Enriched whole genome sequencing identified compensatory mutations in the RNA polymerase gene of rifampicin-resistant Mycobacterium leprae strains.

Despite more than three decades of multidrug therapy (MDT), leprosy remains a major public health issue in several endemic countries, including India. The emergence of drug resistance in Mycobacterium leprae (M. leprae) is a cause of concern and poses a threat to the leprosy-control program, which might ultimately dampen the achievement of the elimination program of the country. Rifampicin resistance in clinical strains of M. leprae are supposed to arise from harboring bacterial strains with mutations in the 81-bp rifampicin resistance determining region (RRDR) of the rpoB gene. However, complete dynamics of rifampicin resistance are not explained only by this mutation in leprosy strains. To understand the role of other compensatory mutations and transmission dynamics of drug-resistant leprosy, a genome-wide sequencing of 11 M. leprae strains - comprising five rifampicin-resistant strains, five sensitive strains, and one reference strain - was done in this study. We observed the presence of compensatory mutations in two rifampicin-resistant strains in rpoC and mmpL7 genes, along with rpoB, that may additionally be responsible for conferring resistance in those strains. Our findings support the role for compensatory mutation(s) in RNA polymerase gene(s), resulting in rifampicin resistance in relapsed leprosy patients.

Molecular typing of Mycobacterium leprae strains from northern India using short tandem repeats.

BACKGROUND & OBJECTIVES: Due to the inability to cultivate Mycobacterium leprae in vitro and most cases being paucibacillary, it has been difficult to apply classical genotyping methods to this organism. The objective of this study was therefore, to analyze the diversity among M. leprae strains from Uttar Pradesh, north India, by targeting ten short tandem repeats (STRs) as molecular markers. METHODS: Ninety specimens including 20 biopsies and 70 slit scrappings were collected in TE buffer from leprosy patients, who attended the OPD of National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, and from villages of Model Rural Health Research Unit (MRHRU) at Ghatampur, Kanpur, Uttar Pradesh. DNA was extracted from these specimens and ten STRs loci were amplified by using published and in-house designed primers. The copy numbers were determined by electrophoretic mobility as well as sequence analysis. Phylogenetic analysis was done on variable number of tandem repeats (VNTRs) data sets using start software. RESULTS: Diversity was observed in the cross-sectional survey of isolates obtained from 90 patients. Allelic index for different loci was found to vary from 0.7 to 0.8 except for rpoT for which allelic index was 0.186. Similarity in fingerprinting profiles observed in specimens from the cases from same house or nearby locations indicated a possible common source of infection. Such analysis was also found to be useful in discriminating the relapse from possible reinfection. INTERPRETATION & CONCLUSIONS: This study led to identification of STRs eliciting polymorphism in north Indian strains of M. leprae. The data suggest that these STRs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci.

Detection of viable Mycobacterium leprae in soil samples: insights into possible sources of transmission of leprosy.

Leprosy has ceased to be a public health problem world wide, after the successful implementation of effective chemotherapy (MDT) and use of control measures. However, new cases of leprosy continue to occur. Mycobacterium leprae cannot be grown in any acceptable culture medium and besides the wild armadillos, there is no known animal reservoir for leprosy. The transmission of leprosy is believed to be due to a large extent by droplet discharge of bacilli through nose and mouth and to a lesser extent by direct contact of susceptible host with a patient for long duration. The exact role of the environment in the transmission dynamics is still speculative. In the present study, we have tried to detect viable M. leprae from soil samples in endemic areas by using molecular methods. Eighty soil samples were collected from villages of this area, DNA and RNA of M. leprae extracted and identified using specific M. leprae primers. PCR amplification was done and real-time RT-PCR was used to detect viable M. leprae. DNA targeting the 16S region of M. leprae was detected in 37.5%, whereas M. leprae RNA targeting the same region was detected in 35% of these samples. Of the total 80 samples, 40 were collected from residential areas of leprosy patients whereas 40 samples were from no-patient areas. Fifty-five percent positivity for 16S rRNA of M. leprae was observed from the "patient" area in comparison to 15% positivity from the "no-patient" area (p < 0.001). This study thus provides valuable information of presence of viable M. leprae in soil specimens, which would be of use in investigating the transmission dynamics in leprosy.

Predominance of three copies of tandem repeats in rpoT gene of Mycobacterium leprae from Northern India.

This study has been carried out to get understanding of the origin among the strains of Mycobacterium leprae in patients from Northern India by using number of tandem repeats in rpoT gene as marker. Biopsies were collected from hundred leprosy cases (paucibacillary (PB) as well as multibacillary (MB)) across the spectrum from patients attending clinic at JALMA or diagnosed in Field Unit at Ghatampur (Kanpur). These biopsies were homogenized and DNA was extracted by a physiochemical procedure. rpoT region was amplified by using the primers and conditions earlier published. Among 100 strains from Northern Indian patients, 89% exhibited the presence of three copies of the 6bp tandem repeat in the rpoT gene, while 11% contained four copies. These profiles along with other genotyping data may help in studying the historical spread of leprosy by strains of M. leprae disseminated by various human races that migrated to Northern India from other places of Asian continent.