Cost-effectiveness of incorporating Mycobacterium indicus pranii vaccine to multidrug therapy in newly diagnosed leprosy cases for better treatment outcomes & immunoprophylaxis in contacts as leprosy control measures for National Leprosy Eradication Programme in India.

Background & objectives: The elimination goal for leprosy as a public health problem at the national level was achieved in 2005 in India. However, the number of new cases reporting annually remained nearly the same during the last 10-15 years. Moreover, a substantial number of these new cases reported disabilities for the first time. Therefore, besides multidrug therapy (MDT), newer strategies with focus on effectively decreasing the number of new cases, optimizing the treatment of detected cases, averting disabilities and arresting the transmission of the disease are required. So the objective of this study was to assess the cost-effectiveness of Mycobacterium indicus pranii (MIP) vaccine implementation in National Leprosy Eradication Programme (NLEP) for newly diagnosed leprosy patients as well as their contacts to arrest/decrease the transmission and occurrence of new cases. Methods: This was a model-based estimation of incremental costs, total quality-adjusted life years (QALYs) gained, new cases averted, deaths averted, incremental cost-effectiveness ratio (ICER) and budget impact of the vaccination intervention. This model included the addition of MIP treatment intervention to the newly detected leprosy patients as well as vaccination with MIP to their contacts. Results: Using the societal perspective, discounted ICER was estimated to be ₹73,790 per QALY gained over a five-year time period. Probabilistic sensitivity analysis (PSA) was assessed by varying the values of input parameters. Majority (96%) of simulations fell in North East quadrant of cost-effectiveness plane, which were all below the willingness to pay threshold. Interpretation & conclusions: Introduction of MIP vaccination in the NLEP appears to be a cost-effective strategy for India. Significant health gains were reduction in the number of new leprosy cases, decreased incidence and severity of reactions during treatment, and after release from treatment, prevention of disabilities, thus reducing the cost as well as stigma of the disease.

Autoimmunity to Tropomyosin-Specific Peptides Induced by Mycobacterium leprae in Leprosy Patients: Identification of Mimicking Proteins.

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 < 0.05) with myosin in all types of leprosy patients compared to HC. Further, hyperimmunization of inbred BALB/c strain of female mice and rabbit with MLSA revealed that both hyperimmunized rabbit and mice evoked heightened levels of antibodies against myosin and this autoimmune response could be adoptively transferred from hyperimmunized to naïve mice. Tropomyosin was found to be mimicking with ATP-dependent Clp protease ATP-binding subunit of M. leprae. We found four mimicking epitopes between these sequences. Conclusion: These data suggest that these mimicking proteins tropomyosin and ATP-dependent Clp protease ATP-binding subunit of M. leprae or more precisely mimicking epitopes (four B cell epitopes) might be responsible for extensive tissue damage during type1 reaction in leprosy.

Development of a potent invigorator of immune responses endowed with both preventive and therapeutic properties.

This article reviews briefly the making of an immunoprophylactic-cum-immunotherapeutic vaccine against leprosy. The vaccine is based on cultivable, heat-killed atypical mycobacteria, whose gene sequence is now known. It has been named Mycobacterium indicus pranii. It has received the approval of the Drug Controller General of India and the US Food and Drug Administration. Besides leprosy, M. indicus pranii has found utility in the treatment of category II ("difficult to treat") tuberculosis. It also heals ugly anogenital warts. It has preventive and therapeutic action against SP2/O myelomas. It is proving to be a potent adjuvant for enhancing antibody titers of a recombinant vaccine against human chorionic gonadotropin, with the potential of preventing pregnancy without derangement of ovulation and menstrual regularity in sexually active women.

National sample survey to assess the new case disease burden of leprosy in India.

