The number and species of Non-tubercular mycobacteria (NTM) isolated from clinical specimens is continuously increasing with the advancement of microbiological detection methods. Numbers of reports have confirmed the pathogenic role, morbidity and mortality caused by NTMs in AIDS patients 1234. The infection due to NTM is difficult to treat because of their intrinsic resistance to the major classes of drugs, probably due to their habitat 56. Therefore to choose an effective therapeutic drug, anti mycobacterial susceptibility testing becomes a primary step for the management of the NTM disease. Though there are specific recommendations and guidelines prescribed by the World Health Organization (WHO) and Centre for Disease Control (CDC) regarding anti-tubercular drug susceptibility methods for Mycobacterium tuberculosis there are no such guidelines for NTMs. As routine drug susceptibility testing of NTMs are discouraged, there are circumstances where susceptibility testing is warranted 7.

Traditionally drug susceptibility testing in mycobacterial isolates is performed in agar and Lowenstein Jensen (LJ) medium which is considered as a 'gold standard' but it is cumbersome and available only in few reference laboratories in a country like India where tuberculosis is highly endemic. Even though automated systems dramatically reduced the time for detecting drug resistance in mycobacterial isolates but these are more expensive and not feasible especially in a low resource setting 89101112.

Since there was a need for rapid, easy to perform and qualitative method for predicting the Minimal Inhibitory Concentration (MICs) for NTM isolates, Mshana et al 13 developed a rapid colorimetric method employing oxidation-reduction indicator Tetrazolium bromide to perform drug susceptibility testing for Mycobacterium tuberculosis which was found to be promising and cost effective 14.

Therefore, we intended to assess the performance and efficiency of Tetrazolium Microplate Assay (TEMA) for determining MICs of Streptomycin (STR), Rifampicin (RIF), Ethambutol (ETH), Ciprofloxacin (CIP), Ofloxacin (OFL), Azithromycin (ATH) and Clarithromycin (CLA) against clinically isolated NTMs. The results obtained by TEMA were compared with Agar Proportion Method (APM) on Middle brook 7H10 agar plates.

The sensitivity and resistance patterns against STR, RIF, ETH, CIP, OFL, ATH and CLA by both TEMA and APM are described in Table 1. All the standard strains of NTMs were sensitive to all the tested drugs. The mean MICs values of the drugs (Table 2) tested by TEMA varied between clinical isolates belonging to different species. Of the 55 isolates, 23 (41.8%) were sensitive to all the drugs and the remaining 32 (58.2%) were resistant to at least one drug (Fig. 2).

When the efficiency of TEMA and APM were compared for detecting susceptibility patterns in NTM isolates, they showed 96.4% concordance for STR (Fig. 3), 98% concordance for RIF (Fig. 4), 100% for ETH (Fig. 5), and CIP (Fig. 6), and 98% for OFL (Fig. 7), ATH (Fig. 8) and CLA (Fig. 9). The Area Under Curve (AUC) by ROC curve analysis for STR was 0.943, RIF was 0.950, ETH was 1.000, CIP was 1.000, OFL was 0.750, ATH was 0.958 and CLA was 0.950. NTM isolates showed 90.09% and 98.18% of susceptibility to Ciprofloxacin and Ofloxacin, respectively. TEMA was rapid and susceptibility results could be obtained in a median time period of 5 to 9 days as compared to 12 to 25 days by APM.

The epidemiology of mycobacterial infections had changed drastically in the past few decades and in-turn changing the treatment regimen and management practices, especially after AIDS epidemic 18. In the terminal stages of AIDS, where CD₄⁺ lymphocyte counts falls below 100, not only M. tuberculosis but several NTM also cause infection and can be isolated from blood, tissue, sputum and fecal samples 19. The NTM disease not necessarily remains localized, but cause disseminated infection such as M. avium-intracellulare complex (MAC) 20. The prognosis for the MAC infected AIDS patient is usually poor, largely due to the inherent resistance of these organisms to most of the available anti-tubercular drugs and have high toxicity.

Seventeen (30.9%) of the 55 NTM isolates studied here were isolated from patients who were earlier treated with anti-tuberculosis therapy (ATT) without any clinical improvement. In clinical settings, these non-responsive cases are later labeled as Multi-drug resistant tuberculosis (MDR) by missing NTM infections. Similar observations were reported with other studies carried out in other countries such as Iran 21 and Brazil 22. The clinical and radiological features of NTM infected patients like abnormal chest roentgenograms with infiltrations, nodular abscesses, cavities, lymphoadenopathy resembling TB can mislead the physician.

TEMA was effectively used to detect drug resistance in NTM isolates and was compared with the APM to which it had very good concordance (table 2). Previously similar results were obtained with M. tuberculosis (Data not published), prompted us to evaluate with other mycobacterial species. In this regard, the TEMA results were accurate, highly reproducible and rapid to determine the MICs of clinically significant mycobacteria.

However, before applying the TEMA as a routine drug susceptibility testing method, a multicentric and inter laboratory testing must be carried out with a supervision of international agencies such as World Health Organisation to validate our findings and to implement it in resource poor settings. We recommend that the TEMA, which is a calorimetric method, is more appropriate for level 3 reference laboratories to manipulate small volume of liquid cultures in a microplate format.

The authors declare that they have no competing interests.

MMS participated in the collection and revival of strains, susceptibility testing, drafted the manuscript and performed the statistical analysis. KG participated in the design of the study, collection and characterization of strains. RS provided the clinical samples. SS conceptualized and designed the study. He also coordinated the study, critically evaluated the paper and arranged financial support. All authors have read and approved the final manuscript.