Comparative analysis of a novel N-butanol-prepared antigen vs thermo-resistant and sonicated antigens for human leptospirosis detection.

The diagnosis of human leptospirosis is mainly based on serological assays. Since the extraction by N-butanol has only been studied as an antigen for the diagnosis of cattle leptospirosis, this study aimed to investigate the feasibility of the N-butanol preparation for the diagnosis of human leptospirosis and compare it with sonicated and thermo-resistant antigens in IgM dot-blot test. Paired serum samples from 147 laboratory-confirmed leptospirosis cases were tested. The control group consisted of 148 serum samples from healthy individuals and nonleptospirosis cases. N-butanol antigens from serovar Copenhageni (ButC3) and serovar Patoc (ButP3) showed reactivity with antileptospiral antibodies from patients with confirmed leptospirosis. In the acute phase, sensitivities of IgM dot-blot assay with ButC3 and ButP3 antigens were 47.6% and 51.0%, respectively. In the convalescent phase, sensitivities were 95.9% (ButC3) and 93.2% (ButP3), and no significant differences were observed among the IgM dot-blot tests with other antigens. The specificity of the IgM dot-blot test with ButC3 antigen was good (92.6%), but with ButP3 (83.1%), it was significantly lower than with the other tests. The IgM dot-blot test described in this study is simple to perform and presents reliable visual results. Antigens prepared by N-butanol proved to be valuable diagnostic markers of leptospirosis.

Population Structure Analysis of Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates from Brazil Reveals Predominance of Clonal Complexes 1, 15, and 79.

The population structure of 71 carbapenem-resistantAcinetobacter baumanniiclinical isolates from several hospitals in Brazil was investigated by ApaI pulsed-field gel electrophoresis,blaOXA-51-like subtyping, and multilocus sequence typing (Institute Pasteur scheme). In addition to the predominance of strains carryingblaOXA-23, we detected the presence ofblaOXA-72andblaOXA-231 We observed a predominance of clonal complex 1 (CC1), CC15, and CC79 and representative strains of the worldwide-disseminated international clone I.

The novel leptospiral surface adhesin Lsa20 binds laminin and human plasminogen and is probably expressed during infection.

Leptospirosis is an emerging infectious disease caused by pathogenic species of Leptospira. In this work, we report the cloning, expression, purification, and characterization of two predicted leptospiral outer membrane proteins, LIC11469 and LIC11030. The LIC11469 protein is well conserved among leptospiral strains, while LIC11030 was identified only in Leptospira interrogans. We confirmed by surface proteolysis of intact leptospires with proteinase K that these proteins are most likely new surface leptospiral proteins. The recombinant proteins were evaluated for their capacity to attach to extracellular matrix (ECM) components and to plasminogen. The leptospiral protein encoded by LIC11469, named Lsa20 (leptospiral surface adhesin of 20 kDa), binds to laminin and to plasminogen. The binding with both components was not detected when Lsa20 was previously denatured or blocked with anti-Lsa20 antibodies. Moreover, Lsa20 binding to laminin was also confirmed by surface plasmon resonance (SPR). Laminin competes with plasminogen for binding to Lsa20, suggesting the same ligand-binding site. Lsa20-bound plasminogen could be converted to enzymatically active plasmin, capable of cleaving plasmin substrate d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Lsa20 was recognized by antibodies in confirmed-leptospirosis serum samples, suggesting that this protein is expressed during infection. Taken together, our results indicate that Lsa20 is a novel leptospiral adhesin that in concert with the host-derived plasmin may help the bacteria to adhere and to spread through the hosts.

Aseptic meningitis caused by Leptospira spp diagnosed by polymerase chain reaction.

