Subclinical Infection in Leprosy

Lymphocyte transformation has been used to study the immune response to Mycobacterium leprae among contacts and non-contacts of leprosy patients. Of 26 subjects living in a leprosy endemic area for less than two months none responded to M. leprae; 24% of subjects who had lived in an endemic area for more than a year gave a positive response to M. leprae; more than 50% of individuals with occupational contact of leprosy for more than a year responded; and about 50% of contacts of tuberculoid and treated lepromatous patients responded to M. leprae, while only 22% (4/18) of contacts of lepromatous patients treated for less than six months responded.It seems that leprosy is more highly infectious than is indicated by the prevalence of the disease and that a subclinical infection commonly follows exposure to M. leprae. The relatively low response found in contacts of active lepromatous patients suggests that in these contacts “superexposure” to M. leprae can bring about a decrease in host resistance.     

New biomarkers with relevance to leprosy diagnosis applicable in areas hyperendemic for leprosy

Leprosy is not eradicable with currently available diagnostics or interventions, as evidenced by its stable incidence. Early diagnosis of Mycobacterium leprae infection should therefore be emphasized in leprosy research. It remains challenging to develop tests based on immunological biomarkers that distinguish individuals controlling bacterial replication from those developing disease. To identify biomarkers for field-applicable diagnostics, we determined cytokines/chemokines induced by M. leprae proteins in blood of leprosy patients and endemic controls (EC) from high leprosy-prevalence areas (Bangladesh, Brazil, Ethiopia) and from South Korea, where leprosy is not endemic anymore. M. leprae-sonicate-induced IFN-γ was similar for all groups, excluding M. leprae/IFN-γ as a diagnostic readout. By contrast, ML2478 and ML0840 induced high IFN-γ concentrations in Bangladeshi EC, which were completely absent for South Korean controls. Importantly, ML2478/IFN-γ could indicate distinct degrees of M. leprae exposure, and thereby the risk of infection and transmission, in different parts of Brazilian and Ethiopian cities. Notwithstanding these discriminatory responses, M. leprae proteins did not distinguish patients from EC in one leprosy-endemic area based on IFN-γ. Analyses of additional cytokines/chemokines showed that M. leprae and ML2478 induced significantly higher concentrations of MCP-1, MIP-1β, and IL-1β in patients compared with EC, whereas IFN-inducible protein-10, like IFN-γ, differed between EC from areas with dissimilar leprosy prevalence. This study identifies M. leprae-unique Ags, particularly ML2478, as biomarker tools to measure M. leprae exposure using IFN-γ or IFN-inducible protein-10, and also shows that MCP-1, MIP-1β, and IL-1β can potentially distinguish pathogenic immune responses from those induced during asymptomatic exposure to M. leprae.     

LSHGD: A database for human leprosy susceptible genes

Studies aiming to explore the involvement of host genetic factors to determine susceptibility to develop disease and individual's response to the infection with Mycobacterium leprae have increased in recent years. To address this issue, we have developed a Leprosy Susceptible Human Gene Database (LSHGD) to integrate leprosy and human associated 45 genes by profound literature search. This will serve as a user-friendly and interactive platform to understand the involvement of human polymorphisms (SNPs) in leprosy, independent genetic control over both susceptibility to leprosy and its association with multi-drug resistance of M. leprae. As the first human genetic database in leprosy it aims to provide information about the associated genes, corresponding protein sequences, available three dimensional structures and polymorphism related to leprosy. In conclusion, this will serve as a multifunctional valuable tool and convenient information platform which is freely available at http://www.vit.ac.in/leprosy/leprosy.htm and enables the user to retrieve information of their interest.     

Signaling lymphocytic activation molecule expression and regulation in human intracellular infection correlate with Th1 cytokine patterns.

