Leptospirose: Características da enfermidade em humanos e principais técnicas de diagnóstico laboratorial / Leptospirosis: Characteristics of the human disease and main laboratory diagnostic techniques

A leptospirose é a zoonose de maior distribuição geográfica, com estimativa de cerca de 60.000 mortes por ano. A doença é causada por bactérias do gênero Leptospira, que possui mais de 300 diferentes sorovares e 64 espécies já identificadas, sendo o ambiente a principal fonte de contaminação. A doença em humanos apresenta manifestações clínicas variadas e caráter bifásico, devendo ser confirmada por meio do diagnóstico laboratorial. O objetivo deste trabalho foi reunir conceitos atualizados sobre a leptospirose humana e as principais técnicas de diagnóstico laboratorial empregadas. A MAT é considerada o padrão-ouro para o diagnóstico da leptospirose, mas devido à baixa sensibilidade na fase inicial da doença é necessário o emprego de técnicas mais sensíveis neste período. Baseado em diversos estudos, as metodologias de PCR, ELISA-IgM e teste rápido apresentaram sensibilidade satisfatória nos primeiros dias após o início dos sintomas. Na segunda semana, a MAT apresentou 100% de sensibilidade, mantendo sua alta especificidade em ambas as fases. No geral, os testes sorológicos de ELISA-IgM e teste rápido apresentaram resultados satisfatórios como métodos de diagnóstico precoce, principalmente tratando-se de locais com pouca infraestrutura, diferente dos laboratórios de referência onde é possível empregar as técnicas de PCR e MAT.

Leptospirosis is the most widespread zoonosis, which has a balance of almost 60,000 deaths per year. Bacteria of Leptospira genus, which has more than 300 different serovars and 64 species already identified, cause the disease, being the environment the main source of contamination. The human disease presents a large set of clinical manifestations, showing biphasic presentation, the reason why leptospirosis must be confirmed by laboratory diagnosis. This study aimed to group recent concepts concerning human leptospirosis and the main diagnosis techniques employed at the laboratory. MAT is considered the gold standard for leptospirosis diagnosis, but has low sensitivity on the onset of disease, leading to the use of techniques with higher sensitivity on this period. Based on several studies, PCR, ELISA-IgM and rapid test presented satisfactory sensitivity on the onset of symptoms. In the second week, MAT showed 100% of sensitivity, maintaining its high specificity in both phases. In general, the ELISA-IgM and rapid serological tests showed satisfactory results as methods for early diagnosis, especially in the case of places with poor infrastructure, different from the reference laboratories where it is possible to use the PCR and MAT techniques.

Molecular and serological characterization of Leptospira kirschneri serogroup Pomona isolated from a human case in a Brazilian rural area

Abstract INTRODUCTION: Leptospirosis is an important health concern in Brazil. Currently, information on the epidemiology of the disease in the rural areas of the country is lacking. METHODS: Serological and molecular techniques were used to characterize a clinical isolate of Leptospira. RESULTS: The strain CLEP 00060, isolated from a 59-year-old man in a rural area of Rio Grande do Sul state, Brazil, was identified as belonging to L. kirschneri serogroup Pomona serovar Mozdok. CONCLUSIONS: This study contributes to the local epidemiological knowledge of leptospirosis, prevention of the disease by vaccines, and improvements in its diagnosis.

Multiplex PCR-based detection of Leptospira in environmental water samples obtained from a slum settlement

The aim of this study was to apply a molecular protocol to detect leptospiral DNA in environmental water samples. The study was carried out in a peri-urban settlement in Petrópolis, state of Rio de Janeiro. A multiplex PCR method employing the primers LipL32 and 16SrRNA was used. Three out of 100 analysed samples were positive in the multiplex PCR, two were considered to have saprophytic leptospires and one had pathogenic leptospires. The results obtained supported the idea that multiplex PCR can be used to detect Leptospira spp in water samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much more easily than conventional methodologies.

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.

The involvement of tetA and tetE tetracycline resistance genes in plasmid and chromosomal resistance of Aeromonas in Brazilian strains

This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 strains of genus Aeromonas, isolated from clinical and food sources. Polymerase chain reactions revealed that 37.5 percent of the samples were positive for tetA, and also 37.5 percent were tetE positive. One isolate was positive for both genes. Only the isolate A. caviae 5.2 had its resistance associated to the presence of a plasmid, pSS2. The molecular characterization of pSS2 involved the construction of its restriction map and the determination of its size. The digestion of pSS2 with HindIII originated two fragments (A and B) that were cloned separately into the pUC18 vector. The tetA gene was shown to be located on the HindIII-A fragment by PCR. After transforming a tetracycline-sensitive strain with pSS2, the transformants expressed the resistance phenotype and harbored a plasmid whose size was identical to that of pSS2. The results confirmed the association between pSS2 and the tetracycline resistance phenotype, and suggest a feasible dissemination of tetA and tetE among strains of Aeromonas. This study suggests the spreading tetA and tetE genes in Aeromonas in Brazil and describes a resistance plasmid that probably contributes to the dissemination of the resistance.