Leptospirosis is a zoonotic disease caused by the pathogenic Leptospira spp. The clinical presentations are diverse, ranging from undifferentiated fever to fulminant disease including meningeal forms. The neurological leptospirosis forms are usually neglected. The aim of this study was to investigate leptospirosis as the cause of aseptic meningitis using different diagnostic techniques including the polymerase chain reaction (PCR). Thirty-nine cerebrospinal fluid (CSF) samples from patients presenting with meningeal abnormalities, predominance of lymphocytes and negative results by traditional microbiological tests were processed by leptospiral culture, anti-leptospiral antibody response and PCR. Leptospira spp DNA was detected in 23 (58.97%) of the CSF samples. Anti-leptospiral antibodies were found in 13 (33.33%) CSF samples. Twelve CSF samples were positive by PCR assay and negative by microscopic agglutination test (MAT) assay. Two CSF samples were positive by MAT and negative by PCR. The positive and negative agreement between both tests was 11 and 14, respectively. CSF samples from six cases of unknown diagnosis were positive by PCR assay. Eight cases showed positive results using PCR and MAT. Leptospirosis could be detected by PCR assay from the 3rd-26th day after illness onset. The sensitivity of the PCR was assessed with confirmed cases of leptospirosis (by MAT) and found to be 89.5%. All CSFs were negative by culture. PCR was found to be a powerful tool for diagnosing meningitis cases of leptospirosis. We recommend that it may be used as a supplementary diagnostic tool, especially in the early stages of the disease, when other diagnostic techniques such as serology are not sensitive.

In LipL32, the major leptospiral lipoprotein, the C terminus is the primary immunogenic domain and mediates interaction with collagen IV and plasma fibronectin.

LipL32 is the major leptospiral outer membrane lipoprotein expressed during infection and is the immunodominant antigen recognized during the humoral immune response to leptospirosis in humans. In this study, we investigated novel aspects of LipL32. In order to define the immunodominant domains(s) of the molecule, subfragments corresponding to the N-terminal, intermediate, and C-terminal portions of the LipL32 gene were cloned and the proteins were expressed and purified by metal affinity chromatography. Our immunoblot results indicate that the C-terminal and intermediate domains of LipL32 are recognized by sera of patients with laboratory-confirmed leptospirosis. An immunoglobulin M response was detected exclusively against the LipL32 C-terminal fragment in both the acute and convalescent phases of illness. We also evaluated the capacity of LipL32 to interact with extracellular matrix (ECM) components. Dose-dependent, specific binding of LipL32 to collagen type IV and plasma fibronectin was observed, and the binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin-binding and 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. Taken together, our results provide evidence that the LipL32 C terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins.

Is the microagglutination test (MAT) good for predicting the infecting serogroup for leptospirosis in Brazil?

Leptospirosis is a zoonotic infection caused by pathogenic members of the genus Leptospira spp. Knowledge of the prevalent serovars and their maintenance hosts is essential to understand the disease. The aim of this study was to evaluate the ability of serology by the microscopic agglutination test (MAT) to predict the serogroups compared with results of identification of leptospires in São Paulo, Brazil. MAT correctly assigned the serogroup of the infecting isolate in 49/52 cases (94.23%). The serogroup Icterohaemorrhagiae was the predominant serogroup (88.46%). This study showed the usefulness of the MAT to correctly identify the infecting serogroup with a good overall agreement between the serologically-identified infecting serogroup and by identification of the isolate and can be used in epidemiological surveys in São Paulo. However, it should be complemented by the identification of Leptospira isolates.

Human leptospirosis: a twenty-nine-year serological study in São Paulo, Brazil.

A retrospective study of 9,335 cases of human leptospirosis in the state of São Paulo, Brazil, diagnosed between 1969 and 1997 showed that the disease is endemic throughout the state. Middle-aged adults, with a range of 20-39 years, were most frequently infected (32.40%). The mean annual incidence was 0.53 per 100,000 population and the disease was more frequent in males (87.0%). Cases occurred mainly in January to April each year. A peak was observed in 1991 and 1996 which rainfall average was 159.9 and 160.3, respectively. These data emphasize the potential public health importance of leptospirosis in the state of São Paulo, Brazil.

Evaluation of nested polymerase chain reaction for the early detection of Leptospira spp. DNA in serum samples from patients with leptospirosis.