Induction of Th1 cytokines, those associated with cell-mediated immunity, is critical for host defense against infection by intracellular pathogens, including mycobacteria. Signaling lymphocytic activation molecule (SLAM, CD150) is a transmembrane protein expressed on lymphocytes that promotes T cell proliferation and IFN-gamma production. The expression and role of SLAM in human infectious disease were investigated using leprosy as a model. We found that SLAM mRNA and protein were more strongly expressed in skin lesions of tuberculoid patients, those with measurable CMI to the pathogen, Mycobacterium leprae, compared with lepromatous patients, who have weak CMI against M. leprae. Peripheral blood T cells from tuberculoid patients showed a striking increase in the level of SLAM expression after stimulation with M. leprae, whereas the expression of SLAM on T cells from lepromatous patients show little change by M. leprae stimulation. Engagement of SLAM by an agonistic mAb up-regulated IFN-gamma production from tuberculoid patients and slightly increased the levels of IFN-gamma in lepromatous patients. In addition, IFN-gamma augmented SLAM expression on M. leprae-stimulated peripheral blood T cells from leprosy patients. Signaling through SLAM after IFN-gamma treatment of Ag-stimulated cells enhanced IFN-gamma production in lepromatous patients to the levels of tuberculoid patients. Our data suggest that the local release of IFN-gamma by M. leprae-activated T cells in tuberculoid leprosy lesions leads to up-regulation of SLAM expression. Ligation of SLAM augments IFN-gamma production in the local microenvironment, creating a positive feedback loop. Failure of T cells from lepromatous leprosy patients to produce IFN-gamma in response to M. leprae contributes to reduced expression of SLAM. Therefore, the activation of SLAM may promote the cell-mediated immune response to intracellular bacterial pathogens.     

Molecular basis for the peripheral nerve predilection of Mycobacterium leprae

Mycobacterium leprae, the causative organism of leprosy, has a unique predilection for Schwann cells, the glial cells of the peripheral nervous system. M. leprae invasion of Schwann cells leads to the neurological damage that underlies the sensory motor loss and subsequent deformity and disability associated with this disease. Recent studies have begun to elucidate the early events of M. leprae infection of Schwann cells on a molecular level, and the host and bacterial factors that determine the neural predilection of this bacterium. These advances have now provided novel insights into the mechanisms of bacterial interactions with host cells.     

Diagnostic test systems based on the immunoenzyme method in leprosy

Diagnostic test systems for the detection of IgG and IgM to Mycobacterium leprae in the blood sera of leprosy patients and armadillos experimentally infected with M. leprae have been developed on the basis of the indirect immunoperoxidase assay. The possibility has been shown of prognosing the activity of the leprotic process in leprosy patients and the results of the experimental infection of armadillos by the dynamic increase of antibody reactions with the development of the infection.     

Endothelial cells and the pathogenesis of lepromatous neuritis:insights from the armadillo model

Selective infection of peripheral nerves is a unique property of Mycobacterium leprae that results in serious injury, but its basis is unexplained. Recent evidence from infected armadillos suggests that endothelial cells of peripheral nerve vasculature may be the gatekeepers by which M. leprae infects nerves. The pathogenesis of neuropathy in leprosy may thus entail a dynamic sequence of adhesion, immunologic, and inflammatory processes involving peripheral nerve endothelial cells.     

Effect of recombinant interferon-gamma on hydrogen peroxide-releasing capacity of monocyte-derived macrophages from patients with lepromatous leprosy

Monocyte-derived macrophages from 14 patients with lepromatous leprosy respond to rIFN-gamma with an enhanced secretion of H2O2 in a fashion similar to that of cells obtained from normal donors. The activation is not dependent on the cutaneous bacterial index, the length of treatment, or the stage and activity of the disease. H2O2 release can be triggered in these cells both by phorbol myristate acetate and by intact irradiated Mycobacterium leprae. Uptake of M. leprae by both normal donors' and patients' macrophages is proportional to the number of bacilli added. Prior ingestion of M. leprae does not interfere with the ability of macrophages to respond to IFN-gamma by the production of oxygen intermediates. We conclude that the immune defect in lepromatous leprosy probably results from a lack of response to M. leprae by the patients' T cells rather than an inability of mononuclear phagocytes to respond to IFN-gamma.     

