Caracterização de cepas de referência de Leptospira sp utilizando a técnica de pulsed field gel electrophoresis / Characterization of Leptospira sp reference strains using the pulsed field gel electrophoresis technique

INTRODUÇÃO: A leptospirose é uma zoonose endêmica, mundialmente distribuída, causada por bactérias do gênero Leptospira. Este gênero compreende espécies patogênicas e saprofíticas, com mais de 200 sorovares distintos, dificultando sua caracterização. A técnica de pulsed field gel electrophoresis tem sido empregada como uma ferramenta para auxiliar nesta caracterização. Os objetivos deste trabalho foram padronizar a técnica de PFGE, determinar os perfis moleculares das cepas de referência utilizadas pelo Laboratório de Referência Nacional para Leptospirose/Centro Colaborador da Organização Mundial de Saúde para Leptospirose e criar um banco de dados com estes perfis. MÉTODOS: Foram analisadas, por PFGE, dezenove cepas utilizando a enzima de restrição NotI. RESULTADOS: Cada cepa apresentou um perfil único que pode ser considerado como uma identidade genômica específica, com exceção dos sorovares Icterohaemorrhagiae e Copenhageni, cujos perfis foram indistinguíveis. CONCLUSÕES: Dessa forma, foi possível a criação de um banco de perfis moleculares que está sendo utilizado no Laboratório para a comparação e identificação de cepas isoladas de quadros clínicos.

INTRODUCTION: Leptospirosis is an endemic zoonosis of worldwide distribution, caused by bacteria of the genus Leptospira. This genus includes pathogenic and saprophytic species, with more than 200 different serovars, thus making it difficult to characterize. The technique of pulsed field gel electrophoresis has been used as a tool to aid in this characterization. The aims of this study were to standardize the PFGE technique, determine the molecular profiles of reference strains used at the National Reference Laboratory for Leptospirosis/World Health Organization Collaborating Center for Leptospirosis and create a database with these profiles. METHODS: Nineteen strains were analyzed by means of PFGE, using the restriction enzyme NotI. RESULTS: Each strain presented a unique profile that could be considered to be a specific genomic identity, with the exception of the serovars Icterohaemorrhagiae and Copenhageni, whose profiles were indistinguishable. CONCLUSIONS: It was possible to create a database of molecular profiles, which are being used in the Laboratory for comparing and identifying strains isolated from clinical cases.

Método de la reacción en cadena de la polimerasa en la detección temprana de Leptospira spp. en hemocultivos de pacientes con sospecha de leptospirosis / PCR method for the early detection of Leptospira spp. in hemocultures from patients suspected of leptospirosis

Se aplicó un método de la reacción en cadena de la polimerasa para la detección temprana de Leptospiras spp. en hemocultivos procedentes de pacientes con sospecha de leptospirosis humana. Los métodos moleculares, y en particular la amplificación del ADN por la reacción en cadena de la polimerasa y sus variantes, constituyen herramientas muy útiles y específicas, utilizadas en la actualidad con esta finalidad. Se lograron identificar por la reacción en cadena de la polimerasa Leptospiras spp. en 41,3 por ciento (33/80) de los hemocultivos a los 7 d de incubación, los que también se clasificaron como positivos por los métodos convencionales. Sin embargo, hubo 20 por ciento (16/80) en los que también por este método se lograron identificar Leptospiras spp., pero por los métodos convencionales resultaron ser negativos. De los hemocultivos 38,7 por ciento (31/80) resultó negativo tanto por la reacción en cadena de la polimerasa como por los métodos convencionales. El uso del método de la reacción en cadena de la polimerasa a partir de hemocultivos, es una alternativa valiosa para la detección temprana y el diagnóstico rápido de Leptospiras spp.

The PCR method for the early detection of Leptospiras spp. in hemocultures from patients suspected of human leptospiros was applied. Molecular methods and, particularly, the amplification of DNA by PCR, and its variants, are very useful and specific tools used nowdays to this end. Leptospiras spp. were identied by means of PCR in 41.3 percent(33/80) of the hemocultures at 7 days of incubation.They were also classified as positive by the conventional methods. However, it was also possible to identify Leptospiras spp. in 20 percent (16/80) by this method, but they proved to be negative by the conventional methods. Of the hemocultures, 38.7 percent (31/80) yielded negative by PCR and by the conventional methods. The use of PCR starting from hemocultures is a valuable alternative for the early detection and rapid diagnosis of Leptospiras spp

Amino acid sequences of proteins from Leptospira serovar pomona

This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.