Genomic characterization of Mycobacterium lepromatosis from ENL patients from India.

BACKGROUND: Leprosy is caused by Mycobacterium leprae and Mycobacterium lepromatosis. Both organisms cannot be cultured in vitro. M. lepromatosis was found to be associated mainly with diffuse lepromatous leprosy and with Lucio's phenomena initially. Later, M. lepromatosis was observed in borderline leprosy cases (BL), lepromatous leprosy cases (LL) and leprosy reactional cases (T1R and ENL). Although many cases are being reported with similar clinical features like Lucio phenomenon in India but M. lepromatosis was not isolated from these cases. The aim of this study was to screen MB patients and patients with type 2 reaction for the presence of M. lepromatosis. METHODOLOGY: We recruited a total of 75 multibacillary leprosy cases (45 MB cases without reaction and 30 type 2 reaction (ENL) cases) from TLM hospitals Purulia (West Bengal), Barabanki (Uttar Pradesh), Shahdara (Delhi) and PGIMER (Chandigarh), India. Punch biopsies of 5 mm were collected in 70% ethanol from all the study subjects. DNA was extracted followed by Hemi-nested PCR targeting 16S rRNA gene specific for M. lepromatosis. Further, PCR products were processed for Sanger sequencing for an absolute confirmation of M. lepromatosis. Whole genome sequencing was done to confirm the presence of M. lepromatosis. RESULT: We observed presence of M. lepromatosis in 4 necrotic ENL patients by heminested PCR. There was 100% 16S rRNA sequence similarity with M. lepromatosis FJ924 in one case, 98.96% in two cases and in one case it was 90.9% similarity by nucleotide BLAST (BLASTn) by using the NCBI website. On the basis of Sanger sequencing, we noted presence of M. lepromatosis in 3 necrotic ENL patients as one sample only gave 90.9% similarity by BLASTn. On the basis of de novo assembly and genome obtained, only one sample S4 with a 2.9 mb genome size was qualified for downstream analysis. Sixteen M. lepromatosis- specific proteins were identified in this case and the closest species was M. lepromatosis strain FJ924 based on whole genome level phylogeny. CONCLUSION: These results provide valuable insights into the prevalence of M. lepromatosis in ENL patients in different regions of India and contribute to our understanding of the genetic characteristics of this pathogen in the context of leprosy.

Efficacy of fixed duration multidrug therapy for the treatment of multibacillary leprosy: A prospective observational study from Northern India.

BACKGROUND: In endemic regions of several countries, the prevalence of leprosy has not come down to the level of elimination. On the contrary, new cases are being detected in large numbers. Clinically, it is frequently noted that despite completion of multibacillary multidrug therapy for 12 months, the lesions remain active, especially in cases with high bacteriological indices. AIM: The present study focused on finding out the viable number of Mycobacterium leprae during the 12-month regimen of multibacillary multidrug therapy, at six and 12 months intervals and, attempting to determine their role in disease transmission. METHODS: Seventy eight cases of multibacillary leprosy cases were recruited from leprosy patients registered at The Leprosy Mission hospitals at Shahdara (Delhi), Naini (Uttar Pradesh) and Champa (Chhattisgarh), respectively. Slit skin smears were collected from these patients which were transported to the laboratory for further processing. Ribonucleic acid was extracted by TRIzol method. Total Ribonucleic acid was used for real-time reverse transcription-polymerase chain reaction (two-step reactions). A standard sample with a known copy number was run along with unknown samples for a reverse transcription-polymerase chain reaction. Patients were further assessed for their clinical and molecular parameters during 6th month and 12th month of therapy. RESULTS: All 78 new cases showed the presence of a viable load of bacilli at the time of recruitment, but we were able to follow up only on 36 of these patients for one year. Among these, using three different genes, 20/36 for esxA, 22/36 for hsp18 and 24/36 for 16S rRNA cases showed viability of M. leprae at the time of completion of 12 months of multidrug therapy treatment. All these positive patients were histopathologically active and had bacillary indexes ranging between 3+ and 4+. Patients with a high copy number of the Mycobacterium leprae gene, even after completion of treatment as per WHO recommended fixed-dose multidrug therapy, indicated the presence of live bacilli. LIMITATIONS: Follow up for one year was difficult, especially in Delhi because of the migratory nature of the population. Patients who defaulted for scheduled sampling were not included in the study. CONCLUSION: The presence of a viable load of bacilli even after completion of therapy may be one of the reasons for relapse and continued transmission of leprosy in the community.

