Determination of mycobacterial phylogeny on the basis of immunological relatedness of superoxide dismutases.

Sixteen strains of cultivable mycobacteria were grown in Sauton's medium, and Mycobacterium leprae was purified from armadillo liver. Cell extracts were prepared from log-phase growths of each of the cultivable mycobacterial strains. Superoxide dismutase (SOD) enzyme was purified from all cultivable mycobacterial strains included in the study, and antibodies against purified SOD enzyme were raised in rabbits. Immunological distances (ImDs) between these anti-SOD antibodies and SOD antigens were determined by a previously described immunoprecipitation method and by a recently developed enzyme-linked immunosorbent assay (ELISA) technique. The reciprocal ImDs among mycobacterial strains were constant, reproducible and consistent by these two methods. An evolutionary tree was constructed on the basis of estimated ImDs. Except for M. duvalii and M. terrae, slowly and rapidly growing mycobacterial species appeared to be separately grouped by this analysis. Rapid growers clustered into a group which is near that of some slow-growing mycobacteria. M. avium falls almost in the middle of the evolutionary tree and the position of M. leprae was found to be between those of M. avium and M. bovis BCG. Measurement of immunological relatedness of SODs provides an alternative system with which to study the taxonomical relatedness among mycobacteria.

Development of a SOD ELISA to determine the immunological relatedness among mycobacteria.

This study reports on the standardization of an enzyme-linked immunosorbent assay (ELISA) system for the measurement of immunological distances (ImDs) of the superoxide dismutase (SOD) molecule among the cultivable mycobacteria, namely, Mycobacterium vaccae, M. phlei, M. smegmatis, M. avium, M. scrofulaceum, M. tuberculosis H37Ra, M. tuberculosis H37Rv, and M. bovis BCG, and M. leprae. SODs from cultivable mycobacteria were purified, antibodies were raised against these molecules, and ImDs between these anti-SOD antibodies and antigen (SODs) were determined by an immunoprecipitation technique standardized earlier and by the ELISA technique developed in this study. The ELISA system developed in this study showed higher sensitivity and consistent and reproducible ImDs among various mycobacteria, including pathogens such as M. tuberculosis, M. leprae and M. avium. These values were comparable with the values derived by the immunoprecipitation technique. Our ELISA technique appears to be a sensitive and rapidly reproducible method with the additional advantage of the stability of reagents, and holds promise in the taxonomy as well as in the development of diagnostics for leprosy and other mycobacterial infections.

Treatment of bacilliferous BL/LL cases with combined chemotherapy and immunotherapy.

Thirty-six, untreated borderline lepromatous/lepromatous (BL/LL) leprosy patients with an initial bacterial index (BI) of 4+ to 6+ were serially allocated to three treatment groups. Group I patients received a slightly modified WHO regimen (rifampin once a month, clofazimine and dapsone daily) and BCG intradermally (i.d.) (0.1 mg/per dose). Group II patients were administered the same MDT and Mycobacterium w (2 x 10(8)) killed bacilli/dose i.d., and Group III received the same MDT with 0.1 ml of distilled water i.d. Vaccination was repeated every 6 months. Biopsies were taken from the local site of vaccination and from a distant site, i.e., the back. The progress was monitored periodically by clinical, histopathological and bacterial (BI, mouse foot pad, ATP) parameters. Twenty-five patients had completed a follow up of more than 2 years. These included: 7 in Group I, 10 in Group II, and 8 in Group III. One patient of the MDT + BCG group who was progressing well dropped out after 28 months. In cases on combined chemotherapy and immunotherapy, no viable bacilli were demonstrable by mouse foot pad and ATP measurement after 6 months (at 12 months or afterward). However, in come of the control cases on MDT alone, viable bacilli could be detected even up to 18 months (by mouse foot pad) and 2 years (by ATP estimation). With 36 months of treatment, the mean BI decreased from 4.64+ to 1.66+ in the group on MDT alone (controls), 4.9+ to 0.08+ in the MDT + BCG group, and 4.75+ to 0 in the MDT+Mycobacterium w group. Compared with the MDT and MDT + BCG groups, the fall in the BI was significantly more in the MDT + Mycobacterium w group at 12, 18, and 24 months. While all of the cases in the Mycobacterium w groups became smear negative by 36 months, it took 42 months for all of the BCG group to achieve negativity. Immunotherapy appears to have a significant effect on the killing and clearance of bacilli and should be considered as an adjunct to chemotherapy, especially in bacilliferous lepromatous cases.

Prevention and early detection of leprosy in children.

India with its 4 million cases of leprosy, accounts for one-third of the world's population of leprosy patients. One-fourth of them are below 15 years of age. We report a 5-year follow-up study of healthy children who were close contacts of leprosy patients, in order to: 1. detect subclinical infection and observe the development of overt disease by using the Fluorescent Leprosy Antibody Absorption Technique (FLA-ABS) and the lepromin test which assess the humoral and cell-mediated immunity (CMI), respectively; 2. evaluate the efficacy of dapsone as a chemoprophylactic agent in the 'at risk' contacts. Four-hundred-and-fifty-five healthy contacts were studied. Majority of the contacts of multibacillary patients (303) were FLA-ABS positive (75 percent) and lepromin negative (55 percent) showing that although most of them had been infected, the lepromin status was negative (P < 0.01). On the other hand, the majority of the contacts of paucibacillary patients (152) were lepromin positive (57 percent) (P < 0.05). Furthermore, only 61 percent of contacts of paucibacillary patients were FLA-ABS positive as compared to 75 percent of contacts of multibacillary patients demonstrating that the former had been exposed to a lesser quantum of infection (P < 0.05). On the basis of results of FLA-ABS and lepromin tests, these 455 contacts were classified into four groups, viz. Group I comprising children who were FLA-ABS positive and lepromin positive; Group II, who were FLA-ABS positive and lepromin negative; Group III, who were FLA-ABS negative and lepromin positive; and Group IV who were FLA-ABS negative and lepromin negative.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement in the histological diagnosis of indeterminate leprosy by demonstration of mycobacterial antigens.