A national sample survey of leprosy was undertaken in partnership with Indian Council of Medical Research (ICMR) institutions, National Leprosy Eradication Programme (NLEP), Panchayati Raj members, and treated leprosy patients to detect new cases of leprosy in India. The objectives of the survey were to estimate the new leprosy case load; record both Grade 1 and Grade 2 disabilities in the new cases; and to assess the magnitude of stigma and discrimination prevalent in the society. A cluster based, cross-sectional survey involving all States was used for the door-to-door survey using inverse sampling methodology. Rural and urban clusters were sampled separately. The population screened for detecting 28 new cases in rural and 30 in urban clusters was enumerated, recorded and analyzed. Data capture and analysis in different schedules were the main tools used. For quality control three tiers of experts were utilized for the confirmation of cases and disabilities. Self-stigma was assessed in more than half of the total new patients detected with disabilities by the approved questionnaire. A different questionnaire was used to assess the stigma in the community. A population of 14,725,525 (10,302,443 rural; 4,423,082 urban) was screened and 2161 new cases - 1300 paucibacillary (PB) and 861 multibacillary (MB) were detected. New case estimates for leprosy was 330,346 (95% Confidence limits, 287,445-380,851). Disabilities observed in these cases were 2.05/100,000 population and 13.9 per cent (302/2161) in new cases. Self-stigma in patients with disabilities was reduced, and the patients were well accepted by the spouse, neighbour, at workplace and in social functions.

Viability of Mycobacterium leprae in the environment and its role in leprosy dissemination.

BACKGROUND: Leprosy, a chronic disease caused by Mycobacterium leprae, is a public health concern in certain countries, including India. Although the prevalence of the disease has fallen drastically over time, new cases continue to occur at nearly the same rate in many regions. Several endemic pockets have been observed in India and elsewhere. The precise dynamics of leprosy transmission are still not clearly understood. Both live bacilli as well as M. leprae DNA have been detected in the soil and water of endemic areas; they possibly play an important role in disease transmission. AIMS: To study the occurrence of viable M. leprae in environmental samples collected from areas of residence of patients with active leprosy. METHODS: The study was conducted on 169 newly diagnosed leprosy patients in Ghatampur, Uttar Pradesh, India. Soil and water samples were collected from their areas of residence using a standardized protocol. An equal number of soil and water samples were also collected from non-patient areas of the same or adjoining villages. The environmental samples collected from the patients surroundings were subjected to 16S ribosomal RNA gene analysis after obtaining informed consent. RESULTS: About a quarter of the environmental samples collected from patient areas, (25.4% of soil samples and 24.2% of water samples) were found to be positive for specific 16S ribosomal RNA genes of M. leprae. Environmental samples collected from non-patient areas were all found negative for M. leprae 16S ribosomal RNA genes. LIMITATIONS: The major limitation of the study was that the sample size was small. CONCLUSION: The study demonstrated the presence of viable strains of M. leprae in skin smear samples of paucibacillary patients and multibacillary patients, as well as in the environmental samples obtained from around their houses. This could play an important role in the continued transmission of leprosy.

TlyA protein of Mycobacterium leprae: a probable bio-marker of active infection.

The extent of pathogenicity of the mycobacterial infections depends on virulence factors that mediate survival inside macrophages. Virulence factors are generally believed to be specific for pathogenic species and mutated/non-functional in nonpathogenic strains. Mycobacterial TlyA can modulate the phagolysosome maturation pathway, immediately after entry into macrophages. Over-expression of open reading frame (ORF) ML1358 (tlyA) in tissues of leprosy patients by partial DNA chip and real time PCR analysis during active infection attracted our interest to explore the properties of this gene at molecular and serological levels, to understand its role in the host. Molecular properties were studied by cloning and expression of the corresponding gene in pASK-iba 43(þ) expression vector in E. coli and bioinformatics tools while sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA were applied to investigate the serological significance of rTlyA protein in different clinical states of leprosy. We observed that TlyA has a close relation among mycobacteria with specific protein domains in slow growing intracellular adapted pathogenic species. The presence of trans-membrane domains indicates its association to the cell membrane. The study revealed its highly significant sero-reactivity (P value , 0·001) in borderline lepromatous (BL) patients, and those with reversal reaction (RR) and erythema nodosum leprosum (ENL). Its role in active infection, association with the cell membrane, presence in pathogenic species and high sero-reactivity, suggested the tlyA gene as a strong disease progression marker.

International open trial of uniform multidrug therapy regimen for leprosy patients: Findings & implications for national leprosy programmes.

