Keywords: Tuberculosis, drug resistance, molecular detection, rapid methods, rifampicin, fluoroquinolones
The treatment success of drug resistant tuberculosis depends largely on an early diagnosis. A system is proposed for the rapid identification of relevant mutations associated to anti-tuberculosis drug resistance. The platform will be developed for the detection of resistance to rifampicin because the associated mutations are well-defined and circumscribed to a small DNA region. However, it is expected to function as a flexible system able to incorporate the recognition of mutations associated with resistance to other key antituberculosis drugs. A microplate strip format will be used to capture the amplified product of specific target sequences. The technique will be validated for reproducibility and proof of principle in several laboratories under different conditions. A small pre-clinical trial is planned for preliminary evaluation. The application of the developed system to the detection of resistance to fluoroquinolones will also be approached.
The management and control of multi-drug resistant tuberculosis (MDR TB) relies upon a solid laboratory support. In this project, a versatile and userfriendly molecular platform will be developed and field-tested for the identification of M. tuberculosis and the simultaneous detection of resistance to key anti-tuberculosis agents directly in clinical specimens and/or liquid cultures. The treatment outcome of MDR TB improves consistently when it is recognised and treated early. To maximise the efficacy of a combined drug regimen, drug resistance to first- and second-line drugs should be investigated. A tool on which it would be possible to count simultaneously the anticipated failure of first-line chemotherapy and susceptibility to a major second-line drug would be useful. Phenotypic drug susceptibility assays entail subculture of the isolate in the presence of a set of anti-tuberculosis agents. By requiring only small amounts of bacterial nucleic acids, genotypic approaches may circumvent this hindrance, thus shortening the turnaround time. Moreover, DNA-based technologies can, in principle, be applied directly to the clinical specimen, provided it contains enough bacilli.
To develop a combined molecular platform for simultaneous identification and rifampicin resistance detection in M. tuberculosis. The second broad aim of the project is to detect resistance to fluoroquinolones.
The platform will be initially developed for the detection of resistance to the first-line drug rifampicin because the associated mutations are well-defined and their prevalence is sufficiently known worldwide. To detect resistance to fluoroquinolones, a collection of M. tuberculosis clinical isolates with known phenotypical susceptibility to these agents will be gathered by all participant laboratories, the gyrA gene will be sequenced, and relevant wild as well as mutated segments will be added as probes to the platform. Conveniently enough, the gyrA gene will serve also as a source for species-specific target segments. A four-month pre-clinical trial will be performed in three laboratories to evaluate the bundled platform directly in clinical specimens and early liquid cultures.
The accomplishment of a combined molecular platform for M. tuberculosis identification and drug resistance detection will add value to the leadership of European initiative in biotechnology research and development and therefore strengthen the European competitiveness. In this respect, the development of this novel and versatile platform is a sharp progress beyond the current stateof- the-art regarding both generation of knowledge and tool development. Firstly, the analysis of genes involved in the resistance to key anti-tuberculosis agents will enhance the understanding of microbial genetic events leading to TB treatment failure. Secondly, mutated sequences will become available for eventual use in drug target research or tool development. Finally, this project would confront the emergency of MDR TB through the proposal of a promising targeted intervention.
1 - Françoise Portaels
Institute of Tropical Medicine
Tel: +32 3 247 6317
Fax: +32 3 247 6333
|Official Address||Other Information|
|2||Dick Van Soolingen||
National Institute of Public Health |
and the Environment,
|Tel: +31 302 742363 |
Fax: +31 302 744418
Swedish Institute for Infectious |
|Tel: +46 8 4572431 |
Fax: +46 8 301797
|4||Patricia Del Portillo||
Corporación CorpoGen, |
|Tel: +571 34 84606 |
Fax: +571 34 84607
E-mail: firstname.lastname@example.org and email@example.com
National Institute for Infectious Diseases |
|Tel: +54 11 4302 7635 |
Fax: +54 11 4302 7635
Hospital Dr. Cetrángolo |
|Tel: +54 11 479 70165 |
Fax: +54 11 472 19153