Unveiling the role of NK cells, NKT-like cells, and γδ cells in pathogenesis of type 1 reactions in leprosy.

Leprosy is a disease with spectral clinical manifestations along with two types of reactions, type 1 reaction (T1R) and type 2 reaction (T2R). T1R especially occurs because of the defensive upgradation of cell-mediated immunity (CMI) to M. leprae antigens. T1R is the main cause of disability in leprosy. The role of conventional adaptive T cells has been well studied to understand T1R. A comprehensive understanding of the role of unconventional T cells in the manifestation of inflammation during T1R is crucial and has not been studied. In our study, we found significantly higher plasma levels of TNFα, IL1ß, IL17, and IP10 in T1R when compared to non-reaction (NR). Gene expression for cytokines in blood circulation by qPCR showed significantly higher expression of IFNγ, IP10, TNFα, IL6, IL17A and chemokines CCL3, CCR1, CCR5, and CXCR3 in T1R as compared to NR. Frequencies of NKT-like cells (48.7 %) and NK cells (22.3 %) were found significantly higher in T1R in comparison to NR (36.9 %, 18.3 %, respectively) (p = 0.0001). Significantly lower levels of γδT cells (3.32 %) were observed in T1R in comparison to NR (5.16 %). The present study has provided evidence for the first time on the role of plausible unconventional T cells in the immunopathogenesis of T1R in leprosy.

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.

Real-time PCR-based quantitation of viable Mycobacterium leprae strain from clinical samples and environmental sources and its genotype in multi-case leprosy families of India.

The potential role of environmental M. leprae in the transmission of leprosy remains unknown. We investigated role of environment as a possible source of viable M. leprae responsible for transmission of leprosy. The samples were collected from 10 multi-case leprosy families comprising, slit skin smear (SSS) from 9 multibacillary (MB), 16 paucibacillary cases (PB), 22 household contacts, and 38 environmental soil samples. The quantum of viable M. leprae was estimated by qRT-PCR using 16S rRNA gene from soil and SSS. Genotypes of M. leprae were determined by gene sequencing. We could observe presence of viable M. leprae in 11 (44%) leprosy cases (M. leprae 16S rRNA gene copies range from 1.78 × 102 to 8.782 × 109) and 4 (18%) household contacts (M. leprae 16S rRNA gene copies range from 2.54 × 103 and 7.47 × 104). Remarkably, presence of viable M. leprae was also noted in 10 (53%) soil samples where in M. leprae 16S rRNA gene copies ranged from 4.36 × 102 to 7.68 × 102. M leprae subtype 1D was noted in most of the leprosy cases their household contacts and in the surrounding soil samples indicating source of infection in household contacts could be from environment or patients. M. leprae 16S rRNA copies were approximately similar in both PB cases and soil samples along with presence of SNP type 1 subtype 1D in both samples indicating source of M. leprae from patients to contacts was either from patients or environment or both.

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.

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.

Mutation at codon 442 in the rpoB gene of Mycobacterium leprae does not confer resistance to rifampicin.

BACKGROUND: Rifampicin is the major drug in the treatment of leprosy. The rifampicin resistance of Mycobacterium leprae results from a mutation in the rpoB gene, encoding the ß subunit of RNA polymerase. As M. leprae is a non-cultivable organism observation of its growth using mouse food-pad (MFP) is the only Gold Standard assay used for confirmation of "in-vivo" drug resistance. OBJECTIVE: Any mutation at molecular level has to be verified by MFP assay for final confirmation of drug resistance in leprosy. MATERIAL AND METHODS: In the present study, M. leprae strains showing a mutation only at codon 442 Gln-His and along with mutation either at codon 424 Val-Gly or at 438 Gln-Val within the Rifampicin Resistance Determining Region (RRDR) confirmed by DNA sequencing and by high resolution melting (HRM) analysis were subjected for its growth in MFP. RESULT AND CONCLUSION: The M. leprae strain having the new mutation at codon 442 Gln-His was found to be sensitive to all the three drugs and strains having additional mutations at 424 Val-Gly and 438 Gln-Val were conferring resistance with Multi drug therapy (MDT) in MFP. These results indicate that MFP is the gold standard method for confirming the mutations detected by molecular techniques.

