Differential TLR recognition of leptospiral lipid A and lipopolysaccharide in murine and human cells

Leptospira interrogans is a spirochete that is responsible for leptospirosis, a zoonotic disease. This bacterium possesses an unusual LPS that has been shown to use TLR2 instead of TLR4 for signaling in human cells. The structure of its lipid A was recently deciphered. Although its overall hexa-acylated disaccharide backbone is a classical feature of all lipid A forms, the lipid A of L. interrogans is peculiar. In this article, the functional characterization of this lipid A was studied in comparison to whole parental leptospiral LPS in terms of cell activation and use of TLR in murine and human cells. Lipid A from L. interrogans did not coagulate the Limulus hemolymph. Although leptospiral lipid A activated strongly murine RAW cells, it did not activate human monocytic cells. Results obtained from stimulation of peritoneal-elicited macrophages from genetically deficient mice for TLR2 or TLR4 clearly showed that lipid A stimulated the cells through TLR4 recognition, whereas highly purified leptospiral LPS utilized TLR2 as well as TLR4. In vitro experiments with transfected human HEK293 cells confirmed that activation by lipid A occurred only through murine TLR4-MD2 but not through human TLR4-MD2, nor murine or human TLR2. Similar studies with parental leptospiral LPS showed that TLR2/TLR1 were the predominant receptors in human cells, whereas TLR2 but also TLR4 contributed to activation in murine cells. Altogether these results highlight important differences between human and mouse specificity in terms of TLR4-MD2 recognition that may have important consequences for leptospiral LPS sensing and subsequent susceptibility to leptospirosis.     

Influence of the mechanism of transmission of the infective agent on the aetiological structure of leptospiral foci

It has been demonstrated that the differences observed in the aetiological structure of the individual foci of leptospirosis can be explained not only by the affinity of leptospiral serogroups to certain animal species, but also by different mechanisms of transmission of the causative agent of leptospiral infection which can be transferred both by sexual and alimentary routes (in water). It has been demonstrated that mostly one serotype of leptospires predominates in natural foci of leptospirosis, but several in anthropurgic ones. In the author's opinion, leptospiral infection in natural foci is mainly spread by the sexual route through the background species of animals--carriers of leptospirosis, and by the alimentary route in the anthropurgic foci. It is presumed that leptospires of the serogroups Javanica, Australis, Icterohaemorrhagiae, transmitted by the shrew-mice, hedgehogs and rats by the sexual route, are by their origin "ancient" serogroups of leptospires while the serogroups of leptospires isolated from domestic animals, showing predominantly the alimentary route of transmission of infection in the focus, are representatives of the "younger" forms of the evolutional development of leptospires.     

Cell aggregation: a mechanism of pathogenic Leptospira to survive in fresh water.

Transmission of leptospirosis is facilitated by the survival of pathogenic leptospires in moist environments outside their mammalian host. In the present study, the survival mechanisms of Leptospira interrogans serovar Canicola in aqueous conditions and lack of nutrients were investigated. In distilled water, leptospires were able to remain motile for 110 days (pH 7.2). However, when incubated in a semi-solid medium composed of distilled water and 0.5% purified agarose (pH 7.2), they survived 347 days. In this viscous environment, aggregates of live spirochetes were observed. Neither antibiotics (e.g. tetracycline and ampicillin) nor nutrients inhibited leptospiral aggregation. Immunoblot analysis suggested that cells incubated in water down-regulate the expression of LipL31, an inner-membrane protein, but retain expression of other membrane proteins. These studies provide insights into the mechanisms by which pathogenic Leptospira survives for prolonged periods of time in natural aqueous environments, a key stage in the leptospiral lifecycle.     

Anti-Leptospira immunoglobulin profiling in mice reveals strain specific IgG and persistent IgM responses associated with virulence and renal colonization