Performance of recombinant ESAT-6 antigen (ML0049) for detection of leprosy patients

AIMS: The study was aimed to evaluate the Mycobacterium leprae recombinant early secreted antigenic target-6 (rESAT-6) for its serological performance in leprosy patients. METHODS AND RESULTS: Employing enzyme-linked immunosorbent assay (ELISA), serum samples were tested for prevalence of immunoglobulin G antibodies against M. leprae rESAT-6. The results revealed that the sensitivity of the assay for smear-positive leprosy patients was 82.4% (14 of 17) while for smear-negative patients it was 19.4% (six of 31). Interestingly, the performance of ESAT-6-based assay was statistically comparable with anti-phenolic glycolipid-I antibody-detecting ELISA, a most widely studied serological assay in leprosy. Regarding specificity, none of the 48 controls was positive indicating that antibody response to ESAT-6 was highly specific. Moreover, a high concordance between bacterial index and anti-ESAT-6 antibody-detecting assay was noted. CONCLUSIONS: Recombinant ESAT-6 seems to be a potential serological reagent for detection of M. leprae infection. SIGNIFICANCE AND IMPACT OF THE STUDY: ESAT-6 serology may have utility for (i) early diagnosis, particularly, of highly infectious form (multibacillary, MB) of leprosy, (ii) monitoring the response in smear-positive leprosy patients during the course of the chemotherapy, (iii) classification of leprosy patients into MB and paucibacillary groups for treatment purpose. Hence, further research on these lines is warranted.     

Field utility of phenolic glycolipid coated latex agglutination test for rapid detection of bacilliferous leprosy cases.

Serum samples were collected from eighty-three leprosy patients and twenty-five healthy controls supposedly not exposed to Mycobacterium leprae infection. Phenolic glycolipid-1 coated latex agglutination test (PGL-LAT) was carried out with the serum samples to detect antibodies specific to M. leprae. Samples showing positive agglutination were 50% in the lepromatous leprosy (LL) group showing no erythema nodosum leprosum (ENL) complications, 66.6% in LL group with ENL complication, 60% in borderline lepromatous (BL) group, 50% in borderline (BB) and 33.3% in borderline tuberculoid (BT). The patients belonging to the tuberculoid (TT) group and most of the long-term treated patients were interestingly negative, and so were sera from all the healthy controls. PGL-LAT developed by us therefore is specific and a fairly sensitive technique to detect antibodies specific to M. leprae and will be very useful in field conditions.     

Effect of Inhibitors on Phenoloxidase of Mycobacterium leprae

Previous results had shown that the human leprosy bacilli possess a phenoloxidase, which, when compared with the enzyme from mammalian and plant sources, seemed unique in the range of substrates utilized and in the nature of the products formed. The effect of several inhibitors on the enzyme in Mycobacterium leprae was tested. Compounds which bind copper were found to be more effective than substrate analogues. Diethyldithiocarbamate penetrated the bacillus and completely suppressed its phenolase activity. Diasone (a derivative of diaminodiphenylsulfone used in the treatment of leprosy) proved to be a potent inhibitor of phenoloxidase of mammalian and plant origin. However, it was less efficient in the case of M. leprae. A biochemical peculiarity of M. leprae was observed in its ability to metabolize mimosine and penicillamine. These compounds produced total inhibition of tyrosinase in melanoma extract and of mushroom tyrosinase. Nontoxic inhibitors of phenoloxidase in the leprosy bacilli may be of value in developing a rational approach to chemotherapy of the disease.     

Morphological and functional characterizations of Schwann cells stimulated with Mycobacterium leprae

Nerve damage, a characteristic of leprosy, is the cause of patient deformities and a consequence of Schwann cells (SC) infection by Mycobacterium leprae. Although function/dysfunction of SC in human diseases like leprosy is difficult to study, many in vitro models, including SC lines derived from rat and/or human Schwannomas, have been employed. ST88-14 is one of the cell lineages used by many researchers as a model for M. leprae/SC interaction. However, it is necessary to establish the values and limitations of the generated data on the effects of M. leprae in these SC. After evaluating the cell line phenotype in the present study, it is close to non-myelinating SC, making this lineage an ideal model for M. leprae/SC interaction. It was also observed that both M. leprae and PGL-1, a mycobacterial cell-wall component, induced low levels of apoptosis in ST88-14 by a mechanism independent of Bcl-2 family members.     

