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Development of a two-approach plate system for the fast and simultaneous detection of MDR and XDR M. tuberculosis

Periodic Report Summary - FAST-XDR-DETECT (Development of a two-approach plate system for the fast and simultaneous detection of MDR and XDR M. tuberculosis)

Description of the work performed

During the second reporting period many activities presented in the work packages (WPs) were continued or started in order to achieve the general project objectives.

WP 1 'Development and standardisation of a phenotypic colorimetric plate system for detection of MDR- and XDR-TB in M. tuberculosis isolates' with participation of ITM (Belgium), SMI (Sweden), LIC (formerly SATLD), (Latvia), CorpoGen (Colombia) and Cetrángolo (Argentina). The main tasks were finished during the previous reporting period. In the current period the final analysis of data generated during the multicenter evaluation performed in several sites was finished. Final cut-off values and critical concentrations of all antibiotics were agreed and interpretation of results was based on these parameters. The final format of the phenotypic colorimetric plate for detection of MDR- and XDR-TB in M. tuberculosis isolates has been completed.

The DIAPOPS platform was initially evaluated in WP 2. Unfortunately, the test did not reach enough sensitivity and specificity to justify further development. For this reason, we designed a new molecular platform based on the rifoligotyping methodology with the innovation that all probes hybridise at the same temperature. This new membrane format was optimised for detection of resistance to rifampicin and isoniazid with very good results and is currently being adapted for detection of resistance to fluoroquinolones, kanamycin and capreomycin. Also, a new slide microarray format has been developed for detection of multidrug resistance that is scheduled to be evaluated by other members of the consortium.

Based on the results obtained in WP1 and starting with a similar plate design, a new format was explored for application in the detection of MDR- XDR-TB directly in sputum smear-positive samples (WP3). Different possibilities were evaluated, including the development of the assay in a new format on a solid culture medium. This new format would also include the possibility for the rapid identification of M. tuberculosis in the same plate by the use of specific reagents. Being able to perform the assay directly on sputum samples will shorten the turnaround time by omitting the pre-isolation step needed in all indirect drug susceptibility testing methods currently in use. A new liquid medium-based approach and candidate new coloured indicators have also been explored.

The database of gene mutations associated with drug resistance in M. tuberculosis (WP4) was updated and maintained. This WP was coordinated by the ITM (beneficiary 1) in close collaboration with beneficiary 8 (Cetrángolo) and participation of the other members of the consortium. Further analysis to assess the geographic distribution of the different mutations associated with drug resistance is under way.

Sequencing activities to look into new mutations responsible for drug resistance to first- and second-line drugs have continued during the present reporting period. Also, genome sequencing activities have been started to investigate new possible candidate genes that could be regulating the emergence of new forms of drug resistance.

Main results achieved so far

1. Development of the final format and evaluation of the colorimetric plate assay for the rapid detection of MDR-TB and XDR-TB in M. tuberculosis isolates.
2. Definition of critical concentrations of drugs responsible for MDR- and XDR-TB used to analyse results of the multicentre evaluation of the proposed test.
3. Validation of the new membrane-format for a new molecular assay for MDR-TB detection.
4. Design of oligonucleotides to detect mutations associated with resistance to ofloxacin, kanamycin and capreomycin.
5. Development of a new microarray slide format for detection of MDR-TB.
6. Development and validation of a new colorimetric plate format for rapid detection of MDR- and XDR-TB in clinical isolates.
7. Optimisation of the new colorimetric plate format.