In vitromethods for determination of viability of mycobacteria: comparison of ATP content, morphological index and FDA-EB fluorescent staining inMycobacterium leprae

Abstract

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

Comparison of radiorespirometric budemeyer assay with ATP assay and mouse foot pad test in detecting viable <i>Mycobacterium leprae </i>from clinical samples

“…Similar lack of correlation between BI/MI and other in vitro viability detection methods like FDA-EB staining, thymidine uptake and ATP assay have been reported. 4,8 Moreover in this study, in few particularly high BI cases, the cpm output was very inconsistent with BI (Table 1). Two likely explanations could be inhibition of metabolic activity by host tissue or suppression of metabolic activity due to competitive inhibition in the medium.…”

“…[4][5][6] Jagannathan and Mahadevan 7 showed a good correlation between ATP and Fc receptor assay. Katoch et al, 8 found ATP, an easy and a better tool for viability determination than morphological index (MI) and ß uorescence di-acetate (FDA-EB) staining. Gupta et al, 6 found a good correlation with gene ampliÞ cation assay.…”

The formation of lipid droplets favors intracellular Mycobacterium leprae survival in SW-10, non-myelinating Schwann cells

“…leprae that is derived from SW-10 cells by measuring its ATP content (Fig 8). Katoch et al [24] have reported that the ATP content of cultivable mycobacteria, M . tuberculosis and M .…”

Rapid characterization of Mycobacterium fortuitum-chelonei complex by restriction fragment length polymorphism of ribosomal RNA genes

“…During recent years members of the M. fortuitum-chelonei complex have emerged as important human pathogens [1][2][3]. The characterization of this complex has been mainly based on analysis of phenotypic characters including biochemical and enzymatic markers, bacteriocin typing, antigens, cell wall lipids, and proteins [4][5][6][7][8][9][10][11][12][13][14][15][16][17].…”