Palmoplantar Involvement in Leprosy: A Clinicopathological Study.

Palmoplantar involvement has been infrequently reported in leprosy and is an easily misdiagnosed entity. The institutional database of leprosy clinic from 2015 to 2018 was accessed. Details pertaining to demography, clinical presentation, comorbidities (if any), treatment received, and outcome were analyzed in leprosy patients with palmoplantar involvement. Among the 520 patients studied, the involvement of palms and/or soles was reported in 49 (9.4%) patients. Isolated palm involvement was the most frequent (26/49, 53.1%), followed by both palm and sole involvement (12/49, 24.5%) and sole involvement alone (11/49, 22.4%). A higher incidence of lepra reactions and disabilities was noted in patients with palmoplantar involvement than in those without (P < 0.0001). Palmoplantar involvement in leprosy, although uncommon, is associated with a higher risk of reactions and disabilities. A knowledge of this aspect of leprosy can help in maintaining a high index of suspicion and reduce misdiagnosis.

BCG-mediated protection against Mycobacterium ulcerans infection in the mouse.

BACKGROUND: Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) is widely used to reduce the risk of childhood tuberculosis and has been reported to have efficacy against two other mycobacterial diseases, leprosy and Buruli ulcer caused by M. ulcerans (Mu). Studies in experimental models have also shown some efficacy against infection caused by Mu. In mice, most studies use the C57BL/6 strain that is known to develop good cell-mediated protective immunity. We hypothesized that there may be differences in vaccination efficacy between C57BL/6 and the less resistant BALB/c strain. METHODS: We evaluated BCG vaccine efficacy against challenge with ∼3×10(5)M. ulcerans in the right hind footpad using three strains: initially, the Australian type strain, designated Mu1617, then, a Malaysian strain, Mu1615, and a recent Ghanaian isolate, Mu1059. The latter two strains both produce mycolactone while the Australian strain has lost that capacity. CFU of both BCG and Mu and splenocyte cytokine production were determined at intervals after infection. Time to footpad swelling was assessed weekly. PRINCIPAL FINDINGS: BCG injection induced visible scars in 95.5% of BALB/c mice but only 43.4% of C57BL/6 mice. BCG persisted at higher levels in spleens of BALB/c than C57BL/6 mice. Vaccination delayed swelling and reduced Mu CFU in BALB/c mice, regardless of challenge strain. However, vaccination was only protective against Mu1615 and Mu1617 in C57BL/6 mice. Possible correlates of the better protection of BALB/c mice included 1) the near universal development of BCG scars in these mice compared to less frequent and smaller scars observed in C57BL/6 mice and 2) the induction of sustained cytokine, e.g., IL17, production as detected in the spleens of BALB/c mice whereas cytokine production was significantly reduced, e.g., IL17, or transient, e.g., Ifnγ, in the spleens of C57BL/6 mice. CONCLUSIONS: The efficacy of BCG against M. ulcerans, in particular, and possibly mycobacteria in general, may vary due to differences in both host and pathogen.

Development of a mouse food pad model for detection of sub clinical leprosy.

Early diagnosis of leprosy and a multi-drug therapy (MDT) regimen will block the trajectory of nerve damage, disability and deformity that are the hallmarks of this chronic disease. However, the diagnosis of leprosy is made solely by recognition of clinical signs and symptoms, requiring special expertise. These limitations also result in the under reporting of worldwide prevalence and incidence rates for leprosy. Sorely needed is an objective laboratory test for detecting early leprosy. As the antigenic burden of M. leprae can be virtually undetectable in early clinical leprosy, cell mediated immunity and antibody responses will likely be weak. So the sensitivity of new diagnostic tests is as important as specificity. Major efforts are underway employing recombinant M. leprae antigens and synthetic peptides, to develop diagnostic assays for early leprosy infection, using in vitro T cell reactivity or serological tests. We have used the initial phase of the mouse foot pad model as an 'early' model of leprosy infection to screen T cell responses against M. leprae specific antigens and synthetic peptides. Unlike human disease in animal models we can control infection progress and monitor bacillary growth relative to time course of development of T cell response to specific M. leprae antigens. The study employed splenic T cells instead of draining lymph node T cells to model the systemic response as opposed to a local one. We found that 10(5) live M. leprae is the minimum dose required for any meaningful and consistent in vitro splenic IFN-gamma response against M. leprae antigens 3 months after foot pad inoculation. Using this model we found that several M. leprae recombinant proteins, ML0840, ML2028, ML2307, ML2346, ML2478, and ML2532, induced significant levels of IFN-gamma secretion. By controlling for variables that can be confounding factors in the sensitivity of human testing, this mouse model provides an interface between M. leprae diagnostic antigen development and the screening of these antigens in humans under field conditions.