Ofloxacin resistance in multibacillary new leprosy cases from Purulia, West Bengal: a threat to effective secondary line treatment for rifampicin-resistant leprosy cases.

OBJECTIVES: Purulia is one of the high-endemic districts for leprosy in West Bengal (the eastern part of India). The annual new case detection rate (ANCDR) of leprosy in West Bengal is 6.04/100000 (DGHS 2019-20). Our earlier report provided evidence of secondary drug resistance in relapse cases of leprosy. The aim of the current study was to observe primary drug resistance patterns for dapsone, rifampicin, and ofloxacin amongst new leprosy patients from Purulia, West Bengal in order to better understand the emergence of primary resistance to these drugs. METHODS: In the present study, slit-skin smear samples were collected from 145 newly diagnosed leprosy cases from The Leprosy Mission (TLM) Purulia hospital between 2017 and 2018. DNA was extracted from these samples and the Mycobacterium leprae genome was analyzed for genes associated with drug resistance by polymerase chain reaction (PCR), followed by Sanger sequencing. Wild-type strain (Thai-53) and mouse footpad-derived drug-resistant strain (Z-4) were used as reference strains. RESULTS: Of 145 cases, 25 cases showed mutations in genes associated with resistance to rifampicin, dapsone, and ofloxacin (as described by the World Health Organization, rpoB, folP, and gyrA, respectively) through Sanger sequencing. Of these 25 cases, 16 cases showed mutations in ofloxacin, two cases showed mutations in combinations of ofloxacin and rifampicin, four cases showed a mutation only in rifampicin, one case showed mutations in combinations of rifampicin and dapsone, and two cases showed mutations only in dapsone. CONCLUSION: Results from this study indicated the emergence of resistance to antileprosy drugs in new cases of leprosy. As ofloxacin is the alternate drug for the treatment of rifampicin-resistant cases, the emergence of new cases with resistance to ofloxacin indicates that ofloxacin-resistant M. leprae strains are actively circulating in this endemic region (i.e., Purulia, West Bengal), posing challenges for the effective treatment of rifampicin-resistant cases.

Zosteriform multidermatomal nodules and plaques in a case of lepromatous leprosy: an uncommon presentation.

Leprosy is caused by the obligate intracellular organism Mycobacterium leprae which mainly affects the skin and nervous system. The course of the disease is determined by host immunity, it is thus believed that in lepromatous leprosy (LL), all manifestations are bilaterally symmetrical. This is because of the inability of the host to mount an adequate cell-mediated immune response, resulting in widespread haematogenous dissemination of bacilli. Varied manifestations of LL have been reported; however, a multidermatomal pattern of nodules is hitherto unreported and we suggest a hypothesis for its presentation.

Late leprosy reaction presenting as erythema multiforme-like erythema nodosum leprosum with underlying rifampicin resistance and its potential implications.

Erythema multiforme (EM)-like erythema nodosum leprosum (ENL) is a rare atypical presentation, and its late appearance after the completion of multidrug therapy (MDT) is unusual. We describe the case of a lepromatous leprosy patient who after the completion of MDT presented to us with late EM-like ENL and was found to be resistant to rifampicin. We discuss the implications of this finding and the potential role of resistant bacilli in causing reactions with atypical presentations.

Appropriately Selected Nerve in Suspected Leprous Neuropathy Yields High Positive Results for Mycobacterium leprae DNA by Polymerase Chain Reaction Method.