The aim of this study was to analyse the nested polymerase chain reaction (nested PCR) in human serum samples of patients with clinical manifestations of leptospirosis. The cases of leptospirosis were defined by the microagglutination test (MAT). The samples were collected in 2010. Of 1042 serum samples collected from 521 patients, 28 (5.4%) were considered positive cases of leptospirosis, and 493 (94.6%) were negative. Twenty-three confirmed cases had no MAT-detectable antibodies in the acute sample (mean of 5.6 days after onset). Nested PCR was positive in 22/23 (95.7%) patients during the acute phase of the disease, with negative results by MAT. Nested PCR was negative in all convalescent serum samples with positive results by MAT. All negative cases of leptospirosis were negative by nested PCR. The nested PCR is an alternative diagnostic tool for early detection of leptospires in sera during the first 7 days of the disease.

Padronização de teste para quantificar anticorpos anti-poliovírus utilizando vírus vacinal / Standardization of a test for quantifying the antibodies against poliovirus using the vaccine virus

A poliomielite é uma doença endêmica no Afeganistão e no Paquistão, apesar dos esforços para ser erradicada, representando uma ameaça para outros países principalmente devido às viagens internacionais. A Organização Mundial da Saúde (OMS) tem como objetivo erradicar a poliomielite causada pelo poliovírus selvagem no mundo. O requisito essencial para a erradicação da poliomielite é a eliminação da cepa do poliovírus selvagem, que é empregada no teste padrão-ouro. Com o intuito de auxiliar na erradicação do poliovírus selvagem, o objetivo deste estudo foi modificar o teste padrão-ouro usando o poliovírus derivado da vacina oral atenuada. Foram testados 63 soros pelo ensaio de neutralização utilizando-se antígenos vacinais. A concordância do sorotipo 1 (k=0,74) foi considerada substancial, enquanto o sorotipo 2 (k=1,00) e sorotipo 3 (k= 0,95) foram consideradas quase perfeitas. A sensibilidade dos testes de soroneutralização utilizando os sorotipos 1, 2 e 3 foi de 94,83%, 100,00% e 100,00%, respectivamente. Em conclusão, o ensaio com antígenos vacinais pode ser usado como procedimento laboratorial seguro, especialmente em estudos de vigilância em larga escala. (AU)

Poliomyelitis is an endemic disease in Afghanistan and Pakistan in despite of the efforts to eradicate it, and it represents a threat to other countries mainly due to the international trips. The World Health Organization (WHO) aims at eradicating the polio disease worldwide. An essential requirement for eradicating the poliomyelitis is the elimination of the wild poliovirus strain, which is employed in the gold standard test. As a support for the eradication of wild poliovirus, the present study aimed at modifying the gold standard test by using poliovirus derived from the oral attenuated vaccine. Sixty-three sera samples were tested by neutralization assay using vaccine antigens. The degree of agreement of the serotype 1 (k=0.74) was considered substantial, while the serotype 2 (k=1.00) and 3 (k= 0.95) showed almost perfect agreement. The sensitivity of serotypes 1, 2 and 3 was 94.83%, 100.00% and 100.00%, respectively. In conclusion, the assay with the vaccine antigens can be used as a safe application, especially for large-scale surveillance studies. (AU)

Padronização de teste para quantificar anticorpos anti-poliovírus utilizando vírus vacinal / Standardization of a test for quantifying the antibodies against poliovirus using the vaccine virus

A poliomielite é uma doença endêmica no Afeganistão e no Paquistão, apesar dos esforços para ser erradicada, representando uma ameaça para outros países principalmente devido às viagens internacionais. A Organização Mundial da Saúde (OMS) tem como objetivo erradicar a poliomielite causada pelo poliovírus selvagem no mundo. O requisito essencial para a erradicação da poliomielite é a eliminação da cepa do poliovírus selvagem, que é empregada no teste padrão-ouro. Com o intuito de auxiliar na erradicação do poliovírus selvagem, o objetivo deste estudo foi modificar o teste padrão-ouro usando o poliovírus derivado da vacina oral atenuada. Foram testados 63 soros pelo ensaio de neutralização utilizando-se antígenos vacinais. A concordância do sorotipo 1 (k=0,74) foi considerada substancial, enquanto o sorotipo 2 (k=1,00) e sorotipo 3 (k= 0,95) foram consideradas quase perfeitas. A sensibilidade dos testes de soroneutralização utilizando os sorotipos 1, 2 e 3 foi de 94,83%, 100,00% e 100,00%, respectivamente. Em conclusão, o ensaio com antígenos vacinais pode ser usado como procedimento laboratorial seguro, especialmente em estudos de vigilância em larga escala.

