ArticleLiterature Review

The mouse foot-pad technique for cultivation of Mycobacterium leprae

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Abstract

Although multiplication of Mycobacterium leprae in the foot pads of immune-competent mice is limited, and no leprosy-like lesions are produced in these animals, the mouse foot-pad system represents the first truly useful and reproducible animal model of M. leprae infection. Its employment has enabled research into basic questions with respect to the microbiology of M. leprae, and the epidemiology, treatment and control of leprosy. The mouse foot-pad technique is labour-intensive and time-consuming, and is expensive in terms of the costs of animal purchase and maintenance. In addition, the technique appears to be rather imprecise and insensitive, compared with the techniques employed in working with cultivable micro-organisms. For these reasons, and also as a by-product of the success of multi-drug therapy, the technique has been abandoned in many research centres. Nevertheless, until a more simple and sensitive technique for demonstrating the viability of M. leprae is developed, the mouse foot-pad system remains an essential tool for leprosy research. In this review, we discuss the mouse foot-pad technique in detail, analyse its precision, point out its shortcomings, describe its most important applications, and prescribe a method by which to assess the ability of an alternative technique to serve in place of this established technique.

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... Experimental animal models for leprosy and Jorge Lobo's disease are commonly used to elucidate the pathogenesis of both infectious diseases, in drug susceptibility testing, and maintenance of strains. [1][2][3] Mycobacterium leprae, the etiologic agent of leprosy, is the only species of the genus that has not yet been cultivated in vitro. 4 The inability of M. leprae to grow in artificial culture media prevents the provision of large quantities of bacilli, in addition to impeding the use of bacilli in routine diagnostic procedures and research, such as determining bacillary viability and verifying the presence of viable bacilli in the lesions of patients after dismissal from treatment and during reactional episodes. ...
... For passage into other animals or for collection of material to be studied, it is necessary to sacrifice the animal host to obtain enough bacilli or fungal cells. 2,8 FNA eliminates the need for sacrificing animals to collect samples; in addition, it facilitates the preparation of cell suspensions. By using FNA, M. leprae and L. loboi are harvested from lesions almost without tissue debris. ...
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Procedures involving the use of Mycobacterium leprae and Lacazia loboi, uncultivated organisms, depend on the collection of material from the lesions of patients or experimental animals. This study compared fine-needle aspiration (FNA) and skin biopsy methods for obtaining bacilli and fungal cells to experimentally infect animals. Lepromas from one armadillo and one enlarged footpad of a mouse previously inoculated with L. loboi were submitted to FNA and biopsy. Materials collected were processed for inoculation in mice. Acid-fast bacilli (AFB) collected by two FNA procedures yielded 7.2×10(7) and 5.3×10(6) AFB/ml and biopsies yielded 1.58×10(8) and 3.5×10(8) AFB/ml from each leproma. Yeast-like cells of L. loboi collected by FNA yielded 1.0×10(6) fungal cells/ml and biopsy 1.0×10(7) fungal cells/ml. After 8 months, inoculated animals were sacrificed and the inoculated footpads submitted to histopathological examination and counting of AFB and fungal cells. The results obtained by the two methods were comparable for both microorganisms. Biopsy may be replaced by FNA during harvesting of material for different purposes, especially for experimental inoculation of mice in leprosy and Jorge Lobo's disease, with the advantage of FNA being a simpler, less invasive, and less costly method.
... Despite its clinical importance, the extraction and purification of the native PGL-I is restricted to the growth of M. leprae in mice and armadillos (Levy and Ji, 2006), due to the natural inability of the pathogen to grow in vitro (Youn et al., 2004), leading to a limited availability of the antigen. This problem led researchers to seek for alternatives to native PGL-I using synthetic antigens, such as ND-O-HSA (natural disaccharide with octyl linkage to human serum albumin) and NT-P-HSA (natural trisaccharide with phenolic ring linkage to HSA) (Fujiwara and Izumi, 1987). ...
... In this investigation, we have developed a chimeric peptide that mimic the native PGL-I with important implications in leprosy diagnosis and in monitoring programs of household contacts, and it may become a good substitute for PGL-I. Our novel chimeric peptide was developed due to the production problems of the native molecule that relies on extensive work with armadillo infection and extraction (Levy and Ji, 2006). Alternatives with synthetic molecules have also been used by producing modified carbohydrates that can perform similar functions of the native PGL-I (Lobato et al., 2011), but these methods are also complex, time-consuming, and demand excessive manipulation of reagents (Fujiwara and Izumi, 1987). ...
Article
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Phenolic glycolipid I (PGL-I) is an abundant antigen on the Mycobacterium leprae cell wall, commonly used for operational classification of leprosy patients. Our aim was to develop PGL-I mimotopes with similar characteristics and functions of the native antigen. We have used a random peptide phage display (PD) library for selections against the monoclonal antibody anti-PGL-I. After three selection cycles, six peptides were identified. All sequences were interspersed by a spacer generating a chimeric peptide (PGLI-M3) that was artificially synthesized. The highly reactive peptide was submitted to a reverse PD selection with a single-chain Fv (scFv) antibody fragment combinatorial library. The most reactive scFv was then validated by enzyme-linked immunosorbent assay (ELISA) against both native PGL-I and two derived synthetic (NDO and ND-O-HSA). We have further proved the scFv specificity by detecting M. leprae bacilli in leprosy lesions through immunohistochemistry. We then described its applicability in ELISA for all clinical forms and household contacts (HC). Afterward, we showed differential binding affinities of PGLI-M3 to sera (anti-PGL-I IgM) from all leprosy clinical forms through surface plasmon resonance (SPR). ELISA IgM detection showed 89.1% sensitivity and 100% specificity, considering all clinical forms. Positivity for anti-PGL-I IgM was twofold higher in both HC and patients with paucibacillary forms in hyperendemic regions than in endemic ones. The SPR immunosensor was able to differentiate clinical forms with 100% accuracy. This is the first time that a PGL-I mimotope has efficiently mimicked the carbohydrate group of the M. leprae antigen with successful immunoassay applications and may become a substitute for the native antigen.
... Live animal models are required for bacterial cultivation. Limited growth occurs in the footpads (FPs) of conventional mice [1,2], whereas more prolific growth is attained in immunosuppressed rodents [3][4][5][6] and armadillos [7]. In these models bacterial multiplication is measured in terms of months to years. ...
... Microscopic counting of acid fast bacilli (AFB) is used to enumerate M. leprae [2,8,9]. Although this method is considered the gold standard, it is time consuming, labor intensive and restricted with regard to specificity. ...
Article
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The inability of Mycobacterium leprae to grow on axenic media has necessitated specialized techniques in order to determine viability of this organism. The purpose of this study was to develop a simple and sensitive molecular assay for determining M. leprae viability directly from infected tissues. Two M. leprae-specific quantitative reverse transcription PCR (qRT-PCR) assays based on the expression levels of esxA, encoding the ESAT-6 protein, and hsp18, encoding the heat shock 18 kDa protein, were developed and tested using infected footpad (FP) tissues of both immunocompetent and immunocompromised (athymic nu/nu) mice. In addition, the ability of these assays to detect the effects of anti-leprosy drug treatment on M. leprae viability was determined using rifampin and rifapentine, each at 10 mg/kg for 1, 5, or 20 daily doses, in the athymic nu/nu FP model. Molecular enumeration (RLEP PCR) and viability determinations (qRT-PCR) were performed via Taqman methodology on DNA and RNA, respectively, purified from ethanol-fixed FP tissue and compared with conventional enumeration (microscopic counting of acid fast bacilli) and viability assays (radiorespirometry, viability staining) which utilized bacilli freshly harvested from the contralateral FP. Both molecular and conventional assays demonstrated growth and high viability of M. leprae in nu/nu FPs over a 4 month infection period. In contrast, viability was markedly decreased by 8 weeks in immunocompetent mice. Rifapentine significantly reduced bacterial viability after 5 treatments, whereas rifampin required up to 20 treatments for the same efficacy. Neither drug was effective after a single treatment. In addition, host gene expression was monitored with the same RNA preparations. hsp18 and esxA qRT-PCR are sensitive molecular indicators, reliably detecting viability of M. leprae in tissues without the need for bacterial isolation or immediate processing, making these assays applicable for in vivo drug screening and promising for clinical and field applications.
... In 2000, research focusing on cultivation of the bacillus in vitro was unsuccessful, although some studies have shown evidence of metabolic activity in vitro (Levy and Ji, 2006). ...
... Moreover, the genome has undergone progressive reduction, accompanied by genetic degradation and a decrease in size. These evolutionary changes originated with the elimination of important metabolic pathways and related ancillary functions of M. leprae, particularly those involved in catabolism (Levy and Ji, 2006). ...
Article
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The purpose of this review was to study the presence of Mycobacterium leprae in the environment and its relation with meteorological variables such as temperature and humidity. There are reports, which provide evidence that meteorological factors such as temperature and soil humidity can influence the dynamics of M. leprae. However, leprosy cases are registered both in the rainy and dry seasons, indicating that M. leprae remains viable in different environmental conditions. Therefore, it is difficult to establish the meteorological pattern(s) required to maintain the bacilli in the environment. The extensive area of endemic countries, endemicity in the bordering countries, diversity of biomes, and lack of urban infrastructure together with weather features are possible factors that influence transmission of the disease.
... Mouse Foot pad and Molecular Methods a. Mouse Foot Pad Technique: Bacilli were extracted from the skin biopsy by manual homogenization (Shepard 1960) and resistance to rifampicin in varying concentrations was determined using technique described by Levy and Ji, 2006. b. Molecular Technique: DNA was extracted from the skin biopsy using DNeasy Blood and Tissue Kit (Cat No: 69506, Qiagen Inc. USA) and primers corresponding to the rifampicin resistance determining region (RRDR) of M. leprae were used to amplify rpoB gene through PCR as described earlier by Matsuoka ( 2010). ...
... At the end of 12 months M. leprae were harvested from the hind foot pads of sub inoculated mice, enumerated (Levy and Ji 2006) and the proportion % of viable bacilli were counted based on Spearman and Karber calculations (Shepard 1982). The p value for statistical significance was calculated using z test of proportions. ...
Article
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Leprosy, a debilitating disease of the skin and peripheral nerves is caused by Mycobacterium leprae (M. leprae) and is treated by multidrug therapy (MDT) comprising of Dapsone, Rifampicin and Clofazimine. Resistance to any of these drugs poses a threat to the current disease control strategies. With the emergence of Rifampicin resistance in leprosy, it is important that alternative drugs need to be tested to develop a treatment strategy to combat drug resistant leprosy. In the current study, we have investigated WHO MDT, Rifapentine, Clarithromycin, Minocycline, Moxifloxacin, Ofloxacin and their combinations in intermittent and daily dose regimens in rifampicin resistant strains of M. leprae through mouse foot pad experiments in order to determine the loss in viability of M. leprae in response to these drugs and their combinations. Our findings suggest that WHO MDT is still the best combination in Rifampicin resistance cases. Combination of Moxifloxacin with Minocycline and Clarithromycin may also be taken up for clinical trials in cases with Rifampicin resistant leprosy. Rifapentine and Moxifloxacin can be effective alternative drugs to replace Rifampicin where required either in daily dose shorter duration regimens or intermittent dose longer regimen to treat resistant strains.
... Thus, immunocompetent mice are by and large resistant to disease development with M. leprae. Although growth was limited, this ability to "culture" M. leprae was a breakthrough which launched numerous studies (reviewed in Levy & Ji 2006), including the isolation and propagation of bacilli from human lesions, evaluation of new leprosy therapeutics and development of assays for the detection of drug resistant strains of M. leprae and diagnostics. In addition, it allowed for fundamental investigations of host resistance and vaccine evaluation. ...
