Effects of activated macrophages on Mycobacterium leprae.

Five alternative methods were used to explore in vitro the effects of normal and activated murine macrophages on the metabolic well-being of intracellular Mycobacterium leprae: fluorescein diacetate-ethidium bromide staining, ATP content, synthesis of phenolic glycolipid 1, and two techniques to quantitate oxidation of palmitic acid. In relatively short-term experiments (7 to 10 days), each of these procedures provided strong evidence that activated macrophages exerted a deleterious effect on the leprosy bacillus. These findings appear to confirm the contention that activated macrophages underlie host resistance to clinical leprosy and limitation of M. leprae growth in paucibacillary leprosy.

Fluorescein diacetate and ethidium bromide staining to determine the viability of Mycobacterium smegmatis and Escherichia coli.

The ability of the fluorescein diacetate and ethidium bromide fluorescent staining method to assess the percentage of viable bacterial cells in suspension was compared with the plate counting method. Mycobacterium smegmatis and Escherichia coli bacterial cell suspensions were incubated at 60 degrees C. At different time intervals samples were taken and the percentage of viable cells in each sample was assessed by the fluorescent staining method and compared with the plate counting method. The fluorescent staining method showed a positive correlation with the plate counting method. However, the viable counts by the plate counting method were lower than the staining method when incubated at 60 degrees C, indicating a lag period in the decay of enzymes after bacterial death. Hence, the fluorescent staining technique can be used to assess the trend of bacterial death rather than to assess to exact number of viable bacilli.

Significance of neither green nor red NGR bacilli in FDA-EB staining.

This Study deals with determination of viability by FDA-EB method. It has been observed that some of the bacilli do not take any colour in FDA-EB preparations. These can be called "neither green nor red" (NGR) bacilli. These non-staining bacilli should be taken into account when reporting viability by FDA-EB method.

Assessing the viability of Mycobacterium leprae by the fluorescein diacetate/ethidium bromide staining technique.

In the present study we have evaluated the Fluorescein Diacetate/Ethidium Bromide (FDA/EB) staining technique to assess the viability of Mycobacterium leprae obtained from biopsies of leprosy patients under different periods of treatment. Bacillary suspensions were obtained from skin punch biopsies and stained with the FDA/EB solution. The average percentage of green cells seen which were deemed to be viable were: 67.2% of green cells in patients without previous treatment; 45.6% in patients with 1 to 6 months of treatment; 25.9% for patients with 7 to 12 months of treatment and 10.5% in patients with 13 to 24 months of treatment. All the patients studied were on multidrug therapy. The differences obtained in the percentages of green cells in the different groups of patients were statistically significant as determined by the Wilcoxon's test. The decrease in the percentage of green cells observed with increasing periods of treatment suggests that the FDA/EB technique correlates with the actual viability of M. leprae. The application of this technique in the routine procedures performed with Hansen's disease patients could be very useful for monitoring the effectiveness of treatment in leprosy patients.

Susceptibilities of Mycobacterium leprae and M. avium complex to the H2O2-Fe-mediated halogenation system supplemented with antimicrobial agents.

The susceptibilities of Mycobacterium leprae and M. avium complex (MAC) to the H2-O2-Fe-mediated halogenation system supplemented with antimicrobial agents were evaluated by fluorescein diacetate-ethidium bromide (FDA/EB) staining. In the case of M. leprae, the number of greenstained bacteria (intact cells) was reduced in the presence of the H2O2-Fe-mediated halogenation system supplemented with agents possessing antileprosy activity, such as rifampin, 4,4'-diaminodiphenylsulfone (dapsone), clofazimine, and ofloxacin. In the case of the MAC strain, although viable units of the organisms were reduced by the halogenation system alone, the number of greenstained cells in the FDA/EB stain was not reduced, even when the halogenation system was used in combination with ofloxacin. Because stainability of the cells is related to structural and functional intactness of the membrane, differences between M. leprae and the MAC strain imply possible differences in the rigidity of the cell membrane.

Effect of chemotherapy on viability of Mycobacterium leprae as determined by ATP content, morphological index and FDA-EB fluorescent staining.