In a clinico-pathological study of Indeterminate leprosy, fifty-six cases were chosen based on specified clinical criteria. Their clinical features were noted, the smears for acid fast bacilli (AFB) were prepared from lesions, lepromin inoculation and biopsies were performed from the lesional edges. They were subsequently treated with a modified extended WHO regimen for paucibacillary leprosy. On routine hematoxylin eosin (HE) and Fite-Faraco staining of paraffin embedded sections, histopathological confirmation of Indeterminate leprosy was observed in only 17/56 (31%) of the clinically diagnosed cases whereas the remaining were labelled as non-specific pathology. Histometric analysis of all HE stained sections did not show any characteristic finding which could be considered as characteristic and discriminatory for Indeterminate leprosy. Immunoperoxidase staining for demonstration of mycobacterial antigen by direct staining procedure using conjugated rabbit anti-BCG and indirect three step procedure using primary rabbit anti-BCG and avidin biotin complex, was next performed on the sections exhibiting non-specific pathology. With the direct immunoperoxidase method, antigen was demonstrable in (11/35) 31% of the cases. The more sensitive indirect method could demonstrate the presence of antigen in (21/35) 60% of the cases. This study thus shows that demonstration of mycobacterial antigen by simple and inexpensive immunoperoxidase techniques enhances the histopathologic diagnosis of Indeterminate leprosy.

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.

A preliminary study of correlation of immuno-histological and ultrastructural characteristics of neural granuloma in leprosy patients.

With an aim to better understand the pathogenesis of nerve damage in leprosy, peripheral nerve biopsies from six untreated leprosy cases (3 BT/TT and 3 BL/LL) were studied by electronmicroscopy and immuno-histology. In addition to routine histopathology for diagnosis, infiltrating cells of granuloma were characterized after preparation of single cell suspension. The lymphocytes in the lesion were characterized by E and EAC rosetting and macrophage phagocytic system (MPS) cells were studied using histochemical markers like esterase and peroxidase. The results indicate that the lymphocyte content was significantly greater in tuberculoid neural granuloma compared to lepromatous nerves and these formed rosettes with sheep erythrocytes (E) and expressed HLA-DR antigen suggesting that they are activated T cells. Infiltrating macrophages in both the tuberculoid and lepromatous neural granuloma were esterase positive, peroxidase negative and did not form rosettes with sheep erythrocytes or EAC. Ultrathin sections of tuberculoid granuloma showed lymphocytes clearly associated to epithelioid macrophages having well developed Golgi apparatus and rough endoplasmic reticulum. Correlation of these immunological and ultrastructural characters suggests that hypersensitivity mechanisms are possibly responsible for nerve damage in tuberculoid leprosy. Ultrastructural examination of lepromatous nerves, on the other hand, showed the predominance of macrophages with large nucleus, heavily bacillated Schwann cells, and a few lymphocytes. The correlation of immuno-histological and ultrastructural characters indicates that the mechanism(s) of nerve damage in lepromatous leprosy are basically different wherein hypersensitivity appears to play a very limited role.

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.

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.

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).

Studies on energy synthesis in M. leprae.

ATP measurements have been earlier used to study the effect of various nutrients on the growth and multiplication of M. leprae. In a preliminary study, we had observed that glycerol and asparagine stimulated the ATP synthesis by M. leprae but this was marginal and not sustained. We have extended the study to investigate the role of various environmental factors which could affect this ATP synthesis. It has been observed that ATP synthesis was better and sustained for a longer period i.e. upto 2 weeks if the M. leprae were incubated at pH 6-6.5 and at 30-33 degrees C in the modified Dubos and Sauton's media. The pH and temperature above these values were suboptimal. It is concluded that temperature and pH are important factors for maintenance and synthesis of ATP by M. leprae.

Isolation, purification and quantification of phenolic glycolipid-1 from human leprosy skin tissues.

Phenolic glycolipid-I, a marker lipid of Mycobacterium leprae, was isolated from skin biopsies obtained from untreated lepromatous leprosy patients by silicic acid and florisil column chromatography and purified by thin layer chromatography. Tissues with varying bacillary loads were analysed for their phenolic glycolipid content. A good correlation was observed between the bacillary population of the tissues and the phenolic glycolipid content.

Ultracytochemical studies of lysosomal function in the macrophages of human leprosy--I.

The present study was undertaken to understand the lysosomal status and function in macrophages across the leprosy spectrum, including the reactional states. Acid phosphatase was used as the marker enzyme to demonstrate lysosomes at the electron microscopic level. It was observed that lysosomal morphology was well maintained in the macrophages of tuberculoid, borderline tuberculoid, and borderline tuberculoid leprosy in reaction, while they lost their cellular morphology and cell membrane integrity in lepromatous leprosy and lepromatous leprosy in reaction. The importance of these findings is discussed.