BACKGROUND & OBJECTIVES: Uniform therapy for all leprosy patients will simplify leprosy treatment. In this context, we evaluated six-month multidrug therapy (MDT) currently recommended for multibacillary (MB) patients as uniform MDT (U-MDT) in a single-arm open trial under programme conditions. Primary objective was to determine efficacy to prevent five-year cumulative five per cent relapse. Secondary objectives were to assess acceptability, safety and compliance. METHODS: Newly detected, treatment-naive leprosy patients were enrolled in India (six sites) and P. R. China (two sites). Primary outcome was clinically confirmed relapse of occurrence of one or more new skin patches consistent with leprosy, without evidence of reactions post-treatment. Event rates per 100 person years as well as five-year cumulative risk of relapse, were calculated. RESULTS: A total of 2091 paucibacillary (PB) and 1298 MB leprosy patients were recruited from the 3437 patients screened. Among PB, two relapsed (rate=0.023; risk=0.11%), eight had suspected adverse drug reactions (ADRs) (rate=0.79) and rate of new lesions due toreactions was 0.24 (n=23). Rates of neuritis, type 1 and type 2 reactions were 0.39 (n=37), 0.54 (n=51) and 0.03 (n=3), respectively. Among MB, four relapsed (rate=0.07; risk=0.37%) and 16 had suspected ADR (rate=2.64). Rate of new lesions due to reactions among MB was 1.34 (n=76) and rates of neuritis, type 1 and type 2 reactions were 1.37 (n=78), 2.01 (n=114) and 0.49 (n=28), respectively. Compliance to U-MDT was 99 per cent. Skin pigmentation due to clofazimine was of short duration and acceptable. INTERPRETATION & CONCLUSIONS: We observed low relapse, minimal ADR and other adverse clinical events. Clofazimine-related pigmentation was acceptable. Evidence supports introduction of U-MDT in national leprosy programmes. [CTRI No: 2012/ 05/ 002696].

Molecular mimicry between Mycobacterium leprae proteins (50S ribosomal protein L2 and Lysyl-tRNA synthetase) and myelin basic protein: a possible mechanism of nerve damage in leprosy.

Autoantibodies against various components of host are known to occur in leprosy. Nerve damage is the primary cause of disability associated with leprosy. The aim of this study was to detect the level of autoantibodies and lympho-proliferative response against myelin basic protein (MBP) in leprosy patients (LPs) and their correlation with clinical phenotypes of LPs. Further, probable role of molecular mimicry in nerve damage of LPs was investigated. We observed significantly high level of anti-MBP antibodies in LPs across the spectrum and a positive significant correlation between the level of anti-MBP antibodies and the number of nerves involved in LPs. We report here that 4 B cell epitopes of myelin A1 and Mycobacterium leprae proteins, 50S ribosomal L2 and lysyl tRNA synthetase are cross-reactive. Further, M. leprae sonicated antigen hyperimmunization was responsible for induction of autoantibody response in mice which could be adoptively transferred to naive mice. For the first time our findings suggest the role of molecular mimicry in nerve damage in leprosy.

CD4+, CD8+, CD3+ cell counts and CD4+/CD8+ ratio among patients with mycobacterial diseases (leprosy, tuberculosis), HIV infections, and normal healthy adults: a comparative analysis of studies in different regions of India.

