Evaluation of a new version of the ``RT-TB'' triplex real-time PCR assay for the rapid diagnosis of Mycobacterium tuberculosis in clinical samples

Abstract number: 1732_315

Sougakoff W., Millot G., Truffot-Pernot C., Veziris N., Brossier F., Jarlier V.

Objectives: The ``Real Time TB'' assay (RT-TB) is a new multiplex real time PCR assay that allows the rapid detection and differentiation of the M. tuberculosis complex from other mycobacteria. The assay targets the IS6110 element and the RD9 specific region that differentiate M. tuberculosis (IS6110 positive – RD9 positive) from the other mycobacterial species included in the M. tuberculosis complex (IS6110 positive – RD9 negative). We recently evaluated the first version of the RT-TB kit, demonstrating that the assay is sensitive, particularly in smear negative–culture positive clinical samples. Here, we report on the evaluation of a new version of the RT-TB assay in which the labelling of the IS6110 probe has been modified in order to improve its stability and detection level.

Methods: The new RT-TB BioRad kit includes an IS6110-BRD04 probe (instead of IS6110-Tamra in the previous version) that increases by 10-fold the sensitivity of detection of the IS6110 amplicons. Fifty-two clinical samples collected in routine (sputa, bronchoalveolar and gastric lavages) were included. DNA preparations and RT PCR reactions were carried out according to the manufacturer's instructions. A new specific software developed by the manufacturer was used for the automated analysis of the RT-TB amplification results.

Results: In the present study, 2 samples were found to contain inhibitors (as indicated by the negative amplification signals of the corresponding internal controls). The 8 smear positive samples included in the study were all found to be IS6110-POS and RD9-POS. Interestingly, 4 of them showed <1 acid fast bacilli per field on microscopic examination. The good sensitivity of the test was confirmed by 2 smear negative samples which were both found to be IS6110-POS and RD9-POS by the RT-TB assay. These two samples were confirmed to be positive for M. tuberculosis by the Roche Amplicor assay. Finally, the 40 remaining smear-negative samples were all found to be negative for IS6110 and RD9, suggesting that the increased sensitivity of the new IS6110-BRD04 probe does not impair the specificity of the test.

Conclusion: The results obtained for the first evaluation of the new version of the RT-TB kit suggest that the assay is sensitive and specific and may be very promising for the detection of M. tuberculosis in clinical samples containing few bacilli. Further experiments are in progress to confirm these preliminary data.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Location: ICC, Munich, Germany
Presentation type:
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