Presence of viable Mycobacterium leprae in environmental specimens around houses of leprosy patients.

Purpose: Leprosy is a chronic systemic infectious disease caused by Mycobacterium leprae, one of the first organisms to be established as the cause for disease in humans. Because of high prevalence pockets of leprosy in the endemic regions, it is necessary to identify the possible sources of M. leprae in the environment and its mode of transmission. Materials and Methods: Slit skin smears (SSSs) from lesions were collected in 70% ethanol from 50 leprosy cases staying in the leprosy resettlement village and hospital from a high endemic area. One hundred and sixty soil samples were collected from different areas around the leprosy hospital and from the resettlement village of cured leprosy patients where active cases also resided at the time of sample collection. M. leprae specific gene region (RLEP 129 bp) and 16S rRNA targets were used for polymerase chain reaction (PCR) based detection for the presence and viability of M. leprae. An rpoT region was also amplified to determine presence of numbers of 6 bp tandem repeats. Results: All the SSS samples collected from patients showed three copies of rpoT region (6 bp tandem repeat, an ancient Indian type). Fifty-two soil samples showed presence of M. leprae DNA whereas M. leprae specific 16S rRNA gene was amplified in sixteen of these samples. PCR amplification and fragment length analysis showed 91 bp, i.e., three copies of the rpoT 6 bp tandem repeats from soil samples and similar three copies observed in patient samples. Conclusion: Presence of viable M. leprae in the soil having same rpoT genotype of M. leprae noted in patients suggests that it could be the same strain of M. leprae. M. leprae found in the soil could be the one that is excreted out by the patient. Significance of its viability in the environment and its pathogenicity with respect to transmission needs to be further explored. Findings of this study might provide possible insights for further exploration into understanding transmission patterns in leprosy and also will throw light on identifying potential for existence of extra human source or reservoirs of M. leprae, if any.

Seroprevalence of HIV infection among the foreign students at Agra during a fifteen year period (1988-2002).

The present study reports a retrospective analysis of data of HIV testing of foreign students from Sub-Saharan Africa, South-East Asia and Europe, studying as well as staying at Agra, over a period of 15 yr (1988 to 2002). Of the 2653 [2092 (78.85%) were from the Sub-Saharan African countries, 377 (14.21%) from the South-East Asian countries, and 184 (6.93%) from the European countries], foreign students tested for HIV, only 26 were found to be positive for HIV-1/2 antibodies by the ELISA, rapid and Western Blot assays. Out of 26 HIV-positive, 17 males and 7 females were from Sub-Saharan Africa and 2 males were from the European countries. The range of HIV-positivity over a period of 15 yr varied greatly. When the five-year (1988-1992, 1993-1997 and 1998-2002) results were compared, the HIV-seropositivity showed a decline from 1.85, 0.50 to 0.36 per cent in the first, second and third 5 yr slots, respectively. While the data were not representative of all foreign students in India, this reflected the population tested in this centre was not a growing focus of HIV infection in this part of the country.

Growth, CO2 exchange rate and dry matter partitioning in mungbean (Vigna radiata L.) grown under elevated CO2.

This study was conducted to determine the effects of anticipated future level of CO2 on growth and dry matter partitioning of mungbean (Vigna radiata). Plants were grown from seedlings to maturity inside the open top chamber under amhient CO2 (350 +/- 25 microL L(-1)) and elevated CO2 (600 +/- 50 microL L(-1)) at Indian Agricultural Research Institute, New Delhi (India). Plants were harvested at 20, 35 and 50 days after germination. Mungbean plants grown under elevated CO2 concentration resulted in greater photosynthetic rate on a leaflet area basis and no acclimation in photosynthesis was recorded due to high CO2. Plants grown under CO, enrichmcnt were taller and attained greater leaf area along with more dry matter than ambient CO2 grown plants at all growth stages. Response to high CO, depends upon the growth stage of the plant and it was more at early growth stages compared to maturity stages. The high CO2 grown mungbean plants also exhibited increased root growth along with stem and leaves. There was a substantial increase in pod number and seed number/plant under elevated CO2 conditions. The increase in dry matter and growth of root, stem and leaves proved that CO2 enrichment of the atmosphere can stimulate photosynthetic rate which can ultimately lead to an increase in dry matter and growth.