Identification of Mycobacterium leprae DNA by polymerase chain reaction (PCR) is a reliable and an affordable method to confirm leprosy. DNA from 87 nerve samples (61 from paraffin blocks and 26 fresh samples) was extracted. Mycobacterium leprae DNA was amplified by PCR from 80/87 (92%) specimens. Patients were seen over a period of 11 years (2007-2019), and leprosy was diagnosed based on clinical and characteristic histopathology findings. The clinical diagnostic possibilities were as follows: leprous neuropathy in 73/80 (91.3%), mononeuritis multiplex of unknown etiology in four (5.0%), vasculitic neuropathy in two (2.5%), and distal symmetric sensory motor neuropathy in one (1.3%). The biopsied nerves were as follows: superficial radial = 34 (42.6%), dorsal cutaneous branch of ulnar = 19 (23.8%), sural = 18 (22.5%), and superficial peroneal = 9 (11.3%), and corresponding neurological deficits were recorded in 77 (96.3%) cases. The histopathological diagnoses in total group were as follows: (borderline tuberculoid (BT) = 52, tuberculoid (TT) = 8, borderline lepromatous (BL) = 8, borderline borderline (BB) = 3, nonspecific inflammation = 3, healed/fibrosed = 4, and axonopathy = 2). Acid fast bacilli (AFB) was demonstrated in 11 (13.7%) samples. For comparison, 31 clinically and histopathologically defined non-leprous disease control nerves (inherited neuropathy = 20, vasculitis = 8, and nutritional neuropathy = 3) subjected to PCR were negative for M. leprae DNA. In most instances, there are multiple thickened peripheral nerves in suspected cases of leprosy, but neurological deficits pertaining to the thickened nerve are not as widespread. The current findings emphasize the importance of selecting the most appropriate nerve for biopsy to obtain a positive PCR result. We infer that clinical, histopathological, and PCR tests complement each other to help achieve a definitive diagnosis of leprosy particularly in pure neuritic leprosy and in leprous neuropathy with negative skin smears/biopsy.

Case Report: Multidrug-Resistant Mycobacterium leprae in a Case of Smear-Negative Relapse.

The ongoing transmission of leprosy in India is worrisome, and emerging drug resistance may be one of the factors responsible for the continued transmission of leprosy in India. Emerging cases of multidrug-resistant Mycobacterium leprae pose a great threat to eradication of leprosy and must be addressed with utmost priority. We report a case of multidrug-resistant M. leprae in a case of relapse where slit skin smear (SSS) was negative and histopathology was inconclusive. Drug resistance studies in leprosy are undertaken only in smear-positive relapse cases, and detection of this type of multidrug resistance in a case with negative SSS and innocuous histopathology is rather unusual and highlights the importance of undertaking drug resistance tests even in smear-negative cases of leprosy relapse. Resistance to ofloxacin (OFL) is also a cause for concern as OFL is one of the reserve drugs recommended for treatment of rifampicin-resistant strains.

Evidence for Mycobacterium leprae Drug Resistance in a Large Cohort of Leprous Neuropathy Patients from India.

Resistance to anti-leprosy drugs is on the rise. Several studies have documented resistance to rifampicin, dapsone, and ofloxacin in patients with leprosy. We looked for point mutations within the folP1, rpoB, and gyrA gene regions of the Mycobacterium leprae genome predominantly in the neural form of leprosy. DNA samples from 77 nerve tissue samples were polymerase chain reaction (PCR)-amplified for M leprae DNA and sequenced for drug resistance-determining regions of genes rpoB, folP1, and gyrA. The mean age at presentation and onset was 38.2 ± 13.4 (range 14-71) years and 34.9 ± 12.6 years (range 10-63) years, respectively. The majority had borderline tuberculoid leprosy (53 [68.8%]). Mutations associated with resistance were identified in 6/77 (7.8%) specimens. Mutations seen were those associated with resistance to rifampicin, ofloxacin, and dapsone. All the six patients were drug-naive. The clinical and pathological manifestations in this group did not differ from the drug-sensitive group. This study highlights the occurrence of resistance to the standard multidrug therapy and ofloxacin in leprosy. Among the entire cohort, 1/77 (1.3%) showed resistance to rifampicin, 2/77 (2.6%) to dapsone, and 5/77 (6.4%) to ofloxacin. Six new patients showing infection by mutant strains indicated the emergence of primary resistance. Resistance to ofloxacin could be due to frequent use of quinolones for many bacterial infections. The results of the study indicate the need for development of a robust and strict surveillance system for detecting drug resistance in leprosy in India.

Multidrug resistance as a cause of steroid-nonresponsive downgrading type I reaction in Hansen's disease.

While Type 1 reaction in Hansen's disease is commonly encountered, the triggers and reasons for its persistence are not well understood even though the immunological milieu and cytokine interplay have been studied. Herein, we present a case of Type 1 downgrading reaction in which multidrug resistance was the probable cause of steroid-nonresponsiveness and which responded promptly on starting alternate antileprosy treatment.

