Analysis of LruC lipoprotein and identification of peptides candidates for vaccine development and diagnosis of leptospirosis.

Leptospirosis is a public health concern with lethality around 15% of the total cases. The current vaccines against Leptospira infection based on bacterins have several limitations, which require urgent development of new ones. In this context, groundbreaking approaches such as peptide-vaccines could be used to come around with promising results. Our goal was to identify conserved and immunogenic epitopes from the lipoprotein LruC that could interact with Major Histocompatibility Complex (MHC) I and II. LruC is a conserved lipoprotein expressed during leptospirosis that is considered among vaccine candidates and can be used as source for development of peptide-based vaccines. We searched for peptides that would be recognized by antibodies from either serum of hamsters previously immunized with low-LPS bacterin vaccines or from serum of patients diagnosed with leptospirosis. Immuno properties of seven peptides from LruC protein were evaluated in silico and by Dot Blot assay, and validate by ELISA. Preliminary results pointed one promising peptide that was recognized by the sera. In conclusion, the immunoinformatic approach helps the search and screening of peptides, while the Dot Blot assay, a simple and effective tool, helps to test and validate them. Thus, these prospective techniques together were validated to identify and validate potential peptides for further investigation as peptide-based vaccines or diagnostic methods.

Evaluation of leptospiral recombinant antigens MPL17 and MPL21 for serological diagnosis of leptospirosis by enzyme-linked immunosorbent assays.

Leptospirosis is a zoonosis of multisystem involvement caused by pathogenic strains of the genus Leptospira. In the last few years, intensive studies aimed at the development of a vaccine have provided important knowledge about the nature of the immunological mechanisms of the host. The purpose of this study was to analyze the immune responses to two recombinant proteins, MPL17 and MPL21 (encoded by the genes LIC10765 and LIC13131, respectively) of Leptospira interrogans serovar Copenhageni in individuals during infection. The recombinant proteins were expressed in Escherichia coli as six-His tag fusion proteins and were purified from the soluble bacterial fraction by affinity chromatography with Ni(2+)-charged resin. The recombinant proteins were used to evaluate their ability to bind to immunoglobulin G (IgG) (and IgG subclass) or IgM antibodies in serum samples from patients in the early and convalescent phases of leptospirosis (n = 52) by enzyme-linked immunosorbent assays. The prevalences of total IgG antibodies against MPL17 and MPL21 were 38.5% and 21.2%, respectively. The titers achieved with MPL17 were statistically significantly higher than those obtained by the reference microscopic agglutination test. The specificity of the assay was estimated to be 95.5% for MPL17 and 80.6% for MPL21 when serum samples from individuals with unrelated febrile diseases and control healthy donors were tested. The proteins are conserved among Leptospira strains that cause human and animal diseases. MPL17 and MPL21 are most likely new surface proteins of leptospires, as revealed by liquid-phase immunofluorescence assays with living organisms. Our results demonstrate that these recombinant proteins are highly immunogenic and, when they are used together, might be useful as a means of diagnosing leptospirosis.

Culturas mistas de micobactérias: é importante isolar e identificar? / Mixed mycobacterial cultures: is it important to separate and to identify multiple species?

A investigação de culturas mistas de micobactérias é importante pois geralmente estas incluem ao menosuma espécie patogênica ou potencialmente patogênica. Dentre 8.036 culturas recebidas entre 1999 e 2000,pelo Setor de Micobactérias do Instituto Adolfo Lutz, foram selecionadas 21 (0,26%) com resultados sugestivos de culturas mistas. Após o isolamento em meio 7H11 as colônias foram repicadas em Lõwenstein Jensen e incubadas à 37° C. A identificação dos 32 subcultivos foi feita por métodos fenotípicos e pela analise do perfil de restrição do produto da amplificação de 440 pares de base do gene hsp65. Em oito subcultivos foi encontrada a espécie M. tuberculosis associada com MNT, em 3 subcultivos foramencontradas 2 espécies de MNT e nos demais foi identificado apenas um tipo de micobacteria. O tempo decrescimento lento das micobactérias inviabiliza o plaqueamento de todas as culturas pois este procedimentoacarretaria demora na liberação do resultado final dos testes, além de representar gastos excessivos em áreas endêmicas, geralmente com escassos recursos econômicos. Embora as dificuldades mencionadas, os microbiologistas devem estar atentos quanto à presença de culturas mistas de micobactérias e usar todos os métodos disponíveis para separar e identificar as espécies

Estratégias para a identificaçäo de espécies do complexo mycobacterium fortuitum / Strategies for identification of the mycobacterium fortuitum complex species

As micobactérias de crescimento rápido anteriormente classificadas como complexo micobacterium fourtuitum foram recentemente designadas M.fortuitum, M. peregrinum, M. chenonae e M. abcessus. A identificaçäo dessas micobactérias é importante para estabelecer a terapêutica adequada, visto que possuem diferentes padröes de resistência às drogas. Dentre 3.441 culturas de micobactérias recebidas em 1999 no Setor de micobactérias do Instituto Adolfo Lutz, foram selecionadas 13 cepas, classificadas como complexo M. fortuitum. O estudo incluiu o isolamento das colônias para certificar-se da pureza das culturas, a adiçäo de testes com carboidratos e citrato de sódio que separam as quatro espécies citadas e o uso da técnica de PCR restriction analysis do gene hsp65 (PRA) para elucidaçäo de resultados duvidosos dos tstes fetípicos. Na análise das 13 culturas, observou-se que oito apresentaram dois tipos de colônias (lisa e rugosa) e as cinco restantes apenas um tipo. As identificaçöes feitas com a técnica de PRA e c om os testes fenotípicos foram concordantes na maioria das cepas. Os resultados desse estudo de referência e implementada com testes específicos para cada espécie, que possibilitem a elucidaçäo rápida do diagnóstico

The rapidly growing mycobacteria, previously classified as Mycobacterium fortuitumcomplex, has recently been designated M. fortuitum, M. peregrinum, M. chelonae and M. abscessus. Theidentification of these mycobacteria is important for establishing the proper treatment, because they havedifferent patterns to drugs. Among 3,441 cultures of mycobacteria received in 1999 by the MycobacteriaLaboratory of the Adolfo Lutz Institute, 13 cultures classified as M. fortuitum complex were selected to bestudied. The study included isolation of colonies in order to ensure the purity of the cultures, the additionof tests with carbohydrate and citrate, which differentiate the four previously mentioned species and theuse of the PCR restriction analysis of hsp65 (PRA) for the analysis of dubious results in conventionaltests. In the analysis of the 13 cultures, it was observed that eight showed two types of colonies (smoothand rough) and the remaining five showed only one type. The identification by PRA technique and byconventional tests agreed to the majority of the strains. The results of this study suggest that theidentification of mycobacteria, due to its complexity, should be centralized in Reference Laboratories andimplemented with specific tests for each specie in order to give a rapid diagnosis