Leptospira interrogans is a pathogenic spirochete responsible for leptospirosis, a neglected, zoonotic reemerging disease. Humans are sensitive hosts and may develop severe disease. Some animal species, such as rats and mice can become asymptomatic renal carriers. More than 350 leptospiral serovars have been identified, classified on the basis of the antibody response directed against the lipopolysaccharide (LPS). Similarly to whole inactivated bacteria used as human vaccines, this response is believed to confer only short-term, serogroup-specific protection. The immune response of hosts against leptospires has not been thoroughly studied, which complicates the testing of vaccine candidates. In this work, we studied the immunoglobulin (Ig) profiles in mice infected with L. interrogans over time to determine whether this humoral response confers long-term protection after homologous challenge six months post-infection. Groups of mice were injected intraperitoneally with 2×10⁷ leptospires of one of three pathogenic serovars (Manilae, Copenhageni or Icterohaemorrhagiae), attenuated mutants or heat-killed bacteria. Leptospira-specific immunoglobulin (IgA, IgM, IgG and 4 subclasses) produced in the first weeks up to 6 months post-infection were measured by ELISA. Strikingly, we found sustained high levels of IgM in mice infected with the pathogenic Manilae and Copenhageni strains, both colonizing the kidney. In contrast, the Icterohaemorrhagiae strain did not lead to kidney colonization, even at high dose, and triggered a classical IgM response that peaked at day 8 post-infection and disappeared. The virulent Manilae and Copenhageni serovars elicited high levels and similar profiles of IgG subclasses in contrast to Icterohaemorrhagiae strains that stimulated weaker antibody responses. Inactivated heat-killed Manilae strains elicited very low responses. However, all mice pre-injected with leptospires challenged with high doses of homologous bacteria did not develop acute leptospirosis, and all antibody responses were boosted after challenge. Furthermore, we showed that 2 months post-challenge, mice pre-infected with the attenuated M895 Manilae LPS mutant or heat-killed bacterin were completely protected against renal colonization. In conclusion, we observed a sustained IgM response potentially associated with chronic leptospiral renal infection. We also demonstrated in mice different profiles of protective and cross-reactive antibodies after L. interrogans infection, depending on the serovar and virulence of strains.     

Live Imaging of Bioluminescent Leptospira interrogans in Mice Reveals Renal Colonization as a Stealth Escape from the Blood Defenses and Antibiotics

Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Some animals asymptomatically carry L. interrogans in their kidneys and excrete bacteria in their urine, which contaminates the environment. Humans are infected through skin contact with leptospires and develop mild to severe leptospirosis. Previous attempts to construct fluorescent or bioluminescent leptospires, which would permit in vivo visualization and investigation of host defense mechanisms during infection, have been unsuccessful. Using a firefly luciferase cassette and random transposition tools, we constructed bioluminescent chromosomal transformants in saprophytic and pathogenic leptospires. The kinetics of leptospiral dissemination in mice, after intraperitoneal inoculation with a pathogenic transformant, was tracked by bioluminescence using live imaging. For infective doses of 10⁶ to 10⁷ bacteria, we observed dissemination and exponential growth of leptospires in the blood, followed by apparent clearance of bacteria. However, with 2×10⁸ bacteria, the septicemia led to the death of mice within 3 days post-infection. In surviving mice, one week after infection, pathogenic leptospires reemerged only in the kidneys, where they multiplied and reached a steady state, leading to a sustained chronic renal infection. These experiments reveal that a fraction of the leptospiral population escapes the potent blood defense, and colonizes a defined number of niches in the kidneys, proportional to the infective dose. Antibiotic treatments failed to eradicate leptospires that colonized the kidneys, although they were effective against L. interrogans if administered before or early after infection. To conclude, mice infected with bioluminescent L. interrogans proved to be a novel model to study both acute and chronic leptospirosis, and revealed that, in the kidneys, leptospires are protected from antibiotics. These bioluminescent leptospires represent a powerful new tool to challenge mice treated with drugs or vaccines, and test the survival, dissemination, and transmission of leptospires between environment and hosts.     

Survival rate of Leptospira pomona in the soil at a natural leptospirosis focus

At the area of a natural focus of leptospirosis (caused by L. pomona) in the Mozdok region of the North Occetic ASSR leptospires were detected by the method of dark field microscopy in 22% of intact soil samples. The presence of pathogenic leptospires in this soil was not confirmed by the method of the biological assay on Syrian golden hamsters. In controlled tests lasting 1-277 days, in 30.4% of cases L. pomona retained their viability, pathogenic and antigenic properties for as long as 74 days, while staying in the soil at the focus of infection with humidity being 15.2-31.4% and pH = 6.7-7.2. 11 Leptospira cultures isolated after staying in the soil retained their pathogenic properties and the death of the animals used in the bioassay from the acute form of leptospiral infection.     

