Renal Involvement in Leptospirosis: The Effect of Glycolipoprotein on Renal Water Absorption

on vasopressin (Vp) action in the guinea pig inner medullary collecting duct (IMCD). 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). (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.The IMCD Pf decrease caused by GLP is evidence, at least in part, towards explaining the urinary concentrating incapacity observed in infected guinea-pigs

Standardization and evaluation of the IgM Dot-blot Test for serodiagnosis of human leptospirosis

A leptospirose é uma zoonose com ampla distribuição mundial causada por espiroquetas patogênicas do gênero Leptospira spp. Os sintomas da leptospirose podem se apresentar de forma bastante inespecífica dificultando o diagnóstico clínico e por isso o diagnóstico laboratorial é fundamental para a confirmação dos casos. Portanto, o objetivo desse trabalho foi padronizar um teste de Dot-blot IgM para o sorodiagnóstico da leptospirose humana. O teste de Dot-blot IgM foi avaliado utilizando antígenos extraídos de Leptospira interrogans sorovar Copenhageni e de Leptospira biflexa sorovar Patoc por termoresistência, n-butanol e sonicação para detecção de anticorpos IgM antileptospiras. Além disso, anticorpo anti-IgM humano conjugado com fosfatase alcalina foi comparado a anticorpo anti-IgM humano conjugado com peroxidase. Foram analisadas amostras pareadas de soros de 147 pacientes com leptospirose confirmada pelo teste de aglutinação microscópica (MAT) que apresentaram soroconversão. Foram analisadas também amostras únicas de soro de 88 pacientes com diferentes patologias e 60 amostras de soro de indivíduos sadios, pertencentes ao grupo controle. Os testes de Dot-blot IgM com antígenos extraídos por termo-resistência e com conjugado fosfatase alcalina apresentaram maiores taxas de sensibilidade, principalmente na fase aguda da doença. Assim, foi realizada uma padronização do teste de Dot-blot IgM com conjugado fosfatase alcalina e antígenos extraídos por termoresistência de sorovar Copenhageni (TR2) e de sorovar Patoc (TR19) e foi testado também a mistura desses dois antígenos (TR). Foram analisadas amostras pareadas de 124 casos confirmados de leptospirose pelo MAT que apresentaram soroconversão e como grupo controle, 85 amostras únicas de soro de pacientes com outras doenças e 58 soros de indivíduos sadios foram analisadas. Os resultados foram interpretados por dois observadores independentes. Também foram avaliadas a reprodutibilidade e repetibilidade dos testes. O Dot-blot IgM com antígeno TR19 e conjugado fosfatase alcalina apresentou maior taxa de sensibilidade nos primeiros sete dias após o início dos sintomas (60,5%) e por isso se mostrou melhor que os antígenos TR2 e TR. A sensibilidade desse teste variou de 58,1% a 96,0% entre as primeiras e segundas amostras de soro e a especificidade foi de 93,7%. O teste se mostrou consistente com 100% de repetibilidade e reprodutibilidade e com resultado visual confiável, com concordância quase perfeita entre as interpretações dos resultados realizadas por dois observadores independentes. O teste de Dot-blot IgM com antígeno TR19 e conjugado fosfatase alcalina pode ser utilizado como teste de triagem no diagnóstico sorológico da leptospirose humanaLeptospirosis is a zoonosis with a worldwide distribution caused by pathogenic spirochetes of the genus Leptospira spp. Symptoms of leptospirosis can be nonspecific, making clinical diagnosis difficult, so laboratory diagnosis is essential to confirm cases. Thus, the aim of this work was to standardize an IgM Dot-blot test for the serodiagnosis of human leptospirosis. The IgM Dot-blot test was evaluated using antigens extracted from Leptospira interrogans serovar Copenhageni and Leptospira biflexa serovar Patoc by thermal-resistance, nbutanol and sonication to detect anti-leptospiral IgM antibodies. In addition, antibody anti human IgM conjugated to alkaline phosphatase was compared to antibody anti human IgM conjugated to horseradish peroxidase. Paired serum samples from 147 patients with leptospirosis confirmed by the microscopic agglutination test (MAT) with seroconversion were analyzed. Single serum samples from 88 patients with different pathologies and 60 serum samples from healthy individuals belonging to the control group were also analyzed. The IgM Dot-blot tests with antigens extracted by thermal-resistance and with alkaline phosphatase conjugate showed higher sensitivity rates, especially in the acute phase of the disease. Thus, a standardization of the IgM Dot-blot test with alkaline phosphatase conjugate and antigens extracted by thermal-resistance from serovar Copenhageni (TR2) and from serovar Patoc (TR19) was carried out and the mixture of these two antigens (TR) was also tested. Paired serum samples from 124 confirmed cases of leptospirosis by MAT with seroconversion were analyzed. The control group consisted of 85 single serum samples from patients with other diseases and 58 sera from healthy individuals.The results were interpreted by two independent observers. The reproducibility and repeatability of the tests were also evaluated. IgM Dot-blot with TR19 antigen and alkaline phosphatase conjugate showed a higher sensitivity rate in the first seven days after the onset of illness (60.5%) and therefore proved to be better than TR2 and TR antigens. The sensitivity of the test ranged from 58.1% to 96.0% between the first and second serum samples and the specificity was 93.7%. The test was shown to be consistent with 100% repeatability and reproducibility and with a reliable visual result, with almost perfect agreement between the interpretations of the two independent observers. The IgM Dot-blot test with TR19 antigen and alkaline phosphatase conjugate can be used as a screening test in the serological diagnosis of human leptospirosis

