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.

Extraction and detection of Mycobacterium leprae DNA from ZNCF-stained skin smear slides for better identification of negative skin smears.

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.

Detection of Mycobacterium leprae DNA from soil samples by PCR targeting RLEP sequences.

Despite near elimination of leprosy as a public health problem, several problems in leprosy still remain. These include early detection, determining efficacy of the treatment and differentiating relapses from re-infection. These aspects have important impact on the patients undergoing treatment and also have a bearing on understanding transmission dynamics in the community. While early diagnosis and management do not need major technological inputs, various reports have suggested that M. leprae is found in the environment and may have a role in continued transmission of disease. In earlier studies from other parts of world the presence of M. leprae DNA in the environment has been investigated both by microbiological and molecular studies. In the present study, an attempt was made to extract M. leprae DNA from soil samples, which were collected from eighteen different locations including 3 from our Institute area and 15 from different villages of Ghatampur area. We optimized a protocol for the extraction of DNA and amplified a fragment of M. leprae using specific primers targeting RLEP sequences. It was found that 33.3% of these soil samples collected from areas inhabited by leprosy cases gave positive result for M. leprae specific DNA. The utility of this method needs to be explored on a larger scale to establish the presence of M.leprae in the environment, and its role in the spread of the disease.

Use of reverse transcription polymerase chain reaction for the detection of Mycobacterium leprae in the slit-skin smears of leprosy patients.

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.

Street-level concentrations of suspended particulate matter (SPM), nitrogen dioxide (NO2) and sulphur dioxide (SO2) in Pune City.

The concentration of suspended particulate matter (SPM), nitrogen dioxide (NO,) and sulphur dioxide (SO2) were measured at 13 important traffic intersections in Pune city. In order to study the contribution of these pollutants from motor vehicles, attention was focused on the roadside, street-level concentration. The statistical analysis of the sampling results indicates that there is not only high correlation between SPM and NO2 but the levels of these pollutants are above the National Ambient Air Quality Standards (NAAQS) laid down by the Central Pollution Control Board (CPCB), India. The SO, concentrations are found to be well below the NAAQS.