Poliomyelitis is an endemic disease in Afghanistan and Pakistan in despite of the efforts to eradicate it, and it represents a threat to other countries mainly due to the international trips. The World Health Organization (WHO) aims at eradicating the polio disease worldwide. An essential requirement for eradicating the poliomyelitis is the elimination of the wild poliovirus strain, which is employed in the gold standard test. As a support for the eradication of wild poliovirus, the present study aimed at modifying the gold standard test by using poliovirus derived from the oral attenuated vaccine. Sixty-three sera samples were tested by neutralization assay using vaccine antigens. The degree of agreement of the serotype 1 (k=0.74) was considered substantial, while the serotype 2 (k=1.00) and 3 (k= 0.95) showed almost perfect agreement. The sensitivity of serotypes 1, 2 and 3 was 94.83%, 100.00% and 100.00%, respectively. In conclusion, the assay with the vaccine antigens can be used as a safe application, especially for large-scale surveillance studies.

Renal involvement in leptospirosis: the effect of glycolipoprotein on renal water absorption.

BACKGROUND: Leptospirotic renal lesions frequently produce a polyuric form of acute kidney injury with a urinary concentration defect. Our study investigated a possible effect of the glycolipoprotein, (GLPc) extracted from L. interrogans, on vasopressin (Vp) action in the guinea pig inner medullary collecting duct (IMCD). METHODS: The osmotic water permeability (Pf µm/s) was measured by the microperfusion in vitro technique. AQP2 protein abundance was determined by Western Blot. Three groups were established for study as follows: Group I, IMCD from normal (ngp, n = 5) and from leptospirotic guinea-pigs (lgp-infected with L. interrogans serovar Copenhageni, GLPc, n = 5); Group II, IMCD from normal guinea-pigs in the presence of GLPc (GLPc group, n = 54); Group III, IMCD from injected animals with GLPc ip (n = 8). RESULTS: In Group I, PFS were: ngp--61.8±22.1 and lgp--8.8±12.4, p<0.01 and the urinary osmolalities were: lgp--735±64 mOsm/Kg and ngp--1,632±120 mOsm/Kg. The lgp BUN was higher (176±36 mg%) than the ngp (56±9 mg%). In Group II, the Pf was measured under GLPc (250 µg/ml) applied directly to the bath solution of the microperfused normal guinea-pig IMCDs. GLPc blocked Vp (200 pg/ml, n = 5) action, did not block cAMP (10(-4) M), and Forskolin (Fors--10(-9) M) action, but partially blocked Cholera Toxin (ChT--10(-9) M) action. GLP from L.biflexa serovar patoc (GLPp, non pathogenic, 250 µg) did not alter Vp action. In Group III, GLPc (250 µg) injected intraperitoneally produced a decrease of about 20% in IMCD Aquaporin 2 expression. CONCLUSION: The IMCD Pf decrease caused by GLP is evidence, at least in part, towards explaining the urinary concentrating incapacity observed in infected guinea-pigs.

Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen.

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D) of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

Avaliação do teste de aglutinação microscópica utilizando-se como antígeno leptospiras saprófitas para o diagnóstico da leptospirose humana / Evaluation of microscopic agglutination test using saprophytic leptospires for diagnosis of human leptospirosis

O objetivo deste trabalho foi avaliar a capacidade de efetuar o diagnóstico precoce do teste de aglutinação microscópica (MAT) utilizando-se como antígeno leptospiras saprófitas sorovar Patoc (MAT-Patoc). Neste contexto, os resultados obtidos em amostras de soro de casos confirmados de leptospirose foram comparados com aqueles obtidos no teste ELISA IgM. Cento e cinquenta e oito amostras colhidas na fase aguda da doença foram analisadas pelo MAT-Patoc. Os soros com títulos ≥ 50 foram considerados reagentes. A sensibilidade do MAT-Patoc e do ELISA-IgM foi de 13,29% e 28,48%, respectivamente. Nos sete primeiros dias da doença, o MAT-Patoc foi capaz de detectar 12,29% dos casos, enquanto o ELISA-IgM detectou 24,59%. O MAT-Patoc foi reagente em amostras de casos cujos prováveis sorogrupos infectantes foram Icterohaemorrhagiae, Cynopteri e Ballum. Em conclusão, a utilização do teste MAT-Patoc apresentou menor sensibilidade no diagnóstico quando comparado ao ELISA-IgM, embora tenha sido reagente em algumas amostras com resultado não reagente. O MAT-Patoc deve ser utilizado em combinação com ELISA-IgM para melhorar a sensibilidade do diagnóstico de leptospirose na primeira fase da doença, com subsequente confirmação pelo MAT de referência.

