Comparative molecular study of Mycobacterium tuberculosis strains, in times of antimicrobial drug resistance / Estudio molecular comparativo de cepas de Mycobacterium tuberculosis, en tiempos de resistencia antimicrobiana a los fármacos

Strains of Mycobacterium tuberculosis were compared using two DNA fingerprinting techniques: Restriction Fragment Length Polymorphism (RFLP) and Double-Repetitive-Element-PCR (DRE-PCR). Two of these strains: IH1 (susceptible to isoniazid) and IH2 (resistant to isoniazid) were recovered from cases of pulmonary tuberculosis which occurred in two brothers who lived together. The first one was recognized on July 1999, and the second was diagnosed one year later. IH1 and IH2 showed the same pattern of bands with both molecular tests. These results suggest that single drug chemoprophylaxis may occasionally select resistant strains for that drug, which can eventually cause disease and be recognized through these tests. Strains IH3, IH4 and IH5 were obtained from sputum samples of 3 different patients, and intra-laboratory cross-contamination was suspected when it was realized that the 3 positive materials had been consecutively processed the same day by the same worker in the same biological safety cabinet. Again, the 3 strains revealed identical band patterns with RFLP and DRE-PCR, confirming the posed suspicion. The results with DRE-PCR were obtained after only 8 hours of work, without the need for subcultures. This procedure allows quick correction of treatment conducts, avoiding unnecessary exposure of people and bacteria to antimicrobial drugs.

Se compararon cepas de Mycobacterium tuberculosis utilizando 2 procedimientos de ADN fingerprinting: polimorfismo de los fragmentos de restricción (RFLP) y Double-Repetitive-Element-PCR (DRE-PCR). Dos de las cepas: IH1 (susceptible a isoniazida) e IH2 (resistente a isoniazida) se recuperaron a partir de casos de tuberculosis pulmonar que ocurrieron en dos hermanos convivientes. La primera fue aislada en julio de 1999 y la segunda un año después. IH1 e IH2 mostraron el mismo patrón de bandas por ambos procedimientos. Estos resultados sugieren que la quimioprofilaxis con una sola droga puede ocasionalmente seleccionar mutantes resistentes, las cuales pueden causar enfermedad y ser reconocidas por estos procedimientos. Las cepas IH3, IH4 e IH5 fueron aisladas de 3 pacientes diferentes, y examinadas por probable contaminación cruzada dentro del laboratorio ya que fueron procesadas el mismo día, por el mismo operador y en la misma cabina de seguridad biológica. Nuevamente, las 3 cepas revelaron el mismo patrón de bandas por RFLP y por DRE-PCR, confirmando la sospecha. Los resultados de la DRE-PCR se obtuvieron luego de 8 horas de trabajo, sin necesidad de subcultivos. Esta técnica permitiría la rápida correción de pautas de tratamiento, evitando la exposición innecesaria de personas y bacterias a drogas antimicrobianas.

Falsos cultivos positivos por contaminación cruzada en laboratorios de tuberculosis / False-positive cultures due to cross contamination in tuberculosis laboratories

Fifteen episodes of Mycobacterium tuberculosis laboratory cross-contamination suspected between 1996 and 2001 at 6 laboratories in Buenos Aires City and suburbs were investigated by IS6110 RFLP. Thirteen episodes were confirmed. Even though BACTEC 460 produced the highest number of confirmed episodes in a single laboratory, the most extended one occurred while employing conventional culture procedures in solid medium. The double repetitive element-polymerase chain reaction (DRE-PCR) was applied to 8 of these episodes and produced concordant results with those of the RFLP. The DRE-PCR appears to be a valuable tool for the prompt identification of false positive cultures. The timely rectification of defects in laboratory protocols can avert false diagnoses of tuberculosis and unnecessary prolonged treatments.

Estudio de Mycobacterium bovis en leche mediante métodos bacteriológicos y reacción en cadena de la polimerasa / [Study of Mycobacterium bovis in milk using bacteriological methods and the polymerase chain reaction]

The frequency of Mycobacterium bovis detection in milk samples obtained from infected animals was explored in an intensive dairy area in Argentina. To this end, an [quot ]in house[quot ] polymerase chain reaction (PCR) method was developed using Mycobacterium tuberculosis Complex specific INS1-INS2 primers, and its performance was compared with that of bacteriological methods. The decontamination procedures previous to culture reduced M. bovis viability. The pathogen was identified in milk samples from 1 of 143 infected cows and in none of 43 uninfected ones. Even though PCR sensitivity was found to be 2-20 times higher than that of bacteriology in experimentally inoculated milk samples, all 186 field samples resulted negative by PCR, including the bacteriologically-confirmed one. In spite of the high prevalence of bovine tuberculosis in Argentinian dairy herds, the detection of M. bovis in milk is an unusual finding.