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IntroductionMolecular epidemiology of leprosy is very important to study leprosy transmission dynamics and to enhance our understanding of leprosy in endemic areas by utilizing the molecular typing method. Nowadays our understanding of leprosy transmission dynamics has been refined by SNP typing and VNTR marker analysis of M. leprae strains.ObjectiveThis study was carried out to find out the presence of viable M. leprae in the soil and water samples from residing areas of leprosy patients staying in different blocks of Purulia district of West Bengal, understanding their genotypes and compared with that of M. leprae present in patients.Material and methodsSlit-skin smear (SSS) samples (n=112) were collected from the active multibacillary leprosy patients from different blocks of leprosy endemic area. Soil samples (n=1060) and water samples (n=620) were collected from residing areas of leprosy patients. SNP subtyping was performed by PCR followed by sequencing. Multiplex PCR was performed using fifteen ML-VNTR loci and results were analysed.ResultsWe observed high PCR positivity in soil samples (344 out of 1060; 32%) and water samples (140 out of 620; 23%). These PCR positive samples when further screened for viability, it was observed that 150 soil samples (44%) and 56 water samples (40%) showed presence of 16S rRNA. SNP typing of M. leprae revealed presence of predominantly type 1. SNP subtype 1D (83%) was most prevalent in all the blocks of Purulia followed by subtype 1C (15%) and subtype 1A (2%). SNP subtype 2F was noted in only one sample. SNP and VNTR combination showed presence of similar strain type in certain pockets of Purulia region which was responsible for transmission.ConclusionPresence of viable M. leprae in the environment, and presence of SNP Type 1 M. leprae in patients and environment suggests both environment and patients play a role in disease transmission.

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.

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.

Objective To elucidate the spectrum of findings on neuroimaging in leprosy with special reference to magnetic resonance imaging (MRI). Methods We retrospectively reviewed the neuroimaging findings on MRI in thirty-two patients with leprosy where dedicated plexus, brain and spine imaging had been performed. Results Twenty-two patients had positive findings on imaging. Six patients showed involvement of the peripheral nerves as thickening and formation of micro or macroabscesses. The brachial plexus showed thickening and hyperintensity in ten patients, with associated involvement of the lumbosacral plexus in one patient. Four patients showed spinal cord involvement as discrete T2 hyperintense lesions with postcontrast enhancement. Intracranial lesions involving the nucleus ambiguus and facial colliculus were seen in two patients. Conclusion Neuroimaging in Hansen's disease has brought several new findings to the fore front. Besides peripheral nerves, involvement of the centrally located plexuses, spinal cord and brain has also been seen which thus expands the imaging spectrum of the disease.

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.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.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.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.

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.

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.

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.

India attained the elimination figure of less than 1 case of leprosy per 10,000 people during December 2005. Despite this, India still accounts for the largest number of new leprosy cases in the world, maintaining more than 50 per cent of the leprosy burden of the world, notwithstanding over three decades of use of multidrug therapy. The present review analyzes the process of execution of the elimination program, identifies any lacunae therein and presents corrective measures that could be taken up for elimination of the disease from the country.

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.

The immune system is a dynamic network of cells and cytokines are the major mediators of immune responses which combat pathogens. Based on the cytokine production, effector T cells differentiate into subsets known as Th1, Th2, Th17, or Treg. This system serves as a barrier to intracellular pathogens, bacterial infections and stimulates the production of reactive oxygen species (ROS), reactive nitrogen intermediates, and nitric oxide, which diffuses across membranes and engulfs intracellular pathogens. Oxidative stress occurs when ROS, reactive nitrogen species (RNS) production, and antioxidant defences become imbalanced. Oxidative stress generated by infected cells produces a substantial amount of free radicals which enables the killing of intracellular pathogens. Intracellular pathogens are exposed to endogenous ROS as part of normal aerobic respiration, also exogenous ROS and RNS are generated by the host immune system in response to infection. Nanoparticles which are designed for drug delivery are capable of trapping the desired drug in the particles which protect the drug from enzymatic degradation in a biological system. The subcellular size of nanoparticles enables higher intracellular uptake of the drug which results in the reduction of the concentration of free drugs reducing their toxic effect. Research on the modulation of immune response and oxidative stress using nanoparticles used to encapsulate drugs has yet to be explored fully. In this review, we illustrate the immune activation and generation of oxidative stress properties which are mediated by nanoparticle encapsulated drug delivery systems which can make the therapy more effective in case of diseases caused by intracellular pathogens.