The aim of this study was to evaluate the early detection of antibodies by the microscopic agglutination test (MAT) using saprophytic leptospira serovar Patoc (MAT-Patoc), in serum samples from patients with confirmed leptospirosis. The obtained results were compared with those found in ELISA-IgM. A hundred fifty-eight serum samples collected from patients in acute phase of illness were analysed by MAT-Patoc. Samples with titers ≥ 50 were considered positive. The sensitivity of MAT-Patoc and IgM-ELISA was 13.29 % and 28.48 %, respectively. In the first seven days of the disease, MAT-Patoc was positive in 12.29 % of the cases, while the IgM-ELISA was positive in 24.59 %. The MAT-Patoc was positive in sera from confirmed cases, and the infecting serogroups were Icterohaemorrhagiae, Cynopteri and Ballum. In conclusion, the use of MAT-Patoc test showed lower sensitivity for diagnosing leptospirosis when compared with ELISA-IgM, although positive reactivity was found in some samples with negative results. The MAT-Patoc should be used in combination with ELISA-IgM to improve the sensitivity of the diagnosis of leptospirosis in the first phase of the disease, with subsequent confirmation by standard MAT.

Severe acute renal failure in a child: a rare complication of anicteric leptospirosis.

A 14-year-old male patient was admitted with fever, vomiting, muscular pain, mainly in the lower limbs, oliguria and oedema. The presence of rats in the vicinity of the home was reported. Laboratory tests on admission revealed severely compromised renal function and increased phosphokinase creatine and thrombocytopenia. Although the patient presented an atypical course without jaundice or meningeal involvement, early dialytic treatment was administered concomitant with empiric antibiotic therapy for leptospirosis. The probable infected serogroup by serology was Autumnalis. This atypical case illustrates that, in the presence of fever, renal failure, rhabdomyolysis and thrombocytopenia, leptospirosis should be considered, even in the absence of jaundice or meningitis, especially if there is a history of contact with carrier animals.

Characterization of Novel OmpA-Like Protein of Leptospira interrogans That Binds Extracellular Matrix Molecules and Plasminogen

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (KD, 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a KD of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

Leptospira DNA detection for the diagnosis of human leptospirosis.

This study aims to analyse PCR applicability to the diagnosis of human leptospirosis and to compare the sensitivity of two primer pairs in urine and blood samples. PCR with G1/G2 and LP1/LP2 primers was specific and able to detect 10pg of DNA by agarose gel and 1pg by hybridization. Twenty-one serovars, representing 20 serogroups of pathogenic leptospires, were amplified with G1/G2 primers. DNA from two non-pathogenic serovars, Andamana and Patoc, was not amplified. For hybridization, one probe employing DNA from most prevalent leptospires (serovars Icterohaemorrhagiae, Copenhageni, and Autumnalis) was chosen in accordance with the microagglutination titres in patient samples. It was observed that not all serovars hybridized with the PCR products of G1/G2 and LP1/LP2 primer amplification, suggesting heterogeneity in the sequence amplified by these primers. G1/G2-primed amplifications of blood and/or urine samples were shown to be significantly more sensitive (57.6%) than the LP1/LP2 primers (33.3%), P=0.04, when positivity of patients is considered. When each primer pair and only urine samples were considered, PCR positivity was higher for G1/G2 primers than for LP1/LP2 (P=0.007). G1/G2 presented greater sensitivity in urine than in blood, and LP1/LP2 presented greater sensitivity in blood than in urine, although these differences were not statistically significant. The positivity of PCR per patient using both primers in blood and/or urine samples was 63.6%, with 84.4% efficiency. PCR was useful for patients without microagglutination detectable antibodies, for whom it was able to diagnose nine out of 11 patients (81.8%).

