Rapid diagnosis of Mycobacterium tuberculosis by a triplex real-time PCR assay
Abstract number: p975
Sougakoff W., Truffot-Pernot C., Millot G., Jarlier V.
Sensitive and rapid techniques to detect and identify Mycobacterium tuberculosis directly in clinical specimens are important for the diagnosis and management of patients with tuberculosis. In this study, we report the evaluation of a real-time PCR assay targeting the IS6110 element and the RD9 specific region as a tool to rapidly diagnose and differentiate M. tuberculosis and M. canetti (IS6110 positive or negative RD9 positive) from the other mycobacterial species included in the M. tuberculosis complex (IS6110 positive RD9 negative) such as M. bovis, M. bovis BCG, M. africanum, M. microti, M. caprae and M. pinnipedii.
The performances of the assay were assessed from 166 clinical samples collected from patients over a two months period. DNA preparations and amplifications were carried out with a new kit developed by BioRad (Bio-Rad Real Time TB Assay), following the manufacturer's instructions. An internal control was added before DNA extraction in order to measure the efficiency of DNA recovery and check for the absence of inhibitors.
The BioRad Real Time TB assay correctly identified 17 specimens that were proven by culture to contain M.tuberculosis, of which 16 were positive for IS6110 and RD9 and 1 was positive for RD9 but negative for IS6110. Seven samples IS6110-positive but RD9-negative was shown by culture to contain M. bovis (n = 6) or M. africanum (n = 1). Eight samples that remained culture-negative were found to be positive to either IS6110-based amplification (n = 2) or RD9-based amplification (n = 3) or both (n = 3). Finally, 131 of the 134 samples negative for both IS6110 and RD9-based amplifications remained negative on culture, the three culture-positive amplification-negative samples yielding only a few colonies of M. tuberculosis on culture.
The assay, based on the real-time amplification of three distinct targets (IS6110, RD9 and Internal Control) was found to be robust, sensitive and specific, and is characterised by a complementary pattern of identification: broad for the IS6110-based amplification and specific of M.tuberculosis and M.canetti for the RD9-based amplification.
|Session name:||XXIst ISTH Congress|
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