Indian Cancer Research Centre (ICRC) bacillus strain (C-44), a candidate vaccine against leprosy is cultured in vitro in Dubos medium enriched with amino acids and human serum. The study was conducted to find out whether ICRC bacilli obtained from these cultures are coated with anti mycobacterial antibody. Anti-ICRC antibody raised by intradermal inoculation of sonicated ICRC bacilli in rabbits reacted with both human immunoglobulin G (IgG) and antigens of ICRC. Further, ICRC bacilli could also be fluoresced directly with FITC labelled anti human IgG. Positive fluorescence of ICRC could be abolished by digestion of human IgG with trypsin and carbon tetrachloride (CCL4). It is concluded that ICRC bacilli present in the vaccine are coated with human IgG.

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.

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.

Purpose: Leprosy is a chronic systemic infectious disease caused by Mycobacterium leprae, one of the first organisms to be established as the cause for disease in humans. Because of high prevalence pockets of leprosy in the endemic regions, it is necessary to identify the possible sources of M. leprae in the environment and its mode of transmission. Materials and Methods: Slit skin smears (SSSs) from lesions were collected in 70% ethanol from 50 leprosy cases staying in the leprosy resettlement village and hospital from a high endemic area. One hundred and sixty soil samples were collected from different areas around the leprosy hospital and from the resettlement village of cured leprosy patients where active cases also resided at the time of sample collection. M. leprae specific gene region (RLEP 129 bp) and 16S rRNA targets were used for polymerase chain reaction (PCR) based detection for the presence and viability of M. leprae. An rpoT region was also amplified to determine presence of numbers of 6 bp tandem repeats. Results: All the SSS samples collected from patients showed three copies of rpoT region (6 bp tandem repeat, an ancient Indian type). Fifty-two soil samples showed presence of M. leprae DNA whereas M. leprae specific 16S rRNA gene was amplified in sixteen of these samples. PCR amplification and fragment length analysis showed 91 bp, i.e., three copies of the rpoT 6 bp tandem repeats from soil samples and similar three copies observed in patient samples. Conclusion: Presence of viable M. leprae in the soil having same rpoT genotype of M. leprae noted in patients suggests that it could be the same strain of M. leprae. M. leprae found in the soil could be the one that is excreted out by the patient. Significance of its viability in the environment and its pathogenicity with respect to transmission needs to be further explored. Findings of this study might provide possible insights for further exploration into understanding transmission patterns in leprosy and also will throw light on identifying potential for existence of extra human source or reservoirs of M. leprae, if any.

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.

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.

Background It has been shown earlier that there is a rise in the levels of autoantibodies and T cell response to cytoskeletal proteins in leprosy. Our group recently demonstrated a rise in both T and B cell responses to keratin and myelin basic protein in all types of leprosy patients and their associations in type 1 reaction (T1R) group of leprosy. Objectives In this study, we investigated the association of levels of autoantibodies and lymphoproliferation against myosin in leprosy patients across the spectrum and tried to find out the mimicking proteins or epitopes between host protein and protein/s of Mycobacterium leprae. Methodology One hundred and sixty-nine leprosy patients and 55 healthy controls (HC) were enrolled in the present study. Levels of anti-myosin antibodies and T-cell responses against myosin were measured by ELISA and lymphoproliferation assay, respectively. Using 2-D gel electrophoresis, western blot and MALDI-TOF/TOF antibody-reactive spots were identified. Three-dimensional structure of mimicking proteins was modeled by online server. B cell epitopes of the proteins were predicted by BCPREDS server 1.0 followed by identification of mimicking epitopes. Mice of inbred BALB/c strain were hyperimmunized with M. leprae soluble antigen (MLSA) and splenocytes and lymph node cells of these animals were adoptively transferred to naïve mice. Results Highest level of anti-myosin antibodies was noted in sera of T1R leprosy patients. We observed significantly higher levels of lymphoproliferative response (p

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., Author summary Present study was carried out to find out the association of vitamin D receptor (VDR) gene polymorphism, mRNA gene expression of VDR gene and level of vitamin D with leprosy reactions and leprosy patients. Surprisingly, level of vitamin D in leprosy patients was not found to be associated with the disease and its manifestations. VDR genotypes (Fok1 and Taq1) were found to be associated with leprosy patients. t-F-a haplotype was significantly associated with leprosy patients. Gene expression of vitamin D receptor was lower in leprosy patients in comparison to healthy controls.

