Express detection and differentiation of M. tuberculosis and nontuberculous mycobacteria in samples

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Development of rapid detection of NTM in mycobacteriosis patients is critical. The aim of this study was the development of real-time PCR system, allowing simultaneous detection of MTB and NTM DNA in samples from patients with TB or mycobacteriosis. Materials and methods. NTM cultures were obtained from sputum using BACTEC MGIT 960. NTM species were identified using the Genotype CM/AS kits (HainLifescience, Germany). Primers and probes were designed using Primer 3 and Primer BLAST Software. 1007 NTM cultures isolated from patients admitted to the CTRI with presumed mycobacteriosis in 2013-2017 were analysed. For the MTB/NTM DNA detection system development were selected 18 rapid- and slowgrowing NTM species each of which was obtained from sputum of the same patient more than ones. Developed oligos were added to the PCR system “Amplitub-RV”(Syntol, Russia). The oligos detected all 18 species and did not interfere with MTB DNA amplification. The resulting multiplex PCR system was tested on 14 non-mycobacterium strains, 1007 NTM cultures, 124 sputum samples from 33 patients with established mycobacteriosis, and 3627 sputum samples from patients with verified TB and positive cultures obtained in Bactec MGIT 960. The specificity of NTM DNA detection was 100%, sensitivity of NTM DNA detection from cultures was 100% and from diagnostic samples – 66.7%. All 3627 samples with positive MTB cultures were PCR-positive for MTB DNA and PCR-negative for NTM DNA. Simultaneous detection of MTB and NTM DNA in samples allows to detect the pathogen within 3 hours, differentiating MTB from NTM; this accelerates diagnosing and enrolling in the adequate treatment regimen.