Comparative evaluation of PCR amplification of RLEP, 16S rRNA, rpoT and Sod A gene targets for detection of M. leprae DNA from clinical and environmental samples.

PURPOSE: PCR assay is a highly sensitive, specific and reliable diagnostic tool for the identification of pathogens in many infectious diseases. Genome sequencing Mycobacterium leprae revealed several gene targets that could be used for the detection of DNA from clinical and environmental samples. The PCR sensitivity of particular gene targets for specific clinical and environmental isolates has not yet been established. The present study was conducted to compare the sensitivity of RLEP, rpoT, Sod A and 16S rRNA gene targets in the detection of M. leprae in slit skin smear (SSS), blood, soil samples of leprosy patients and their surroundings. METHOD: Leprosy patients were classified into Paucibacillary (PB) and Multibacillary (MB) types. Ziehl-Neelsen (ZN) staining method for all the SSS samples and Bacteriological Index (BI) was calculated for all patients. Standard laboratory protocol was used for DNA extraction from SSS, blood and soil samples. PCR technique was performed for the detection of M. leprae DNA from all the above-mentioned samples. RESULTS: RLEP gene target was able to detect the presence of M. leprae in 83% of SSS, 100% of blood samples and in 36% of soil samples and was noted to be the best out of all other gene targets (rpoT, Sod A and 16S rRNA). It was noted that the RLEP gene target was able to detect the highest number (53%) of BI-negative leprosy patients amongst all the gene targets used in this study. CONCLUSION: Amongst all the gene targets used in this study, PCR positivity using RLEP gene target was the highest in all the clinical and environmental samples. Further, the RLEP gene target was able to detect 53% of blood samples as positive in BI-negative leprosy cases indicating its future standardization and use for diagnostic purposes.

Genotyping of Mycobacterium leprae strains from a region of high endemic leprosy prevalence in India.

Leprosy is still a major health problem in India which has the highest number of cases. Multiple locus variable number of tandem repeat analysis (MLVA) and single nucleotide polymorphism (SNP) have been proposed as tools of strain typing for tracking the transmission of leprosy. However, empirical data for a defined population from scale and duration were lacking for studying the transmission chain of leprosy. Seventy slit skin scrapings were collected from Purulia (West Bengal), Miraj (Maharashtra), Shahdara (Delhi), and Naini (UP) hospitals of The Leprosy Mission (TLM). SNP subtyping and MLVA on 10 VNTR loci were applied for the strain typing of Mycobacterium leprae. Along with the strain typing conventional epidemiological investigation was also performed to trace the transmission chain. In addition, phylogenetic analysis was done on variable number of tandem repeat (VNTR) data sets using sequence type analysis and recombinational tests (START) software. START software performs analyses to aid in the investigation of bacterial population structure using multilocus sequence data. These analyses include data summary, lineage assignment, and tests for recombination and selection. Diversity was observed in the cross-sectional survey of isolates obtained from 70 patients. Similarity in fingerprinting profiles observed in specimens of cases from the same family or neighborhood locations indicated a possible common source of infection. The data suggest that these VNTRs including subtyping of SNPs 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. The present study strongly indicates that multi-case families might constitute epidemic foci and the main source of M. leprae in villages, causing the predominant strain or cluster infection leading to the spread of leprosy in the community.

A report of rifampin-resistant leprosy from northern and eastern India: identification and in silico analysis of molecular interactions.