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.

Alpha 1 Acid Glycoprotein (AGP) has been identified as a potentially useful marker of clinical outcome in disease having an inflammatory component. The objective of this study is to determine the levels of AGP with the possibility of using as a biomarker for Type 1 reactions (T1R) and Type 2 reactions (T2R) in leprosy. Serum levels of AGP are evaluated in a total of 88 leprosy subjects [T1R; n = 32, T2R; n =17 and Non-Reactional (NR); n = 39] using an Enzyme Linked Immunosorbent Assay (ELISA). Relative quantification of mRNA encoding AGP is also done from skin biopsies of same patients using Real-time PCR. The results revealed that the levels of serum AGP are significantly higher in all leprosy cases with T1R and T2R when compared to NR leprosy cases across the study groups (p < 0.05). The Cross-sectional analysis also revealed that the levels of mRNA expression of AGP are significantly higher (p < 0.05) in leprosy cases with both reactions when compared to non-reactional leprosy Cases (p < 0.05). Comparisons are made using Kruskal-Wallis nonparametric test. Significantly higher levels of AGP in serum and in the lesions of patients may indicate its role as a predictive biomarker for type 1 and 2 reactions in leprosy.

Background: Substantial number of patients with paucibacillary leprosy may still have active skin lesions at the end of MD treatment because of the continuing immune responses, though this does not denote failure of treatment. A clinical trial was conducted to compare efficacy of a homoeopathic medicine with MDT for the cure of leprosy. Aim: To compare the effect of a homoeopathic medicine - Sulphur with the Multi drug in the treatment of Paucibacillary leprosy according to WHO guidelines. Materials and Methods: Out of total 90 patients enlisted, 60 confirmed Borderline Tuberculoid (BT) leprosy patients of the age 14 years to less than 60 years were registered under this trial. All the patients were allotted a precoded number, randomly and equally divided in two groups. One group was treated with Sulphur in 200 potency in liquid orally once in week for two years and the other group received Paucibacillary (PB) regimen as recommended by WHO. At the end of two years findings were corroborated. Results: The study showed that MDT therapy (PB regimen) and homoeopathic medicine were found equally effective as histopathology of skin showed no granulomatous lesion. The most significant clinical sign observed in all cases treated with Sulphur was reappearance of normal skin colour and regain of loss of sensation of the skin lesion. Conclusions: MD therapy (PB regimen) and homoeopathic medicine were found equally effective. The patients of paucibacillary leprosy could get rid of the skin symptoms if treatment with Sulphur (is also given with MDT).

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.

The exact mode of transmission of leprosy is not clearly understood; however, many studies have demonstrated active transmission of leprosy around a source case. Families of five active leprosy cases and their household contacts were chosen from a high endemic area in Purulia. Fifty-two soil samples were also collected from different areas of their houses. DNA was extracted from slit-skin smears (SSS) and soil samples and the Mycobacterium leprae-specific RLEP (129 bp) region was amplified using PCR. Molecular typing of M. leprae was performed for all RLEP PCR-positive samples by single nucleotide polymorphism (SNP) typing and confirmation by DNA sequencing. SSS of these five patients and six out of the total 28 contacts were PCR positive for RLEP whereas 17 soil samples out of 52 showed the presence of M. leprae DNA. SNP typing of M. leprae from all RLEP PCR-positive subjects (patients and smear-positive contacts) and 10 soil samples showed the SNP type 1 genotype. M. leprae DNA from the five leprosy patients and the six contacts was further subtyped and the D subtype was noted in all patients and contacts, except for one contact where the C subtype was identified. Typing followed by subtyping of M. leprae clearly revealed that either the contacts were infected by the patients or both patients and contacts had the same source of infection. It also revealed that the type of M. leprae in the soil in the inhabited areas where patients resided was also of the same type as that found in patients.

