Presence of viable Mycobacterium leprae in environmental specimens around houses of leprosy patients.

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.

Single nucleotide polymorphism-based molecular typing of M. leprae from multicase families of leprosy patients and their surroundings to understand the transmission of leprosy.

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.

Interleukin-17F single-nucleotide polymorphism (7488T>C) and its association with susceptibility to leprosy.

The objective of this study was to investigate the association, if any, between the interleukin-17F (7488T>C) (rs763780) polymorphism and susceptibility to leprosy and to elucidate the relationship between IL-17F genotypes and clinical profile of the disease. DNA was extracted from the peripheral venous blood of leprosy cases (n = 140), which were classified as per WHO classification into paucibacillary (PB) (n = 53) and multibacillary (MB) (n = 87) categories and healthy controls (n = 84) without any signs and symptoms of leprosy. The IL-17F (7488 T/C) polymorphism was genotyped using amplification refractory mutation system - polymerase chain reaction (Allele-specific amplification). In both PB and MB categories of leprosy cases, the homozygous TT genotype frequency was significantly higher than that of the healthy controls (78.70% vs. 29.76%, P < 0.05). The heterozygous TC genotype was higher in the controls than in the leprosy cases (57.14% vs. 17.68%, P < 0.05). TT genotype was more associated with the type 1 reactional states and tuberculoid/borderline tuberculoid groups in leprosy than the TC genotype. This study reveals that the IL-17F (7488T>C) single-nucleotide polymorphism is associated with susceptibility to leprosy and polymorphism confers decrease in risk of contracting leprosy in the north Indian cohort.

Cortisol and proinflammatory cytokine profiles in type 1 (reversal) reactions of leprosy.

PURPOSE: Cortisol levels in the circulation and at the sites of peripheral inflammation regulate type 1 (Reversal) reactions in leprosy akin to delayed type hypersensitivity reactions (DTH). In this study we determine the extent to which the differential mRNA expression of genes encoding cortisone-cortisol shuttle enzymes (11 ß hydroxysteriod dehydrogenase I & II (11 ß HSD I & II)), circulatory levels of proinflammatory cytokines (IL-6, IL-7, IP-10, IL-17F, IL-23, TNF-α, IL-1ß, PDGF BB and CRP) and cortisol are associated with development of type 1 reactions in leprosy. METHODS: Urine, blood and incisional skin biopsy samples from site of lesions were collected from 49 newly diagnosed untreated leprosy cases in T1R and 51 cases not in reaction (NR). mRNA expression levels of genes encoding 11 ß HSD I & II in skin biopsy samples were determined by realtime PCR. Cortisol levels from the lesional skin biopsies, serum and urine samples and serum proinflammatory cytokine levels were measured using ELISA. RESULTS: The mean expression ratios of 11 ß HSD I & II are significantly lower in leprosy cases with T1R when compared to the NR leprosy cases. Cortisol levels in lesional skin biopsies and in urine are significantly lower (p=0.001) in leprosy cases with T1R. Serum cytokine levels of IP-10, IL-17F, IL-IL-6 and TNF-α are significantly higher (p<0.05) in leprosy cases with T1R when compared the NR leprosy cases. CONCLUSION: Our study indicated an association of urinary and lesional skin cortisol levels with the manifestation of T1R in leprosy. IP-10, IL-17F, IL-6 and TNF-α can be potential prognostic serological markers and gene expression markers for early detection of type 1 reactions in leprosy.

Cohort study of the seasonal effect on nasal carriage and the presence of Mycobacterium leprae in an endemic area in the general population.

Leprosy continues to be a significant health problem in certain pockets in developing countries. Better understanding of the transmission and source of the infection would help to decipher the transmission link, leading to control of the spread of the disease. The nose is considered to be a portal of entry, suggesting an aerial route for transmission through droplet infection. The evidence suggests that many individuals from endemic countries carry Mycobacterium leprae in their nasal cavities without having obvious symptoms of leprosy. The objective of the present study was to assess the presence of M. leprae on the nasal mucosa in the general population from a leprosy-endemic pocket. M. leprae detection was carried out using PCR targeting RLEP. Four hundred subjects from an area highly endemic for leprosy were included in the study and followed up during three different seasons--winter, summer, and monsoon--for evidence of nasal exposure to M. leprae. PCR positivity for M. leprae was observed in 29%, 21% and 31% of the samples collected in winter, summer and the monsoon season, respectively. Twenty-six individuals from the cohort showed amplification for M. leprae for all seasons. Our results are consistent with reports in the literature showing widespread exposure to M. leprae in the endemic community. The results also suggest possible association of the environmental conditions (climate) with the transmission pattern and levels of exposure to M. leprae. However, the present study indicated that the population from highly endemic pockets will have exposure to M. leprae irrespective of season.