Evaluation of qPCR-based assays for leprosy diagnosis directly in clinical specimens

The increased reliability and efficiency of the quantitative polymerase chain reaction (qPCR) makes it a promising tool for performing large-scale screening for infectious disease among high-risk individuals. To date, no study has evaluated the specificity and sensitivity of different qPCR assays for leprosy diagnosis using a range of clinical samples that could bias molecular results such as difficult-to-diagnose cases. In this study, qPCR assays amplifying different M. leprae gene targets, sodA, 16S rRNA, RLEP and Ag 85B were compared for leprosy differential diagnosis. qPCR assays were performed on frozen skin biopsy samples from a total of 62 patients: 21 untreated multibacillary (MB), 26 untreated paucibacillary (PB) leprosy patients, as well as 10 patients suffering from other dermatological diseases and 5 healthy donors. To develop standardized protocols and to overcome the bias resulted from using chromosome count cutoffs arbitrarily defined for different assays, decision tree classifiers were used to estimate optimum cutoffs and to evaluate the assays. As a result, we found a decreasing sensitivity for Ag 85B (66.1%), 16S rRNA (62.9%), and sodA (59.7%) optimized assay classifiers, but with similar maximum specificity for leprosy diagnosis. Conversely, the RLEP assay showed to be the most sensitive (87.1%). Moreover, RLEP assay was positive for 3 samples of patients originally not diagnosed as having leprosy, but these patients developed leprosy 5-10 years after the collection of the biopsy. In addition, 4 other samples of patients clinically classified as non-leprosy presented detectable chromosome counts in their samples by the RLEP assay suggesting that those patients either had leprosy that was misdiagnosed or a subclinical state of leprosy. Overall, these results are encouraging and suggest that RLEP assay could be useful as a sensitive diagnostic test to detect M. leprae infection before major clinical manifestations.     

Identification of specific proteins and peptides in Mycobacterium leprae suitable for the selective diagnosis of leprosy

Diagnosis of leprosy is a major obstacle to disease control and has been compromised in the past due to the lack of specific reagents. We have used comparative genome analysis to identify genes that are specific to Mycobacterium leprae and tested both recombinant proteins and synthetic peptides from a subset of these for immunological reactivity. Four unique recombinant proteins (ML0008, ML0126, ML1057, and ML2567) and a panel of 58 peptides (15 and 9 mer) were tested for IFN-gamma responses in PBMC from leprosy patients and contacts, tuberculosis patients, and endemic and nonendemic controls. The responses to the four recombinant proteins gave higher levels of IFN-gamma production, but less specificity, than the peptides. Thirty-five peptides showed IFN-gamma responses only in the paucibacillary leprosy and household contact groups, with no responses in the tuberculosis or endemic control groups. High frequencies of IFN-gamma-producing CD4+ and CD8+ T cells specific for the 15- and 9-mer peptides were observed in the blood of a paucibacillary leprosy patient. 9-mer peptides preferentially activated CD8+ T cells, while the 15-mer peptides were efficient in inducing responses in both the CD4+ and CD8+ T cell subsets. Four of the six 9-mer peptides tested showed promising specificity, indicating that CD8+ T cell epitopes may also have diagnostic potential. Those peptides that provide specific responses in leprosy patients from an endemic setting could potentially be developed into a rapid diagnostic test for the early detection of M. leprae infection and epidemiological surveys of the incidence of leprosy, of which little is known.     

A role for IL-12 receptor expression and signal transduction in host defense in leprosy.

The generation of cell-mediated immunity against intracellular infection involves the production of IL-12, a critical cytokine required for the development of Th1 responses. The biologic activities of IL-12 are mediated through a specific, high affinity IL-12R composed of an IL-12Rbeta1/IL-12Rbeta2 heterodimer, with the IL-12Rbeta2 chain involved in signaling via Stat4. We investigated IL-12R expression and function in human infectious disease, using the clinical/immunologic spectrum of leprosy as a model. T cells from tuberculoid patients, the resistant form of leprosy, are responsive to IL-12; however, T cells from lepromatous patients, the susceptible form of leprosy, do not respond to IL-12. We found that the IL-12Rbeta2 was more highly expressed in tuberculoid lesions compared with lepromatous lesions. In contrast, IL-12Rbeta1 expression was similar in both tuberculoid and lepromatous lesions. The expression of IL-12Rbeta2 on T cells was up-regulated by Mycobacterium leprae in tuberculoid but not in lepromatous patients. Furthermore, IL-12 induced Stat4 phosphorylation and DNA binding in M. leprae-activated T cells from tuberculoid but not from lepromatous patients. Interestingly, IL-12Rbeta2 in lepromatous patients could be up-regulated by stimulation with M. tuberculosis. These data suggest that Th response to M. leprae determines IL-12Rbeta2 expression and function in host defense in leprosy.     