In this study, we estimated the CD4+, CD8+, CD3+ cell counts and the CD4/CD8 ratio among normal healthy controls (adults and children), leprosy patients (without any complications and during reactional states), TB patients (with and without HIV), and HIV-positive patients (early infection and full-blown AIDS) and correlated the changes with disease progression. In our study, it was observed that among adults, CD4+ cell counts ranged from 518-1098, CD8+ from 312-952, whereas CD4/CD8 ratio from 0.75-2.30. Among children, both CD4+ and CD8+ cells were more and the CD4/CD8 ratio varied from 0.91-3.17. With regard to leprosy patients, we observed that CD4+ and CD8+ cell counts were lower among PB (pauci-bacillary) and MB (multi-bacillary) patients. CD4/CD8 ratio was 0.99 ± 0.28 among PB patients while the ratio was lower, 0.78 ± 0.20, among MB patients. CD4+ cell counts were raised during RR (reversal reactions) and ENL (erythema nodosum leprosum) among the PB and MB patients whereas the CD8+ cell counts were lower among PB and MB patients. CD4/CD8 ratio doubled during reactional episodes of RR and ENL. Among the HIV-negative tuberculosis (TB) patients, both the CD4+ and CD8+ cell counts were found to be less and the CD4/CD8 ratio varied between 0.53-1.75. Among the HIV-positive TB patients and HIV-positive patients, both the CD4+ and CD8+ cells were very less and ratio drops significantly. In the initial stages of infection, as CD4+ counts drop, an increase in the CD8+ cell counts was observed and the ratio declines. In full-blown cases, CD4+ cell counts were very low, 3-4 to 54 cells, CD8+ cells from 12-211 and the ratio drops too low. This study is the first of its kind in this region of the country and assumes importance since no other study has reported the values of CD4+ and CD8+ T-lymphocyte counts among patients with mycobacterial diseases (leprosy and TB), HIV infections along with normal healthy individuals of the region, and correlation with clinical presentations of patients.

Linkage disequilibrium pattern and age-at-diagnosis are critical for replicating genetic associations across ethnic groups in leprosy.

One of the persistent challenges of genetic association studies is the replication of genetic marker-disease associations across ethnic groups. Here, we conducted high-density association mapping of PARK2/PACRG SNPs with leprosy and identified 69 SNPs significantly associated with leprosy in 198 single-case Vietnamese leprosy families. A total of 56 associated SNPs localized to the overlapping promoter regions of PARK2/PACRG. For this region, multivariate analysis identified four SNPs belonging to two major SNP bins (rs1333955, rs7744433) and two single SNP bins (rs2023004, rs6936895) that capture the combined statistical evidence (P = 1.1 × 10(-5)) for association among Vietnamese patients. Next, we enrolled a case-control sample of 364 leprosy cases and 370 controls from Northern India. We genotyped all subjects for 149 SNPs that capture >80 % of the genetic variation in the Vietnamese sample and found 24 SNPs significantly associated with leprosy. Multivariate analysis identified three SNPs (rs1333955, rs9356058 and rs2023004) that capture the association with leprosy (P < 10(-8)). Hence, two SNPs (rs1333955 and rs2023004) were replicated by multivariate analysis between both ethnic groups. Marked differences in the linkage disequilibrium pattern explained some of the differences in univariate analysis between the two ethnic groups. In addition, the strength of association for two promoter region SNP bins was significantly stronger among young leprosy patients in the Vietnamese sample. The same trend was observed in the Indian sample, but due to the higher age-at-diagnosis of the patients the age effect was less pronounced.

Expression of CXCL10 (IP-10) and CXCL11 (I-TAC) chemokines during Mycobacterium tuberculosis infection and immunoprophylaxis with Mycobacterium indicus pranii (Mw) in guinea pig.

Mycobacterium indicus pranii (earlier known as Mycobacterium w) has been used as an immunmodulatory agent in leprosy and tuberculosis by mediating the release of various cytokines and chemokines. CXCL10 (IP-10) and CXCL11 (I-TAC) chemokines are involved in T-cell migration and stimulation of natural killer cells in Mycobacterium tuberculosis infection. In this study, the effect of heat killed M. indicus pranii (alone and in conjunction with chemotherapy) on disease progression was determined by colony forming units (CFUs) in guinea pig lung following their aerosol infection and the expression levels of CXCL10 and CXCL11 were studied by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) and in situ RT-PCR. Four groups of animals included; infection only (Rv), immunoprophylaxis (RvMw), chemotherapy (RvCh) and combination of immunoprophylaxis with chemotherapy (RvChMw). In the group where immunoprophylaxis was given in combination with chemotherapy, the CFU counts reduced significantly at 4th week post-infection as compared to animals that received immunoprophylaxis or chemotherapy alone. At the same time, all groups of animals had elevated expression of CXCL 10 which was significantly high only in animals that received Mw with or without chemotherapy. Unlike to CXCL 10, study demonstrated suppressed expression CXCL 11 in both immunoprophylaxis as well as chemotherapy groups that became up-regulated in synergistic response of immunoprophylaxis and chemotherapy. Taken together, data indicates that the expression of CXCL10 and CXCL11 positively correlates with anti-tubercular treatment (at least with combination of immunoprophylaxis and chemotherapy). Therefore, prior immunization with Mw appears to be a good immunomodulator for release of chemokines and augments the effect of chemotherapy.