Article
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A variety of host immunogenetic factors appear to influence both an individual's susceptibility to infection with Mycobacterium leprae and the pathologic course of the disease. Animal models can contribute to a better understanding of the role of immunogenetics in leprosy through comparative studies helping to confirm the significance of various identified traits and in deciphering the underlying mechanisms that may be involved in expression of different disease related phenotypes. Genetically engineered mice, with specific immune or biochemical pathway defects, are particularly useful for investigating granuloma formation and resistance to infection and are shedding new light on borderline areas of the leprosy spectrum which are clinically unstable and have a tendency toward immunological complications. Though armadillos are less developed in this regard, these animals are the only other natural hosts of M. leprae and they present a unique opportunity for comparative study of genetic markers and mechanisms associable with disease susceptibility or resistance, especially the neurological aspects of leprosy. In this paper, we review the recent contributions of genetically engineered mice and armadillos toward our understanding of the immunogenetics of leprosy.
... The protocol was approved by the Experimental Animal Committee, of the National Institute of Infectious Diseases, Tokyo (Permit Number: 210001). M. leprae (Thai-53 strain) is passaged in athymic nu/nu mice (Clea Co, Tokyo) [19]. At 8 to 9 months post-infection, the footpads were processed to recover M. leprae [20]. ...
Article
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Little is known of the direct microbicidal activity of T cells in leprosy, so a lipopeptide consisting of the N-terminal 13 amino acids lipopeptide (LipoK) of a 33-kD lipoprotein of Mycobacterium leprae, was synthesized. LipoK activated M. leprae infected human dendritic cells (DCs) to induce the production of IL-12. These activated DCs stimulated autologous CD4+ or CD8+ T cells towards type 1 immune response by inducing interferon-gamma secretion. T cell proliferation was also evident from the CFSE labeling of target CD4+ or CD8+ T cells. The direct microbicidal activity of T cells in the control of M. leprae multiplication is not well understood. The present study showed significant production of granulysin, granzyme B and perforin from these activated CD4+ and CD8+ T cells when stimulated with LipoK activated, M. leprae infected DCs. Assessment of the viability of M. leprae in DCs indicated LipoK mediated T cell-dependent killing of M. leprae. Remarkably, granulysin as well as granzyme B could directly kill M. leprae in vitro. Our results provide evidence that LipoK could facilitate M. leprae killing through the production of effector molecules granulysin and granzyme B in T cells.
... M. bovis BCG Moreau strain (obtained from Fundação Ataulpho Paiva, Rio de Janeiro, Brazil) was cultured, for about 2 weeks, in Middlebrook 7H9 (Invitrogen, Carlsbad, CA) containing 0.02% glycerol and enriched with 10% ADC Middlebrook and 0.5% Tween-80 at 37uC as described elsewhere [32]. Live M. leprae from Instituto Lauro de Souza Lima (Bauru, São Paulo) was aseptically cultured in footpads of athymic NU/ NU mice, purified and enumerated using methods described previously [33][34][35]. All infections experiments with live M. leprae were conduct at 33uC. ...
Article
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Mycobacterium leprae infects macrophages and Schwann cells inducing a gene expression program to facilitate its replication and progression to disease. MicroRNAs (miRNAs) are key regulators of gene expression and could be involved during the infection. To address the genetic influence of miRNAs in leprosy, we enrolled 1,098 individuals and conducted a case-control analysis in order to study four miRNAs genes containing single nucleotide polymorphism (miRSNP). We tested miRSNP-125a (rs12975333 G.T), miRSNP-223 (rs34952329 *.T), miRSNP-196a-2 (rs11614913 C.T) and miRSNP-146a (rs2910164 G.C). Amongst them, miRSNP-146a was the unique gene associated with risk to leprosy per se (GC OR = 1.44, p = 0.04; CC OR = 2.18, p = 0.0091). We replicated this finding showing that the C-allele was over-transmitted (p = 0.003) using a transmission-disequilibrium test. A functional analysis revealed that live M. leprae (MOI 100:1) was able to induce miR-146a expression in THP-1 (p,0.05). Furthermore, pure neural leprosy biopsies expressed augmented levels of that miRNA as compared to biopsy samples from neuropathies not related with leprosy (p = 0.001). Interestingly, carriers of the risk variant (C-allele) produce higher levels of mature miR-146a in nerves (p = 0.04). From skin biopsies, although we observed augmented levels of miR-146a, we were not able to correlate it with a particular clinical form or neither host genotype. MiR-146a is known to modulate TNF levels, thus we assessed TNF expression (nerve biopsies) and released by peripheral blood mononuclear cells infected with BCG Moreau. In both cases lower TNF levels correlates with subjects carrying the risk C-allele, (p = 0.0453 and p = 0.0352; respectively), which is consistent with an immunomodulatory role of this miRNA in leprosy.
... The mouse MFP method was used to determine the viability of M. leprae by growing the bacilli in the mouse foot-pad [22] for multidrug therapy monitoring. But MFP was labor-intensive, time-consuming and expensive technique, used for mice [21,23]. It is pointed out that molecular approaches may be more useful tools but this method can't distinguish dead and viable bacilli during monitoring. ...
Article
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Mycobacterium tuberculosis complex and non-tuberculosis mycobacterium are still caused serious problems of public health around the world, mainly in resource limited settings. The emergence of drug resistant, failure and relapse of mycobacterium infections treatment made the need of efficient and cost-effective tools for treatment monitoring. In resource limited settings with poor facilities, the transfer and implementation by National Tuberculosis Programs of fluorescein diacetate vitality staining could be used as an alternative method of mycobacteria culture for drug therapy monitoring in district level. We described in this review the potential use of fluorescein diacetate staining in the monitoring of infectious disease caused by mycobacterium strains.
... Si le tatou dé veloppe toujours des lé sions systé miques et ne permet pas l'é tude de la forme « paucibacillaire » [26,27], il devrait constituer un modè le de choix pour l'é tude de la forme dissé miné e mais cette possibilité n'a pas é té exploité e à notre connaissance [28]. Quant à la souris, si la culture et la formation de granulomes sont possibles dans ses coussinets plantaires, elle ne pré sente jamais de lé sion à distance du site d'inoculation [29,30]. La recherche de gè nes candidats s'est donc principalement appuyé e sur l'analogie avec d'autres pathologies infectieuses, par exemple la tuberculose et sur l'é tude de gè nes « gé né ralistes » de l'immunité . ...
Article
Despite a natural reservoir of Mycobacterium leprae limited to humans and free availability of an effective antibiotic treatment, more than 200,000 people develop leprosy each year. This disease remains a major cause of disability and social stigma worldwide. The cause of this constant incidence is currently unknown and indicates that important aspects of the complex relationship between the pathogen and its human host remain to be discovered. An important contribution of host genetics to susceptibility to leprosy has long been suggested to account for the considerable variability between individuals sustainably exposed to M. leprae. Given the inability to cultivate M. leprae in vitro and in the absence of relevant animal model, genetic epidemiology is the main strategy used to identify the genes and, consequently, the immunological pathways involved in protective immunity to M. leprae. Recent genome-wide studies have identified new pathophysiological pathways which importance is only beginning to be understood. In addition, the prism of human genetics placed leprosy at the crossroads of other common diseases such as Crohn's disease, asthma or myocardial infarction. Therefore, novel lights on the pathogenesis of many common diseases could eventually emerge from the detailed understanding of a disease of the shadows.
... Live M. leprae (Thai-53 strain) was propagated in athymic BALB/c-nu/nu mice and was harvested from the mouse footpad after 9 months using previously described methods [18,19], and then thawed and washed in PBS/0.05% Tween 80 (Sigma-Aldrich) [20,21]. ...
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Background: The persistence of Mycobacterium leprae (M. leprae) infection is largely dependent on the types of host immune responses being induced. Macrophage, a crucial modulator of innate and adaptive immune responses, could be directly infected by M. leprae. We therefore postulated that M. leprae-infected macrophages might have altered immune functions. Methodology/principal findings: Here, we treated monocyte-derived macrophages with live or killed M. leprae, and examined their activation status and antigen presentation. We found that macrophages treated with live M. leprae showed committed M2-like function, with decreased interleukin 1 beta (IL-1beta), IL-6, tumor necrosis factor alpha (TNF-alpha) and MHC class II molecule expression and elevated IL-10 and CD163 expression. When incubating with naive T cells, macrophages treated with live M. leprae preferentially primed regulatory T (Treg) cell responses with elevated FoxP3 and IL-10 expression, while interferon gamma (IFN-gamma) expression and CD8+ T cell cytotoxicity were reduced. Chromium release assay also found that live M. leprae-treated macrophages were more resistant to CD8+ T cell-mediated cytotoxicity than sonicated M. leprae-treated monocytes. Ex vivo studies showed that the phenotype and function of monocytes and macrophages had clear differences between L-lep and T-lep patients, consistent with the in vitro findings. Conclusions/significance: Together, our data demonstrate that M. leprae could utilize infected macrophages by two mechanisms: firstly, M. leprae-infected macrophages preferentially primed Treg but not Th1 or cytotoxic T cell responses; secondly, M. leprae-infected macrophages were more effective at evading CD8+ T cell-mediated cytotoxicity.
... Conversely, tuberculoid leprosy, is characterised by reduced count of bacilli, expression of T-helper 1 cytokines and is generally restricted to delimited skin lesions. Compared to TB, leprosy still remains poorly understood, for several limitations, which include the scarce cultivability in axenic conditions 120,121,122 and the limited availability of animal models 123,124 . However, several studies have demonstrated that expression levels of different chemokines, including CXCL8-9-10 and CCL2-3-7, are sensitive to leprosy infection 125,126,127,128,129 . ...
Thesis
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Mycobacterium tuberculosis, the agent of TB, is one of the deadliest human pathogens, infecting one third of the global population. Establishment of infection by mycobacteria relies on complex interactions with host innate immune cells, especially macrophages. Once engulfed by macrophages, mycobacteria “usurp” the host cell machineries to facilitate dissemination and to establish an intracellular niche for survival and replication. To investigate how mycobacteria force the immune cells to support infection, we explored the chemokine pathway, best known for its capability to induce cell migration. To dissect the interplay between immune cells and the pathogen, we modelled human TB using the zebrafish-Mycobacterium marinum natural host-pathogen pair, which is attractive for the excellent optical accessibility of the zebrafish larvae and the possibility to apply genetic tools to impair the chemokine signaling. We show that depletion of either CXCR3 or CXCR4 axes are beneficial to the host. Exploitation of CXCR3 signaling leads to macrophage recruitment and to transcriptional changes in macrophages that make them more permissive for mycobacterial intracellular persistence. Activating CXCR4 signaling triggers instead vascularization of the nascent tuberculous granulomas, which in turn supports expansion of the infection. Therefore, inhibitions of these pathways represent promising host-directed therapeutic avenues to counteract mycobacterial diseases.
... The range of the variable values used for sampling is listed in Table 4. All the parameter value ranges are chosen based on the clinical papers [17,18] and we introduced an uncertainty in y for our computational convenience. The remaining values of the parameters are as in Table 1. ...