Single nucleotide polymorphisms typing of Mycobacterium leprae reveals focal transmission of leprosy in high endemic regions of India.

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.

Extraction and detection of Mycobacterium leprae DNA from ZNCF-stained skin smear slides for better identification of negative skin smears.

Abstract Identification of Mycobacterium leprae, which causes leprosy, is done by Ziehl Neelsen Carbol Fuchsin (ZNCF) stained slit skin smear microscopy that aids in the diagnosis and quantification of approximate bacterial load carried by the patient. We attempted M. leprae DNA extraction from 46 stained slit skin smear negative slides, using Proteinase K and SDS lysis, followed by ethanol precipitation. M. leprae specific primers (16SrRNA) were used for PCR-based amplification of DNA. We could detect M. leprae DNA in 15 (32.6%) samples. The method can be useful in the diagnosis of apparently slit skin smear negative leprosy cases.

Study of cytokine response against panel of purified Mycobacterium leprae antigens by using whole blood assay in subjects residing in a resettlement village of cured leprosy patients.

Mycobacterium leprae being an intracellular pathogen, cell mediated immunity is very important in the clinical outcome of leprosy. Manifestation of the disease is correlated with the level and type of cell mediated immune response. The main objective of this study was to analyse TNF-alpha and IFN-gamma production by T-cells when challenged with different M. leprae purified antigens in subjects with known exposure. 50 subjects residing in resettlement village of cured leprosy patients were included in the study. Whole Blood assay studies were undertaken in which the blood was placed in culture and was challenged with 35-kDa antigen, whole M. leprae cells, M. leprae cell wall antigen and M. leprae soluble antigen minus LAM. T-cell derived cytokines TNF-alpha and IFN-gamma were measured by ELISA. It was observed that challenging the lymphocytes with 35-kDa antigen, the cell wall antigen and M. leprae soluble antigen minus LAM resulted in increased levels of IFN-gamma whereas challenge with 35-kDa antigen and M. leprae cell wall antigen resulted in increased levels of TNF-alpha.

Study of cross-reactivity of Mycobacterium leprae reactive salivary IgA with other environmental mycobacteria.

Majority of the endemic population is exposed to Mycobacterium leprae but very few develop disease. Humoral mucosal immune response mediated through M. leprae reactive salivary antibodies has been suggested to be quite important in the protective immunity. As the endemic population is also exposed to many environmental mycobacteria, we tested saliva from 121 subjects for the cross-reactivity of the M. leprae reactive salivary antibodies to mycobacteria like M. smegmatis and M. phlei. Saliva samples were treated with these two mycobacteria prior to testing M. leprae reactive antibodies by ELISA. In 59 subjects (48.76%), original and cross-reacted saliva showed same absorbance values suggesting no cross-reactivity. 26 subjects (21.49%) showed less than 25% drop in the OD values whereas 21 subjects (17.4%) showed 25% to 50% drop after reacting saliva with the mycobacteria. 15 subjects (12.4%) showed more that 50% drop in OD. The data suggest that though in half of subjects antibodies did not cross-react with mycobacteria tested, there were subjects where antibodies showed cross-reactivity to mycobacteria suggesting that positive salivary M. leprae reactive IgA response could be to some extent due to exposure to environmental mycobacteria and it could also be protective against M. leprae.

Genotypic analysis of Mycobacterium leprae strains from different regions of India on the basis of rpoT.

Mycobacterium leprae strains from Indian leprosy patients were analyzed using the six base tandem repeat, GACATC, in rpoT gene as genetic marker. DNA was extracted from slit-skin smears and nasal swabs of new untreated as well as treated leprosy patients living in different regions of India. PCR amplification of rpoT gene and sequencing of amplicons showed the presence of two genotype of M. leprae in this study, 73.4% having three copies (ancient Indian type) and 26.6% contain 4 copies (considered to be Japanese and Korean). These genotypes along with other short tandem repeats may help in studying the historical spread of disease and the strains of M. leprae disseminated by various human races that migrated to India from other places of Asia and European countries during our history.

