AplicaÃÃo de tÃcnica molecular no desenvolvimento de teste para diagnÃstico de tuberculose paucibacilar e na caracterizaÃÃo genotÃpica do mycobacterium tuberculosis

AUTOR(ES)
DATA DE PUBLICAÇÃO

2003

RESUMO

Tuberculosis (TB) is a disease for which early diagnostic and correct treatment are essential to improve the efficacy of the TB Control Program to cure sick people and avoid transmission of the disease. Laboratory diagnosis involves baciloscopy using Zeehl Neelsen staining, a fast, inexpensive and accessible methodology, with sensitivity of 104 bacilli/ml and a culture that is more sensitive. However it can take between 9-45 days to receive results. For TB diagnosis it is hoped genetic amplification techniques will improve sensitivity and especificity for detection of Mycobacterium. In this study a molecular technique for paucibacilary TB diagnosis was developed and genetic characterization of Mycobacterium tuberculosis (M.tb) strains performed. The Heminested-PCR assay was developed for TB diagnosis based on the IS6110 genetic marker. The optimal conditions for primers, magnesium chloride and melting temperature were determined. The sensitivity of the technique was 30fg and can be concluded in up to three days after sample collection. Heminested PCR in comparison with conventional PCR improves sensitivity, especificity, which is especially important in biological paucibacilary samples. The results of the genetic characterization of 61 M.tb strains obtained in 2000-2002 period, by Double Repetitive Elements-PCR (DREPCR), allowed identification of five groups of patients, designated as I, II, III, IV, and V with 4, 3, 6, 2 and 2 patients respectively, suffering from strains which were 100% similar and showing respectively 5, 5, 1, 2 and 5 bands on the agarose electrophoresis gel. The strains with the same drug resistance profile showed different genetic profiles, except for three strains of group I. The best epidemiological correlation was detected in group I. Three members of this group are relatives, all of whom carry strains resistant to isoniazid and rifampin. The three patients are women and all three suffer from âdiabetes mellitusâ. The first case of TB in these patients was diagnosed in 1999 and the second in 2000. The third case was diagnosed in the daughter of this last case in 2002. The impact of the genetic characterization in these cases was the analysis of the dynamics of transmission and immediate use of the correct therapeutic scheme for the daughter, once the resistance profile to the drugs of first choice was known for the other two patients

ASSUNTO(S)

genetica caracterizaÃÃo genotÃpica tuberculose mycobacterium tuberculosis tÃcnica molecular

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