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Leprosy (Hansen's disease) is an infectious, neglected tropical disease caused by the Mycobacterium Leprae (M. Leprae). Each year there are approximately 2,02,189 new cases are detected globally. In the year 2017 more than half million people were disabled due to leprosy and almost 50000 new cases are added every year world wide. In leprosy, lepra reactions are the major cause for nerve damage leading to disability. Early detection of lepra reactions through study of biomarkers have important role in prevention of subsequent disabilities. To our knowledge there seems to be very limited literature available on within-host modeling at cellular level involving the crucial biomarkers and the possible optimal drug regimen for leprosy disease and lepra reactions. Motivated by these observations, in this study, we have proposed and analyzed a three dimensional mathematical model to capture the dynamics of susceptible schwann cells, infected schwann cells and the bacterial load based on the pathogenesis of leprosy. We estimated the parameters from various clinical papers to make the model more practical. The sensitivity of couple of parameters was evaluated through PRCC method to find out the single most influential parameter and also combination of two most influential parameters was studied using SRCC method. The sensitivity of other remaining parameters was evaluated using Sobol's index. We then have framed and studied an optimal control problem considering the different medication involved in the Multi Drug Therapy (MDT) as control variables. We further studied this optimal control problem along with both MDT and steroid interventions. The finding from this novel and comprehensive study will help the clinicians and public health researchers involved in the process of elimination and eradication of leprosy.
... The protocol was approved by the Experimental Animal Committee of NIID Tokyo (Permit Number: 211002). M. leprae (Thai-53 strain) was propagated in athymic BALB/c-nu/nu mice (Clea Co, Tokyo) [22]. At 8-9 months post-infection, mouse footpads were processed to recover M. leprae [23]. ...
Article
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Background Leprosy is a contagious and chronic systemic granulomatous disease caused by Mycobacterium leprae. In the pathogenesis of leprosy, granulomas play a key role, however, the mechanisms of the formation and maintenance of M. leprae granulomas are still not clearly understood. Methods To better understand the molecular physiology of M. leprae granulomas and the interaction between the bacilli and human host cells, we developed an in vitro model of human granulomas, which mimicked the in vivo granulomas of leprosy. Macrophages were differentiated from human monocytes, and infected with M. leprae, and then cultured with autologous human peripheral blood mononuclear cells (PBMCs). Results Robust granuloma-like aggregates were obtained only when the M. leprae infected macrophages were co-cultured with PBMCs. Histological examination showed M. leprae within the cytoplasmic center of the multinucleated giant cells, and these bacilli were metabolically active. Macrophages of both M1 and M2 types co-existed in the granuloma like aggregates. There was a strong relationship between the formation of granulomas and changes in the expression levels of cell surface antigens on macrophages, cytokine production and the macrophage polarization. The viability of M. leprae isolated from granulomas indicated that the formation of host cell aggregates benefited the host, but the bacilli also remained metabolically active. Conclusions A simple in vitro model of human M. leprae granulomas was established using human monocyte-derived macrophages and PBMCs. This system may be useful to unravel the mechanisms of disease progression, and subsequently develop methods to control leprosy.
... M. leprae can be grown in mouse food pads as well as ninebanded armadillos. M. leprae can be infected into macrophage and Schwann cell (SC) lines to study its immunological behavior [10,11]. M. leprae's survival strategy involves regulation of host genes involved in lipid metabolism, whereas M. tuberculosis is less likely to do it since it has more than 250 genes of its own involved in lipid metabolism. ...
Article
Mycobacterium leprae must adopt a metabolic strategy and undergo various metabolic alterations upon infection to survive inside the human body for years in a dormant state. A change in lipid homeostasis upon infection is highly pronounced in Mycobacterium leprae. Lipids play an essential role in the survival and pathogenesis of mycobacteria. Lipids are present in several forms and serve multiple roles from being a source of nutrition, providing rigidity, evading the host immune response to serving as virulence factors, etc. The synthesis and degradation of lipids is a highly regulated process and is the key to future drug designing and diagnosis for mycobacteria. In the current review, an account of the distinct roles served by lipids, the mechanism of their synthesis and degradation has been elucidated.
... The increased bacterial burden in the pu.1 morphants is likely due to the lack of bacterial killing, rather than bacterial replication. The doubling time of Mlep is approximately 12 days (Levy and Ji, 2006); therefore, most bacteria would not have replicated in the larvae during the 2 day infection. In addition, we assessed the role of macrophages in Mlep dissemination, by infecting animals with fluorescent vascular endothelial cells (kdrl:dsRed). ...
Article
Mycobacterium leprae causes leprosy, and is unique among mycobacterial diseases in producing peripheral neuropathy. This debilitating morbidity is attributed to axon demyelination resulting from direct interactions of the M. leprae-specific phenolic glycolipid 1 (PGL-1) with myelinating glia, and their subsequent infection. Here, we use transparent zebrafish larvae to visualize the earliest events of M. leprae-induced nerve damage. We find that demyelination and axonal damage are not directly initiated by M. leprae but by infected macrophages that patrol axons; demyelination occurs in areas of intimate contact. PGL-1 confers this neurotoxic response on macrophages: macrophages infected with M. marinum expressing PGL-1 also damage axons. PGL-1 induces nitric oxide synthase in infected macrophages, and the resultant increase in reactive nitrogen species damages axons by injuring their mitochondria and inducing demyelination. Our findings implicate the response of innate macrophages to M. leprae PGL-1 in initiating nerve damage in leprosy.
... The increased bacterial burden in the pu.1 morphants is likely due to the lack of bacterial killing, rather than bacterial replication. The doubling time of Mlep is approximately 12 days (Levy and Ji, 2006); therefore, most bacteria would not have replicated in the larvae during the 2 day infection. In addition, we assessed the role of macrophages in Mlep dissemination, by infecting animals with fluorescent vascular endothelial cells (kdrl:dsRed). ...
Article
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Mycobacterium leprae causes leprosy and is unique among mycobacterial diseases in producing peripheral neuropathy. This debilitating morbidity is attributed to axon demyelination resulting from direct interaction of the M. leprae-specific phenolic glycolipid 1 (PGL-1) with myelinating glia and their subsequent infection. Here, we use transparent zebrafish larvae to visualize the earliest events of M. leprae-induced nerve damage. We find that demyelination and axonal damage are not directly initiated by M. leprae but by infected macrophages that patrol axons; demyelination occurs in areas of intimate contact. PGL-1 confers this neurotoxic response on macrophages: macrophages infected with M. marinum-expressing PGL-1 also damage axons. PGL-1 induces nitric oxide synthase in infected macrophages, and the resultant increase in reactive nitrogen species damages axons by injuring their mitochondria and inducing demyelination. Our findings implicate the response of innate macrophages to M. leprae PGL-1 in initiating nerve damage in leprosy.
... The regions of the  subunit that are least impacted by mutations (mutation coolspots) are overlaid with fragment hotspots. The site B (Fig 11), which is in close proximity to the RNA binding which are time consuming, posing the need for effective alternative solutions to decipher the possible impacts of the mutations on drug-resistance outcomes (Levy & Ji, 2006). ...
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In contrast to the situation with tuberculosis, rifampin resistance in leprosy may remain undetected due to the lack of rapid and effective diagnostic methods. A quick and reliable method is essential to determine the impacts of emerging detrimental mutations. The functional consequences of missense mutations within the β-subunit of RNA polymerase in Mycobacterium leprae ( M. leprae ) contribute to phenotypic rifampin resistance outcomes in leprosy. Here we report in-silico saturation mutagenesis of all residues in the β-subunit of RNA polymerase to all other 19 amino acid types and predict their impacts on overall thermodynamic stability, on interactions at subunit interfaces, and on β-subunit-RNA and rifampin affinities using state-of-the-art structure, sequence and normal mode analysis-based methods. A total of 21,394 mutations were analysed, and it was noted that mutations in the conserved residues that line the active-site cleft show largely destabilizing effects, resulting in increased relative solvent accessibility and concomitant decrease in depth of the mutant residues. The mutations at residues S437, G459, H451, P489, K884 and H1035 are identified as extremely detrimental as they induce highly destabilizing effects on the overall stability, nucleic acid and rifampin affinities. Destabilizing effects were predicted for all the experimentally identified rifampin-resistant mutations in M. leprae indicating that this model can be used as a surveillance tool to monitor emerging detrimental mutations conferring rifampin resistance in leprosy. AUTHOR SUMMARY Emergence of primary and secondary drug resistance to rifampin in leprosy is a growing concern and poses threat to the leprosy control and elimination measures globally. In the absence of an effective in-vitro system to detect and monitor phenotypic rifampin resistance in leprosy, most of the diagnosis relies on detecting mutations in the drug resistance determining regions of the rpoB gene that encodes the β subunit of RNA polymerase in M. leprae . Few labs in the world perform mouse food pad propagation of M. leprae in the presence of drugs (rifampin) to determine growth patterns and confirm resistance, however the duration of these methods lasts from 8 to 12 months making them impractical for diagnosis. Understanding molecular mechanisms of drug resistance is vital to associating mutations to clinical resistance outcomes in leprosy. Here we propose an in-silico saturation mutagenesis approach to comprehensively elucidate the structural implications of any mutations that exist or can arise in the β subunit of RNA polymerase in M. leprae . Most of the predicted mutations may not occur in M. leprae due to fitness costs but the information thus generated by this approach help decipher the impacts of mutations across the structure and conversely enable identification of stable regions in the protein that are least impacted by mutations (mutation coolspots) which can be a choice for small molecule binding and structure guided drug discovery.
... In order to demonstrate M. leprae viability, MFP test as gold standard is currently an essential tool in mycobacterium research (Ng et al., 1973;Levy and Ji, 2006). In the current study, after >300 days in culture, both strains of M. leprae were able to replicate in MFP, suggesting that non-propagative culture still allows for maintenance of viable bacilli. ...
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Leprosy is a zoonosis in the southern United States involving humans and wild armadillos. The majority of patients presenting with zoonotic strains of Mycobacterium leprae note extensive outdoor activity but only rarely report any history of direct contact with wild armadillos. Whether M. leprae is transmitted to new vertebrate hosts through the environment independently or with the aid of other organisms, e.g., arthropod vectors, is a fundamental question in leprosy transmission. The objectives of this study were to assess the potential for ticks to transmit M. leprae and to test if viable M. leprae can be maintained in tick-derived cells. To evaluate tick transmission, nymphal Amblyomma maculatum ticks were injected with isolated M. leprae. Infection and transmission were assessed by qPCR. Ticks infected as nymphs harbored M. leprae through vertical transmission events (nymph to adult and adult to progeny); and, horizontal transmission of M. leprae to a vertebrate host was observed. Mycobacterium leprae DNA was detected in multiple tick life cycle stages. Likewise, freshly isolated M. leprae (Thai-53) was used to infect a tick-derived cell line, and enumeration and bacterial viability were assessed at individual time points for up to 49 days. Evaluations of the viability of long-term cultured M. leprae (Thai-53 and Br4923) were also assessed in a mouse model. Tick-derived cells were able to maintain viable M. leprae over the 49-day course of infection and M. leprae remained infectious within tick cells for at least 300 days. The results of this study suggest that ticks themselves might serve as a vector for the transmission of M. leprae and that tick cells are suitable for maintenance of viable M. leprae for an extended period of time.
... However, the resistance of M. leprae to anti-leprosy drugs has been reported in several leprosy endemic region [3,4]. The drug susceptibility testing was done either by the mouse footpad method [5] which is cumbersome and time-consuming or by detecting mutation after direct sequencing of the drug resistance determining region (DRDR) [6]. Studies on anti-leprosy drug susceptibility testing had shown that drug resistance can occur either by transmission of a strain resistant (primary resistance) or most often by mutation of the wildtype drug-susceptible strain during therapy [7]. ...