Expression and immunologic characterization of recombinant heat shock protein 58 of Leptospira species: a major target antigen of the humoral immune response

A clone of Leptospira interrogans serovar lai that was isolated by immunoscreening of a genomic lambda library with sera from convalescent patients with leptospirosis directed expression of a unique 62-kDa protein in Escherichia coli. When examined by SDS-PAGE, the protein comigrated with an immunodominant protein present in leptospiral cell lysate. Determination of the nucleotide sequence of the 2.7-kb insert DNA identified two genes homologous to the hsp58 and hsp10 of L. interrogans serovar copenhageni reported previously. The overexpressed recombinant Hsp58 protein was purified and used to immunize a rabbit to produce a polyclonal antibody. Immunoblot analysis using the rabbit anti-Hsp58 G antibodies showed that the 62-kDa protein was commonly present in lysates of other serovars of leptospires, consistent with the strong sequence conservation between the hsp58 genes of the two serovars. Immunoglobulin G antibodies to the Hsp58 were specifically detected by ELISA in 82% of sera (18/22) from patients with leptospirosis. Deletion analysis of the recombinant Hsp58 protein indicated that a strong antigenic determinant for humoral immune response is located between amino acids 360 and 380 (DREKLQERLAKLAGGVAVIHV) of Hsp58, which are highly conserved among the GroEL family. The strong sequence conservation of the Hsp58 among leptospires and its importance as a major target for the humoral immune response warrant further studies of its potential pathogenetic role.     

Flying foxes as carriers of pathogenic Leptospira species

Recent serologic studies have identified flying foxes (Pteropus spp.) as carriers of leptospirosis; however, little is known about the role of flying foxes as carriers of pathogenic Leptospira spp. To determine if Australian Pteropus spp. are carriers of pathogenic Leptospira spp., TaqMan real-time polymerase chain reaction (PCR) was used to detect leptospiral DNA in kidney and urine specimens from four species of flying fox, including the spectacled flying fox (Pteropus conspicillatus), black flying fox (Pteropus alecto), grey-headed flying fox (Pteropus poliocephalus), and little red flying fox (Pteropus scapulatus). Of the 173 kidney samples tested, 19 (11%) were positive for leptospiral DNA. Positive individuals were detected in all four species; significant differences in prevalence were not detected between species, between species within the same geographic area, or between geographically separated samples from the same species. Of the 46 urine samples tested, 18 (39%) tested positive by PCR, confirming that flying foxes shed leptospires into the environment. The detection of leptospiral DNA in the kidneys and urine of flying foxes suggests that flying foxes are carriers of pathogenic Leptospira spp. No evidence collected in the present study, however, suggests that flying foxes pose a significant risk of leptospirosis to the wider community or that humans who are in regular, close contact with flying foxes are at risk for leptospirosis.     

Potent innate immune response to pathogenic leptospira in human whole blood

Background

Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. The bacteria enter the human body via abraded skin or mucous membranes and may disseminate throughout. In general the clinical picture is mild but some patients develop rapidly progressive, severe disease with a high case fatality rate. Not much is known about the innate immune response to leptospires during haematogenous dissemination. Previous work showed that a human THP-1 cell line recognized heat-killed leptospires and leptospiral LPS through TLR2 instead of TLR4. The LPS of virulent leptospires displayed a lower potency to trigger TNF production by THP-1 cells compared to LPS of non-virulent leptospires.

Methodology/Principal Findings

We investigated the host response and killing of virulent and non-virulent Leptospira of different serovars by human THP-1 cells, human PBMC''s and human whole blood. Virulence of each leptospiral strain was tested in a well accepted standard guinea pig model. Virulent leptospires displayed complement resistance in human serum and whole blood while in-vitro attenuated non-virulent leptospires were rapidly killed in a complement dependent manner. In vitro stimulation of THP-1 and PBMC''s with heat-killed and living leptospires showed differential serovar and cell type dependence of cytokine induction. However, at low, physiological, leptospiral dose, living virulent complement resistant strains were consistently more potent in whole blood stimulations than the corresponding non-virulent complement sensitive strains. At higher dose living virulent and non-virulent leptospires were equipotent in whole blood. Inhibition of different TLRs indicated that both TLR2 and TLR4 as well as TLR5 play a role in the whole blood cytokine response to living leptospires.

Conclusions/Significance

Thus, in a minimally altered system as human whole blood, highly virulent Leptospira are potent inducers of the cytokine response.     