Efforts in diagnosing early leprosy using serological techniques.

Skin scrapings obtained from the lesions of leprosy patients of all types showed 96% positivity to the serum antibody competition test using monoclonal antibody (ML04)to 35 kDa antigen of Mycobacterium leprae. Further, in vitro culture of full thickness skin biopsies from lepromatous patients were noted to release IgG antibodies to M. leprae with a peak antibody response at 48 h. The significance of this local antibody response to Μ. leprae in skin has been discussed for its possible use in diagnosing early leprosy.

Group specific component (Gc) in erythema nodosum leprosum (ENL).

The distribution of phenotypes of group specific component (Gc) was examined in 71 lepromatous leprosy (LL) patients without any history of ENL reaction and 65 LL patients with history of frequent episodes of ENL reaction. The distribution of none of the phenotypes of Gc (Gc 1-1, Gc 2-1, Gc 2-2) was statistically significant among these groups.

An appraisal of enzyme linked immunosorbent assay (ELISA) and serum antibody competition test (SACT) in leprosy.

Seventy-eight untreated leprosy patients, 104 treated patients and 105 healthy contacts were tested using two serological tests, SACT (serum antibody competition test based on competitive inhibition of monoclonal antibody binding to the MY2a determinant of M. leprae) and ELISA (measurement of IgM antibodies to the neoglycoproteins D-BSA and ND-BSA representing the phenolic-glycolipid antigen of M. leprae). The controls included normal healthy individuals, patients with sputum positive pulmonary tuberculosis, and active cases of rheumatoid arthritis from the department of rheumatology. The specificity of SACT was found to be very high. ELISA was found to be positive in two patients with rheumatoid arthritis, one each for D-BSA and ND-BSA ELISA. Both tests had a high sensitivity in BL and lepromatous patients. The sensitivity to both tests was considerably lower in tuberculoid and BT patients i.e., below 40%. Therefore the diagnostic value of a negative test in suspected cases of leprosy was very low employing either of the two tests. A proportion of patients with paucibacillary tuberculoid and BT leprosy were positive after six months or longer after therapy. Similarly a large number of BL and lepromatous patients were positive after considerably longer periods of treatment. The use of either tests for determining the duration of therapy is therefore limited. SACT appears to be more sensitive than ELISA with ND-BSA in detecting subclinical infection. The cumulative positivity of the two tests may be used as a measure of the infectivity of the disease in the community and for evaluating disease control methods.

Studies on lepromin and soluble antigens of M.leprae: their classification standardization and use.

Before the discovery of armadillo as a susceptible animal the source of M.leprae was limited and hence the use of lepromin was not common in the field. In recent times, the soluble antigens of armadillo-derived M.leprae have been used extensively in the field. Although the results of the study show that these antigens do not differentiate always a susceptible form from the resistant form, they are able to segregate the polar forms of leprosy. In a given field situation the criteria for diagnosis is so stressed that leprosy is overdiagnosed and within one year of follow up nearly half the number of cases are noted as not leprosy. Hence, in such situations lepromin reaction would be definitely a poor correlate with the type of leprosy. However, in hospital based studies the lepromin reaction has always been and would remain useful in confirming the classification (Sengupta et al 1984). Lepromins and M.leprae soluble antigens have gone through extensive standardization procedures. As these antigens contain mostly common mycobacterial antigens along with the M.leprae-specific antigens, these antigens are unable to specifically diagnose M.leprae infection. After purification of M.leprae from infected armadillo tissue, it was expected that the soluble antigen of M.leprae would probably be as useful as tuberculin. However, this was not found to be true in case of lepromin. Specificity for M.leprae has been noted in the epitopes (antigenic sites) on cross reacting molecules (12 kd, 18 kd, 28 kd, 35 kd, 36 kd) of mycobacteria (Ivanyi et al 1983; Watson 1989). These specific epitopes, if synthesized, could be of use as skin test antigens for determining M.leprae infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Coupling of proteins to liposomes and their role in understanding delayed type of hypersensitivity in human and mice.

Liposome-coupled lepromin was found to elicit a 3-week skin reaction in leprosy patients similar to that elicited by whole Mycobacterium leprae. The present study suggests that the presentation of antigens in a specific orientation is necessary for evoking delayed type hypersensitivity response in humans.

