate data were extracted for each municipality and stored in a spatial database. Spatial clusters were identified using kernel density estimations and quantified using local autocorrelation statistics. Associations between the incidence of leptospirosis and the local environment were determined using Simultaneous Autoregressive Models (SAR) explicitly modelling spatial dependence of the model residuals. Leptospirosis incidence rates were found to be spatially clustered, showing a marked spatial pattern. Fitting a spatial autoregressive model significantly improved model fit and revealed significant association between leptospirosis and the coverage of arable land, built up area, grassland and sabulous clay soils. The incidence of leptospirosis in the Netherlands could effectively be modelled using a combination of soil and land-use variables accounting for spatial dependence of incidence rates per municipality. The resulting spatially explicit risk predictions provide an important source of information which will benefit clinical awareness on potential leptospirosis infections in endemic areas.

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

BACKGROUND: Leptospira 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. RESULTS: We 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. CONCLUSION: The 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.