A Borderline Lepromatous (BL) leprosy case was treated with Mercurius solubilis , a homoeopathic medicine for two years. The clinical, immunological and histological assessment of the patient before and after treatment was recorded. The patient showed appreciable improvement in clinical signs and symptoms. Lesions on the skin almost disappeared; no swelling of hand and feet was noted along with disappearance of infiltration on eye brows. Nodular lesions on both the ear lobes diminished remarkably. Regain of sensation to touch, pain and pressure were noted and their test responses were normal. Slit skin smears were negative from all sites with 0 Bacteriological Index [BI] which was positive for acid fast bacilli [AFB] with an average of 4+ BI before treatment. Histologic study revealed absence of granuloma in dermis except the presence of occasional clusters of lymphocytes. Lepromin reaction which was negative before treatment was positive with 14 mm after 2 years of treatment. Estimation of different cytokines after completion of treatment showed appreciable changes with remarkable reduction in the levels of proinflammatory cytokines (IFN and TNF).

Earlier studies indicate that genotyping of Mycobaterium leprae based on single-nucleotide polymorphisms (SNPs) is useful for analysis of the global spread of leprosy. In the present study, we investigated the diversity of M. leprae at eight SNP loci using 180 clinical isolates obtained from patients with leprosy residing mainly in Delhi and Purulia (West Bengal) regions. It was observed that the frequency of SNP type 1 and subtype D was most predominant in the Indian population. Further, the SNP type 2 subtype E was noted only from East Delhi region and SNP type 2 subtype G was noted only from the nearby areas of Hoogly district of West Bengal. These results indicate the occurrence of focal transmission of M. leprae infection and demonstrate that analysis by SNP typing has great potential to help researchers in understanding the transmission of M. leprae infection in the community.

Brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3) and glial cell line derived neurotrophic factor (GDNF) play critical role in growth, differentiation, maintenance and synaptic plasticity in neuronal systems which is more relevant in adolescence. The present study was undertaken to verify the 'neurotrophin hypothesis' in adolescent depression by (i) comparing serum concentrations of neurotrophic factors in depression patients and healthy control, and (ii) analyzing correlations between clinical severity and serum neurotrophin levels.Eighty four adolescent (aged 13-18 years) depressed patients (56 males; 60 medication free/naive) and 64 healthy controls (39 males) were recruited. Severity of depression was measured by Beck's depression inventory, and anxiety by state-trait anxiety inventory. Measurement of serum neurotrophins was done by ELISA.Adolescents with depression had significantly lower levels of BDNF: mean diff. (95% C.I.): 2093.9 (1074.0, 3113.9), NGF: 813.3 (343.1, 1283.6) and GDNF: 158.8 (77.2, 240.4) compared to controls. On gender based analysis female controls had significantly increased trait anxiety scores [-1.1 (-1.8, -0.1)], as compared to control males. In the patient group, female patients had far lower level of NGF: 919.6 (210.9, 1628.3) and NT-3: 1288.8 (145.4, 2432.3) compared to male. BDI-II score showed a statistically significant (p0.01) negative correlation with all four neurotrophins in male patients while in female patients such negative correlation was observed only with NGF and GDNF (p0.01).The study is cross-sectional from a tertiary care hospital.The novelty of the study lies in its large number of exclusively adolescent depression patients showing significant reduction of BDNF, NGF and GDNF serum levels as compared to controls. A gender bias with much reduction in female has also been recorded.