Correlation between renal distribution of leptospires during the acute phase and chronic renal dysfunction in a hamster model of infection with Leptospira interrogans

BackgroundLeptospirosis has been described as a biphasic disease consisting of hematogenous dissemination to major organs in the acute phase and asymptomatic renal colonization in the chronic phase. Several observational studies have suggested an association between leptospirosis and chronic kidney disease (CKD). We investigated the dynamics of leptospires and histopathological changes in the kidney to understand the relationship between them, and also investigated the extent of renal dysfunction in the acute and chronic phases of leptospirosis using a hamster model.FindingsHamsters (n = 68) were subcutaneously infected with 1 × 10⁴ cells of the Leptospira interrogans serovar Manilae strain UP-MMC-SM. A total of 53 infected hamsters developed fatal acute leptospirosis, and the remaining 15 hamsters recovered from the acute phase, 13 of which showed Leptospira colonization in the kidneys in the chronic phase. Five asymptomatic hamsters also had renal colonization in the chronic phase. Immunofluorescence staining showed that leptospires were locally distributed in the renal interstitium in the early acute phase and then spread continuously into the surrounding interstitium. The kidneys of the surviving hamsters in the chronic phase showed patchy lesions of atrophic tubules, a finding of chronic tubulointerstitial nephritis, which were substantially consistent with the distribution of leptospires in the renal interstitium. The degree of atrophic tubules in kidney sections correlated statistically with the serum creatinine level in the chronic phase (rs = 0.78, p = 0.01).ConclusionSubcutaneous infection with pathogenic leptospires could cause acute death or chronic leptospirosis in hamsters after surviving the acute phase. We suggest that the renal distribution of leptospires during the acute phase probably affected the extent of tubular atrophy, leading to CKD.     

Detection and ecology of leptospirosis in Iowa wildlife

To gain additional information on the extent of leptospirosis in wildlife following a human outbreak in Iowa, wild mammals and lower forms of life were collected. Isolation, darkfield microscopic, serologic and pathologic procedures were used to identify past or present evidence of leptospiral infection. Leptospires were isolated from 7 of 75 (9%) mammals. Serotype grippotyphosa was isolated from three raccoons (procyon lotor) and one Western Harvest Mouse (Reithrodontomys megalotis). Serotype ballum was isolated from three opossums (Didelphis marsupialis). Leptospires, unidentified to date, were isolated from frog (Rana pipiens) kidneys. Other positive serologic and pathologic tests gave evidence of infection or previous infection. Utilization of Darkfield microscopic and silver staining techniques did not detect all cases of leptospiral infection. Macroscopic and microscopic serologic methods failed to identify evidence of leptospirosis in all mammals from which leptospires were isolated. Pathologic lesions could only be considered presumptive evidence for leptospirosis. These findings indicate that detection of leptospirosis in wildlife cannot be limited to a single diagnostic test. A combination of diagnostic procedures and clinical evaluation is necessary. Although serotype pomona was implicated as the predominant infecting leptospire in the human cases and domestic animals and was isolated from water at a swimming site, only serotypes grippotyphosa, ballum and ICF (frog isolate) were isolated from wild mammals and lower forms of life in the same vicinity.     

Neurotoxic activity induced by a haemolytic substance in the extract of the marine sponge Geodia corticostylifera

In our search for marine bioactive compounds we chose a Brazilian Coast sponge, Geodia corticostylifera (Demospongiae), whose extracts showed previously antibacterial and antifungal activities. In the present work we studied the following toxic properties of G. corticostylifera extract: neurotoxic (in mouse neuromuscular junction); mouse acute toxicity (IP) and haemolytic (against mouse and frog erythrocytes). Insertion of ionic channels in planar lipid bilayers in presence of a haemolytic purified fraction of the extract was observed. The toxic activities of G. corticostylifera crude extract are related to the formation of ionic pores in the cell membrane, which induce the release of haemoglobin from erythrocytes, and depolarization of nerve and muscle membranes. These last physiological effects cause the blockade of the diaphragm contractions, leading to death through respiratory arrest.     

