Pyrazinamide (PZA) is an important first-line antituberculosis drug because of its sterilizing activity against semidormant tubercle bacilli. In spite of its very high in vivo activity, its in vitro activity is not apparent unless an acidic environment is available, which makes PZA susceptibility testing difficult by conventional methods. The present study was, therefore, planned to assess the performance of the colorimetric BacT/ALERT 3D system and compare the results with those from conventional tests, i.e., the Löwenstein-Jensen (LJ) proportion method (pH 4.85) and Wayne's pyrazinamidase (PZase) assay, using 107 clinical isolates. The concordance among all of these tests was 89.71% after the first round of testing and reached 92.52% after resolution of the discordant results by retesting. Prolonged incubation of the PZase tube for up to 10 days was found to increase the specificity of the PZase test. The concordances between LJ proportion and BacT/ALERT 3D, LJ proportion and the PZase assay, and BacT/ALERT 3D and the PZase assay were found to be 99.06%, 93.46%, and 92.52%, respectively. Using the LJ results as the gold standard, the sensitivities of BacT/ALERT 3D and the PZase assay were 100 and 82.85%, respectively, while the specificity was 98.61% for both of the tests. The difference between the sensitivities of BacT/ALERT 3D and the PZase assay was significant (P ‫؍‬ 0.025). The mean turnaround times for the detection of resistant and susceptible results by BacT/ALERT 3D were 8.04 and 11.32 days, respectively. While the major limitations associated with the PZase assay and the LJ proportion method are lower sensitivity in previously treated patients and a longer time requirement, respectively, the BacT/ ALERT 3D system was found to be rapid, highly sensitive, and specific.

REMA can be used as a rapid and inexpensive method for mycobacterial drug susceptibility testing against ethambutol. In comparison with the agar method, the MICs determined by REMA can more accurately be correlated with achievable plasma concentrations of antimycobacterial agents.

The occurrence of otitis externa in dogs in relation to ear types, age‐group, sex, months and seasons of the year and microbiology of canine ears was studied.

For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast-and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.Several species of environmental mycobacteria have been known to be important human pathogens (12). Further exposure to them is believed to alter immunity to vaccines like Mycobacterium bovis BCG (11). Isolation of mycobacteria from environmental samples is difficult because other microbes are also present in the environment. All mycobacterial species are not equally resistant to the different decontamination procedures. For the isolation of mycobacteria from environmental samples, such as soil and water, different methods have been described by various workers (1-10). These are not universally applicable because of differences between floras. No studies of this issue had previously been carried out in the northern parts of India. For this reason, the present study was undertaken to select or develop an improved, appropriate decontamination method(s) for the isolation of mycobacteria from the predominantly hot, dry environment of Agra, India (annual temperature, maximum of 16 to 47°C and minimum of 3 to 30°C; humidity, maximum of 49 to 100% and minimum of 28 to 70%).After we tried different permutations and combinations in controlled experiments, the following procedure was standardized ( Fig. 1). Wet soil samples of approximately 5 g were collected from a depth of 3 cm, and 50-ml water samples were collected from ditches, ponds, lakes, and rivers in the Agra region throughout the year. Soil was suspended in 20 ml of double-distilled autoclaved water (D/W) in polycarbonate centrifuge tubes. After being shaken manually for 60 s, the suspension was centrifuged at 600 ϫ g for 5 min at 4°C to pellet the soil particles. The turbid supernatant (10 ml) was transferred into other sterile centrifuge tubes and centrifuged at 8,000 ϫ g for 15 min at 4°C. Water samples were centrifuged at 8,000 ϫ g for 15 min at 4°C. Pellets from the soil and water samples were resuspended in 20 ml of treatment solution (3% sodium dodecyl sulfate [SDS] plus 4% NaOH) and then divided into two parts: A and B. Part A was incubated at room temperature (RT) for 15 min to obtain the growth of rapid growers, and part B was incubated at RT for 30 min to obtain the growth of slow growers. After incubation, both the suspensions were centrifuged at 8,000 ϫ g for 15 min at 4°C, and then the supernatants were decanted. Sediments were processed for cetrimide treatment. In the initial pilot experiments, various incubation periods with 2% cetrimide treatment were tried for slow and rapid growers. The pellets were resuspended in 20 ml of 2% cetrimide. Part A was incubated at RT for 5 min to obtain the g...

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Bacilli were purified from the 23 cases of multi bacillary type of leprosy. The ATP content of these bacilli was assayed by a firefly bioluminescent technique which is capable of detecting a very small number of cultivable mycobacteria as assessed by colony counts. The A TP content was compared with morphological index (MI) and FDA-EB staining of bacilli from the same specimens. It was observed that when MI was I % or more, the A TP content/solid bacillus was fairly constant in 15 cases studied. It ranged from 2•02 x 10-1 5 g to 5•60 X-I S g/solid bacillus (mean 3•46 x 10-1 5 g) and was in the same range as A TP content of viable cultivable mycobacteria. In the same IS cases, when the green-staining bacilli was considered as 'supposedly viable bacilli', ATP content/green-staining bacillus varied up to 9-fold (0•22-1 5 x g to 1•98 X 10-15 g/green-staining bacillus) and this did not correlate. The percentage of green-staining bacilli (FDA-EB) and solid staining bacilli (MI) was different in all the cases. In 2 cases with 0% MI in which ATP levels were also zero, 7•5% and 21•5% green-staining bacilli were present which implies that the enzymes responsible for green-staining character may persist for some time after death. Three cases with 0% MI had also 0% green staining bacilli and zero A TP levels, whereas in another 3 cases with zero MI significantly high levels of A TP were detected. It is inferred that solid-staining bacilli may be the viable bacilli but when MI is 0% (I % or less) sampling error or clumping may be responsible for missing out the solid bacilli in some cases. It is concluded that the ATP content of M. /eprae appears to be an easy, rapid and sensitive tool for determining the viability fo r monitoring the therapy. On the other hand MI and FDA-EB staining appear to have their limitations. As My cobacterium /eprae can not be cultivated in in vitro systems, there is a strong need to develop other rapid screening methods for monitoring the effect of therapy as growth in the mouse fo otpad

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