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.

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.