Utility of multiplex PCR for early diagnosis and household contact surveillance for leprosy.

Early diagnosis of leprosy is important for limiting the severity of disease, which may lead to disabilities and deformities if not treated timely. Multiplex PCR employing more than one gene, specific to target DNA, is more efficient detection tool. In the present study, slit skin scrapings, blood, nasal swabs and saliva from Paucibacillary (PB) and Multibacillary (MB) cases as well as household contacts of PB cases were tested by multiplex PCR using three different gene targets namely RLEP, 16SrRNA and sodA. We found an increase in overall diagnostic positivity for M. leprae DNA detection by M-PCR as compared to individual PCR. In case of nasal swabs using M-PCR the PPV, NPV were 0.5454, 0.8333 respectively. There is remarkable increase in PPV in SSS of PB cases and nasal swabs of HHCs using M-PCR. Conclusively, our finding suggests the utility of M-PCR for early diagnosis and household contact surveillance for leprosy.

Brain and Spinal Cord Lesions in Leprosy: A Magnetic Resonance Imaging-Based Study.

Neurotropism and infiltration by Mycobacterium leprae of peripheral nerves causing neuropathy are well established, but reports of central nervous system (CNS) damage are exceptional. We report CNS magnetic resonance imaging (MRI) abnormalities of the brain and spinal cord as well as lesions in nerve roots and plexus in leprosy patients. Eight patients aged between 17 and 41 years underwent detailed clinical, histopathological, and MRI evaluation. All had prominent sensory-motor deficits with hypopigmented and hypo/anesthetic skin patches and thickened peripheral nerves. All demonstrated M. Leprae DNA in affected peripheral nerve tissue. All received multidrug therapy (MDT). Two patients had brainstem lesions with enhancing facial nuclei and nerves, and one patient had a lesion in the nucleus ambiguus. Two patients had enhancing spinal cord lesions. Follow-up MRI performed in four cases showed resolution of brainstem and cord lesions after starting on MDT. Thickened brachial and lumbosacral plexus nerves were observed in six and two patients, respectively, which partially resolved on follow-up MRI in the two cases who had reimaging. The site and side of the MRI lesions corresponded with the location and side of neurological deficits. This precise clinico-radiological correlation of proximal lesions could be explained by an immune reaction in the gray matter corresponding to the involved peripheral nerves, retrograde axonal and gray matter changes, or infection of the CNS and plexus by lepra bacilli. Further study of the CNS in patients with leprous neuropathy is needed to establish the exact nature of these CNS MRI findings.

Survival of Mycobacterium leprae and association with Acanthamoeba from environmental samples in the inhabitant areas of active leprosy cases: A cross sectional study from endemic pockets of Purulia, West Bengal.

BACKGROUND: Mycobacterium leprae being an obligate intracellular parasite cannot be cultured in any artificial culture media but it has been shown to reside in wild armadillos in North America. Many studies suggested that M. leprae could be found in the environment and may have a role in continuing transmission of the disease. The exact role of the environment in the transmission dynamics is still speculative. The present study was undertaken to find out the presence of viable M. leprae around patients' environment like soil and water and association of free living pathogenic protozoa, Acanthamoeba which might play an important role in transmission of the disease. METHODS: Seven hundred soil and 400 water samples were collected from the surroundings of the houses of leprosy patients from endemic villages. Two hundred soil and 80 water samples were also collected from the surroundings of normal inhabitants from non-endemic villages as controls. These samples were screened for the presence of M. leprae and Acanthamoeba using DNA PCR. RNA was extracted from the PCR positive samples and Reverse Transcriptase - PCR targeting 16S rRNA gene region was performed for detection of viable M. leprae. RESULTS: We observed high PCR positivity in soil samples (218 out of 700; 31%) and water samples (73 out of 400; 18%). These samples when further screened for viability, it was observed that 106 soil samples (15% of total) and 34 water samples (8% of total) showed presence of 16S rRNA. We observed 18.3% of soil and 20.5% of water samples were PCR positive for Acanthamoeba. Soil samples from the control area, where no active leprosy case resided in the last 5 years, showed PCR positivity in 4 samples (2%) for M. leprae DNA in only soil samples with all water samples being negative. RT-PCR for all PCR positive soil samples was negative. Of the 106 soil samples positive for M. leprae RT-PCR, 30 samples were also positive for Acanthamoeba whereas out of 112 M. leprae RT-PCR negative but PCR positive samples only 10 samples were Acanthamoeba positive showing association of viability with presence of Acanthamoeba (p = .0021). Similarly, for water samples also, association of M. leprae viability with presence of Acanthamoeba was seen (p = .0009). CONCLUSION: This study suggests that the surrounding environment (soil and water) of leprosy patients contain viable M. leprae and the viability has association with Acanthamoeba which may provide a protective niche for M. leprae. This could play an important role in the focal transmission of the disease.

