The main advantage of the described method is the possibility of detecting rifampicin and isoniazid resistance within 48-72 h after sample collection, with a sensitivity of nearly 100% in smear-positive samples if the chosen target is responsible for the resistance.

A new mycobacteriophage-based technique (PhageTek MB)Tuberculosis continues to be a global health problem, with more than 8 million new cases and 2 million deaths each year (24). The spread of human immunodeficiency virus (HIV) infection and the breakdown in health services have contributed to a dramatic rise in the incidence of tuberculosis. The great majority of the new tuberculosis cases (95%) and tuberculosis deaths (98%) are in developing regions such as Southeast Asia, sub-Saharan Africa, Latin America, and Eastern Europe (15,20). One of the principles of tuberculosis control is rapid and accurate diagnosis of infected patients in order to allow prompt initiation of antibiotic therapy and to prevent transmission. Although the conventional procedures are irreplaceable diagnostic tools, detection of acid-fast bacilli by microscopy shows poor sensitivity, and solid culture methods can take as long as 8 weeks (liquid culture methods are faster but unaffordable for laboratories in low-income areas). In addition, coinfection with HIV has changed the clinical presentation of tuberculosis and reduced the sensitivity of classical microbiology methods (5, 9, 17). Therefore, in developing countries, it is especially important to have an inexpensive and rapid test for tuberculosis identification so that infected individuals can be isolated and treated immediately (10).Mycobacteriophages constitute a potentially useful approach for detecting viable Mycobacterium tuberculosis bacilli as well as for susceptibility studies (22). Several mycobacteriophages have been reported to be highly specific for all M. tuberculosis complex species (11,13,25). These mycobacteriophages have been important in the development of phagebased technology, as shown in some studies (7,14,28). Their relatively rapid replication (which compensates for the otherwise slow growth of their hosts), the simplicity of the methodology, and the relatively inexpensive equipment required make the use of mycobacteriophages a suitable tool for the rapid diagnosis of tuberculosis.The PhageTek MB assay (Organon Teknika Corporation, Durham, N.C.; manufactured by Biotec Laboratories Ltd., Ipswich, United Kingdom, as a variant of its FASTPlaqueTB test) is a phenotypic assay that uses M. tuberculosis complex-specific mycobacteriophages to report the presence of live M. tuberculosis complex organisms within a sample. The target bacteria in a decontaminated respiratory specimen are rapidly infected by the target-specific bacteriophage. A selective virucide is added which, without affecting the cells, destroys all exogenous phages that have not infected the M. tuberculosis complex bacilli. The phages protected within the M. tuberculosis complex organisms replicate and form clear areas (plaques) in a lawn of rapidly growing host helper cells. Although the number of plaques generated from a given sample is related to the number of viable M. tuberculosis complex cells containing mycobacteriophage, this test is qualitative. Results from specimens can be read by eye after ...

We studied the presence of mutations in the whole katG gene and specific regions of the oxyR-ahpC and mabAinhA regulatory region in 61 Mycobacterium tuberculosis isoniazid-resistant isolates. An 81-bp region of the rpoB gene was also sequenced in 17 rifampin-resistant strains. Alterations in the katG gene were detected in 55% of the isolates. Mutation in codon 315 was the most prevalent (32%). Strains showed a high level of resistance, and most maintained a substantial catalase-peroxidase activity. Three strains with an isoniazid MIC of Ն32 g/ml lacked catalase-peroxidase activity. Two of them had deletions in the catalytic domain of the KatG protein. One strain with deletion and three strains with mutations in the C-terminal domain showed low-level resistance and conserved the catalase-peroxidase activity. Mutations in the mabA-inhA regulatory region were identified in 32% of the isolates. All had low-level resistance, and the vast majority conserved catalase-peroxidase activity. Seventeen percent of the isoniazid-resistant isolates had no detectable alterations at the studied loci. Resistance to rifampin was associated with mutations in the 81-bp of the rpoB gene in all cases. IS6110 analysis indicated that recent transmission contributed substantially to the emergence of isoniazid-resistant tuberculosis in Barcelona through short transmission chains. A rapid genotypic assay, including the 315-katG codon and the ؊15 nucleotide of the mabA-inhA regulatory region, may cover 62% of isoniazid-resistant strains in Barcelona. In contrast, the targeting of the 81-bp region of rpoB would detect all our rifampin-resistant isolates. 107

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