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AIMS: Non-Tuberculous Mycobacteria (NTM) are ubiquitous in nature. The data on prevalence of NTM under the RNTCP is scarce. Many NTM species have clinical significance, and hence their identification and speciation are important. METHODS AND RESULTS: It is a cross-sectional study conducted at the five RNTCP accredited culture and drug susceptibility testing (CDST) laboratory. The culture isolates from AFB positive but Immunochromatographic test negative samples were taken for identification and speciation using HPLC. Of the total 266 isolates only 164 isolates had a second sample received at the laboratory. The speciation was done using HPLC for those isolates. The type of species identified are: 26·8% (44) were Mycobacterium chelonae, 12·8% (21) were Mycobacterium fortuitum, 9% (15) were Mycobacterium gordonae, 9% (15) were Mycobacterium tuberculosis complex, 6·1% (10) were Mycobacterium kansasii, 4·9% (8) were Mycobacterium simiae, 2·4% (4) were Mycobacterium thermophile, 1·2% (2) were Mycobacterium gastri, 0·6% (1) were Mycobacterium scrofulaceum, 0·6% (1) were Mycobacterium avium and 4·9% (8) isolates had chromatogram which was un-interpretable. CONCLUSION: Identification and its speciation of NTM are not routinely done under TB control programme. Since HPLC could identify 95% of isolates belonging to 10 species, the speciation of NTM using HPLC should gain importance in the diagnosis of disease caused by NTM. SIGNIFICANCE AND IMPACT OF STUDY: NTM are emerging as important causative agents of pulmonary and extra pulmonary disease, the ability to recognize disease caused by NTM and subsequently treat such disease has become increasingly important. The identification of NTM up to its species level should gain importance in all TB reference Laboratories.

Aims: To facilitate efficient identification of commonly encountered mycobacteria species (Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium fortuitum complex, Mycobacterium chelonae/abscessus, Mycobacterium kansasii, Mycobacterium gordonae) in high throughput laboratories, a 16s rDNA sequence based real-time PCR assay was developed and evaluated. Methods and Results: Oligonucleotide primers and hybridization probes were designed based on sequence differences of the mycobacterial 16S rDNA gene. This assay was evaluated with 1649 suspected non-tuberculosis mycobacterial isolates. Apart from 3 out of 40 M. avium isolates that showed false signal with M. intracellulare specific probe, 100% specificity was obtained for all tested probes. Assay sensitivity varied from 88·9 to 100% depending on species. Average cost for obtaining a definite identification was only USD 1·1 with an average turn around time of less than 3 days. Conclusions: A rapid, simple and inexpensive real-time PCR assay was developed for the identification of common encountered mycobacteria in a high throughput laboratory setting. Significance and Impact of the Study: With this assay, more than 80% of the clinically isolated nontuberculous mycobacteria could be identified in a highly cost effective manner. This helped to save resources for other laboratory activities especially in high throughput mycobacterial laboratories.

A study was performed to evaluate the prevalence of non-tubercular mycobacteria in swimming pool environments. The bacteria in question were found in 88.2% of pool water samples. The most frequent species were Mycobacterium gordonae (73.5% of samples; range 1-840 cfu 100 ml - 1), M. chelonei (38.2% 2-360 cfu 100 ml - 1) and M. fortuitum (35.3% 2-250 cfu 100 ml - 1). The same species were also recovered from the water at the different phases of the treatment cycle, with relative percentages similar to those of the pool water. Shower floors and pool edges also presented high concentrations of the mycobacteria (100% of samples) and M. marinum was isolated from the surfaces of pool edges on two occasions (4.5% of samples). The swimming pool environment provides a suitable habitat for the survival and reproduction of mycobacteria. Although mycobacteria are common in swimming pools, human mycobacterial disease associated with their use is rare. Apart from superficial infections with M. marinum, the risk of more serious diseases in subjects with weakened immune systems should not be underestimated, given the widespread presence of mycobacteria that are possible opportunistic pathogens and the direct contact bathers have with the water and aerosol.