Presence of point mutations within the drug resistance determining regions of Mycobacterium leprae (M. leprae) genome confers molecular basis of drug resistance to dapsone, rifampin and ofloxacin in leprosy. This study is focused on the identification of mutations within the rpoB gene region of M. leprae that are specific for rifampin interaction, and further in silico analysis was carried out to determine the variations in the interactions. DNA and RNA were isolated from slit skin scrapings of 60 relapsed leprosy patients. PCR targeting rpoB gene region and amplicon sequencing was performed to determine point mutations. mRNA expression levels of rpoB and high-resolution melt analysis of mutants were performed using Rotor Gene Q Realtime PCR. Molecular docking was performed using LigandFit Software. Ten cases having point mutations within the rpoB gene region were identified and were clinically confirmed to be resistant to rifampin. A new mutation at codon position Gln442His has been identified. There is a 9.44-fold upregulation in the mRNA expression of rpoB gene in mutant/resistant samples when compared with the wild/sensitive samples. In silico docking analysis of rifampin with wild-type and Gln442His mutant RpoB proteins revealed a variation in the hydrogen-bonding pattern leading to a difference in the total interaction energy and conformational change at position Asp441. These preliminary downstream functional observations revealed that the presence of point mutations within the rifampin resistance determining regions of rpoB gene plays a vital role in conferring genetic and molecular basis of resistance to rifampin in leprosy.

Increased serum circulatory levels of interleukin 17F in type 1 reactions of leprosy.

PURPOSE: Leprosy is a chronic infectious disease caused by Mycobacterium leprae affecting mainly skin and peripheral nerves. Acute inflammatory episodes in the borderline immunological spectrum of the disease cause severe nerve and tissue damage leading to deformities. Finding of any serological marker for leprosy reactions will help in prediction of reactions and in early treatment intervention. The objective of this study was to measure the serum circulatory levels of Interleukin 17F (IL 17F) and to correlate the levels with type 1 and type 2 reactional states and with clinico-histopathological spectrum of leprosy. We studied IL 17F to delineate its role and its clinical implications in leprosy reactions. METHODS: Patients were classified based on the Ridley DS and Jopling WH Classification and blood samples (5 ml each) were collected from 80 active untreated leprosy cases in Type 1 reaction (T1R), 21 cases in Type 2 (Erythema Nodosum Leprosum ENL) reaction (T2R), 80 cases without reaction (NR), and 94 non-leprosy cases (NL). Serum was separated and measured for IL 17F levels using ELISA (Commercial Kits, R&D Systems Inc., USA). RESULTS: IL 17F levels were significantly higher in the T1R group when compared to the NR group (p < 0.001). The borderline lepromatous group showed the highest levels of IL 17F among the other groups in the disease spectrum. Bacteriological index (BI) showed negative correlation with the IL 17F levels. CONCLUSION: The results specify that serum circulatory levels of IL 17F are elevated during T1Rs in the borderline spectrum of the disease and thus may play a role in the regulation of inflammatory responses associated with reactions in leprosy.

Dynamics of Mycobacterium leprae transmission in environmental context: deciphering the role of environment as a potential reservoir.

Leprosy is a disease caused by Mycobacterium leprae. Various modes of transmission have been suggested for this disease. Transmission and risk of the infection is perhaps related to presence of the infectious cases and is controlled by environmental factors. Evidence suggests that humidity may favor survival of M. leprae in the environment. Several reports show that non-human sources like 'naturally' infected armadillos or monkeys could act as reservoir for M. leprae. Inanimate objects or fomites like articles used by infectious patients may theoretically spread infection. However, it is only through detailed knowledge of the biodiversity and ecology that the importance of this mode of transmission can be fully assessed. Our study focuses here to decipher the role of environment in the transmission of the disease. Two hundred and seven soil samples were collected from a village in endemic area where active cases also resided at the time of sample collection. Slit skin smears were collected from 13 multibacillary (MB) leprosy patients and 12 household contacts of the patients suspected to be hidden cases. DNA and RNA of M. leprae were extracted and amplified using M. leprae specific primers. Seventy-one soil samples showed presence of M. leprae DNA whereas 16S rRNA could be detected in twenty-eight of these samples. Samples, both from the environment and the patients, exhibited the same genotype when tested by single nucleotide polymorphism (SNP) typing. Genotype of M. leprae found in the soil and the patients residing in the same area could help in understanding the transmission link in leprosy.