Viable bacterial populations were estimated in bacilli purified from 105 biopsies from 40 untreated and 65 multibacillary leprosy patients treated with multidrug therapy (MDT) for varying periods. The bacilli were purified and viability was determined by ATP content, morphological index (MI), and fluorescein diacetate-ethidium bromide (FDA-EB) staining. Viable populations were calculated, taking 3.58 x 10(-15) g/solid bacillus as the mean ATP content of a viable unit of Mycobacterium leprae. The proportion of viable bacilli was also estimated in the same specimens using solid-staining (MI) and green-staining bacilli by the FDA-EB method. In the untreated cases, the positive viability by ATP assay was 100%, 92% by MI, and 100% by FDA-EB. ATP content per solid bacillus was relatively constant, which was not the case with ATP content per green-staining bacillus. While the MI was zero in all cases, viable bacilli could still be detected by ATP estimations in 5 of the 32 (16%) patients after 2 years of MDT and in 1 of the 20 (5%) patients after 3 years of MDT. No viable bacilli could be detected even by this method beyond 3 years of MDT. On the other hand, green-staining bacilli were demonstrable in 7/32 (22%) of cases after 2 years of MDT, 2/20 (10%) after 3 years of MDT, and 1/13 (8%) after more than 3 years of treatment, indicating that the FDA-EB staining and ATP assay did not detect the same populations. A determination of the ATP content of M. leprae could be used as a reliable and sensitive tool for determining viability of the bacilli.

Correlation of in vitro Fc receptor assay with in vivo mouse foot pad method.

Resistant strains of M. leprae have been reported to the various antileprosy drugs. There is currently no accepted test to identify the susceptibility pattern of M. leprae to the drugs in a short period. The only accepted test is the mouse foot method which takes a long period to yield results. The Fc receptor assay using the ability of viable M. leprae to alter the membrane of the macrophage is well established. It takes only ten days and is inexpensive. In 6 cases of leprosy patients the susceptibility pattern was worked out both with the in vitro Fc receptor assay and the vivo in mouse foot method The results correlated very well leading to the fact that the assay system is reliable. Hence it can be used not only to study the status of a patient, but also to shortlist the number of compounds to be tested on the mouse foot pad as anti-leprosy drug candidates.

Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus Calmette-Guérin. An in vitro comparison of leprosy vaccine components.

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.

Reduction of tellurite and deesterification of fluorescein diacetate are not well correlated with the viability of mycobacteria.

Both Mycobacterium leprae and M. lepraemurium (MLM) were capable of reducing tellurium as tellurite ion (Te4+) to elemental tellurium (Te), seen by electron microscopy as fine crystals within the bacterial cells. There appeared to be close correspondence between the capacity to reduce tellurite, bright green fluorescence after staining with fluorescein diacetate (FDA) and the ability of M. smegmatis to multiply in culture. Likewise, there appeared to be correspondence between tellurite reduction and fluorescence after FDA staining for MLM subjected to prolonged storage in the cold or to heating at 70 degrees C. However, correspondence with tellurite-reduction or fluorescence after FDA staining was not observed when death of MLM occurred in vivo.

Epithelioid cell granuloma induction in the guinea pig by haptenated Mycobacterium leprae.

Fluorescein isothiocyanate (FITC)-conjugated Mycobacterium leprae (FITC-M. leprae) was injected intradermally into the ears of guinea pigs and granuloma formation in the draining postauricular lymph nodes was studied. At 2 weeks, there was an increase in weights and infiltration of the draining lymph nodes in half of the animals injected with FITC-M. leprae. At 5 weeks, there was a significant increase in the weights and infiltration of these draining lymph nodes in the guinea pigs injected with haptenated M. leprae compared with those injected with unconjugated M. leprae. At 5 weeks, there was also a significant increase in delayed type hypersensitivity responses to 25 micrograms purified protein derivative. Histologically, epithelioid cell granulomas were seen in these lymph nodes as early as 2 weeks when FITC-M. leprae was used as the source of antigen. Enhancement in the immunogenicity of M. leprae by conjugation with FITC has been postulated.

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.

A rapid method for viability and drug sensitivity of Mycobacterium leprae cultured in macrophages and using fluorescein diacetate.

The ability of viable M. leprae to hydrolyze Fluorescein diacetate and retain fluorescein inside the bacteria was used to identify viable M. leprae inside the cultured in vitro macrophages. The subjective microscopic count of the FDA test was demonstrated as useful routine test by confirming the results obtained therein with a quantitative and non subjective measurement of fluorescence in spectrofluorimeter. Using this method loss of viability of M. leprae in presence of dapsone and rifampicin was demonstrated. Such an assay, was well correlated with another in vitro assay, the Fc receptor test and also the in vivo mouse foot test. The drug resistance of clinical isolates of M. leprae demonstrated by mouse foot pad was also correlated with FDA test system. Thus we have reported a reliable, consistent and rapid in vitro test system for determining viability and drug sensitivity of M. leprae.

