Diagnostic value of gene probes and its correlation with clinical profile of leprosy in children.

Clinico-bacteriological profile of 73 leprosy patients below 16 years of age was studied. Majority of the patients were males and fell in 11-16 years age group (p < 0.05). Skin lesions were present in all cases on both exposed as well as unexposed areas and their number increased with advancing age. Cutaneous sensations were affected in most of the patients while nerve thickening was observed in 41. As age increased, the disease moved from the tuberculoid end of spectrum towards the lepromatous end (p < 0.05) and the positivity of the skin smears increased (p < 0.05). Majority of the paucibacillary cases were lepromin positive while most multibacillary cases were lepromin negative (p < 0.01). Two M. leprae specific gene probes were applied in 42 cases to assess their diagnostic value. Eighty one per cent cases were picked up by the probes indicating presence of active bacilli. These included all lepromin positive cases, all smear positive cases, and most of smear negative cases (p < 0.05). Seven children with inconclusive histology were also positive. Drug treatment and inadequate size of biopsy sample could explain the negative probe results in 19% cases. This study highlights the immense potential of gene probes in diagnosing leprosy in children.

Adenosine deaminase activity in leprosy.

Adenosine deaminase (ADA) activity was studied in serum and peripheral blood lymphocytes of leprosy patients and healthy controls. Serum ADA levels were found to be elevated in tuberculoid as well as lepromatous cases compared to control subjects. Serum ADA activity was significantly higher in tuberculoid cases than in the lepromatous group. Lymphocyte adenosine deaminase activity showed a similar trend. These results suggest that, since the overall activity of the enzyme is not deficient in leprosy, the cellular immune abberation seen in the different types of leprosy may be due to abnormal proliferation of different subsets of lymphocytes in response to M. leprae.

Lymphocyte arginase activity in leprosy--a preliminary report.

Arginase activity was estimated in serum and lymphocytes of 22 healthy controls and 50 untreated leprosy patients across the spectrum. The patients included 21 lepromatous/borderline lepromatous (LL/BL); 20 borderline borderline/borderline tuberculoid (BB/BT) and 9 tuberculoid (TT) cases. Mean serum arginase levels were 1.51 +/- 0.43, 1.41 +/- 0.43, 1.24 +/- 0.43 and 1.10 +/- 0.026 mu moles/min/ml in LL/BL, BB/BT and TT patients and healthy controls respectively. The lymphocyte arginase activity showed a similar increasing trend from TT to LL/BL. The mean lymphocyte arginase levels were 0.87 +/- 0.31 mu moles/min/10(6) cells in healthy controls and 1.81 +/- 0.40, 2.54 +/- 0.60 and 5.48 +/- 0.56 mu moles/min/10(6) cells in TT, BB/BT and LL/BL patients respectively. The increasing trend specially in lymphocyte arginase levels across the spectrum of leprosy correlated with the degree of impairment in the protective cell mediated immune response and also the extent of disease. The role of these pathophysiological alterations in relation to defect in immune response calls for investigation.

Detection of subclinical infection in leprosy: an 8 years follow-up study.

A follow-up study has been carried out using Fluorescent Leprosy Antibody Absorption (FLA-ABS) test in 1069 healthy contacts of multi and pauci-bacillary leprosy patients. Simultaneously lepromin testing with Dharmendra antigen has also been done to determine their delayed type hypersensitivity. In nearly 8 years of follow-up, 46 contacts have developed disease and of these 41 contacts were FLA-ABS positive and lepromin negative. It is inferred that test (along with lepromin) can be used to identify the contacts who are at higher risk of developing the disease. FLA-ABS test has also been found to be highly sensitive for detection of subclinical infection specially in younger age groups. This test could therefore serve as a very sensitive epidemiological tool for assessing the extent of disease in the community and for monitoring the transmission of disease especially after MDT and other intervention measures.

Application of ATP assay for in vitro drug screening testing against human derived M. leprae.

