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 & BACTEC 
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 BACTEC 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 BACTEC 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.