Association of non-tuberculous mycobacteria with Mycobacterium leprae in environment of leprosy endemic regions in India.

Non-tuberculous mycobacteria (NTM) are environmental mycobacteria found ubiquitously in nature. The present study was conducted to find out the presence of various species of NTM in leprosy endemic region along with Mycobacterium (M) leprae. Water and wet soil samples from the periphery of ponds used by the community were collected from districts of Purulia of West Bengal and Champa of Chhattisgarh, India. Samples were processed and decontaminated followed by culturing on Lowenstein Jensen (LJ) media. Polymerase chain reaction (PCR) was performed using 16S rRNA gene target of mycobacteria and species was confirmed by sequencing method. Indirect immune-fluorescent staining of M. leprae from soil was performed using M. leprae-PGL-1 rabbit polyclonal antibody. The phylogenetic tree was constructed by using MEGA-X software. From 380 soil samples 86 NTM were isolated, out of which 34(40%) isolates were rapid growing mycobacteria (RGM) and 52(60%) isolates were slow growing mycobacteria (SGM). Seventy-seven NTM isolates were obtained from 250 water samples, out of which 35(45%) were RGM and 42(55%) were SGM. Amongst all the RGM, we isolated M. porcinum, M. psychrotolerans, M. alsenase, M. arabiense and M. asiaticum from Indian environmental samples. M. fortuitum was the most commonly isolated species of all RGM. Out of all SGM, M. holsaticum, M. yongonense, M. seoulense, M. szulgai, M. europaeum, M. simiae and M. chimaera were isolated for the first time from Indian environment. M. intracellulare was the commonest of all isolated SGM. Presence of M. leprae was confirmed by indirect immunofluorescent microcopy and PCR method from the same environmental samples. Phylogenetic tree was showing a close association between these NTMs and M. leprae in these samples. Several NTM species of pathogenic and nonpathogenic in nature along with M. leprae were isolated from soil and pond water samples from leprosy endemic regions and these might be playing a role in causing disease and maintaining leprosy endemicity in India.

VDR polymorphism, gene expression and vitamin D levels in leprosy patients from North Indian population.

BACKGROUND: Leprosy is a chronic infectious disease caused by Mycobacterium leprae and mainly affects skin, peripheral nerves. Vitamin D receptor (VDR) gene polymorphism has been found to be associated with leprosy. Vitamin D has been shown to control several host immunomodulating properties through VDR gene. Vitamin D deficiency was also found to be linked to an increased risk for several infections and metabolic diseases. OBJECTIVE: In the present study, we investigated the association of VDR gene polymorphism, mRNA gene expression of VDR and the vitamin D levels with leprosy and its reactional states. METHODOLOGY: A total of 305 leprosy patients consisting of tuberculoid (TT), borderline tuberculoid (BT), borderline lepromatous (BL), lepromatous leprosy (LL), as well as 200 healthy controls were enrolled in the study. We identified single nucleotide polymorphisms (SNPs) of VDR Taq1, Fok1 and Apa1, as well as the expression of VDR mRNA gene using PCR-based restriction fragment length polymorphism (RFLP) analysis and real-time PCR respectively. We also performed ELISA to measure vitamin D levels. RESULT: We observed that SNP of VDR gene (Fok1 and Taq1) are associated with the leprosy disease. The allelic frequency distribution of T and t allele (p = 0.0037), F and f allele (p = 0.0024) was significantly higher in leprosy patients and healthy controls. ff genotype of Fok1 was found to be associated with leprosy patients [p = 0.0004; OR (95% CI) 3.148 (1.662-5.965)]. The recessive model of Fok1 genotype was also found to be significantly associated in leprosy patients in comparison to healthy controls [p = 0.00004; OR (95% CI) 2.85 (1.56-5.22)]. Leprosy patients are significantly associated with t-F-a haplotype. Further, VDR gene expression was found to be lower in non-reaction group compared to that of reaction group of leprosy and healthy controls. Paradoxically, we noted no difference in the levels of vitamin D between leprosy patients and healthy controls. CONCLUSION: Blood levels of vitamin D do not play any role in clinical manifestations of any forms of leprosy. ff genotype of Fok1 and tt genotype of Taq1 was found to be associated with leprosy per se. Association of t-F-a haplotype with leprosy was found to be significant and could be used as a genetic marker to identify individuals at high risk for developing leprosy. VDR gene expression was lower in TT/BT and BL/LL groups of leprosy in comparison to that of healthy controls.

