Uptake of purine and pyrimidine nucleosides by macrophage-resident Mycobacterium leprae: 3H-adenosine as an indicator of viability and antimicrobial activity.

Freshly extracted human- and armadillo-derived Mycobacterium leprae maintained within murine macrophages incorporated significant levels (p less than 0.05 to p less than 0.001) of 3H-adenosine and 3H-hypoxanthine by 6 and 9 days of the culture period. The incorporation of 3H-adenosine was twofold or more higher than 3H-thymidine in 10 out of 15 human-derived M. leprae isolates. Macrophage-adapted bacilli incorporated 10-14-fold higher levels of 3H-adenosine compared to the same bacilli maintained in axenic cultures. The incorporation of these two labels was inhibited by dapsone and rifampin, indicating the utility of in vitro radiometric assays for screening antileprosy drugs and drug sensitivity/resistance in patients.

Comparison of radiometric macrophage assay and fluorescein diacetate/ethidium bromide staining for evaluation of M. leprae viability.

Earlier studies from our laboratory reported that a radiometric Mycobacterium leprae resident macrophage assay was a useful in vitro indicator of bacillary viability with good correlation with the established mouse foot pad model. The present study compares our assay with the recently described fluorescein diacetate/ethidium bromide (FDA/EB) method. M. leprae extracted from the dermal lesions of 73 bacilliferous leprosy patients were tested concurrently by both techniques. Good correlation (r = 0.52, p less than 0.001) was found between the radiometric assay evaluating DNA synthesis and the FDA/EB staining reflecting the presence of active esterase enzyme. In addition, the utility of the FDA/EB staining in the monitoring of therapy was established. Twenty-two patients treated for greater than 1 year showed lower numbers of green fluorescing bacilli when compared to 19 untreated or short-term-treated individuals.