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


The treatment success and the containment of drug-resistant tuberculosis rely on a timely laboratory diagnosis. In view of this, a versatile and user-friendly molecular platform is proposed for the identification of Mycobacterium tuberculosis in clinical s pecimens and the simultaneous detection of resistance to two key anti- tuberculosis agents: rifampicin and fluoroquinolones. The platform will be initially developed for the detection of resistance to rifampicin because the associated mutations are well de fined 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 w ill 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 o f 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 tr ial 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 setting s 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 pro mising deliverable useful to support targeted interventions.

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