In this study, the ATP content of M. leprae exposed to various antimicrobial agents has been measured to evaluate its usefulness in drug sensitivity screening. Purified M. leprae suspensions from human biopsies have been incubated at 30 degrees C in a modified Dubos medium in the presence of different concentrations of various drugs viz., Rifampicin, Ethionamide, Ethambutol, Cycloserine, Dapsone, Clofazimine, Erythromycin and Tetracycline. ATP levels were estimated at 0, 7 days, 14 days of incubation by the procedures modified and standardised at this laboratory. ATP decay was accelerated by ethionamide, rifampicin, clofazimine, dapsone, erythromycin and to a lesser extent by cycloserine, whereas ethambutol and tetracycline did not have any significant effect. The rate of decay depended on the concentrations of these drugs. ATP assay promises to be a useful system for in vitro drug sensitivity screening against M. leprae isolated from patients.

A clinico-bacteriological profile of leprosy in children.

Clinico-bacteriological profile of 106 leprosy patients below 15 years of age was studied. Majority of the patients were males and fell in the 10-15 years age group (p less than 0.01). Nearly 89% had not received any prior treatment because of financial constraints. Seventy per cent gave a positive history of contact with adult patients who were mainly of the lepromatous variety (p less than 0.01). Skin lesions were present in 103 cases, mainly on the exposed areas and their number was found to increase significantly with advancing age (p less than 0.01). These lesions were hypopigmented patches in 71% of the children and erythematous in the rest. Cutaneous sensations were affected in most of the patients while nerve thickening was observed in 45. Positivity of the skin smears increased significantly as the number of skin lesions per patient increased (p less than 0.05). With advancing age, the disease moved from the tuberculoid end of the spectrum towards the lepromatous end (p less than 0.01).

Metabolic studies on mycobacteria. III. Demonstration of key enzymes of TCA cycle in M. leprae.

Cell free extracts of armadillo derived M. leprae, M. phlei, M. smegmatis and normal armadillo liver were analysed for the two key enzymes of TCA cycle. Aconitase activity was assayed in the presence of inhibitor fluorocitrate and it was observed that cell free extracts from cultivable mycobacteria as well as aramadillo derived M. leprae had this enzyme activity and 66-82% of this activity was inhibited by 0.1 mM fluorocitrate. 74% of M. leprae derived enzyme activity was inhibited by fluorocitrate in contrast with armadillo derived enzyme which was only 29% inhibited by fluorocitrate. PAGE separation of cell free extracts and staining for Isocitrate dehydrogenase (ICD) activity showed that an additional bond of ICD activity was demonstrable in the cell free extracts of armadillo derived M. leprae and this was NADP dependent. The mobility (ef) of this band of activity was in the same range as ICD from cultivable mycobacteria and much lower than ICD from normal armadillo liver. From this study and from the previously reported work, it is concluded that like other mycobacteria TCA cycle is operative in M. leprae.

Metabolic studies on mycobacteria. IV. Assay of isocitrate lyase and malate synthase activity in M. leprae.

Cell free extracts from M. tuberculosis H37 Rv, M. smegmatis armadillo derived M. leprae and normal armadillo liver homogenates were assayed for the presence of isocitrate lyase and malate synthase activity. It was observed that significant amount of isocitrate lyase and malate synthase activity was present in M. tuberculosis H37 Rv, M. smegmatis and armadillo derived M. leprae. No such activity was demonstrable in cell free extracts of normal armadillo liver. It is concluded that M. leprae like other mycobacteria has the capability to metabolise via glyoxylate bypass of TCA cycle. These findings may be relevant for understanding the energy metabolism of M. leprae under stress conditions and possibly the 'persister' stage.

Metabolic studies on mycobacteria. V. A preliminary report on the ATP synthesis by mycobacteria including M. leprae by using different substrates.

