Multiplex PCR assay for simultaneous detection and differentiation ofMycobacterium tuberculosis,Mycobacterium aviumcomplexes and other Mycobacterial species directly from clinical specimens

Abstract

Aims: Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management. Methods and Results: Two hundred and thirty‐five samples from 145 clinically suspected cases of tuberculosis were processed for the detection of Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L‐J & BACTECTM MGIT‐960 culture and multiplex PCR tests. The multiplex PCR comprised of genus‐specific primers targeting hsp65 gene, Mycobacterium tuberculosis complex‐specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium avium complex‐specific primer pairs targeting 16S–23S Internal Transcribed Spacer sequences. The multiplex PCR developed had an analytical sensitivity of 10 fg (3–4 cells) of mycobacterial DNA. The multiplex PCR test showed the highest (77·24%) detection rate, while ZN smear examination had the lowest (20%) detection rate, which was bettered by L‐J culture (34·4%) and BACTECTM MGIT‐960 culture (50·34%) methods. The mean isolation time for M. tuberculosis was 19·03 days in L‐J culture and 8·7 days in BACTECTM MGIT‐960 culture. Using the multiplex PCR, we could establish M. tuberculosis + M. avium co‐infection in 1·3% HIV‐negative and 2·9% HIV‐positive patients. The multiplex PCR was also highly useful in diagnosing mycobacteraemia in 38·09% HIV‐positive and 15·38% HIV‐negative cases. Conclusions: The developed in‐house multiplex PCR could identify and differentiate the M. tuberculosis and M. avium complexes from other Mycobacterial species directly from clinical specimens. Significance and Impact of the Study: The triplex PCR developed by us could be used to detect and differentiate M. tuberculosis, M. avium and other mycobacteria in a single reaction tube.

Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay

“…Previously reported PCR methods for genus detection seem to have different drawbacks apparently avoided by this new PCR. As alignment between published primers/probes and genomic targets used by other researchers (16S rRNA, ITS, and heat shock protein 65) suggested, some might not detect DNA from various mycobacteria (37,38), others would detect nonmycobacterial DNA (20,21,23,25,32), and others might combine both sensitivity and specificity limitations (22,24,26,39). For M. avium subspecies detection, we used the multicopy element IS1311, a target previously used in multiplex real-time PCR assays (25) that, as far as we know, is M. avium subspecies specific (5).…”

“…Multiplexing strategies allow for further improvement of diagnostics in terms of rapidity and efficiency, and multiplex real-time PCR represents a reliable alternative. Among the various multiplex PCR assays reported thus far (20)(21)(22)(23)(24)(25)(26), many are conventional PCRs or use melting curve analysis and need additional interpretation; some have not been used directly on clinical samples, and others seem to have some specificity or sensitivity issues. A recently published duplex real-time PCR assay for the detection of M. tuberculosis and M. avium complexes on human respiratory specimens has been shown to be reliable and cost-effective (27), but it could have an added value by including an additional target for genus detection.…”

PCR as a Diagnostic Tool for Extra-Pulmonary Tuberculosis

“…The conventional microbiological methods of diagnosis are less reliable, and the diagnosis is often delayed for up to many weeks in the conventional culture method [4,5]. Since the disease usually responds to the standard antituberculosis therapy, an early and an accurate diagnosis is crucial.…”

Isolated tuberculous liver abscess in immunocompetent children – Report of two cases

“…Multiplex PCR assay was used to differentiate M. tuberculosis from non-tuberculous mycobacterium as per our protocol. 14 In the second case, pus examination revealed AFB and PCR assay of the aspirate was positive for M. tuberculosis, along with positive culture results (BACTEC MGIT-960).…”