T cell defect is a common feature in lepromatous or borderline lepromatous leprosy (LL/BL) patients in contrast to tuberculoid or borderline tuberculoid type (TT/BT) patients. Tuberculoid leprosy is characterized by strong Th1-type cell response with localized lesions whereas lepromatous leprosy is hallmarked by its selective Mycobacterium leprae specific T cell anergy leading to disseminated and progressive disease. FoxP3+ Regulatory T cells (Treg) which are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases also dampen proinflammatory T cells that include T helper 17 (Th17) cells. This study is aimed at evaluating the role of Treg cells in influencing other effector T cells and its relationship with the cytokine polarized state in leprosy patients. Peripheral blood mononuclear cells from of BT/TT (n = 15) and BL/LL (n = 15) patients were stimulated with M. leprae antigen (WCL) in presence of golgi transport inhibitor monensin for FACS based intracellular cytokine estimation. The frequency of Treg cells showed >5-fold increase in BL/LL in comparison to BT/TT and healthy contacts. These cells produced suppressive cytokine, IL-10 in BL/LL as opposed to BT/TT (p = 0.0200) indicating their suppressive function. The frequency of Th17 cells (CD4, CD45RO, IL-17) was, however, higher in BT/TT. Significant negative correlation (r = -0.68, P = 0.03) was also found between IL-10 of Treg cells and IL-17+ T cells in BL/LL. Blocking IL-10/TGF-β restored the IL-17+ T cells in BL/LL patients. Simultaneously, presence of Th17 related cytokines (TGF-β, IL-6, IL-17 and IL-23) decreased the number of FoxP3+ Treg cells concomitantly increasing IL-17 producing CD4+ cells in lepromatous leprosy. Higher frequency of Programmed Death-1/PD-1+ Treg cells and its ligand, PDL-1 in antigen presenting cells (APCs) was found in BL/LL patients. Inhibition of this pathway led to rescue of IFN-γ and IL-17 producing T cells. Results indicate that Treg cells are largely responsible for the kind of immunosuppression observed in BL/LL patients. This study also proves that Treg cells are profoundly affected by the cytokine milieu and this property may be utilized for benefit of the host., Author Summary Polarized T cell response (Th1/Th2 biased) to Mycobacterium leprae (M. leprae) is believed to be a critical element in the pathogenesis of leprosy and its varied clinical manifestations. However, immune response at the pathologic sites of leprosy is an extremely complex process, particularly in the light of recently evidenced heterogeneity of T cell subsets. FoxP3 positive regulatory T cells (Treg) are one of the most potent hierarchic cell types suppressing the effector T cell function with eventual regulation of immune response elicited by the host during intracellular infections. This study shows the recovery of the cell mediated response by CD4+ T cells by inhibiting the suppressive cytokines, IL-10 and TGF-β and also by blocking of the Programmed Death-1 pathway in cells isolated from lepromatous leprosy patients. Reversal of IL-17 immune response was also achieved by modulating the cytokine milieu of in vitro cell culture and hence provides us cues to counter the M. leprae unresponsiveness in leprosy patients.

Introduction: Mycobacterium gilvum is a fast grower with smooth growth having pale yellow colour. This strain was first isolated from the sediment of the Grand Calumet River in North-western Indiana based on its ability to utilize pyrene, a toxic polycyclic hydrocarbon, as a growth substrate. Methods: The identification and characterization of this isolate was done by various conventional and molecular tests including 16S rDNA sequencing. Sequencing of the nearly complete 16S rRNA gene revealed a unique organism M.glivum, distantly related to M.vaccae group. Blast results showed similarity of these sequences with M.gilvum. Conclusion: Results might shed further light and its association with amoeba in the leprosy endemic area of this rare Mycobacterium species.

Mycobacteria are known to induce autoimmune response in the host. Anti-host keratrin antibodies (AkAbs) might be responsible for the autoimmune phenomena in leprosy patients as majority of leprosy lesions are manifested in the skin and occurrence of keratosis is not an uncommon feature. The aim of this study was to find out the level of AkAbs in leprosy patients across the spectrum and to explore its correlation with the clinical manifestation of the disease. Further, mimicking epitopes of keratin and Mycobacterium leprae components were characterized. We screened 140 leprosy patients (27 BT, 28 BL, 41 LL, 25 T1R, 19 ENL), 74 healthy controls (HC) and 3 psoriasis patients as positive control. Highest AkAbs level was observed in the psoriasis patients followed by T1R, LL, BL, ENL, TT/BT. AkAbs level was significantly (p

Resurgence of mycobacterial diseases particularly tuberculosis has caused a renewed interest to unravel the strategies employed by mycobacteria for intracellular survival. In spite of advancement in mycobacterial research, our knowledge about genes and their corresponding functional proteins involved during the interaction of mycobacterium with host’s macrophages is fragmentary. This study pertains to development of a suitable in vitro model using murine macrophages and Mycobacterium bovis BCG to study proteins expressed during macrophage-myco bacterium interactions. Peritoneal macrophages from BALB/c mice were infected with M. bovis BCG and intracellular replication was assessed by {3H} thymi- dine uptake assay which was maximal when macrophage to mycobacterium ratio was 1:10. SDS-PAGE was employed to study the proteins expressed and selected proteins were subjected to mass spectrometry. Seven proteins found to be upregulated during macrophage-mycobacterium interaction were identified by MALDI-TOF. The results indicate that the present in vitro infection model was able to support the growth of M. bovis BCG in murine macrophages and is an ideal model to determine the pattern of functions of gene expression during the interaction of mycobacterium with macrophages. The differentially expressed proteins will help in understanding the mycobacterial molecular basis of adaptation to intracellular macrophage environment.