The novel leptospiral surface adhesin Lsa20 binds laminin and human plasminogen and is probably expressed during infection

Leptospirosis is an emerging infectious disease caused by pathogenic species of Leptospira. In this work, we report the cloning, expression, purification, and characterization of two predicted leptospiral outer membrane proteins, LIC11469 and LIC11030. The LIC11469 protein is well conserved among leptospiral strains, while LIC11030 was identified only in Leptospira interrogans. We confirmed by surface proteolysis of intact leptospires with proteinase K that these proteins are most likely new surface leptospiral proteins. The recombinant proteins were evaluated for their capacity to attach to extracellular matrix (ECM) components and to plasminogen. The leptospiral protein encoded by LIC11469, named Lsa20 (leptospiral surface adhesin of 20 kDa), binds to laminin and to plasminogen. The binding with both components was not detected when Lsa20 was previously denatured or blocked with anti-Lsa20 antibodies. Moreover, Lsa20 binding to laminin was also confirmed by surface plasmon resonance (SPR). Laminin competes with plasminogen for binding to Lsa20, suggesting the same ligand-binding site. Lsa20-bound plasminogen could be converted to enzymatically active plasmin, capable of cleaving plasmin substrate D-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Lsa20 was recognized by antibodies in confirmed-leptospirosis serum samples, suggesting that this protein is expressed during infection. Taken together, our results indicate that Lsa20 is a novel leptospiral adhesin that in concert with the host-derived plasmin may help the bacteria to adhere and to spread through the hosts.

Aseptic meningitis caused by Leptospira spp diagnosed by polymerase chain reaction

Leptospirosis is a zoonotic disease caused by the pathogenic Leptospira spp. The clinical presentations are diverse, ranging from undifferentiated fever to fulminant disease including meningeal forms. The neurological leptospirosis forms are usually neglected. The aim of this study was to investigate leptospirosis as the cause of aseptic meningitis using different diagnostic techniques including the polymerase chain reaction (PCR). Thirty-nine cerebrospinal fluid (CSF) samples from patients presenting with meningeal abnormalities, predominance of lymphocytes and negative results by traditional microbiological tests were processed by leptospiral culture, anti-leptospiral antibody response and PCR. Leptospira spp DNA was detected in 23 (58.97 percent) of the CSF samples. Anti-leptospiral antibodies were found in 13 (33.33 percent) CSF samples. Twelve CSF samples were positive by PCR assay and negative by microscopic agglutination test (MAT) assay. Two CSF samples were positive by MAT and negative by PCR. The positive and negative agreement between both tests was 11 and 14, respectively. CSF samples from six cases of unknown diagnosis were positive by PCR assay. Eight cases showed positive results using PCR and MAT. Leptospirosis could be detected by PCR assay from the 3rd-26th day after illness onset. The sensitivity of the PCR was assessed with confirmed cases of leptospirosis (by MAT) and found to be 89.5 percent. All CSFs were negative by culture. PCR was found to be a powerful tool for diagnosing meningitis cases of leptospirosis. We recommend that it may be used as a supplementary diagnostic tool, especially in the early stages of the disease, when other diagnostic techniques such as serology are not sensitive.

Expression and characterization of HlyX hemolysin from Leptospira interrogans serovar Copenhageni: potentiation of hemolytic activity by LipL32.

The HlyX, a putative hemolysin identified from the Leptospira genomes, was cloned, expressed in Escherichia coli, purified, and its hemolytic activity was confirmed. Mouse polyclonal antiserum against the recombinant HlyX recognized HlyX-related antigens in a panel of Leptospira species extracts and it was also able to abolish the hemolytic activity of HlyX. A mixture of HlyX and LipL32, a known hemolysin from Leptospira, induced hemolysis in a synergistic way that was fully inhibited by antiserum against either protein. Moreover, sera from patients with leptospirosis also recognized the recombinant HlyX, showing that it is presented to the host immune system during Leptospira infection.