Comparative proteomic analysis of sequential isolates of Mycobacterium tuberculosis from a patient with pulmonary tuberculosis turning from drug sensitive to multidrug resistant.

Background & objectives: Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). Methods: In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: The genotypes of all four isolates remained homologous, indicating no re-infection. The initial isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and rpoB. the intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF resistant isolates. The most prominent and overexpressed proteins found during the development of drug resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c. Interpretation & conclusions: This preliminary proteomic study provides an insight about the proteins that are upregulated during drug resistance development. These upregulated proteins, identified here, could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more studies are required to confirm these findings.

Evaluation of an immunochromatographic test for discrimination between Mycobacterium tuberculosis complex & non tuberculous mycobacteria in clinical isolates from extra-pulmonary tuberculosis.

Background & objectives: Accurate diagnosis of tuberculosis (TB) is crucial to facilitate early treatment of the patients, and to reduce its spread. Clinical presentation of Mycobacterium tuberculosis complex (MTBC) and non tuberculous mycobacteria (NTM) may or may not be the same, but the treatment regimen is always different for both the infections. Differentiation between MTBC and NTM by routine laboratory methods is time consuming and cumbersome. This study was aimed to evaluate an immunochromatographic test (ICT), based on mouse monoclonal anti-MPT64, for simple and rapid discrimination between MTBC and NTM in clinical isolates from extra-pulmonary tuberculosis cases. Methods: A total of 800 clinical samples were collected from patients suspected to have extra-pulmonary tuberculosis. Preliminary diagnosis has been done by direct Ziehl–Neelsen (ZN) staining followed by culture in BACTEC system. A total of 150 clinical isolates, which were found positive in BD 460 TB system during September 2009 to September 2010 were selected for the screening by ICT test. p-nitro-α-acetylamino- β-hydroxy propiophenone (NAP) test was performed for differentiation of MTBC and NTM. M. tuberculosis complex was further confirmed by IS6110 PCR of BACTEC culture positive isolates, this served as the reference method for MTBC identification and comparative evaluation of the ICT kit. Results: Of the 150 BACTEC culture positive isolates tested by ICT kit, 101 (67.3%) were found positive for MTBC and remaining 49 (32.7%) were considered as NTM. These results were further confirmed by IS6110 PCR that served as the reference method for detection of MTBC. H37Rv reference strain was taken as a control for ICT test and IS6110 PCR. The reference strain showed the presence of MPT64 antigen band in the ICT test. Similar bands were formed in 101 of 102 MTBC isolates tested, proving 99.1 per cent sensitivity and no bands were detected in 48 (100%) NTM isolates tested, proving 100 per cent specificity of the ICT kit. Interpretation & conclusions: Our findings show that ICT test can be used on direct culture positive specimens. It does not require any special equipment, is simple and less time consuming. It can easily discriminate between MTBC and NTM and thus can help in appropriate management of tuberculosis.

Acción del agua del Volcán Copahue (Neuquén, Argentina) sobre micobacterias / Action of the Copahue volcano waters on mycobacteries

Se efectúa un estudio de la acción del agua del Volcán Copahue (AVC) sobre 14 cepas de micobacterias. Los tres ensayos utilizados para determinar resistencia o sensibilidad al agua dieron resultados coincidentes. Las Micobacterias no tuberculosas presentaron distintos grados de resistencia, en cambio M.tuberculosis, M. bovis y M.marinum mayor sensibilidad. Se complementa con breves estudios de la acción del AVC sobre el efecto pH, destrucción del "factor cuerda" de los bacilos, y pérdida de su actividad enzimática (catalasa y peroxidasa)

Viabilidad del M. tuberculosis expuesto al fosfato trisodico en medio de Ogawa-Kudoh a temperatura ambiente / Viability of the M. tuberculosis exposed to trisodic phosphate in Ogawa-Kudoh media at room temperature

Se practico un estudio en donde se demostro que la viabilidad del M. Tuberculosis en esputo no resulta afectada por la adicion de fosfato trosodico (FTS) al 10% en partes iguales, al ser utilizado como decontaminante hasta por 48 horas y mantenido a temperatura ambiente; tampoco disminuyo dicha viabilidad despues de sembrar el esputo en medio de Ogawa-Kudoh (O-K) y mantenerlo a temperatura ambiente (20+ o - 2 grados C) durante un mes