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Leprosy is a chronic tropical infectious skin disease caused by an obligate intracellular pathogen called Mycobacterium leprae. Until now no vaccine was available so early diagnosis and treatment were the basic strategy for leprosy control. The treatment is based on combined drug therapy including Dapsone, Rifampicin, and Ofloxacin according protocols recommended by the WHO. However, anti-leprosy drugs resistance has been reported in several leprosy endemic region. The drug susceptibility testing was done by detecting mutation after sequencing of the drug resistance determining region. Côte d'Ivoire like many African countries has reaching the threshold of elimination of the disease and the PCT is available nationwide. On the basis of recurrences of therapeutic failures that could be due to misobservance of patients drug therapy or eventually due to circulating resistant strains, we evaluated the drug susceptibility in 155 patients from a leprosy care center in Côte d'Ivoire. Patients were previously diagnosed by clinicians and confirmed by PCR then the genetic drug susceptibility was done by PCR-direct sequencing of the drug resistance determining region of rifampicin, dapsone and ofloxacin used in the treatment. Our results showed multiple cases of multiresistance to anti-leprosy drugs in Côte d'Ivoire. This should be an alert for antibiotic resistance observatories, and policies so that more active surveillance was carried out for the control and surveillance of M. leprae resistance to drugs.
... Computational simulations indicate that the mechanisms of rifampin resistance in leprosy and tuberculosis are similar (Vedithi et al., 2018). In the absence of an experimental method to culture M. leprae in the lab, drug resistance in leprosy is determined by in vivo propagation in mouse footpads (Levy and Ji, 2006) and by associating mutations in the drug-resistance-determining regions of the target coding genes with clinical manifestations. Missense mutations noted in rifampin, dapsone and ofloxacin-resistant strains of M. leprae are associated with clinical drug-resistance outcomes in leprosy (Williams et al., 2013). ...
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Tuberculosis (TB) and leprosy are mycobacterial infections caused by Mycobacterium tuberculosis and Mycobacterium leprae respectively. These diseases continue to be endemic in developing countries where the cost of new medicines presents major challenges. The situation is further exacerbated by the emergence of resistance to many front-line antibiotics. A priority now is to design new antimycobacterials that are not only effective in combatting the diseases but are also less likely to give rise to resistance. In both these respects understanding the structure of drug targets in M. tuberculosis and M. leprae is crucial. In this review we describe structure-guided approaches to understanding the impacts of mutations that give rise to antimycobacterial resistance and the use of this information in the design of new medicines.
... We have cultivated M.leprae by using the mouse foot-pad technique [17]. M.leprae Thai-53 was donated by Dr. Kenji Kohsaka, Sasakawa Research Center, Soi Bamrasnaradoon Hospital, Thailand. ...
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Although Mycobacterium leprae (M.leprae) is usually found in macrophages and nerves of the dermis of patients with multibacillary leprosy, it is also present in all layers of the epidermis, basal, suprabasal, prickle cells, and keratin layers. However, the mechanism by which M.leprae invades the dermis remains unknown, whereas the underlying mechanism by which M.leprae invades peripheral nerves, especially Schwann cells, is well defined. M. leprae binds to the α-dystroglycan (DG) of Schwann cells via the interaction of α-DG and laminin (LN) -α2 in the basal lamina, thus permitting it to become attached to and invade peripheral nerves. In the current study, we investigated the issue of how M.leprae is phagocytosed by human epidermal keratinocytes, neonatal (HEKn). LN-5 is the predominant form of laminin in the epidermis and allows the epidermis to be stably attached to the dermis via its interaction with α/β-DG as well as integrins that are produced by keratinocytes. We therefore focused on the role of LN-5 when M. leprae is internalized by HEKn cells. Our results show that M.leprae preferentially binds to LN-5-coated slides and this binding to LN-5 enhances its binding to HEKn cells. The findings also show that pre-treatment with an antibody against α-DG, integrin-β1, or -β4 inhibited the binding of LN-5-coated M.leprae to HEKn cells. These results suggest that M. leprae binds to keratinocytes by taking advantage of the interaction of LN-5 in the basal lamina of the epidermis and a surface receptor of keratinocytes, such as α-DG, integrin-β1, or -β4.
... It is estimated that M. leprae has lost approximately 2000 genes from its genome [14]. M. leprae can only grow as a parasite in animals with lower body temperature, such as nine-banded armadillos, footpads of immunocompromised mice, or the extremities of a human body [17][18]. The loss of genes involved in vital metabolic pathways such as energy metabolism, limiting the carbon sources the bacteria can use, and interruption in respiration pathways could explain the uncultivable nature of M. leprae [19]. ...
... However, there is no accepted alternative for cultivating M leprae/leprosum. 3 Another method to assess treatment response is the comparison of skin biopsies performed at regular (1-2 year) intervals and evaluating reduction of inflammation and decline of bacilli in the tissues. In our patient, persistent infection was unlikely due to the decrease and degeneration of mycobacteria observed on skin biopsy after prolonged treatment. ...
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The distinction between persistent infection and immunologic reactions in leprosy is often difficult but critically important since their management is different. We present the case of a 51-year-old Vietnamese female who presented in 2015 with areas of erythema and skin infiltration on face and chest, as well as edema on her hands and feet. Skin biopsy was consistent with lepromatous leprosy. She was treated with rifampin, clarithromycin, and levofloxacin for 2 years. Her lower extremity edema was attributed to type 2 immunological reaction for which she was started on prednisone and methotrexate, but she was lost to follow-up for 19 months. She presented with new skin lesions and pain on her extremities. New biopsies revealed an intense neutrophilic infiltrate in the dermis and acid-fast bacilli focally within cutaneous nerve twigs. As compared with the initial biopsy, the inflammatory infiltrates were diminished and the bacilli had a degenerating appearance. These findings were consistent with type 2 immunological reaction. The patient was treated with thalidomide with improvement in the appearance of the skin lesions. A follow-up biopsy showed lack of neutrophilic infiltrates and decreased number of bacilli. This case illustrates the importance of differentiating between persistent infection and immunologic reactions in leprosy. Clinicians should be aware of these complications. A high index of suspicion and accurate interpretation of skin biopsy results are essential for appropriate diagnosis.
... For appropriate treatment, early assessment of drug susceptibility is essential; however, M. leprae cannot be cultivated on artificial media and a drug susceptibility test depending on in vitro growth is not available. Consequently, antibiotic susceptibility tests have relied on the mouse footpad leprosy model, requiring 8 to 12 months because of the slow growth of M. leprae (18). Recently, genetic analysis of drug-resistant M. leprae substantiated the correlation of DDS, RIF, and OFX resistance with mutations in folP1, encoding dihydropteroate synthetase (5,15,19,(23)(24)(25)35); rpoB (4,6,12,19,(23)(24)(25)33), encoding the beta subunit of RNA polymerase; and gyrA, encoding the A subunit of DNA gyrase (4,19,24,26,40), respectively. ...
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Amino acid substitutions at position 89 or 91 in GyrA of fluoroquinolone-resistant Mycobacterium leprae clinical isolates have been reported. In contrast, those at position 94 in M. tuberculosis, equivalent to position 95 in M. leprae, have been identified most frequently. To verify the possible contribution of amino acid substitutions at position 95 in M. leprae to fluoroquinolone resistance, we conducted an in vitro assay using wild-type and mutant recombinant DNA gyrases. Fluoroquinolone-mediated supercoiling activity inhibition assay and DNA cleavage assay revealed the potent contribution of an amino acid substitution of Asp to Gly or Asn at position 95 to fluoroquinolone resistance. These results suggested the possible future emergence of quinolone-resistant M. leprae isolates with these amino acid substitutions and the usefulness of detecting these mutations for the rapid identification of fluoroquinolone resistance in leprosy.
Article
Despite a natural reservoir of Mycobacterium leprae limited to humans and free availability of an effective antibiotic treatment, more than 200,000 people develop leprosy each year. This disease remains a major cause of disability and social stigma worldwide. The cause of this constant incidence is currently unknown and indicates that important aspects of the complex relationship between the pathogen and its human host remain to be discovered. An important contribution of host genetics to susceptibility to leprosy has long been suggested to account for the considerable variability between individuals sustainably exposed to M. leprae. Given the inability to cultivate M. leprae in vitro and in the absence of relevant animal model, genetic epidemiology is the main strategy used to identify the genes and, consequently, the immunological pathways involved in protective immunity to M. leprae. Recent genome-wide studies have identified new pathophysiological pathways which importance is only beginning to be understood. In addition, the prism of human genetics placed leprosy at the crossroads of other common diseases such as Crohn's disease, asthma or myocardial infarction. Therefore, novel lights on the pathogenesis of many common diseases could eventually emerge from the detailed understanding of a disease of the shadows. Copyright © 2013 Elsevier Masson SAS. All rights reserved.
Article
The recognition of the vast numbers of bacteriophages in the biosphere has prompted a renewal of interest in understanding their morphological and genetic diversity, and elucidating the evolutionary mechanisms that give rise to them. We have approached these questions by isolating and characterizing a collection of mycobacteriophages that infect a common bacterial host, Mycobacterium smegmatis. Comparative genomic analysis of 50 mycobacteriophages shows that they are highly diverse, although not uniformly so, that they are pervasively mosaic with a multitude of single gene modules, and that this mosaicism is generated through illegitimate recombination.
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Leprosy, due to Mycobacterium leprae, is an infectious disease which did not totally disappear in France. Indeed, in French metropolis about 20 new cases, all non autochtonous, are diagnosed annually and in French overseas departments, localized in the historic endemic areas, new native (autochtonous) cases are still regularly diagnosed. The diagnosis of the disease is not difficult provided that it is evoked. It always remains based on classic clinical, histological and bacteriological criteria. The new techniques of detection of bacilli by M. leprae DNA amplification by polymerase chain reaction (PCR) and of serology are certainly specific but stay of a partial sensibility. They are not at present considered as indispensable to the diagnosis.
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Leprosy is caused by Mycobacterium leprae (M. leprae) and is unique in terms of the chronicity of the disease and its prolonged treatment protocol. Even after the introduction of multidrug therapy (MDT) by World health organization (WHO), large numbers of new cases (nearly 200,000) of leprosy are reported yearly, indicating active transmission, especially in developing countries. Recurrent clinical manifestations after MDT can occur due to leprosy reactions, relapse or reinfection. It is very difficult to differentiate reaction, relapse and reinfection. Here we categorized a recent case of reoccurrence of leprosy as reinfection by differentiating it from reaction and relapse based on evidence and by analysing the clinical data of the patient.
Article
Introduction: Leprosy is a slowly progressing bacterial infection caused by Mycobacterium leprae. The World Health Organization recommended multidrug therapy (MDT) which is extremely effective and halts the progress of the disease. Even though the objective of eliminating leprosy as a public health problem has been achieved successfully, leprosy is not yet eradicated. Furthermore, the long-term use of MDT results in single- and multidrug resistance. Therefore, there is still a need for new drug discovery for leprosy. Areas covered: The authors explain the importance of discovery of new drug to leprosy and the significance of homology modeling to drug discovery. This review highlights the principle steps, applications, and the resources of homology modeling. Finally, the authors emphasize the application of different structure-based drug design (SBDD) approaches to design novel therapeutics for leprosy. Expert opinion: MDT has proved to be effective in controlling infection, with prevalence of leprosy now predominantly isolated to the developing countries. The emergence of single- and multidrug-resistant strains of M. leprae has, however, provided some concern with the need for newer antibacterial agents. Drug resistance can be overcome by multi-targeted therapy. SBDD approaches, which reported many successful drugs, depend predominantly on the three-dimensional (3D) structure of drug targets. As of 2013, only very few experimental structures are available for M. leprae proteins. Hence, SBDD, in leprosy research, relies heavily on homology modeling to predict the 3D structure of drug targets and to design better therapeutics.