Objective: To report HIV and sexually transmitted infection (STI) prevalence and sexual behaviour of long-distance truckers on four national highway routes from a large, cross-sectional, national-level trucker survey in India. Methods: Seven trans-shipment locations covering the bulk of India’s transport volume along four routes, north-west (NW), north-south (NS), north-east (NE) and south-east (SE) were identified as survey sites. A total of 2066 long-distance truckers were selected using a two-stage, time–location cluster sampling approach and, after consent, interviewed about their sexual behaviour. Urine and blood sample were tested for selected STIs. Results: Overall, HIV prevalence among truckers was found to be 4.6%, with prevalence highest on the SE route (6.8%) and lowest on the NS (2.4%). Positive HSV-2 serology, which was tested in a 10% subsample, was low along three routes, 10.0%, 12.8% and 6.7% for the NE, NS and NW, respectively, but 38.7% in the SE. The truckers from the SE were found to be more likely to have sex with paid partners than the NE route. Moreover, truckers who owned their trucks were more likely than those who did not use condoms consistently with paid partners, and truckers who drive trucks owned by their relatives/friends are more likely than others to have any STI. Conclusions: Low self-risk perception for HIV (9.9%), low consistent condom use with non-paid partners (18.6%) and wives (3%), low reported exposure to any interventions (25.6%) and low levels of ever having taken an HIV test (16.5%) make truckers an important bridge population requiring strengthened interventions.

Edwardsiella tarda is an important cause for hemorrhagic septicemia in fish and also for gastro intestinal infections in humans. Sodium lauryl sarcosinate extraction method was used for the isolation of outer membrane proteins (OMPs) from 16 isolates of E. tarda, recovered from snakeheads (Ophiocephalus punctatus). Sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles of OMPs from E. tarda isolates had one major and common protein band of 44 kilodalton (kDa) and 9 other variable protein bands. Rabbit polyclonal antibodies (RPAbs) against OMPs of E. tarda ET-7 detected mainly one common 44 kDa protein in all isolates by western blotting. RPAbs strongly reacted with all isolates of E. tarda by indirect enzyme linked immunosorbent assay (ELISA). These polyclonal antibodies showed no cross reactivity with OMPs or whole cells of other selected aquatic bacteria, i.e., Escherichia coli, Pseudomonas fluorescens, Aeromonas hydrophila, Vibrio cholerae and Flavobacterium ferrugineum. This study indicated that RPAbs against OMPs is useful for detection of E. tarda by ELISA.

We describe here a method, potentially suitable for field applications, for semi-quantitative detection of Mycobacterium leprae antigens in skin scrapings, which are taken normally for smear microscopy. Thirty acid-fast bacilli-negative paucibacillary (PB) leprosy patients comprised the main study group; eight acid-fast bacilli-positive multibacillary (MB) patients and five healthy laboratory workers served as controls. Samples in saline were spotted on nitrocellulose paper and probed with mycobacterium-specific polyclonal or M. leprae-specific mAbs against 12, 35 and 65kDa protein antigens, using a dot-ELISA format. Spot densities were read through a densitometer and also graded visually. The polyclonal antibody produced the best sensitivity, resulting in densitometric detection of mycobacterial antigen in 100% MB, 76% multiple-lesion PB and 62% single-lesion PB patients. None of the healthy volunteers showed antigen positivity. A correlation was noted between the densitometric and visual estimates of the antigen. Determination of antigen in the lesion and an apparently uninvolved area of skin in a subset of PB patients provided clues to the state of the underlying infection. Serological positivity of PB patients for M. leprae-specific antibodies against the 35kDa and phenolic glycolipid-I antigens was too low (20%) for any diagnostic significance.