Use of microcapsular leptospiral antigens for detecting specific antibodies

The sensitivity of microcapsular leptospiral antigens, produced by Japan Lyophilization Laboratory and intended for use in tests for the detection of antibodies to leptospires in the sera of experimentally immunized laboratory animals, were studied. The comparative study of the microcapsular agglutination (MCA) test and other serological tests, such as the microagglutination (MA) test and the indirect enzyme immunoassay (EIA), was made. The leptospiral antigens under study were found to actively react with serospecific and group-specific antibodies. In infected guinea pigs and rabbits specific antibodies could be detected from days 3-4 in the MCA test and only from days 5-7 in the MA test. The average antibody level determined by titration in the MCA test was 3.3 times higher and in indirect EIA, 4.3 times higher than that determined by titration in the MA test. These data make it possible to recommend the use of microcapsular leptospiral antigens for the early diagnosis of leptospirosis.     

Leptospira strains kept at the National Centre for Leptospirosis in Rome, Italy

Since the National Centre for Leptospirosis (Department of Bacteriology and Medical Mycology, Istituto Superiore di Sanità, Rome) was established in 1956 by B. Babudieri, efforts have been devoted to identifying new Leptospira isolates and maintaining a collection of strains that today comprises 670 strains, 550 of which have been totally or partially classified, and 120 are still under study. This collection includes 23 serogroups and 156 serovars of pathogenic leptospires, and 32 serogroups and 54 serovars of saprophytic leptospires. The conventional serogroup and serovar identification, mainly based on antigenic relatedness, is tedious and time-consuming, requiring the maintenance of a comprehensive collection of serovar reference strains and the preparation of the corresponding rabbit antisera. Although considerable difficulties are encountered in the classification of leptospires at the serogroup and serovar level, this classification system is essential to obtain information on the epidemiology of leptospirosis in the different geographical areas. Serovar identification has become faster with the introduction of pulsed-field gel electrophoresis (PFGE) of large DNA fragments obtained after digestion of leptospiral DNAs with rare-cutting restriction enzymes. This technique has been successfully utilized to discriminate between closely related serovars of the Leptospira interrogans complex. We have recently used PFGE to characterize several Italian leptospiral isolates, confirming that PFGE analysis combined with microscopic agglutination test (MAT) with monoclonal and polyclonal antibodies can be used as an accurate and reliable method to compare and classify leptospires.     

LPS regulation of the immune response: separate mechanisms for murine B cell activation by lipid A (direct) and polysaccharide (macrophage-dependent) derived from Bacteroides LPS

Lipopolysaccharide (LPS) derived from Bacteroides fragilis has been reported to stimulate mitogenic responses in spleen cell cultures from the classical LPS-hyporesponsive C3H/HeJ mouse strain; however, we have shown that purified splenic B cells from C3H/HeJ mice are hyporesponsive to phenol-water extracted LPS from B. fragilis ATCC 25285 (B-LPS). In the present study, B-LPS and its purified lipid A and polysaccharide components were tested for their ability to induce mitogenic and polyclonal IgM synthesis in spleen cell and purified splenic B cell cultures from classical LPS-responsive and -hyporesponsive mice. Mitogenic responses to B-LPS and E. coli K235 LPS(Ph) of whole spleen cells (2 X 10(5) cells/culture) or purified B cells (5 X 10(5) cells/culture) from classical LPS-responsive mouse strains (C3H/HeN, BALB/c, C57BL/6J, C57BL/10Sn, and DBA/2), F1 mice (derived from crosses between LPS responsive and C3H/HeJ mice), and classical LPS-hyporesponsive mice (C3H/HeJ and C57BL/10ScN) were high, intermediate, and low, respectively. When a higher number of whole spleen cells (5 X 10(5) cells/well) were cultured, B-LPS induced high mitogenic responses in C3H/HeN, intermediate responses in F1, and lower but significant responses in C3H/HeJ cultures. Similar results were obtained when polyclonal IgM synthesis was assessed in cultures containing 1 X 10(6) cells/culture. In contrast, the purified lipid A component of B-LPS failed to induce mitogenic responses in either whole spleen or purified B cell cultures. The addition of purified splenic B cells from C3H/HeJ mice to C3H/HeN or C3H/HeJ splenic adherent cells resulted in mitogenic responses to B-LPS, implying that the hyporesponsiveness to B-LPS seen in whole spleen cell cultures from C3H/HeJ mice at the lower cell concentration was due to limiting numbers of M phi. When splenic B cells and M phi from either C3H/HeN or C3H/HeJ mice were incubated with the lipid A or the polysaccharide moiety of B-LPS, lipid A induced mitogenic responses only in C3H/HeN cultures, whereas the polysaccharide moiety induced similar responses in both C3H/HeN and C3H/HeJ cultures. These results suggest that Bacteroides lipid A does not stimulate B cells from the classical LPS-hyporesponsive C3H/HeJ mouse strain, whereas the polysaccharide moiety of B-LPS is biologically active and mediates B cell stimulation via M phi.     

