In vitro methods for determination of viability of mycobacteria: Comparison of ATP content, morphological index and FDA-EB fluorescent staining in Mycobacterium leprae

Abstract

Bacilli were purified from the 23 cases of multibacillary 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 ATP content was compared with morphological index (MI) and FDA-EB staining of bacilli from the same specimens. It was observed that when MI was 1% or more, the ATP content/solid bacillus was fairly constant in 15 cases studied. It ranged from 2.02 x 10-15 g to 5.60 x -15 g/solid bacillus (mean 3.46 x 10-15 g) and was in the same range as ATP content of viable cultivable mycobacteria. In the same 15 cases, when the green-staining bacilli was considered as 'supposedly viable bacilli', ATP content/green-staining bacillus varied up to 9-fold (0.22-15 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 ATP levels, whereas in another 3 cases with zero MI significantly high levels of ATP were detected. It is inferred that solid-staining bacilli may be the viable bacilli but when MI is 0% (1% 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. leprae appears to be an easy, rapid and sensitive tool for determining the viability for monitoring the therapy. On the other hand MI and FDA-EB staining appear to have their limitations.