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Ergebnis dieser Arbeit ist ein einfacher und sensitiver Kriterien-Score zur Optimierung der Diagnostik von MB-Lepra-Rückfällen in konventionellen Lepra-Kontrollprogrammen. Da es bisher keine praktikablen, allerorts verfügbaren Methoden zum Nachweis einer erneuten bakteriellen Replikation gibt, bereitet der Ausschluss einer isolierten späten Reaktion als relevante Differential-Diagnose Schwierigkeiten. Durch eine umfangreiche Literaturauswertung wurden Kriterien zur Rückfall-Diagnose ermittelt, in einem Score zusammengefasst und an zwei verschiedenen Patientenkollektiven getestet. Als Goldstandard zur Gegentestung dienten die Daten von 43 von Shetty et al. durch Mouse-Foot-Pad-Untersuchungen bestätigten Rückfällen. Zum anderen wurde der Score auf 57 Patienten angewendet, die aufgrund einer erneuten Lepra-Symptomatik in Karachi, Pakistan in den Jahren 1998 – 2004 als Rückfälle diagnostiziert worden waren. Die Daten waren im Rahmen eines Forschungsaufenthaltes in Karachi aufgenommen worden. Die Beobachtung, dass Rückfälle später als isolierte späte Reaktionen auftreten, wurde durch einen Vergleich des zeitlichen Auftretens von Mouse –Foot – Pad – bestätigten Rückfällen und späten Reaktionen objektiviert und als wichtiges Unterscheidungskriterium im Diagnose-Score berücksichtigt. Zusätzlich wurde ein hoher initialer Bakterien Index von >/=3+ als Risikofaktor identifiziert und berücksichtigt. Als weitere Kriterien für einen Rückfall wurden verfügbare diagnostische Mittel in Form eines Anstiegs des Bakterien Index über einen zu erwartenden Wert sowie nicht vorhandene Reaktionszeichen miteinbezogen. Die Testung dieses Rückfall-Scores am Kollektiv der Mouse –Foot – Pad – bestätigten Rückfälle ergab eine Sensitivität von 95%. Diese war signifikant höher als die der WHO-Kriterien, die lediglich eine Sensitivität von 65% erzielten. Auch die Anwendung beider Kriterien-Sets auf das Rückfall-Kollektiv aus Karachi ergab eine höhere Sensitivität des neuen Linder-Scores (72% vs. 54%). Eine abschließende Evaluation der in Karachi diagnostizierten Rückfälle zeigte eine Überdiagnose von Rückfällen in den frühen Jahren nach Behandlungs-Ende. Grund war vor allem eine Überbewertung hoher BI-Werte.
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Mice as a species are susceptible to tuberculosis infection while mouse inbred strains present wide spectrum of susceptibility/resistance to this infection. However, non-tuberculosis Mycobacterial infections usually cannot be modeled in mice of common inbred strains. Introduction of specific properties, such as gene mutations, recombinants, targeted gene knockouts significantly extended the use of mice to mimic human Mycobacterial infections, including non-tuberculosis ones. This review describes the available mouse models of tuberculosis and non-tuberculosis infections and drug therapy in these models. Mouse models of non-tuberculosis infections are significantly less developed than tuberculosis models, hampering the development of therapies.
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La enfermedad de Hansen o lepra es la segunda micobacteriosis mundial después de la tuberculosis, con una incidencia aproximada de 255.000 nuevos casos en 2007. Según la Organización Mundial de la Salud (OMS), la lepra ya no constituye un problema de salud pública a escala mundial (menos de 1 caso por 10.000 habitantes), pero todavía persisten zonas con una elevada endemicidad. En Francia metropolitana, la enfermedad ya no existe de forma autóctona, pero regularmente se diagnostican nuevos casos procedentes de los DOM-TOM (departamentos y territorios de ultramar) o importados de otros países. Es fundamental que los dermatólogos sepan reconocer los primeros signos, esencialmente cutáneos, porque aunque la poliquimioterapia (PQT) antibacilar introducida en 1982, hoy día permite curar a los pacientes, sólo un diagnóstico precoz evita la aparición de secuelas neurotróficas que agravan notablemente el pronóstico de la enfermedad.
Chapter
Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae, principally affecting the peripheral nerves and skin. Leprosy is a serious health issue in a number of low socioeconomic classes and overcrowded countries. Although it seldom kills, leprosy represents a deforming, disabling, and stigmatizing disease. It has a wide spectrum of clinical findings, including lepromatous, tuberculoid, borderline, and indeterminate poles. The diagnosis is usually made by characteristic clinical findings (typical skin lesions, skin anesthesia, or thickened nerves), slit-skin smears, skin and nerve biopsy, and lepromin test. Antibacterial treatment (multidrug regimen) for leprosy is highly effective, with low relapse rate, but needs to be taken over many months. If left untreated, borderline patients will downgrade toward the lepromatous end of the spectrum, and lepromatous patients will suffer from the numerous complications of bacillary invasion.
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Mycobacterium leprae, the etiologic agent of leprosy, cannot be cultured on artificial media. This characteristic, coupled with its long generation time, presents a number of unique challenges to studying this pathogen. One of the difficulties facing both researchers and clinicians is the absence of a rapid test to measure the viability of M. leprae in clinical or experimental specimens. The lack of such a tool limits the understanding of M. leprae immunopathogenesis and makes determining the efficacy of drug treatments difficult. With this in mind, we developed a robust two‐step molecular viability assay (MVA) that first enumerates the M. leprae in the tissue; then, this data is used to normalize bacterial RNA quantities for the second step, in which the expression of M. leprae esxA and hsp18 are measured. This assay is specific and sensitive enough to be used on most clinical samples. This protocol describes the steps required to extract DNA and RNA from M. leprae‐infected tissue, enumerate M. leprae, and measure M. leprae viability based on the normalized expression of two M. leprae‐specific genes (hsp18 and esxA). This protocol also outlines an optimal laboratory design and workflow for performing this assay. © 2022 The Leprosy Mission Nepal. Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: DNA and RNA P purification from M. leprae‐infected tissue Basic Protocol 2: Enumeration of M. leprae by RLEP qPCR on the DNA fraction Basic Protocol 3: Calculation of M. leprae per tissue and normalization of RNA Basic Protocol 4: Reverse‐transcription of normalized RNA to generate cDNA Basic Protocol 5: Determination of M. leprae viability using HSP18 and ESXA qPCR on the cDNA Support Protocol 1: M. leprae qPCR primer/probe stock preparation Support Protocol 2: Preparation of plasmid stocks and standard curves
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Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1nu). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.
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Leprosy is a chronic infectious condition caused by Mycobacterium leprae(M. leprae). It is endemic in many regions of the world and a public health problem in Brazil. Additionally, it presents a wide spectrum of clinical manifestations, which are dependent on the interaction between M. leprae and host, and are related to the degree of immunity to the bacillus. The diagnosis of this disease is a clinical one. However, in some situations laboratory exams are necessary to confirm the diagnosis of leprosy or classify its clinical form. This article aims to update dermatologists on leprosy, through a review of complementary laboratory techniques that can be employed for the diagnosis of leprosy, including Mitsuda intradermal reaction, skin smear microscopy, histopathology, serology, immunohistochemistry, polymerase chain reaction, imaging tests, electromyography, and blood tests. It also aims to explain standard multidrug therapy regimens, the treatment of reactions and resistant cases, immunotherapy with bacillus Calmette-Guérin (BCG) vaccine and chemoprophylaxis.
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Ultrastructural Pathology, Second Edition is a comprehensive reference on electron microscopy of pathologic tissue in animals and humans. Now presented in an atlas format for easier identification of organelles, the text is designed to bridge the gap between what is seen in the electron microscope at the cellular level and what the pathologist encounters in the postmortem room. New to this edition are sections on diagnostic electron microscopy, providing information on specialized technologies for electron microscopy, and invertebrate pathology. Emphasizing comparative pathology, the book explains and integrates all aspects of cellular changes in lesions occurring from natural or experimental disease.
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Leprosy is a chronic granulomatous infectious disease caused by the pathogen, Mycobacteriu M. lepromatosis, and the more recently discovered, M. lepromatosis. Described in 1873, M. leprae was among the first microorganisms to be proposed as a cause of a human infectious disease. As an obligate intracellular bacterium, it has still not thus far been reproducibly cultivated in axenic medium or cell cultures. Shepard's mouse footpad assay, therefore, was truly a breakthrough in leprosy research. The generation of immunosuppressed and genetically engineered mice, along with advances in molecular and cellular techniques, has since offered more tools for the study of the M. leprae–induced granuloma. While far from perfect, these new mouse models have provided insights into the immunoregulatory mechanisms responsible for the spectrum of this complex disease.
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The objective of the research is to test the efficacy of new drugs and drug combinations in mice infected with Mycobacterium leprae (M. leprae) as alternative to current WHO MDT. Individual drugs tested were Rifampicin (RMP), Rifapentine (RPT) and Moxifloxacin (MOXI). Drug combinations were RMP, Clarithromycin (CLARI), Minocycline (MINO) and RMP, MINO and Ofloxacin (OFLO). RPT drug combinations were RPT, CLARI, MINO and RPT, OFLO, MINO. Both the drugs and drug combinations were used as daily regimen and intermittent regimen. WHO MB MDT served as a positive control. Mice pre-inoculated with M. leprae were allotted to daily and intermittent groups and administered selected drugs and drug combinations. At the end of 12 months post sub-inoculation, mice were sacrificed and the proportion % of viable bacilli were counted using Spearman and Karber method. It was noted that RMP, RPT and Moxifloxacin indicated a range of 89.99% to 99.99% bactericidal effect when used in daily or intermittent doses in both normal and TR mice. Drug combinations showed bactericidal effect comparable to that of WHO MDT. From the study it was concluded that if the present duration of MDT has to be shortened then daily dose regimen with RMP/MINO/OFLO or RPT/CLARI/MINO are recommended for a clinical trial.
Chapter
Structure of the Bacterial Cell Phylum Tenericutes, Order Mycoplasmatales Order Entomoplasmatales Order Incertae Sedis Phylum Proteobacteriae, Order Rickettsiales Order Rhizobiales Order Burkholderiales Order Neisseriales Order Campylobacterales Order Aeromonadales Order Enterobacteriales Order Legionellales Order Pasteurellales Order Pseudomonadales Order Thiotricales Order Vibrionales Phylum Chlamydiae, Order Chlamydiales Order Actinomycetales Phylum Firmicutes, Order Bacillales Order Lactobacillales Order Clostridiales Order Spriochaetales Order Bacteroidales Order Fusobacteriales Other Bacteria
Chapter
Leprosy (synonyms: Hansen’s disease, Hanseniasis, Hansenosis, Lepra) (from the Latin word lepra, which means “scaly” and the Greek lepo meaning “to scale”) is a chronic infective granulomatous disease caused by the bacillus Mycobacterium leprae, an intracytoplasmic parasite of Schwann cells and macrophages. It mainly affects the peripheral nervous system, skin, and other tissues such as the reticuloendothelial system, mucous membranes, the eyes, testes, bones and joints, muscles, and adrenal glands [1–10]. Among communicable diseases, leprosy is one of the leading causes of permanent physical disability worldwide. The disease and resulting visible deformities contribute to the intense social stigma associated with this affliction, that provokes discrimination and avoidance of any contact with patients and their families. Pathogenic transmission occurs via person-to-person contact, especially through the nasorespiratory route, although there is some possibility of transplacental infection [11]. Recently, zoonotic infections have also been reported [12, 13].