Molecular mimicry between HSP 65 of Mycobacterium leprae and cytokeratin 10 of the host keratin; role in pathogenesis of leprosy.

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<0.05) higher in all the groups of leprosy patients except TT/BT in comparison to HC. Significant positive correlation was found between number of lesions and level of AkAbs in leprosy patients. Highest lympho-proliferation for keratin protein was observed in T1R, followed by BL/LL, TT/BT, ENL. Lympho-proliferation was significantly (p<0.05) higher in all groups of leprosy patients except ENL in comparison to HC. Interestingly, it was noted that hyperimmunization of inbred strains of female BALB/c mice and rabbit with M. leprae soluble antigen (MLSA) induce higher level of AkAbs. The percentage of FoxP3(+) expressing Treg cells (total CD4(+)CD25(+)FoxP3(+) andCD4(+)CD25(+hi)FoxP3(+)) in splenocytes and lymph nodes of hyperimmunized mice were declined in comparison to control mice. Further, it was found that this autoimmune response can be adoptively transferred in naïve mice by splenocytes and lymph node cells as well as T cells. Comparative molecular characterization between keratin and MLSA noted a cross-reactivity/similarity between these two antigens. The cross-reactive protein of keratin was found to be in molecular weight range ≈74-51kDa and at pI 4.5 while the cross-reactive protein of MLSA was found to be in molecular weight ≈65kDa and at pI 4-4.5. Cross-reactive protein of keratin and MLSA was identified and characterized by MALDI-TOF/TOF analysis and Mascot software. It was found that the keratin (host protein) which reacted with anti-M. leprae sera is cytokeratin-10 and MLSA which reacted with anti-keratin sera is heat shock protein 65 (HSP 65). Seven B-cell epitopes of cytokeratin-10 and HSP 65 was found to be similar by multiple sequence alignment using ClustalW server and out of which 6 B-cell epitopes were found to be on the surface of HSP 65. In conclusion, our study provides evidence for the existence of molecular mimicry between cytokeratin-10 of keratin (host protein) and 65kDa HSP (groEL2) of M. leprae. Presence of heightened CMI response of leprosy patients to keratin and positive correlation of AkAbs level with number of lesions of leprosy patients showed the clinical evidence for its role in the pathogenesis in leprosy.

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

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

Human leukocyte antigen class I region single-nucleotide polymorphisms are associated with leprosy susceptibility in Vietnam and India.

Experimental evidence suggested the existence of unidentified leprosy susceptibility loci in the human leukocyte antigen (HLA) complex. To identify such genetic risk factors, a high-density association scan of a 1.9-mega-base (Mb) region in the HLA complex was performed. Among 682 single-nucleotide polymorphisms (SNPs), 59 were associated with leprosy (P <.01) in 198 Vietnamese single-case leprosy families. Genotyping of these SNPs in an independent sample of 292 Vietnamese single-case leprosy families replicated the association of 12 SNPs (P <.01). Multivariate analysis of these 12 SNPs showed that the association information could be captured by 2 intergenic HLA class I region SNPs (P = 9.4 × 10⁻9)-rs2394885 and rs2922997 (marginal multivariate P = 2.1 × 10⁻7 and P = .0016, respectively). SNP rs2394885 tagged the HLA-C*15:05 allele in the Vietnamese population. The identical associations were validated in a third sample of 364 patients with leprosy and 371 control subjects from North India. These results implicated class I alleles in leprosy pathogenesis.

Induction of lepromin reactivity in cured lepromatous leprosy patients: impaired chemokine response dissociates protective immunity from delayed type hypersensitivity.

