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Identificação de micobactérias não tuberculosas através de métodos moleculares não comerciais
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Embora Mycobacterium tuberculosis, o agente etiológico da tuberculose humana, seja a principal causa de micobacteriose no Homem, outras micobactérias não tuberculosas (MNT), podem também causar infecção em seres humanos, sendo cada vez mais frequentemente isoladas no laboratório de micobacteriologia. Dada a morosidade da identificação de MNT por métodos convencionais, torna-se essencial o desenvolvimento de métodos rápidos e fiáveis para a sua identificação. Neste estudo foram comparados três métodos moleculares para a identificação de MNT, baseados na análise de restrição enzimática após amplificação de: (i) região rRNA 16S-23S “Internal Transcribed Spacer” (ITS), (ii) gene hsp65, e (iii) zona ITS e regiões adjacentes, 16S e 23S rDNA. Para este fim, avaliamos 50 isolados, correspondendo a 47 isolados clínicos de MNT e três estirpes de referência, representando as 11 espécies de MNT mais frequentemente isoladas no laboratório de Micobactérias do Instituto de Higiene e Medicina Tropical (IHMT, UNL) e uma estirpe referência de M. tuberculosis, e identificadas anteriormente utilizando o sistema comercial GenoType® Mycobacterium CM/AS (Hain Lifescience).
O PCR-RFLP do gene hsp65 proporcionou os melhores resultados, identificando correctamente 42 dos 49 isolados de MNT, pertencentes a M. avium, M. gordonae, M. intracellulare, M. kansasii, M. chelonae, M. fortuitum, M. abscessus, M. szulgai, M. peregrinum e M. xenopi. O PCR-RFLP da região ITS identificou correctamente 28 dos 49 isolados testados, não distinguindo M. intracellulare/M. scrofulaceum, M. avium/M. bohemicum e M. kansasii/M. szulgai. Finalmente, o PCR-RFLP da região ITS e regiões adjacentes mostrou o pior desempenho, com apenas oito isolados correctamente identificados e falhando na amplificação de diversos isolados. Dos três métodos testados, o PCR-RFLP do gene hsp65 e da região ITS mostraram maior capacidade de identificação e reprodutibilidade. No entanto, a sua aplicação exige uma optimização cuidadosa das condições de análise e a sua aplicabilidade depende, em grande parte, da diversidade
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de MNT em cada laboratório. Verificaram-se também várias dificuldades a nível da interpretação dos resultados. Assim, apesar das vantagens referidas na literatura para estes três métodos, verificou-se que o grau de variabilidade e dificuldade de interpretação associados aos padrões obtidos, limitam a sua implementação no laboratório de diagnóstico de micobacteriologia.
Although Mycobacterium tuberculosis, the etiologic agent of human tuberculosis is the main cause of mycobacteriosis in Man, other nontuberculous mycobacteria (NTM), may also cause infection in humans, being increasingly isolated in the mycobacteriolgy laboratory. The NTM identification by conventional methods is time consuming, demanding forfaster and reliable identification methods.In this study we compared three molecular methods for the identification of NTM based on the restriction enzyme analysisofthe amplification products of: (i) the 16S-23S rRNA InternalTranscribed Spacer (ITS) region, (ii) the hsp65gene and (iii) the ITS and its flanking regions, 16S and 23S rDNA. For this purpose, we evaluated a collection of 50 isolates, comprising 47 clinical NTM isolatesand threereference strains, representing the 11 NTM species most frequently isolated in the laboratory of Micobactérias,Instituto de Higiene e Medicina Tropical (IHMT, UNL) and one M. tuberculosisreference strain, and previously identified using the GenoType® Mycobacterium CM/AS (Hain Lifescience).The methodbased in the PCR-RFLP of hsp65provided the best results, identifying correctly 42of the 49NTM isolates, corresponding to M. avium, M. gordonae,M. intracellulare, M. kansasii,M. chelonae, M. fortuitum, M. abscessus, M. szulgai, M. peregrinum andM. xenopi. The PCR-RFLP ITS identified correctly 28of the 49isolates tested. However, it could not distinguish between M. intracellulare/M. scrofulaceum, M. avium/M. bohemicumandM. kansasii/M. szulgai. Finally, the method based on the ITS and flanking regions showed the lowest identification performance, with only eight isolates correctly identified and failure to obtain amplification product for several isolates.From the three methods tested, the PCR-RFLP of hsp65and ITS showed the highest identification capacity and reproducibility. However, their application requires a careful optimization of the analysis conditions and their applicability depends largely on the NTM species diversity on each laboratory. We also experienced several difficulties on results interpretation.In conclusion, in spite the advantages referred in the literature for these three methods, the variability associated with the patterns obtained and the difficulties in their interpretationlimit the implementation of these methods in the mycobacteriology laboratory.
Although Mycobacterium tuberculosis, the etiologic agent of human tuberculosis is the main cause of mycobacteriosis in Man, other nontuberculous mycobacteria (NTM), may also cause infection in humans, being increasingly isolated in the mycobacteriolgy laboratory. The NTM identification by conventional methods is time consuming, demanding forfaster and reliable identification methods.In this study we compared three molecular methods for the identification of NTM based on the restriction enzyme analysisofthe amplification products of: (i) the 16S-23S rRNA InternalTranscribed Spacer (ITS) region, (ii) the hsp65gene and (iii) the ITS and its flanking regions, 16S and 23S rDNA. For this purpose, we evaluated a collection of 50 isolates, comprising 47 clinical NTM isolatesand threereference strains, representing the 11 NTM species most frequently isolated in the laboratory of Micobactérias,Instituto de Higiene e Medicina Tropical (IHMT, UNL) and one M. tuberculosisreference strain, and previously identified using the GenoType® Mycobacterium CM/AS (Hain Lifescience).The methodbased in the PCR-RFLP of hsp65provided the best results, identifying correctly 42of the 49NTM isolates, corresponding to M. avium, M. gordonae,M. intracellulare, M. kansasii,M. chelonae, M. fortuitum, M. abscessus, M. szulgai, M. peregrinum andM. xenopi. The PCR-RFLP ITS identified correctly 28of the 49isolates tested. However, it could not distinguish between M. intracellulare/M. scrofulaceum, M. avium/M. bohemicumandM. kansasii/M. szulgai. Finally, the method based on the ITS and flanking regions showed the lowest identification performance, with only eight isolates correctly identified and failure to obtain amplification product for several isolates.From the three methods tested, the PCR-RFLP of hsp65and ITS showed the highest identification capacity and reproducibility. However, their application requires a careful optimization of the analysis conditions and their applicability depends largely on the NTM species diversity on each laboratory. We also experienced several difficulties on results interpretation.In conclusion, in spite the advantages referred in the literature for these three methods, the variability associated with the patterns obtained and the difficulties in their interpretationlimit the implementation of these methods in the mycobacteriology laboratory.
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Ciências biomédicas Biologia molecular Micobactérias Diagnóstico Tuberculose
Contexto Educativo
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Instituto de Higiene e Medicina Tropical