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Abstract Identification of Mycobacterium leprae, which causes leprosy, is done by Ziehl Neelsen Carbol Fuchsin (ZNCF) stained slit skin smear microscopy that aids in the diagnosis and quantification of approximate bacterial load carried by the patient. We attempted M. leprae DNA extraction from 46 stained slit skin smear negative slides, using Proteinase K and SDS lysis, followed by ethanol precipitation. M. leprae specific primers (16SrRNA) were used for PCR-based amplification of DNA. We could detect M. leprae DNA in 15 (32.6%) samples. The method can be useful in the diagnosis of apparently slit skin smear negative leprosy cases.

Mycobacterium leprae being an intracellular pathogen, cell mediated immunity is very important in the clinical outcome of leprosy. Manifestation of the disease is correlated with the level and type of cell mediated immune response. The main objective of this study was to analyse TNF-alpha and IFN-gamma production by T-cells when challenged with different M. leprae purified antigens in subjects with known exposure. 50 subjects residing in resettlement village of cured leprosy patients were included in the study. Whole Blood assay studies were undertaken in which the blood was placed in culture and was challenged with 35-kDa antigen, whole M. leprae cells, M. leprae cell wall antigen and M. leprae soluble antigen minus LAM. T-cell derived cytokines TNF-alpha and IFN-gamma were measured by ELISA. It was observed that challenging the lymphocytes with 35-kDa antigen, the cell wall antigen and M. leprae soluble antigen minus LAM resulted in increased levels of IFN-gamma whereas challenge with 35-kDa antigen and M. leprae cell wall antigen resulted in increased levels of TNF-alpha.

Majority of the endemic population is exposed to Mycobacterium leprae but very few develop disease. Humoral mucosal immune response mediated through M. leprae reactive salivary antibodies has been suggested to be quite important in the protective immunity. As the endemic population is also exposed to many environmental mycobacteria, we tested saliva from 121 subjects for the cross-reactivity of the M. leprae reactive salivary antibodies to mycobacteria like M. smegmatis and M. phlei. Saliva samples were treated with these two mycobacteria prior to testing M. leprae reactive antibodies by ELISA. In 59 subjects (48.76%), original and cross-reacted saliva showed same absorbance values suggesting no cross-reactivity. 26 subjects (21.49%) showed less than 25% drop in the OD values whereas 21 subjects (17.4%) showed 25% to 50% drop after reacting saliva with the mycobacteria. 15 subjects (12.4%) showed more that 50% drop in OD. The data suggest that though in half of subjects antibodies did not cross-react with mycobacteria tested, there were subjects where antibodies showed cross-reactivity to mycobacteria suggesting that positive salivary M. leprae reactive IgA response could be to some extent due to exposure to environmental mycobacteria and it could also be protective against M. leprae.

The relevance of bacterial index (BI) for understanding the prognosis of leprosy patients on treatment has been extensively debated, as it does not give a very clear idea of the viability of the bacteria in patients under treatment. Here we used slit-skin smear samples of leprosy patients to test the suitability for studying viability of Mycobacterium leprae using reverse transcription polymerase chain reaction (RT-PCR). For this purpose, we recruited 13 multibacillary (MB) leprosy patients (8 lepromatous and 5 borderline lepromatous). Of these, 7 were relapse cases, 3 were under treatment (MB-MDT), 2 were new cases and 1 had completed treatment. We carried out extraction of RNA using Trizol reagent (Life Technologies, UK) from the slit-skin smear samples from these patients. The RNA preparation was then used for the RT-PCR using Mycobacterium leprae-specific primers for the fragment of 16s ribosomal RNA gene. Samples from both the new cases, 4 suspected relapse cases and 1 patient under treatment showed positive RT-PCR results. Other 6 patients whose smear samples did not show any amplification by RT-PCR were on MB-MDT from 8 to 30 months. The usefulness of the technique needs to be validated using mouse footpad technique and also should be more extensively explored for studying the viability of M. leprae, the efficacy of treatment and the presence of other mycobacteria in the slit-skin smear samples.