Delayed Type Hypersensitivity (DTH) and protective immunity are thought to be tightly linked. Remarkable similarity exists between their cellular and immune mechanisms. However, their dissociation is also well known. Here we investigate the immunological mechanisms relevant for their dissociation in a group of non-relapsing cured lepromatous leprosy (CLL) patients. In these patients, using lepromin reaction as a model system of DTH we report critical role of tissue chemokine response in synchronous manifestation of these linked phenomena. Results indicate elevation of the threshold of tissue chemokine induction thus dissociating DTH from protective immunity in lepromin -ive CLL patients. We also show that the DTH anergy in these subjects is not an absolute one but depends on the strength of the stimulus. Our data provide insights into the intricate relationship between DTH and immunity and highlight the persistent presence of effector immune mechanisms involving these two pathways in apparently unresponsive lepromatous leprosy patients.

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

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

Serum beta-glucuronidase levels in children with leprosy.

A study was undertaken to find out the usefulness of determining the circulating levels of beta-glucuronidase, a lysosomal enzyme in leprosy affected children of less than 15 years of age. The serum enzyme levels were significantly higher in BB/BL patients compared to healthy control children as well as children with skin diseases other than leprosy. Treatment with Multidrug regimen advocated by WHO for multi/paucibacillary leprosy resulted in a significant fall in the serum enzyme levels in BB and BL cases. The findings suggest that serum beta-glucuronidase may be a useful parameter for the activity and extent of pathogenesis in leprosy.

Serum samples from patients with mycobacterial infections cross-react with HIV structural proteins Gp41, p55 and p18.

BACKGROUND: Infection with Mycobacterium leprae is associated with a high frequency of false positive results in a variety of serological assays. Our studies have found cross-reactivity to HIV structural proteins in serum samples from leprosy patients, irrespective of the type of disease, treatment duration, age and gender and from a few patients with active TB disease. METHODS: Western blot (WB) analysis revealed that sera from HIV negative leprosy patients across the spectrum showed high reactivity with p18, Gp41 and p55 and lower reactivity with other HIV proteins. The reactivity appeared to be specific; western blot-positive samples were negative in ELISA and in several rapid tests for HIV. Cross-reactivity was not found in sera from patients with leishmaniasis or from normal healthy individuals. RESULTS: None of the WB reactive leprosy patients seroconverted to HIV positivity within 6 months to 1 year after Western blot testing. BLAST analysis revealed that envelope antigens of HIV (Gp41, Gp120 and Gp160) contained amino acid sequences similar to M. leprae ML0470, putative integral membrane protein, Rv0740, mmpL9 (M. tuberculosis). Core (gag) antigens (p18) had similarities to ML0406, but polymerase antigens (p52) had similarities to PE_PGRS (M. tuberculosis, H37Rv). Nucleotide sequence analysis, on the other hand, did not reveal any significant homology between M. leprae or M. tuberculosis and HIV. CONCLUSIONS: The occurrence of these high false-positive rates in M. leprae-infected individuals suggests a possible complication of serodiagnosis of HIV in regions where mycobacterial infections are endemic. There is a need for caution in reporting HIV infection among leprosy patients. Our observations emphasise the value of the various rapid assay kits for HIV, where this false positivity is not observed.

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

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

Leprosy-specific B-cells within cellular infiltrates in active leprosy lesions.

Leprosy is a spectral disease with polar lepromatous and tuberculoid forms correlating with enhanced humoral and cell-mediated immunity, respectively, against Mycobacterium leprae and the borderline forms, borderline lepromatous, midborderline, and borderline tuberculoid showing in-between clinical and immunological characteristics. Histopathologically, the cellular infiltrates of leprosy lesions show predominantly the presence of interacting T-cells and antigen presenting cells like macrophages, whereas the presence of B-cells has only been sporadically reported. The present study demonstrates by immunohistochemical techniques the presence of B-cells, including plasma cells, in active lesions from lepromatous leprosy, skin smear negative borderline lepromatous, and paucibacillary borderline tuberculoid leprosy. Furthermore, the study demonstrates the in situ production of M leprae-specific antibodies from BT lesions using an organotypic skin explant culture model. Finally, analysis of the cytokine release profile in supernatants of lesional organotypic skin cultures showed a microenvironment conducive to the differentiation and maturation of B-cells. The results demonstrate the presence of different functionally active B-cell stages within lesions of patients with leprosy, including borderline tuberculoid patients, which could secrete anti-M leprae-specific antibodies. However, their role in leprosy pathology remains to be elucidated.