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Leprosy, an important infectious disease in humans caused by Mycobacterium leprae (Mle), remains endemic in many countries. Notably, the pathogen cannot be cultured in vitro, except in mouse footpads in vivo. The molecular basis of these characteristics and the mechanisms remain unknown. Consequently, analysis of Mle growth and survival is urgently needed to develop novel therapies against leprosy, including rapid, simple, and specific methods to detect infection. Here, we demonstrated the functional role and contribution of Mle-DNA gyrase, which regulates DNA topology, DNA replication, and chromosome segregation to promote bacterial growth and survival, in Mle growth and survival in vitro and in vivo. The optimum temperature for Mle-DNA gyrase activity was 30 °C. When the DNA gyrB-gyrA genes in Mycobacterium smegmatis were replaced with the Mle gyrase genes by allelic exchange, the recombinants could not grow at 37 °C. Moreover, using radiorespirometry analysis for viability of Mle bacilli, we found that Mle growth was more vigorous at 25–30 °C than at 37 °C, but was inhibited above 40 °C. These results propose that DNA gyrase is a crucial factor for Mle growth and survival and its sensitivity to temperature may be exploited in heat-based treatment of leprosy.
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Background: Subclinical infection with Mycobacterium leprae is one potential source of leprosy transmission, and post-exposure prophylaxis (PEP) regimens have been proposed to control this source. Because PEP trials require considerable investment, we applied a sensitive variation of the kinetic mouse footpad (MFP) screening assay to aid in the choice of drugs and regimens for clinical trials. Methodology/principal findings: Athymic nude mice were inoculated in the footpad (FP) with 6 x 103 viable M. leprae and treated by gastric gavage with a single dose of Rifampin (SDR), Rifampin + Ofloxacin + Minocycline (SD-ROM), or Rifapentine + Minocycline + Moxifloxacin (SD-PMM) or with the proposed PEP++ regimen of three once-monthly doses of Rifampin + Moxifloxacin (RM), Rifampin + Clarithromycin (RC), Rifapentine + Moxifloxacin (PM), or Rifapentine + Clarithromycin (PC). At various times post-treatment, DNA was purified from the FP, and M. leprae were enumerated by RLEP quantitative PCR. A regression analysis was calculated to determine the expected RLEP value if 99.9% of the bacilli were killed after the administration of each regimen. SDR and SD-ROM induced little growth delay in this highly susceptible murine model of subclinical infection. In contrast, SD-PMM delayed measurable M. leprae growth above the inoculum by 8 months. The four multi-dose regimens delayed bacterial growth for >9months post-treatment cessation. Conclusions/significance: The delay in discernable M. leprae growth post-treatment was an excellent indicator of drug efficacy for both early (3-4 months) and late (8-9 months) drug efficacy. Our data indicates that multi-dose PEP may be required to control infection in highly susceptible individuals with subclinical leprosy to prevent disease and decrease transmission.
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The rifampin resistance of Mycobacterium leprae is due to missense mutations in the rpoB gene encoding the beta-subunit of the essential enzyme RNA polymerase. A rapid and very simple method has been developed to detect rifampin resistance in small numbers of M. leprae present in biopsies. It involves polymerase chain reaction amplification of a defined region of the rpoB gene followed by single-strand conformational polymorphism analysis (PCR-SSCP). The reliability of the method has been tested on a sample of known drug-resistant and susceptible isolates of M. leprae.
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Rifampin is currently the most potent drug used in leprosy control programs. We show that the rifampin resistance which emerged in nine patients with lepromatous leprosy, who had received rifampin monotherapy, stemmed from mutations in the rpoB gene, which encodes the beta subunit of RNA polymerase of Mycobacterium leprae. In eight cases missense mutations were found to affect a serine residue, Ser-425, while in the remaining mutant a small insertion was found close to this site. These findings will be of use for the development of a rapid screening procedure, involving the polymerase chain reaction, for monitoring the emergence of rifampin-resistant M. leprae strains.
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Fifty patients with newly diagnosed lepromatous leprosy were allocated randomly to one of five groups and treated with either a month-long standard regimen of multidrug therapy (MDT) for multibacillary leprosy, a single dose of 600 mg of rifampin, a month-long regimen with the dapsone (DDS) and clofazimine (CLO) components of the standard MDT, or a single dose of 2,000 mg of clarithromycin (CLARI) plus 200 mg of minocycline (MINO), with or without the addition of 800 mg of ofloxacin (OFLO). At the end of 1 month, clinical improvement accompanied by significant decreases of morphological indexes in skin smears was observed in about half of the patients of each group. A significant bactericidal effect was demonstrated in the great majority of patients in all five groups by inoculating the footpads of mice with organisms recovered from biopsy samples obtained before and after treatment. Rifampin proved to be a bactericidal drug against Mycobacterium leprae more potent than any combination of the other drugs. A single dose of CLARI-MINO, with or without OFLO, displayed a degree of bactericidal activity similar to that of a regimen daily of doses of DDS-CLO for 1 month, suggesting that it may be possible to replace the DDS and CLO components of the MDT with a monthly dose of CLARI-MINO, with or without OFLO. However, gastrointestinal adverse events were quite frequent among patients treated with CLARI-MINO, with or without OFLO, and may be attributed to the higher dosage of CLARI or MINO or to the combination of CLARI-MINO plus OFLO. In future trials, therefore, we propose to reduce the dosages of the drugs to 1,000 mg of CLARI, 100 mg of MINO, and 400 mg of OFLO.
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Fifty-one lepromatous leprosy patients, all of whom had relapsed after previous dapsone (DDS) monotherapy, were treated between 1990 and 1991 with 600 mg of rifampin (RMP) plus 400 mg of ofloxacin (OFLO) daily for 4 weeks, and the great majority of the patients were followed up at least once a year after completion of the treatment. After only 173 patient-years of follow-up, 5 relapses had been detected; the overall relapse rate was 10.0% (confidence limits, 1.7 and 18.3%), or 2.9 relapses (confidence limits, 0.4 and 5.4) per 100 patient-years. The unacceptably high relapse rate indicated that 4 weeks of treatment with daily RMP-OFLO was unable to reduce the number of viable Mycobacterium leprae organisms to a negligible level. In addition, the M. leprae from one of the relapses were proved to have multiple resistance to DDS, RMP, and OFLO. To avoid further relapses, the follow-up was terminated and the great majority of the patients were retreated with the standard 2-year multidrug therapy from 1994. No further relapse has been diagnosed since the beginning of retreatment.
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To develop a fully supervisable, monthly administered regimen for treatment of leprosy, the bactericidal effect of a single-dose combination of ofloxacin (OFLO) and minocycline (MINO), with or without rifampin (RMP), against Mycobacterium leprae was studied in the mouse footpad system and in previously untreated lepromatous leprosy patients. Bactericidal activity was measured by the proportional bactericidal method. In mouse experiments, the activity of a single dose of the combination OFLO-MINO was dosage related; the higher dosage of the combination displayed bactericidal activity which was significantly inferior to that of a single dose of RMP, whereas the lower dosage did not exhibit a bactericidal effect. In the clinical trial, 20 patients with previously untreated lepromatous leprosy were treated with a single dose consisting of either 600 mg of RMP plus 400 mg of OFLO and 100 mg of MINO or 400 mg of OFLO plus 100 mg of MINO. The OFLO-MINO combination exhibited definite bactericidal activity in 7 of 10 patients but was less bactericidal than the RMP-OFLO-MINO combination. Both combinations were well tolerated. Because of these promising results, a test of the efficacy of multiple doses of ROM in a larger clinical trial appears justified.
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Two Mycobacterium leprae genes, folP1 andfolP2, encoding putative dihydropteroate synthases (DHPS), were studied for enzymatic activity and for the presence of mutations associated with dapsone resistance. Each gene was cloned and expressed in a folP knockout mutant of Escherichia coli(C600ΔfolP::Kmr). Expression ofM. leprae folP1 in C600ΔfolP::Kmr conferred growth on a folate-deficient medium, and bacterial lysates exhibited DHPS activity. This recombinant displayed a 256-fold-greater sensitivity to dapsone (measured by the MIC) than wild-type E. coli C600, and 50-fold less dapsone was required to block (expressed as the 50% inhibitory concentration [IC50]) the DHPS activity of this recombinant. When the folP1 genes of several dapsone-resistant M. leprae clinical isolates were sequenced, two missense mutations were identified. One mutation occurred at codon 53, substituting an isoleucine for a threonine residue (T53I) in the DHPS-1, and a second mutation occurred in codon 55, substituting an arginine for a proline residue (P55R). Transformation of the C600ΔfolP::Kmr knockout with plasmids carrying either the T53I or the P55R mutant allele did not substantially alter the DHPS activity compared to levels produced by recombinants containing wild-type M. leprae folP1. However, both mutations increased dapsone resistance, with P55R having the greatest affect on dapsone resistance by increasing the MIC 64-fold and the IC50 68-fold. These results prove that thefolP1 of M. leprae encodes a functional DHPS and that mutations within this gene are associated with the development of dapsone resistance in clinical isolates of M. leprae. Transformants created with M. leprae folP2 did not confer growth on the C600ΔfolP::Kmrknockout strain, and DNA sequences of folP2 from dapsone-susceptible and -resistant M. leprae strains were identical, indicating that this gene does not encode a functional DHPS and is not involved in dapsone resistance in M. leprae.
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Bactericidal activities of HMR 3647 (HMR), moxifloxacin (MXFX), and rifapentine (RPT) against Mycobacterium leprae, measured by the proportional bactericidal technique in the mouse footpad system, were compared with those of the established antileprosy drugs clarithromycin (CLARI), ofloxacin (OFLO), and rifampin (RMP). Administered in five daily doses of 100 mg/kg of body weight, HMR appeared slightly more bactericidal than CLARI. In a single dose, MXFX at 150 mg/kg was more active than the same dose of OFLO and displayed exactly the same level of activity as RMP at 10 mg/kg; the combination MXFX-minocycline (MINO) (MM) was more bactericidal than the combination OFLO-MINO (OM); RPT at 10 mg/kg was more bactericidal than the same dose of RMP and even more active than the combination RMP-OFLO-MINO (ROM); the combination RPT-MXFX-MINO (PMM) killed 99.9% of viableM. leprae and was slightly more bactericidal than RPT alone, indicating that the combination PMM showed an additive effect against M. leprae.
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Leprosy, a chronic human neurological disease, results from infection with the obligate intracellular pathogen Mycobacterium leprae, a close relative of the tubercle bacillus. Mycobacterium leprae has the longest doubling time of all known bacteria and has thwarted every effort at culture in the laboratory. Comparing the 3.27-megabase (Mb) genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus with that of Mycobacterium tuberculosis (4.41 Mb) provides clear explanations for these properties and reveals an extreme case of reductive evolution. Less than half of the genome contains functional genes but pseudogenes, with intact counterparts in M. tuberculosis, abound. Genome downsizing and the current mosaic arrangement appear to have resulted from extensive recombination events between dispersed repetitive sequences. Gene deletion and decay have eliminated many important metabolic activities including siderophore production, part of the oxidative and most of the microaerophilic and anaerobic respiratory chains, and numerous catabolic systems and their regulatory circuits.
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Everything that we need to know about Mycobacterium leprae, a close relative of the tubercle bacillus, is encrypted in its genome. Inspection of the 3.27 Mb genome sequence of an armadillo-derived Indian isolate of the leprosy bacillus identified 1,605 genes encoding proteins and 50 genes for stable RNA species. Comparison with the genome sequence of Mycobacterium tuberculosis revealed an extreme case of reductive evolution, since less than half of the genome contains functional genes while inactivated or pseudogenes are highly abundant. The level of gene duplication was approximately 34% and, on classification of the proteins into families, the largest functional groups were found to be involved in the metabolism and modification of fatty acids and polyketides, transport of metabolites, cell envelope synthesis and gene regulation. Reductive evolution, gene decay and genome downsizing have eliminated entire metabolic pathways, together with their regulatory circuits and accessory functions, particularly those involved in catabolism. This may explain the unusually long generation time and account for our inability to culture the leprosy bacillus.