By deletion and addition of various substrates in Sauton's and Dubos media, an experimental system has been standardised in which the role of various nutrients in the energy synthesis of mycobacteria can be determined. By using this system with cultivable mycobacteria it was observed that glycerol and asparagine are the important ingredients for ATP synthesis by mycobacteria. Glucose further enhanced the ATP synthesis and growth of these mycobacteria. In the media containing asparagine or glycerol, there was marginal increase in the ATP in the M. leprae suspensions initially but this was not sustained and there was no progressive increase in biomass or multiplication. When M. leprae was incubated in the media from which both these substrates were deleted, there was progressive decline in ATP levels right from the beginning. From these preliminary results, it appears that asparagine and glycerol may be useful as substrates for ATP synthesis by M. leprae and need to be investigated further. In depth studies are necessary to find out the factors which results in the inability of M. leprae to utilise these and other substrates in a substrained manner for its multiplication and growth in artificial media.

Seroepidemiological studies in subsided cases of multi and paucibacillary types of leprosy using FLA-ABS test.

FLA-ABS test was carried out in 145 subsided cases of multibacillary (BL/LL) and 197 subsided cases of paucibacillary (TT/BT/I) types. The period of subsidence varied from 0-5 years or more. It was observed that percentage positivity decreases with the subsidence of the disease. In multibacillary cases, FLA-ABS positivity was 95% (active cases), 50% (after 1 year of subsidence) and 36% (after 5 years of subsidence) whereas in paucibacillary types FLA-ABS positivity was 80% (active cases), 34% (after 1 year of subsidence) and 19% (after 5 years of subsidence). A significant proportion of multibacillary subsided cases continued to be positive at high titres (1:160 and 1:640) even after 5 years of clinical subsidence. This implies the presence of continuous antigenic stimulus resulting in high antibody titres even after several years of clinical subsidence. On the other hand percentage positivity at high titres gradually decreased with the increased duration in paucibacillary cases.

Studies on healthy contacts of leprosy patients--a preliminary report.

91 healthy contacts of leprosy patients were studied for subclinical infection and possibly the pre-clinical stage of the disease using a battery of tests. It was observed that the test based on competitive inhibition of monoclonal antibody binding to the MY2 a determinant of M. leprae identifies a preclinical stage of the disease.

Metabolic studies on mycobacteria--II. Glyoxylate by-pass (TCA cycle) enzymes of slow and fast growing mycobacteria.

Glyoxylate by-pass of tricarboxylic acid cycle (TCA) comes into prominence during survival of microorganisms under oxygen limitations and study of these enzymes may contribute to understanding of physiology of 'persisters' in various mycobacterial diseases. The enzymes of glyoxylate by-pass have been assayed in the extracts of various mycobacterial species, namely, M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. flavescens, M. vaccae, M. smegmatis and Mycobacteria strain w (M.w.). M.w. has been included because of its close antigenic resemblance to M. leprae. It has been found that all of the above investigated species possess isocitrate lyase and malate synthetase, the key enzymes of glyoxylate by-pass. The presence of the enzymes is being reported for the first time in M. flavescens, M. vaccae and M.w. whereas these were earlier shown to be present in M. tuberculosis and M. smegmatis. It was also demonstrated in M.w. where acetate alone could not serve as sole source of carbon, but in the presence of glycerol stimulates the activity of glyoxylate pathway enzymes. The importance of these findings has been discussed.

Metabolic studies on mycobacteria-I. Demonstration of key enzymes of glycolysis and tricarboxylic acid cycle on polyacrylamide gels.

Polyacrylamide gel electrophoresis (PAGE) technique was standardised to demonstrate some key enzymes of glycolysis, hexose mono phosphate (HMP) pathway and tricarboxylic acid cycle in slow growing mycobacteria (M. avium. M. gastri) as well as in fast growing mycobacteria (M. vaccae, M. phlei). The enzymes studied were lactate dehydrogenase (LDH) glucose-6-phosphate dehydrogenase (G6PD), aconitase, isocitrate dehydrogenase (ICD), succinic dehydrogenase (SDH), fumerase and malate dehydrogenase (MDH). All the three pathways were found to be operative in slow as well as fast growing mycobacteria. Using this technique M. leprae specific MDH activity was demonstrated in the cell free extract of M. leprae. It's (MDH) electrophoretic mobility on gels lies in the range shown by other mycobacterial species studied and was distinct from that of host MDH. It appears that PAGE offers a useful tool for metabolic characterization of M. leprae using infected tissues.