Evaluation of glycolipoprotein antigen for serodiagnosis of leptospirosis

Este trabalho visa investigar a resposta sorológica para glicolipoproteína (GLP) de leptospiras com a finalidade de padronizar o uso deste antígeno em testes sorológicos para o diagnóstico da leptospirose humana. Dentre as proteínas envolvidas na patogenicidade das leptospiras, a GLP ainda não foi estudada quanto a imunogenicidade e quanto ao seu emprego como antígeno na detecção de anticorpos específicos. Assim, dot-ELISA para detecção de anticorpos IgG e IgM, com o emprego de GLP de leptospiras patogênica e não patogênica, foi padronizado. Foram testadas amostras pareadas de soros de 90 pacientes com leptospirose confirmada sorologicamente por MAT, sendo as primeiras amostras colhidas na fase aguda da doença e as segundas amostras após 10 a 15 dias. Foram testadas também amostras únicas de 30 pacientes de diferentes patologias, que apresentaram resultados negativos no MAT, pertencentes ao grupo controle. A detecção de anticorpos IgG não mostrou resultados satisfatórios, tendo sido, o seu estudo, descontinuado. Os resultados do dot-ELISA IgM mostraram sensibilidade de 100% nas amostras colhidas após soroconversão no MAT e a precocidade de detecção foi demonstrada pela alta positividade nas amostras colhidas na fase aguda da doença (76,6% para dot-ELISA com antígeno de Leptospira interrogans sorovar Copenhageni e 90% com antígeno de Leptospira biflexa sorovar Patoc). A especificidade foi alta (96,6%), porém o dot-ELISA utilizando ambos os antígenos apresentou 3,3% de resultados falso-positivos. Este estudo demonstrou a importância da resposta imune humoral ao antígeno GLP de leptospiras. A utilização da GLP, no teste dot-ELISA para detecção de anticorpos IgM permitiu realizar o diagnóstico sorológico de forma simples, rápida e com alta eficiência diagnóstica.The aim of this work was the study of serologic response against leptospira glycolipoprotein (GLP) for the purpose of standardizing its use as an antigen on serological tests to diagnose human leptospirosis. Among the proteins involved in the pathogenicity, GLP is yet to be studied regarding its immunogenicity and its use as an antigen in the detection of specific antibodies. That led to our standardization of dot-ELISA for detecting IgG and IgM antibodies, using GLP extracted from either pathogenic Leptospira interrogans serovar Copenhageni or nonpathogenic Leptospira biflexa serovar Patoc. Paired serum samples were taken from 90 patients with serologically confirmed leptospirosis, by MAT, with the first samples collected on the disease\'s acute phase and the second samples after a period of 10 to 15 days. We also tested single samples taken from 30 patients with other diseases, MAT negative, belonging to the control group. Detection rates for IgG antibodies were unsatisfactory, so the study for that use was discontinued. The dot-ELISA IgM results yielded 100% sensitivity on the samples taken after MAT seroconversion. The early detection pattern was evidenced by the high positivity rate on the samples taken during the disease\'s acute phase (76,6% dot-ELISA using Leptospira interrogans serovar Copenhageni antigen and 90% using Leptospira biflexa sorovar Patoc antigen). Specificity rates were high (96.6%), although a 3.3% rate of false-positive results was observed for dot-ELISA using both antigens. The present study revealed the importance of the humoral immune response against the GLP antigen. The use of GLP, on the dot-ELISA test for IgM antibodies afforded a simple, quick and effective diagnosis

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

Data from normal and leptospirotic guinea pigs.

<p>Values are expressed as mean±SEM. Pf- Osmotic Water Permeability, µm/s; UOsm- Urinary Osmolality, mOsm/Kg H₂O; UV- Urinary Volume ml; BUN-Blood Urea Nitrogen mg%; ngp- normal and lgp leptospirotic guinea pigs.</p>**<p>-p<0.05;</p>*<p>p<0.01 vs ngp values.</p

IMCD water permeability.

<p>Effects of GLPc (250 µg/ml) on: 2A - Vasopressin action (Vp, 200 pg/ml, n = 5), 2B - cAMP activity (10⁻⁵ M), 2C - Forskolin action (Fors 10⁻⁹ M, n = 5), 2D - Cholera Toxin action (ChT 10⁻⁹ M, n = 6), 2E - effect of GLPp on Vp action (GLPp- 250 µg/ml, n = 5). Values are expressed as mean ± SEM. Lines connect the averages of the data for each different period of the experiments. Significant differences: * p<0.02, **p<0.01. £ p<0.05, # p<0.001.</p

Leptospirotic guinea pig data.

<p>A- Urinary Volume (ml) and Urinary Osmolality (mOsm/Kg H₂O). Dotted bar-UOsm; Open bar-UV. B- Water permeability. Pf µm/s. Values are expressed as mean ± SEM. Significant differences: * p<0.01; ** p<0.05.</p

Western blot analysis of water transporter protein in IMCD from normal (n = 3) and injected i.p. with GLPc (n = 5) guinea pigs.

<p>3A- Western blot analysis of AQP2 protein expression showing the 29 KDa and 35–50 KDa bands and the actin band; 3B- Quantitative densitometric analysis of AQP2 protein abundance. * p<0.05 vs. control.</p