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When leprosy bacilli from human patients are inoculated into the foot-pads of CFW mice, a microscopic granuloma containing acid-fast bacilli develops in a characteristic manner. This has been seen in 22 of 22 instances with leprosy bacilli from nasal washings, in 12 of 16 instances with leprosy bacilli from skin biopsies, and in none of 16 cases where the nasal washings were not observed to contain leprosy bacilli. Quantitative studies revealed a relationship between the number of bacilli inoculated and the time required for the appearance of the lesions. The incubation period was usually 1 to 2 months when the dose was 105.5 to 106.0 bacilli and about 6 months when the dose was about 103 organisms. After the development of the lesion, the number of bacilli harvested was usually in the range 104.5 to 106.0, regardless of the number inoculated. When the inoculum has contained 102.0 to 103.5 acid-fast bacilli, and harvests were reasonably prompt, there were regular increases of 50- to 1000-fold. Passage to new groups of mice has been successful 11 of 12 times. Most of these were second passages. One strain has been maintained in 3 passages with a total increase in acid-fast bacilli of 4 x 104-fold. Another strain has been through 4 passages with a total increase of about 4 x 106-fold. Cultures on bacteriological media favorable for the growth of most known mycobacterial species have not shown growth of mycobacteria.
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Acid fast staining of mycobacteria in the form of beadings is obtained by means of a carbolfuchsin solution (Ziehl-Neelsen stain) prepared from pararosaniline or from certain kinds of basic fuchsin. After such acid-fast stains, the intensity of the bacilli's colouring was rather poor and unstable, so that some bacilli lost their acid-fast stain. In contrast, an acid-fast staining of mycobacteria in rod form results by using a carbolfuchsin prepared from rosaniline or from other basic fuchsins included new fuchsin. The spectrophotometric and thin-layer chromatographic data indicate that the main component of those basic fuchsins showing beady staining may be pararosaniline, whereas the main ingredient of basic fuchsin with staining the bacteria in rod form may be its higher homologues. Neither chloride nor acetate of the fuchsin could affect the appearance and number of stained bacilli. The commercially available "basic fuchsin" is either the chloride or acetate of pure pararosaniline or consists of variable mixtures of it with higher homologues. Consequently, only a basic fuchsin which has an absorption maximum at lambda greater than or equal to 552 nm could be employed for the acid-fast stain of mycobacteria in a stable manner. Pararosaniline included some basic fuchsins, composed mainly from pararosaniline, should not be selected for the preparation of the carbolfuchsin formula.
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A new method for assessing the bactericidal activity of antileprous drugs against Mycobacterium leprae using the mouse footpad technique is described. This approach, referred to as the 'proportional bactericidal test', has been devised to overcome some of the problems of interpretation caused by drug persistence or prolonged bacteriostasis after drug administration has ended. The bactericidal activity of several drugs against M.leprae has been determined using this approach and the results obtained compared with those previously reported using alternative methods.
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The doubling time of a strain of Mycobacterium leprae during logarithmic multiplication in the mouse foot pad was estimated by inoculating mice with serial dilutions of a bacterial suspension and measuring the time from inoculation to multiplication to 106 organisms per foot pad. The doubling time was found to be 11.1±1.92 (mean ±95% conficence limits) days, about 15% shorter than an earlier estimate based on measurements of the slopes of many single growth curves of M. leprae.
Article
THE failure to grow the leprosy bacillus, Mycobacterium leprae, in vitro means that there is a requirement for a highly susceptible experimental animal model. The only animal confirmed so far as naturally highly susceptible is the nine-banded armadillo, of which at least some specimens develop, after inoculation, a disseminated disease which resembles human lepromatous leprosy1. The inoculation of M. leprae into the footpads of mice results in a localised, self-limiting infection, with a maximum bacillary yield of about 106.5 organisms per footpad2. Suppression of the immune response of the mouse, by thymectomy and irradiation, results in enhancement of the footpad infection, and dissemination of M. leprae bacilli to internal organs and peripheral tissue such as ears, nose and tail3. This ability to enhance infection by nonspecific depression of T-cell function suggests that the congenitally athymic, or nude, mouse might provide a suitable, naturally susceptible host for M. leprae. In spite of the severe impairment of the cellular immune response of nude mice4-7, the only report of the growth of M. leprae in such mice concluded that there was no significant increase in bacillary proliferation, and no tendency to promote a generalised infection8. This conclusion was based, however, on observations made only 6 months after inoculation-an interval that is almost certainly too short for detection of significant enhancement or dissemination.
Article
An analysis of data generated by harvests of Mycobacterium leprae from the foot pads of mice is presented. Acid fast bacteria (AFB) were randomly distributed within the circles of a counting slide in fewer than half of the preparations; the AFB were more likely to be distributed randomly in those preparations containing fewer organisms. The mean coefficient of variation 100 x (standard deviation/mean) of the number of AFB was 29% for the 3 circles on a counting slide, 60% for the 4 foot pads normally pooled for a harvest, and 48% for harvests from 4 replicate pools of 4 to 8 foot pads. The doubling time of M. leprae during logarithmic multiplication in mice averaged 10.7 days, confirming an almost identical estimate made in an earlier study by a different technique. Finally, multiplication of M. leprae was found to be a little slower in mice inoculated in both hind foot pads than in mice inoculated in only one. This analysis confirms the precision of data generated by work with Shepard's foot pad technique. Except for the case of foot by foot harvests, differences among measurements equivalent in time or numbers of AFB to 2 doublings of M. leprae appear certainly to be meaningful.
Article
Previous studies of the protection of mice by prior infection with Mycobacterium leprae in one hind footpad against challenge with M.leprae in the opposite hind footpad had produced conflicting results; therefore, the problem was restudied. In several experiments, BALB/c mice were inoculated first in the right hind footpad with 5,000 M. leprae and then challenged in the left hind footpad with 5,000 M. leprae of the same strain at intervals after primary infection, at the same time that uninfected mice were inoculated. Multiplication of the M. leprae of the secondary challenge inoculum occurred at the same rate and to the same level as multiplication in uninfected mice when challenges were made soon after primary infection. Multiplication was slowed but proceeded to the same level in previously infected as in uninfected mice when the challenges were administered between 76 and 106 days after primary infection (47 to 17 days before the M. leprae of the primary inoculum had multiplied to the level of 10-6 organisms per footpad). Finally, the M. leprae of a secondary challenge administered at the time that the organisms of the primary inoculum had multiplied to 10-6 per footpad or later not only multiplied more slowly in previously infected than in control animals, but multiplication in the previously infected animals reached a lower maximum. These results are similar to those observed when mice previously infected with M. bovis (BCG), M. marinum, Toxoplasma gondii, or Besnoitia jellisoni were challenged with M. leprae.
Article
The susceptibility of Mycobacterium leprae to clinical and experimental antileprosy agents was assessed in the BACTEC 460 system. Nude-mouse-derived M. leprae (10(7) cells), incubated in BACTEC 12B medium at 33 degrees C under reduced oxygen, maintained a fairly constant growth index (14CO2 evolution) for 2 to 3 weeks. At concentrations ranging from 0.031 to 2.0 micrograms/ml, dapsone, rifampin, clofazimine, ethionamide, ofloxacin, clarithromycin, and minocycline all effected reductions in the growth index within 1 to 2 weeks, the extent of inhibition increasing with the incubation time. An in vivo rifampin-resistant isolate displayed markedly reduced susceptibility to rifampin compared with an in vivo-susceptible strain. This system appears to be highly suitable for in vitro drug susceptibility testing of M. leprae.
Article
The purpose of these experiments was to study the early response of mouse foot pads to Mycobacterium leprae. To accomplish this, mice were inoculated in both foot pads with large and small numbers of organisms. The animals were sacrificed at intervals from 2 hr to 27 days after inoculation. The microscopical results, which utilized normal BALB/c and thymectomized-irradiated B6C3F(1) mice, showed that the tissue responded first with an influx of polymorphonuclear cells and later lymphocytes and monocytes. The latter formed a diffuse infiltrate in the tissues. Under conditions where growth normally occurred, the mononuclear cell infiltrate did not persist. The organisms were found within phagocytic cells and the interstitial space. They were always contained within a phagosome and often fused with lysosomes. Most of the organisms appeared to be degenerating at all of the times studied. No organisms were observed in striated muscle fibers of tissues studied.
Article
The possibility of loss from the mouse foot pad of a large fraction of an inoculum of M. leprae was suggested by a preliminary experiment, and a systematic investigation of this problem was undertaken. A series of experiments with various inocula, including freshly harvested M. leprae, M. leprae stored at 4°C, M. marinum, and suspensions of 99Tc(m) sulfur colloid all yielded much the same result: 60% to 90% of the inoculum could not be recovered by a harvest performed soon after foot pad inoculation. The recovery of organisms added to foot pad tissue harvested from uninoculated mice was nearly complete, excluding the possibility of an inherent deficiency of the harvesting procedure. Recovery of the inoculum was improved somewhat when a more extensive harvest was done, but much of the inoculum remained unaccounted for. Inoculum was lost even when dead mice were inoculated, and when anesthetized mice were inoculated to minimize the possibility of leakage. When radioactive colloidal particles of about the same size as M. leprae were inoculated, traces were found in the blood, liver, spleen and inguinal lymph nodes, but the quantities of the radioactive material in these organs were smaller than expected if that fraction of the inoculum lost from the foot pad were distributed uniformly among all of the tissues of the mouse. Loss of a large fraction of inoculated M. leprae from the mouse foot pad occurs regularly, does not represent an artifact, and is not the result of leakage of the inoculum. Loss occurs probably by way of the circulation. The organisms lost from the foot pad do not appear to be uniformly distributed in the mouse, suggesting that they may be taken up preferentially from the blood by some organ such as the bone marrow.
Article
ADMINISTRATION of heterologous antilymphocytic serum (ALS) to mice enhances their susceptibility to experimental infection with Mycobacterium tuberculosis and Myco. lepraemurium (rat ``leprosy'' bacillus)1. Here, I examine the ability of ALS to enhance the much more chronic Myco. leprae (human leprosy bacillus) infection in thymectomized mice.
Article
If the assumptions be valid that the lag phase of bacterial multiplication is constant when M. leprae are repeatedly harvested from untreated mice and passed to other mice of the same inbred strain, and that those M. leprae capable of multiplying in the mouse foot pad do so always at the same rate, then the results of these experiments may be interpreted to show that once the peak of bacterial multiplication has been reached, death of M. leprae ensues. Death of M. leprae appears to have occurred in mice during DDS treatment at the same rate as in the untreated mice, but the lag phase of bacterial growth was uniformly prolonged as a result of treatment.
Article
Neonatal thymectomy and neonatal thymectomy plus ATS resulted in a marked increase in susceptibility of both Buffalo and Lewis rats to infection with M. leprae. In Buffalo rats, increase in susceptibility was limited to footpad infection. In Lewis rats, this was extended to include testis infection where the organisms in thymectomized—ATS-treated rats were found to be still in the logarithmic phase of growth 15 months after inoculation.