Dermatoglyphics in leprosy: (III) Creases of palm.

Palmar configurations of triradii and creases of 100 leprosy patients [50 lepromatous (BL/LL) and 50 tuberculoid (BT/LL)] were compared with those of 100 normal persons selected from families of these patients. The patterns of position of triradii were similar in controls and leprosy patients as such. But, the patterns in the two types of leprosy patients were different. As for palmar creases patterns, there was significant difference between those of controls and patients, double radial base crease occurring more often in patients. However, the differences between the two types of patients were not statistically significant.

Follow up of BL/LL patients on a slightly modified WHO regimen of multidrug therapy.

56 lepromatous leprosy patients with an initial average BI of 4.45 were administered once a month 600 mg of Rifampicin, 100 mg of Clofazimine on alternate days and 100 mg of Dapsone daily. None of these patients became smear negative in 2 years, and the same regimen was continued further. Two patients have become negative in 3 years and treatment has been stopped in them. The study indicates that highly bacilliferous LL/BL patients are likely to need 3 years or more of MDT for achieving bacterial negativity.

Studies on serum proteins in leprosy by polyacrylamide gel electrophoresis (page)--I.

Serum protein pattern was studied in the leprosy spectrum, their contacts and in normal individuals by employing polyacrylamide gel electrophoresis. Sera from 80% of untreated BL/LL, 70% of untreated TT/BT patients and 67% of contacts have shown dysproteinaemia either for 232 kD or for 175 kD or for both these proteins together. Tendency of these proteins to return to normal levels was observed after treatment. But both these proteins come back to normal levels only after subsidence of the disease.

Dermatoglyphics in leprosy (II Metric analysis of palms).

A group of 100 leprosy patients consisting of 50 lepromatous (BL/LL) and 50 tuberculoid (BT/TT) were investigated for metric analysis of the patterns present on their palms. Hundred normal persons were also selected from the families of patients to serve as controls. BT/TT patients and controls did not show any significant difference in their palmar patterns. On the other hand, significant differences were observed in the patterns between BL/LL patients and controls.

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.

In situ characterization of cells in the dermal infiltrates of lepromin reaction using monoclonal antibodies.

A study was made on the in situ characteristics of dermal infiltrates in the early and late lepromin reaction with monoclonal antibodies defining T cell subsets, Langerhan cells and Ia like antigens. The early reaction (24 hrs) was elicited either with standard Dharmendra lepromin or leprosin-A and the late reaction (3-4 weeks) was elicited with standard Dharmendra lepromin. In all, 15 biopsies were studied. Most lymphocytes in the infiltrates of both the lepromin and leprosin reactions were positive for OKT 11, Leu 3a, OKT 8 and Ia like antigens indicating thereby the presence of activated T cells. A high proportion of OKT6 + cells were also noticed in the infiltrates of these reactions. In the late reaction, the lymphocytes in the granulomas were predominantly activated T lymphocytes expressing OKT 11, Leu 3a, OKT 8 and Ia like antigens. Leu 3a + cells were scattered diffusely amidst the epithelioid cells. In contrast, OKT 8 + cells were present mainly in the peripheral region of the granuloma. A small proportion of OKT6 + cells were also seen in these granulomas. Ia like antigens and T6 antigens were not discernible on the epithelioid cells. No difference in the number of OKT6 + epidermal langerhan cells was observed in the various types of reactions.

Demonstration of Mycobacterium leprae specific antigens in leprosy lesions using monoclonal antibodies.

Cryostat sections of dermal lesions from 13 untreated patients of leprosy were studied by indirect immunoperoxidase using monoclonal antibodies (MLO4 & MLO6), defining M. leprae specific antigens. The lymphocytes and macrophages in both the tuberculoid and lepromatous granulomas showed membranous staining with the above antibodies. M. leprae organisms in the lepromatous granulomas and the cells in the section of lymph nodes of patients with tuberculosis, or sections of normal skin or psoriatic lesions did not show any staining with these antibodies. These observations suggest that M. leprae specific antigens are present and expressed on the cells infiltrating the granulomas of leprosy lesions.