Viability of Mycobacterium leprae: a comparison of morphological index and fluorescent staining techniques in slit-skin smears and M. leprae suspensions.

In a comparison of the estimation of Mycobacterium leprae viability by morphology and the fluorescent vital dyes FDA/EB and R123/EB, the latter techniques were more satisfactory using suspensions and slit-skin smears of M. leprae bacilli. Both FDA/EB and R123/EB seem to more accurately reflect viability after freeze/thaw cycles and heating, and are able to detect lower percentages of viable bacilli. In addition, the fluorescent vital dye techniques are both simple and less open to subjective interpretation than the conventional estimation of the morphological index.

Staining tissue-derived Mycobacterium leprae with fluorescein diacetate and ethidium bromide.

A fluorescent staining procedure incorporating the use of fluorescein diacetate (FDA) and ethidium bromide (EB) has previously been shown to accurately measure the viability of saprophytic mycobacterial cells. Green-stained cells were shown to be viable and red-stained cells, dead. Staining Mycobacterium leprae cells with FDA/EB, however, was complicated by interfering tissue components which masked the presence of stained bacteria. A petroleum ether separation technique enables M. leprae to be segregated from armadillo liver tissue components and permitted M. leprae to be stained qualitatively equal to the saprophytic mycobacteria. An alternative and technically simpler method of staining M. leprae from human skin biopsies and mouse foot pads was developed which permitted the initiation of a clinical assessment of the staining method. Preliminary data indicate that patients who have undergone three or 24 months of chemotherapy possess a significantly lower percentage of green-stained M. leprae in their tissues than untreated patients. This would be expected if the FDA/EB staining method was providing an accurate measure of viability. M. leprae cells obtained from mouse foot pads which were harvested 5-13 months post-infection displayed more than 90% green-stained cells. There was no correlation between the FDA/EB staining method and the morphological index.

A fluorescent staining procedure for determining the viability of mycobacterial cells.

A fluorescent staining procedure has been developed which rapidly, accurately, and economically measures the viability of mycobacterial cells. M. smegmatis and M. phlei have served as prototype organisms to establish conditions which ensure optimal staining. The staining method incorporates the use of the fatty acid ester fluorescein diacetate (FDA) and ethidium bromide (EB). Non-polar, non-fluorescent FDA enters live cells where it is enzymatically hydrolyzed by acetylesterase to polar, fluorescent fluorescein which rapidly accumulates in the cytoplasm. These cells appear green when viewed under incident ultraviolet illumination. Ethidium bromide enters dead cells and intercalates between the bases of DNA molecules. These cells appear red-orange under UV illumination. Live cells are, therefore, identified on the basis of possessing acetylesterase and their ability to exclude EB; whereas dead cells are identified on the basis of lacking acetylesterase and their inability to exclude EB. The feasibility of applying the staining procedure of M. leprae has been investigated and the results are encouraging. Our findings reveal that armadillo-derived M. leprae possess acetylesterase and, therefore, stain green. M. leprae cell suspensions exposed to adverse physico-chemical conditions give rise to high proportions of red-stained cells as would be expected if the cells are being killed. An alternative means of determining the viability of M. leprae appears to be feasible.

The therapeutic effects of phthalic acid salts

The magnesium, calcium, potassium-hydrogen and cotarnine salts of phthalic acid have been tried out, on twenty patients each, over a period of six months by intravenous injections twice a week. Intravenous injections of magnesium, calcium and potassium-hydrogen phthalate appear to have no therapeutic effect in leprosy in doses of 20 cc. of a 1 per cent solution. Out of nineteen cases treated with intravenous injections of cotarnine phthalate, in 20 cc. doses, first of a 1 per cent solution and later a 2 per cent solution, twice a week, twelve showed marked improvement, four slight improvement and three no change. The inprovement in these cases occurred only during the first three months of treatment. The clinical improvement appears to be accompanied by a general increase in resistance as indicated by a drop of over 25 per cent in the sedimentation rate of twelve of the patients. It is suggested that the effect of the phthalate group in leprosy is dependent to some extent on the length of time the drug is retained in the body.