Article
Intravenous and footpad infections with Mycobacterium marinum and footpad infections with M. leprae were compared in the following mouse strains: A/He, BALB/C, CBA, C3H, C57BL, C57L, DBA, 101, and CFW. The results varied a great deal according to mouse strain used. Intravenous injection of high doses of M. marinum resulted in deaths after 28 days of 100% of strain A/He, and none of strain 101; 27 days after injection, the feet and noses of all strain CBA mice, but few of the C57BL, 101, or CFW mice, were involved. Injection of a small dose of M. marinum into the footpad produced visible disease in 5 days in all of the C57BL and 101 mice, but in not more than 60% of the A/He, DBA, and CFW mice; the average amount of swelling at 17 days varied from 4.40 mm in strain C57L to 0.92 in strain 101. After footpad injection of M. leprae, the average plateau harvests varied from 1.3 x 10(7) acid-fast bacteria in strain CBA to 6.5 x 10(5) in strain C57L. The infections in CBA mice extended from the site of inoculation throughout the foot. The temperature was measured rectally, in the footpad, and in the tail. Analysis of all the results revealed little correlation among the three types of infection. There was a strong negative correlation between the tail temperature and the death rate after intravenous injection of M. marinum, and a strong positive correlation between footpad temperature and plateau harvest of M. leprae.
Article
Previous studies have demonstrated that congenitally athymic, nude mice are highly susceptible to infection with Mycobacterium leprae. In this study, we showed that footpad inoculation of nude mice with different inoculum sizes of M. leprae resulted in exponential growth of bacilli until bacillary numbers reached approximately 10(10) bacilli per footpad. There was dissemination of the infection from approximately 10 months after inoculation. When nude mice were compared with thymectomized and irradiated mice and normal intact mice for the ability to detect growth from large inocula of low viability, nude mice were the most sensitive, permitting the detection of 10(2) viable M. leprae among 10(7) irradiation-killed organisms. There was widespread dissemination of the infection throughout the reticuloendothelial system and the tissues of the cooler body sites from approximately 10 months after inoculation. Histologically, the lesions resembled those seen in lepromatous leprosy, although the bacillary load appeared larger and was similar to that seen in heavily infected tissues of the nine-banded armadillo. An unusual feature was the presence of numerous foci of neutrophil polymorphs in the footpads and liver of infected nude mice.
Article
As a part of the programme of the Therapy of Leprosy (THELEP) Scientific Working Group, a number of compounds with potential activity against Mycobacterium leprae were prepared in other laboratories. The authors report here the results of studies of their activity against M. leprae with the use of the kinetic method in mice. A modified protocol is described that facilitates comparison of drugs in the same experiment. Two analogues of cycloserine, glycylhydroxamic acid and beta-analylhydroxamic acid were inactive in a dosage of 0.1% in the diet. Isoetam, (D-2,2'-(ethylendiimino)-di-l-butanol)di-isoniazid methane sulphonate was also inactive at this dosage. Three compounds related to dapsone, 4-nitro-N'-phenylsulphonamide, 4-amino-N'-phenylsulphonamide, and 4,4'-diaminobenzene sulphonic acid phenyl ester, had little or no activity at dosages of 0.01% in the diet in experiments with strains shown to have normal susceptibility to dapsone. Two thiosemicarbazones, pyridinal-4-thiosemicarbazone and pyridinal-2-thiosemicarbazone, were inactive in dosages of 0.01%; the latter was inactive at 0.01% in an experiment where thiacetazone was shown to have bactericidal-type activity at a dosage of 0.1% and marginal activity at 0.01%. Brodimoprim, a dihydrofolate reductase inhibitor, which is related to trimethoprim but has a longer half-life, was inactive in a dosage of 0.1%; it had no synergistic effect with 0.01% dapsone against a dapsone-susceptible strain. It was also inactive against a dapsone-resistant strain, alone or in combination with dapsone. The cyanimino analogs of ethionamide and prothionamide were inactive in a dosage of 0.1% against an ethionamide-susceptible strain. Experiments with a series of compounds related to chaulmoogric acid were unsuccessful because the compounds were too toxic. Experiments with a series of compounds related to clofazimine were unsuccessful because their pharmacokinetics were unfavourable for study at dosages where clofazimine itself was active. The limitations imposed by the mouse-foot-pad system are discussed and related to those in other experimental systems.
Article
To measure the rate at which Mycobacterium leprae are killed in the course of the mouse footpad infection after the maximum of multiplication has been achieved, M. leprae were harvested shortly before and at intervals after multiplication had reached the level of 10(6) organisms per footpad, serially diluted, and inoculated into the footpads of passage mice. Beginning 1 year later, foot-by-foot harvests of M. leprae were performed from passage mice, and the proportion of viable organisms in the passage inocula was calculated by means of a most-probable-number calculation. In addition, the proportion of solidly staining M. leprae was measured in the passage inocula. The proportion of viable M. leprae in the passage inocula was found to decrease with the time after multiplication to 10(6) organisms per footpad of donor mice; the half-time of loss of viable M. leprae was 25 days. The proportion of solidly staining organisms appeared to be directly related to the proportion of viable organisms, as measured by mouse passage, and inversely proportional to the time after multiplication to 10(6) organisms per footpad.
Article
The methods used for the study of antileprosy drugs are briefly reviewed. The two chief methods used for the measurement of activity against Mycobacterium leprae are the kinetic method and the proportional bactericidal method. Methods for the statistical analysis of the results are now described for those two methods and their use is illustrated by examples.
Article
Thirty-five multibacillary (MB) leprosy patients were treated with 2 years of multidrug therapy (MDT) and followed up regularly for relapse. Relapse was defined as: a) an increase of the bacterial index (BI) by 2+ over the previous value from any single site of old lesions and b) the occurrence of definite new skin lesion(s) which demonstrated a higher BI than any pre-existing lesion. After a mean duration of 72.7 +/- 17.3 months of follow up per patient, seven relapses were diagnosed; the mean incubation period of relapse was 62.7 +/- 18.7 months. The overall relapse rate was 20.0% (or 3.3 per 100 patient-years), very significantly higher than the figures obtained from the same group of patients analyzed 2 1/2 years earlier, indicating that relapses occurred late (at least 5 +/- 2 years) after stopping MDT. Further analysis indicated that the relapse rate was closely correlated with the bacterial load of the patient, occurring far more frequently among patients with a BI of > or = 4.0 before MDT or with a BI of > or = 3.0 at the end of MDT. To avoid the alarmingly high relapse rate, it is proposed that the duration of MDT be doubled to 4 years in patients with an average BI of > or = 4.0 before MDT.
Article
The anti-Mycobacterium leprae activities of single doses of rifampin (RMP), clarithromycin (CLARI), or minocycline (MINO) alone, and various combinations of CLARI + MINO were determined in immunocompetent mice by the kinetic method. A single dose of RMP 10 mg/kg, CLARI 100 mg/kg or 200 mg/kg, MINO 25 mg/kg or 50 mg/kg alone, or various combinations of CLARI & MINO were active. RMP was more active than the other treatments; the activity of CLARI 100 mg/kg was greater than that of 50 mg/kg, but did not differ significantly from that of 200 mg/kg; MINO 50 mg/kg was more active than 25 mg/kg; and none of the combinations of CLARI + MINO was more active than any of the stronger components administered alone. Therefore, both CLARI and MINO may be applied, either alone or in combination, as components of monthly administered, fully supervised, multidrug regimens for the treatment of multibacillary leprosy. Taking into account the effectiveness of the drugs and the comparative pharmacokinetic data, we propose that the optimal dosage in human trials is CLARI 1000 mg per month or MINO 200 mg per month.
Article
Results from animal and in vitro studies suggest that essential fatty acid (EFA) deficiency enhances cell-mediated immunity by reducing production of prostaglandins with immunosuppressive actions. However, direct experimental evidence that EFA deficiency enhances T-lymphocyte function in vivo has not been obtained. In this study, athymic (nu/nu) mice were infected in the footpads with Mycobacterium leprae and fed a linoleic acid-free diet. These mice, and infected nu/nu mice on control diets, were given an adoptive transfer of M. leprae-primed, T-cell-enriched lymphocytes. After 2 weeks, M. leprae bacilli were harvested from the recipient mice and bacterial viability was determined by the BACTEC system. M. leprae recovered from recipient mice fed control diets displayed little reduction in metabolic activity. In contrast, M. leprae from recipient mice fed the EFA-deficient (EFAD) diet exhibited markedly reduced viability. In vitro, donor cells from M. leprae-primed mice secreted elevated levels of gamma interferon upon exposure to the bacilli. These cells also exhibited an enhanced proliferative response, which was reduced by exogenous prostaglandin E2 (PGE2). In addition, M. leprae-infected granuloma macrophages (Mphi) from EFAD recipient nu/nu mice secreted significantly less PGE2 than granuloma Mphi from mice on control diets. These data suggest that enhanced levels of Mphi-generated PGE2, induced by M. leprae or its constituents, could act as an endogenous negative modulator of the immune response occurring in the microenvironment of the lepromatous granuloma.
Article
The nucleotide sequence analysis of the dihydropteroate synthase (DHPS) gene of six diaminodiphenylsulfone-resistant Mycobacterium leprae strains revealed that the mutation was limited at highly conserved amino acid residues 53 or 55. Though the mutation at amino acid residue 55 or its homologous site has been reported in other bacteria, the mutation at residue 53 is the first case in bacteria. This is the first paper which links the mutations in DHPS and sulfonamide resistance in M. leprae. This finding is medically and socially relevant, since leprosy is still a big problem in certain regions.
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Molecular detection of rifampin resistance (rpoB analysis) in Mycobacterium leprae was determined for 49 patients who experienced relapse of multibacillary leprosy and for 34 untreated patients. Molecular detection of ofloxacin resistance (gyrA analysis) was determined for the 12 patients who experienced relapse and who had received ofloxacin. Results of molecular tests were compared with the reference susceptibility test in the mouse footpad. Overall, the efficiency of molecular detection—that is, positive DNA amplification—was 95%, whereas that of the in vivo test was 55% (P > .001). Results of molecular detection and in vivo test were fully concordant when both were available—that is, for 35 rifampin-sensitive cases of leprosy (no rpoB mutation), 4 ofloxacin-sensitive cases (no gyrA mutation), 11 rifampin-resistant cases (rpoB missense mutations), and 1 ofloxacin-resistant case (gyrA mutation). rpoB and gyrA analysis appears to be an effective method for detection of rifampin and ofloxacin resistance in patients with leprosy.
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A DNA vaccine composed of the gene for the common mycobacterial secreted protein antigen 85B was demonstrated to protect the mouse foot pad against infection with Mycobacterium leprae. The protective effect was demonstrated by a 61%-88% reduction in the bacterial number, a protective effect less than that of BCG. The same DNA vaccine has been shown to protect mice against M. tuberculosis infection, and the importance of testing other candidate tuberculosis vaccines for their potential to protect against leprosy is discussed.
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Mycobacterium leprae infection was evaluated in interferon-γ knockout (GKO) mice. At 4 months, growth of the bacilli in the footpads of GKO mice plateaued a log10 higher than that in control mice. Control mice exhibited mild lymphocytic and histiocytic infiltrates, whereas GKO mice developed large, unorganized infiltrates of epithelioid macrophages and scattered CD4 and CD8 T cells. Flow cytometric analysis of popliteal lymph node cells demonstrated similar profiles of T cells; however, GKO cells exhibited an elevated proliferative response to M. leprae antigen. Expression of inducible nitric oxide synthase mRNA was decreased in GKO mice, whereas macrophage inflammatory protein-1α and interleukin-4 and -10 mRNA expression were augmented. Control and GKO activated macrophages inhibited bacterial metabolism and produced nitrite. Thus, although deficient in an important Th1 cytokine, GKO mice possess compensatory mechanisms to control M. leprae growth and feature elements resembling mid-borderline leprosy in humans.