Rapid detection, identification and drug susceptibility testing of M. tuberculosis is important for better patient management. Delay in diagnosis and treatment is an important contributor for rapid spread of TB in the community  leading to excessive morbidity and mortality especially in HIV infected patients . This delay is also responsible for increased nosocomial outbreaks among patients and health care workers .
Growing M. tuberculosis in liquid medium is much faster when compared to conventional method of detection using solid LJ medium. The major limitation in using liquid medium for the primary isolation of M. tuberculosis is the over growth of normal flora which compete for nutrients and mask the growth of tubercle bacilli. Antibiotics are in use either in form of PANTA or PACT to control the growth of normal flora. But it is recommended that exposure of primary isolates to these selective antibiotics should be limited as these agents affect the growth of mycobacteria in liquid media. As an alternative to antibiotics, bacteriophages and their lysins were used in liquid medium to control the growth of normal flora for the detection of M. tuberculosis.
Use of liquid culture systems such as BACTEC MGIT 960 has significantly reduced the time for detection and drug susceptibility testing of mycobacteria . The higher initial investment for the equipment and further recurring charges for the procurement of MGIT vials is a major limitation for MGIT 960 system especially in resource limited settings . Phage lysin was evaluated initially in comparison with PANTA to control the overgrowth of normal of normal flora for the recovery of M. tuberculosis using BACTEC MGIT 960 system. Phage lysin was found to be comparable to PANTA to control the growth of normal flora.
Mycobacteriophage based methods have been developed for the rapid detection and drug sensitivity testing of M. tuberculosis. Among phage based assays, luciferase reporter phage assay has the potential for the detection, identification and drug sensitivity testing of M. tuberculosis. LRP assay is utilized to study the drug susceptibility profile of tubercle bacilli using primary culture obtained from LJ medium. The presence of mucus strands, normal flora, enzymes and inhibitory factors in sputum specimens forms the major hurdle in developing a standard diagnostic assay.
Since LRP assay involves frequent opening of the culture vials for taking out aliquots at different days for setting up the assay, it requires stringent measures to prevent the overgrowth of non mycobacterial contaminants especially from environmental organisms. Combination of antibiotics (PANTA) was used in most of the liquid culture detection systems available for mycobacteria to decontaminate processed sputum samples. PANTA was also used to control the overgrowth of normal flora in sputum samples processed for M. tuberculosis detection by LRP assay [15, 16]. Earlier, lysin was evaluated in comparison with PANTA to control the overgrowth of normal flora normal in processed sputum samples for detection of M. tuberculosis by BACTEC MGIT 960 system . Here we reported a simple and bio-friendly methodology to control surviving normal flora using phage lysin in place of antibiotics for luciferase reporter phage assay with improved sensitivity and specificity for the detection of M. tuberculosis.
Luciferase reporter phage assay had been recognized for the rapid assessment of drug susceptibility of Mycobacterium tuberculosis. The time required for the identification of antibiotics sensitivity pattern of M. tuberculosis was reduced from weeks to days using LRP assay . Being a viability based test, LRP was also utilized for the rapid screening of new antituberculosis drugs [18, 19]. Mycobacteriophages specific for M. tuberculosis alone were used for simultaneous identification and drug susceptibility testing of M. tuberculosis cultures grown from MGIT 960 system. Luciferase reporter mycobacteriophage phAE142 was utilized for this purpose . Based on the rapidity, specificity and the ability of LRP assay to detect viable tubercle bacilli, the present work is aimed to improve upon the sensitivity of detection using phage lysin and avoiding the use of antibiotics.
In 2003, Bardarov et al.,  reported that LRP can be used for the rapid detection of M. tuberculosis in conventionally processed sputum samples but the assay required a minimum of >107 CFU/ml as a starting inoculum. Allowing a brief period of time to grow M. tuberculosis in liquid media, light can be detected at a rate faster than the doubling time of organisms was also suggested. Accordingly, the samples were incubated at 37°C and tested by LRP infection at each days starting from 1 to 42 to improve the sensitivity of detection. The present approach is also corroborating the earlier methodology but LRP infection by phAE129 is restricted to days 7, 9, 14 and 21 for the detection of M. tuberculosis. The results also suggest that the samples containing higher CFU can be detected in ≤ 7 days and the samples with low CFU can be detected between 7 to 21 days. Incubation of samples for more than 21 days probably would result in increased sensitivity of M. tuberculosis detection especially from paucibacillary samples.
Phage phAE142 was used for the direct detection of M. tuberculosis from processed sputum samples . The results were compared with MGIT 960 and conventional culture on LJ. LRP infection was done on post incubation days 1, 3, 5, 7, 11, 15, 19, 23, 27, 41 and 55. LRP detected M. tuberculosis in a range between 1 and 41 days with a mean time to detection of 7 days. The sensitivity of LRP assay was 76% while 97% and 90% of the samples were positive by MGIT 960 and LJ respectively. The sensitivity of detection by LRP was improved from 76% to 92% when contaminated specimens were excluded. In the present work, though the median TTD is 14.5 days, the sensitivity of detecting M. tuberculosis is as high as 90% with phAE129 using a simple methodology.
In the present work, 12 out of 129 samples were contaminated by DLRPA when the growth of other mycobacteria is included in the comparison. Among these 12 samples, 5 samples were reported as non-tuberculous mycobacteria (NTM) by conventional LJ method. In principle, all the NTMs also will be considered as contamination by conventional LJ. But because of the limitation of DLRPA to confirm all these NTMs, the samples were reported as contamination. If the NTMs were excluded from the comparison, the rate of contamination for DLPRA would be only 5.4%. Similarly, if consider the total mycobacteria isolated the positivity rate for LJ was 62% (80 out of 129) but for DLRPA it was only 55% (71/129). If the NTMs were excluded, the positivity rate for LJ was 54% and for DLRPA it remains 55%.
Luciferase reporter phage assay has certain advantages over the other liquid based rapid diagnostic assays while detecting M. tuberculosis directly from processed sputum samples. The methodology of this rapid assay is very simple and can be easily adopted by any laboratory. The average TTD of “BACTEC MGIT 960”, the current Gold-standard for liquid medium, has been reported to range between 8.5  and 13.3  days. The median TTD of DLRPA is 14.5 as observed in the present study. Among the 10 non-tuberculous mycobacteria grown on LJ, 5 resulted in contamination while growing on liquid medium; the other 5 became negative by DLPRA which reiterates that the phage used in this study is highly specific in infecting M. tuberculosis. As only viable bacterial cell will support phage adsorption, infection, replication and subsequent progeny release, the assay detects the active disease. As an indigenous method, propagation, harvest and maintenance of phages are relatively simple and less laborious. The cost-effectiveness of the assay is highly dependent on the cost of luminometer and the substrate, D-luciferin.