Evaluation of effectiveness of clofazimine therapy. I. Monitoring of absorption of clofazimine from gastrointestinal tract.

The quantity of clofazimine absorbed from the gastrointestinal tract when administered to lepromatous leprosy patients at varying single doses of 600 mg., 400 mg., 300 mg., and 100 mg. has been worked out by determining the amount of clofazimine present in total faecal excreta. Except in 100 mg. dose where the percentage absorption was 62.5 +/- 17 in all other case the values were around 45%. The efficacy of daily administration of 100 mg. clofazimine is discussed in this first article.

Isoenzymes of mycobacteria. II. Relevance of LDH zymograms in taxonomy and identification.

The cell free extracts of mycobacteria namely M. kansasii M. avium, M. tuberculosis, BCG (Glaxo), M. gastri, M. phlei, M. smegmatis, M. vaccae, M. strain w., M. scrofulaceum, M. gordonae, M. nonchromogenicum E. coli, Staph, aureus, and M. leprae infected skin have been electrophoresed and stained for LDH activity. Normal skin tissue was also taken as control. It was found that all the organisms tested showed distinct species specific LDH isoenzyme patterns. There was no extra band but an aberrant zone of LDH activity was seen in M. leprae infected human skin in comparison to LDH isoenzymes from normal skin. No strain variations was found among the different strains of species investigated. Results described in the present paper indicate that LDH isoenzyme patterns of mycobacteria could be of identification value at species level.

Isoenzyme patterns of mycobacteria. I. Factors influencing LDH isoenzymes of mycobacteria.

Cell free extracts of a fast growing mycobacterium (M. phlei) and a slow growing mycobacterium (M. tuberculosis H37Ra) were analysed for lactate dehydrogenase (LDH) isoenzymes under different experimental conditions. It was observed that growth of M. phlei when taken from Lowenstein Jensen (LJ) as well as Sauton's medium showed identical band but for (M. tuberculosis H37Ra the number of bands observed were less when grown on LJ-medium. There was no difference in LDH isoenzyme patterns when the mycobacteria were incubated at 30 degrees C and 37 degrees C and under different pH conditions (6.2-8.2). Actively growing cultures of both the species showed distinct LDH isoenzyme patterns whereas the activity and bands became indistinct in old cultures. The LDH bands from lyophilized growth studied resembled to those of fresh growth. The treatment of growth with 1M NaOH for one hour resulted in marked diminution of LDH activity. Sonication with wet growth weight of 0.5 gm per ml of distilled water was found to give clearer bands as compared to phosphate buffer. No loss of LDH isoenzymes activity was noticed after storing the extracts at -80 degrees C for one month, treating to 58 degrees C for one hour or freezing and thawing for 2 times whereas these isoenzymes were quite unstable at other storage temperatures. Increasing the staining time was found helpful in getting clearer bands when activity was low. It is concluded that the factors studied have important bearing on LDH isoenzyme patterns of mycobacteria and must be kept in mind while studying the LDH zymograms for any taxonomic identification of mycobacteria or for studying the metabolic role. These are important both for sensitivity and reproducibility of LDH zymograms.

Alanine dehydrogenase in mycobacteria--a preliminary report.

Various mycobacterial species namely M. phlei, M. vaccae, M. scrofulaceum, M. avium and M. tuberculosis have been investigated for the presence of enzyme alanine dehydrogenase which could be important for utilization of alanine by TCA cycle. It was found that alanine dehydrogenase was present in all species of mycobacteria tested irrespective of the fact whether they are rapid or slow growers. Electrophoretic mobilities of alanine dehydrogenase from different species of mycobacteria were not found to be significant for taxonomical differentiation of rapid and slow growers.