Leptospirosis: an emerging global public health problem

Leptospirosis has been recognized as an emerging global public health problem because of its increasing incidence in both developing and developed countries. A number of leptospirosis outbreaks have occurred in the past few years in various places such as Nicaragua, Brazil and India. Some of these resulted due to natural calamities such as cyclone and floods. It is a direct zoonotic disease caused by spirochetes belonging to different pathogenic species of the genus Leptospira. Large number of animals acts as carriers or vectors. Human infection results from accidental contact with carrier animals or environment contaminated with leptospires. The primary source of leptospires is the excretor animal, from whose renal tubules leptospires are excreted into the environment with the animal urine. Majority of leptospiral infections are either sub clinical or result in very mild illness and recover without any complications. However, a small proportion develops various complications due to involvement of multiple organ systems. In such patients, the clinical presentation depends upon the predominant organs involved and the case fatality ratio could be about 40% or more. Febrile illness with icterus, splenomegaly and nephritis (known as Weil’s disease), acute febrile illness with severe muscle pain, febrile illness with pulmonary haemorrhages in the form of haemoptysis, jaundice with pulmonary haemorrhages, jaundice with heamaturea, meningitis with haemorrhages including sub conjunctival haemorrhage or febrile illness with cardiac arrhythmias with or without haemorrhages are some of the syndromes. Because of the protean manifestations of leptospirosis it is often misdiagnosed and under-reported. Although the basic principles of prevention such as source reduction, environmental sanitation, more hygienic work-related and personal practices etc., are same everywhere, there is no universal control method applicable to all epidemiological settings. Comprehensive understanding of the eco- epidemiological and cultural characteristics of a community that faces the problem of leptospirosis is an essential prerequisite for evolving an effective and acceptable control measure.     

Detection and quantification of Leptospira interrogans in hamster and rat kidney samples: immunofluorescent imprints versus real-time PCR

A major limitation in the clinical management and experimental research of leptospirosis is the poor performance of the available methods for the direct detection of leptospires. In this study, we compared real-time PCR (qPCR), targeting the lipL32 gene, with the immunofluorescent imprint method (IM) for the detection and quantification of leptospires in kidney samples from the rat and hamster experimental models of leptospirosis. Using a virulent strain of Leptospira interrogans serovar Copenhageni, a chronic infection was established in the rat model, which were euthanized 28 days post-infection, while the hamster model simulated an acute infection and the hamsters were euthanized eight days after inoculation. Leptospires in the kidney samples were detected using culture isolation, qPCR and the IM, and quantified using qPCR and the IM. In both the acute and chronic infection models, the correlation between quantification by qPCR and the IM was found to be positive and statistically significant (P<0.05). Therefore, this study demonstrates that the IM is a viable alternative for not only the detection but also the quantification of leptospires, particularly when the use of qPCR is not feasible.     

Rapid diagnosis of leptospirosis in patients with different clinical manifestations by 16S rRNA gene based nested PCR

Leptospirosis, a zoonosis of global importance and it is underreported in India and more than 50,000 severe cases are reported each year. Here we present the evaluation of 16S rRNA based nested PCR assay for the rapid identification of human leptospires using serum and urine samples. The study includes 261 suspected cases for leptospirosis with different clinical manifestations. 16S rRNA based nested PCR assay was compared and evaluated against the conventional serological methods such as MAT and ELISA. The technique enabled amplification of a 289 bp product with notable percentage of positivity in all sample groups including 94.8 in pediatric cases, 93 in pregnant women, 94.2 in renal failure, 87.8 in jaundice and 94.6 in common febrile cases. The sensitivity and specificity was 94.4% and 100%, respectively. The technique proved to be prompt and effective for the diagnosis of leptospiral infection at the acute phase of the disease. PCR based approach detects leptospiral DNA from the clinical samples both at the acute and leptospiruria phase on comparison with its counter parts where detection is made possible only after 7 days or 7–30 days post-infection. In this regard PCR based diagnosis of leptospirosis should be made available for clinicians for the early diagnosis and prompt treatment of the disease.