Detection of Mycobacterium lepromatosis in patients with leprosy in India.

INTRODUCTION: The most commonly noted reactions in leprosy patients are type 1 reactions and erythema nodosum leprosum, with some rare phenomenon of host response known as Lucio phenomenon or leprosy of Lucio and Latapi which is caused by Mycobacterium lepromatosis. So far, no case of M. lepromatosis has been reported from India. MATERIALS AND METHODS: The main objective of this study was to detect any positive cases of M. lepromatosis in India with such a complication. We screened slit skin smear/biopsy samples from lepromatous leprosy (LL) patients reporting to The Leprosy Mission Community Hospitals across the country. Eighty-eight slit skin smears were collected from leprosy patients in 70% ethanol. DNA was extracted from all these samples. Polymerase chain reaction (PCR) was done for 2 genes; one set was for 16S rRNA and the other set was for coproporphyrinogen III oxidase (hemN) gene. Then, sequencing was done for all positive amplicons. Homology of the sequences was analyzed using the Basic Local Alignment Search Tool at the National Center of Biotechnology Information database. RESULTS: Among 88 isolates, we found 4 positive cases for M. lepromatosis. All 4 were LL cases with a bacteriological index ranging from 2+ to 4+. On the basis of the National Center of Biotechnology Information Basic Local Alignment Search Tool analysis, the sequenced amplicons of both genes matched with the M. lepromatosis 16S rRNA and phosphofructokinase genes but not with hemN gene of lepromatosis. This is the first report for the presence of M. lepromatosis in LL cases from India. CONCLUSION: This new species M. lepromatosis exists beyond Mexico, Singapore and it is the cause of DLL in India also. It may cause dual infections along with M. leprae in endemic areas like India.

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 detection of multidrug-resistant Mycobacterium leprae from Indian leprosy patients.

OBJECTIVES: The emergence of multidrug-resistant (MDR) organisms for any infectious disease is a public health concern. Global efforts to control leprosy by intensive chemotherapy have led to a significant decrease in the number of registered patients. Currently recommended control measures for treating leprosy with multidrug therapy (MDT) were designed to prevent the spread of dapsone-resistant Mycobacterium leprae strains. Here we report the identification of MDR M. leprae from relapse leprosy patients from endemic regions in India. METHODS: Resistance profiles to rifampicin, dapsone and ofloxacin of the isolated strains were confirmed by identification of mutations in genes previously shown to be associated with resistance to each drug. Between 2009-2016, slit-skin smear samples were collected from 239 relapse and 11 new leprosy cases from hospitals of The Leprosy Mission across India. DNA was extracted from the samples and was analysed by PCR targeting the rpoB, folP and gyrA genes associated with resistance to rifampicin, dapsone and ofloxacin, respectively, in M. leprae. M. leprae Thai-53 (wild-type) and Zensho-4 (MDR) were used as reference strains. RESULTS: Fifteen strains showed representative mutations in at least two resistance genes. Two strains showed mutations in all three genes responsible for drug resistance. Seven, seven and one strain, respectively, showed mutations in genes responsible for rifampicin and dapsone resistance, for dapsone and ofloxacin resistance and for rifampicin and ofloxacin resistance. CONCLUSION: This study showed the emergence of MDR M. leprae in MDT-treated leprosy patients from endemic regions of India.