Analysis of apoptosis and Bcl-2 expression in polar forms of leprosy

Apoptosis eliminates pathogen-infected cells. Its modulation can influence the course of infections, permitting the survival of intracellular pathogens. In leprosy, which presents several clinical manifestations related to bacillary burden and host immune status, the mechanisms responsible for the persistence of the bacillus are unknown. Few studies have focused on apoptosis over the disease spectrum and as a defense mechanism against Mycobacterium leprae. We evaluated apoptosis using terminal transferase dUTP nick end labeling and the expression of Bcl-2 by immunohistochemistry in skin lesions from 11 tuberculoid and 15 lepromatous leprosy patients. Each specimen was evaluated by determining the number of positive cells in 10 fields at × 400 magnification. We observed a higher number of apoptotic cells in tuberculoid lesions in comparison with lepromatous leprosy (42.5 cells per 10 fields vs. 11.5 cells per 10 fields, P<0.0001). Expression of Bcl-2, conversely, was larger in lepromatous than in tuberculoid samples (172.0 cells per 10 fields vs. 17.7 cells per 10 fields, P<0.0001). These observations suggest modulation of apoptosis in leprosy, primarily in lepromatous patients, for which the decrease in cell death could support M. leprae survival and contribute to the success of infection. Conversely, in tuberculoid patients, apoptosis could contribute to reducing propagation of the bacillus.     

Novel mechanisms in the immunopathogenesis of leprosy nerve damage: the role of Schwann cells, T cells and Mycobacterium leprae

The major complication of reversal (or type 1) reactions in leprosy is peripheral nerve damage. The pathogenesis of nerve damage remains largely unresolved. In situ analyses suggest an important role for type 1 T cells. Mycobacterium leprae is known to have a remarkable tropism for Schwann cells that surround peripheral axons. Reversal reactions in leprosy are often accompanied by severe and irreversible nerve destruction and are associated with increased cellular immune reactivity against M. leprae. Thus, a likely immunopathogenic mechanism of Schwann cell and nerve damage in leprosy is that infected Schwann cells process and present antigens of M. Leprae to antigen-specific, inflammatory type 1 T cells and that these T cells subsequently damage and lyse infected Schwann cells. Previous studies using rodent CD8+ T cells and Schwann cells have revealed evidence for the existence of such a mechanism. Recently, a similar role has been suggested for human CD4+ T cells. These cells may be more important in causing leprosy nerve damage in vivo, given the predilection of M. leprae for Schwann cells and the dominant role of CD4+ serine esterase+ Th1 cells in leprosy lesions. Antagonism of molecular interactions between M. leprae, Schwann cells and inflammatory T cells may therefore provide a rational strategy to prevent Schwann cell and nerve damage in leprosy.     

Polymerase chain reaction for the detection of Mycobacterium leprae

A polymerase chain reaction (PCR) using heat-stable Taq polymerase is described for the specific detection of Mycobacterium leprae, the causative agent of leprosy. A set of primers was selected on the basis of the nucleotide sequence of a gene encoding the 36 kDa antigen of M. leprae. With this set of primers in the PCR, M. leprae could be detected specifically with a detection limit approximating one bacterium. This PCR appears to meet the criteria of specificity and sensitivity required for a useful tool in epidemiology and eventually for the control of leprosy.     

Implications of genetics for the epidemiology and control of leprosy

This paper reviews the rationale and history of genetic studies related to leprosy, and considers their implications for the epidemiology and control of the disease. A long tradition of genetic studies in leprosy was initiated by early impressions that the disease clusters within families. Investigations were first motivated by an attempt to understand population patterns, and the focus shifted from investigations of racial differences to investigations of families, of twins and ultimately of genetic markers. The strongest evidence for genetic influence has come from studies of HLA segregation patterns within families, and this has led to elegant in vitro work demonstrating the role of HLA-DR alleles in mediating T-cell reactions in conjunction with antigens of Mycobacterium leprae. The epidemiological implications of this work are not yet clear. The emphasis on family-segregation studies may have given a biased impression because of their requirement for multi-case families. There is evidence that the genetic mechanisms underlying leprosy differ within and between populations. One possible application of the current work would be the use of HLA-DR-specific reactions to identify epitopes of M. leprae which should be excluded from future vaccine preparations.