Development of an Assay System for Genotyping Mycobacterium leprae Resistant to Dapsone, Rifampicin, and Ofloxacin

Even after the availability of effective anti-leprosy drugs, leprosy treatment is facing the challenge of emergence of Mycobacterium leprae strains resistant to dapsone, rifampicin and ofloxacin. As the conventional mouse foot pad (MFP) assay is time consuming and requires sufficient bacterial load, it is important to adapt, develop and use molecular assay(s) that can detect M. leprae strains resistant to the drugs. Real-time PCR (rPCR) assay targeting RLEP sequences was used for detection of M.leprae DNA. Further a nested PCR reaction technique was optimized using sets of primers targeting folP, rpoB and gyrA gene target. Amplicons were sequenced to detect mutations. The optimal reactions were applied to slit skin smear samples from 20 leprosy patients and was found to be applicable for slit skin smear samples. The optimized assay could genotype 17 M. leprae strains for drug resistance, all (100%) of these strains were found to be sensitive for dapsone and rifampicin and 5.9% (1/17) resistant for fluoroquinolones (ofloxacin). This genotyping test could be used to detect leprosy drug resistance and may be useful for patient care. It will be also be important to validate the assay developed in this study with mouse foot technique and compare it with other molecular assays developed by investigators from different countries and then choose the best for patient care/surveillance purposes.