Detection of Mycobacterium leprae DNA from soil samples by PCR targeting RLEP sequences.

Despite near elimination of leprosy as a public health problem, several problems in leprosy still remain. These include early detection, determining efficacy of the treatment and differentiating relapses from re-infection. These aspects have important impact on the patients undergoing treatment and also have a bearing on understanding transmission dynamics in the community. While early diagnosis and management do not need major technological inputs, various reports have suggested that M. leprae is found in the environment and may have a role in continued transmission of disease. In earlier studies from other parts of world the presence of M. leprae DNA in the environment has been investigated both by microbiological and molecular studies. In the present study, an attempt was made to extract M. leprae DNA from soil samples, which were collected from eighteen different locations including 3 from our Institute area and 15 from different villages of Ghatampur area. We optimized a protocol for the extraction of DNA and amplified a fragment of M. leprae using specific primers targeting RLEP sequences. It was found that 33.3% of these soil samples collected from areas inhabited by leprosy cases gave positive result for M. leprae specific DNA. The utility of this method needs to be explored on a larger scale to establish the presence of M.leprae in the environment, and its role in the spread of the disease.

Use of reverse transcription polymerase chain reaction for the detection of Mycobacterium leprae in the slit-skin smears of leprosy patients.

The relevance of bacterial index (BI) for understanding the prognosis of leprosy patients on treatment has been extensively debated, as it does not give a very clear idea of the viability of the bacteria in patients under treatment. Here we used slit-skin smear samples of leprosy patients to test the suitability for studying viability of Mycobacterium leprae using reverse transcription polymerase chain reaction (RT-PCR). For this purpose, we recruited 13 multibacillary (MB) leprosy patients (8 lepromatous and 5 borderline lepromatous). Of these, 7 were relapse cases, 3 were under treatment (MB-MDT), 2 were new cases and 1 had completed treatment. We carried out extraction of RNA using Trizol reagent (Life Technologies, UK) from the slit-skin smear samples from these patients. The RNA preparation was then used for the RT-PCR using Mycobacterium leprae-specific primers for the fragment of 16s ribosomal RNA gene. Samples from both the new cases, 4 suspected relapse cases and 1 patient under treatment showed positive RT-PCR results. Other 6 patients whose smear samples did not show any amplification by RT-PCR were on MB-MDT from 8 to 30 months. The usefulness of the technique needs to be validated using mouse footpad technique and also should be more extensively explored for studying the viability of M. leprae, the efficacy of treatment and the presence of other mycobacteria in the slit-skin smear samples.

Simplified PCR detection method for nasal Mycobacterium leprae.

We report here a simplified method for the detection of nasal carriage of Mycobacterium leprae. DNA extracted from nasal swabs was analyzed by PCR, and M. leprae specific amplicons detected by means of a novel peptide-nucleic-acid-ELISA (PNA-ELISA) method. Parameters for the method were established using swabs taken from untreated lepromatous leprosy patients. We have developed this method to study nasal carriage in endemic populations. However, due to the sensitivity of PCR based techniques, we wished to assess the possibility of false positive samples arising in our method. We therefore examined samples taken from individuals in Norway, a country non-endemic for leprosy, using our technique. A total of 219 nasal swabs were collected and tested in our laboratory in London. All of these were found to be negative by our criteria. In order to corroborate our results, and also to assess the specificity of the method, a small number of these samples were randomly selected, and a known amount of M. leprae DNA added to them. All 219 samples were then retested using the same techniques under "double blind" conditions in our laboratory in India. All of the samples to which M. leprae DNA had been added were successfully identified by this method whereas all other swabs were negative. Taken together, these results suggest that the technique described here is simple, sensitive, and specific for use in large-scale epidemiological studies. This study, part of the larger MILEP 2 study, represents the first use of a PNA-PCR method for an epidemiological study of infection. The method using PNA-ELISA is significantly simpler and more rapid than gel based detection methods. The supply of laboratory consumables and overall detection procedure were simplified and standardized by use of PCR Ready-to-Go beads.