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Development of a molecular platform for the simultaneous detection of Mycobacterium tuberculosis resistance to rifampicin and fluoroquinolones
EC contribution
: € 768.856
: 33 months
Starting date
: 01/01/2005
Funding scheme
: Specific Targeted Research Project
: tuberculosis, drug resistance, molecular diagnosis
Contract/Grant agreement number
: LSHP-CT-2004-516028
Project web-site


Treatment success and containment of drug-resistant tuberculosis (TB) rely on a timely laboratory diagnosis. In view of this, a versatile and user-friendly molecular platform was proposed for the identification of Mycobacterium tuberculosis in clinical specimens and the simultaneous detection of resistance to two key anti-TB agents: rifampicin and fluoroquinolones. The platform will be initially developed for the detection of resistance to rifampicin because the associated mutations are well defined and their prevalence is sufficiently known worldwide. A small, single-stranded primer covalently-linked to an activated solid surface will be used to amplify a specific DNA target in a microplate well strip format, and an enzymatic chromogen system will be applied for detection. The technique will be validated for reproducibility and proof-of-principle.

Subsequently, mutated sequences encoding resistance to quinolones will be investigated. To this end, the gyrA gene will be sequenced in a collection of M. tuberculosis clinical isolates with known phenotypical resistance to fluoroquinolones. Relevant segments will be added as probes to the platform. The gyrA gene will serve also as a source for M. tuberculosis-specific target segments. A pre-clinical trial will be performed to evaluate the combined platform directly in clinical specimens and early liquid cultures. In this trial, the performance of the system will be verified by measuring: i) sensitivity, ii) specificity, iii) predictive values in settings with different prevalence rates of drug resistance, and iv) turnaround time to diagnosis. This project will add value to the leadership of the European initiative in biotechnology research and confront the emergency of MDRTB through the proposal of a promising deliverable useful to support targeted interventions.

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