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Development of a molecular platform for the simultaneous detection of Mycobacterium tuberculosis resistance to rifampicin and fluoroquinolones

Final Report Summary - TB-DRUG OLIGOCOLOR (Development of a molecular platform for the simultaneous detection of Mycobacterium tuberculosis resistance to rifampicin and fluoroquinolones)

The ultimate aim of the TB-DRUG OLIGOCOLOR was to address the problem of Multi-drug resistant tuberculosis (MDR-TB) through the development of a versatile and user-friendly molecular platform for the identification of Mycobacterium tuberculosis and the simultaneous detection of resistance to key anti-tuberculosis agents directly in clinical specimens or liquid cultures. The platform would be initially developed for the detection of resistance to rifampicin because the associated mutations are well-defined and their prevalence is sufficiently known worldwide. The project focused on the development of a modification of the Detection of the immobilised amplified product in one phase system (DIAPOPS) technique. This system was successfully applied for the identification of Human leukocyte antigens, HLA-A alleles, and the detection of bovine leukaemia virus. Unlike other solid support-probe systems used for the detection of anti-tuberculosis drug resistance, it allowed the whole reaction to occur in a single well with the hybridisation step being avoided. The visualisation was then accomplished by an enzyme-chromogen colorimetric system. The prototype of this molecular platform was evaluated for reproducibility and proof-of-principle in six laboratories of the project's consortium under local conditions.

The second general objective of the project was the detection of resistance to fluoroquinolones. This was addressed by analysing a collection of M. tuberculosis clinical isolates with known phenotypical susceptibility or resistance to these antibiotics. The gyrA gene was sequenced to assess new mutations associated with quinolone resistance feasible to be added as primers to the molecular platform previously designed. M. tuberculosis specific target segments in the gyrA gene also served for identification purposes.

A third objective of the project was to perform a small pre-clinical evaluation in three laboratories of the consortium to evaluate the combined platform directly on sputum samples and early positive liquid cultures.