Analysis of Mycobacterium leprae gene expression using DNA microarray.

Mycobacterium leprae, the causative agent of leprosy, does not grow under in vitro condition, making molecular analysis of this bacterium difficult. For this reason, bacteriological information regarding M. leprae gene function is limited compared with other mycobacterium species. In this study, we performed DNA microarray analysis to clarify the RNA expression profile of the Thai53 strain of M. leprae grown in footpads of hypertensive nude rats (SHR/NCrj-rnu). Of 1605 M. leprae genes, 315 showed signal intensity twofold higher than the median. These genes include Acyl-CoA metabolic enzymes and drug metabolic enzymes, which might be related to the virulence of M. leprae. In addition, consecutive RNA expression profile and in silico analyses enabled identification of possible operons within the M. leprae genome. The present results will shed light on M. leprae gene function and further our understanding of the pathogenesis of leprosy.

Powerful bactericidal activity of moxifloxacin in human leprosy.

In a clinical trial of moxifloxacin in eight multibacillary leprosy patients, moxifloxacin proved highly effective. In all trial patients, a single 400-mg dose of moxifloxacin resulted in significant killing (P

First cultivation and characterization of Mycobacterium ulcerans from the environment.

BACKGROUND: Mycobacterium ulcerans disease, or Buruli ulcer (BU), is an indolent, necrotizing infection of skin, subcutaneous tissue and, occasionally, bones. It is the third most common human mycobacteriosis worldwide, after tuberculosis and leprosy. There is evidence that M. ulcerans is an environmental pathogen transmitted to humans from aquatic niches; however, well-characterized pure cultures of M. ulcerans from the environment have never been reported. Here we present details of the isolation and characterization of an M. ulcerans strain (00-1441) obtained from an aquatic Hemiptera (common name Water Strider, Gerris sp.) from Benin. METHODOLOGY/PRINCIPAL FINDINGS: One culture from a homogenate of a Gerris sp. in BACTEC became positive for IS2404, an insertion sequence with more than 200 copies in M. ulcerans. A pure culture of M. ulcerans 00-1441 was obtained on Löwenstein-Jensen medium after inoculation of BACTEC culture in mouse footpads followed by two other mouse footpad passages. The phenotypic characteristics of 00-1441 were identical to those of African M. ulcerans, including production of mycolactone A/B. The nucleotide sequence of the 5' end of 16S rRNA gene of 00-1441 was 100% identical to M. ulcerans and M. marinum, and the sequence of the 3' end was identical to that of the African type except for a single nucleotide substitution at position 1317. This mutation in M. ulcerans was recently discovered in BU patients living in the same geographic area. Various genotyping methods confirmed that strain 00-1441 has a profile identical to that of the predominant African type. Strain 00-1441 produced severe progressive infection and disease in mouse footpads with involvement of bone. CONCLUSION: Strain 00-1441 represents the first genetically and phenotypically identified strain of M. ulcerans isolated in pure culture from the environment. This isolation supports the concept that the agent of BU is a human pathogen with an environmental niche.

Effect of cryo preservation on Mycobacterium leprae growth in the footpads of non-immunosuppressed mice.

OBJECTIVE: To investigate using the mouse footpad system, whether the use of cryopreservants help in retaining the viability of Mycobacterium leprae samples stored at three different temperatures of 4 degrees, -20 degrees and -70 degrees C for 30 days. DESIGN: Biopsies from eight untreated lepromatous leprosy cases were homogenised and inoculated into footpads of normal Swiss White mice within 24 hours (control) and remaining homogenates in each case was divided and stored at 4 degrees C, -20 degrees C and -70 degrees C respectively for 1 month, using either 10% skimmed milk (SM) or Roswell Park Memorial Institute media + 10% glycerol (RPMI) (test). Homogenates adjusted to contain 1 x 10(4) M. leprae/footpad was inoculated into 10 mice per set. Harvestings were done at 6th, 7th, 8th and 12th months. Footpad counts showing > 1 x 10(5) M. leprae at 6th month or later were considered as positive yield. RESULTS: Control All the cases showed > 100 fold growth and 100% take. Viability at 4 degrees C: Only one case (SM) showed a 100 fold increase and 23% take. Viability at -20 degrees C: Two cases showed fold growth that was 40-60 fold less with takes of 63% (SM) and 71% (RPMI) respectively. Viability at -70 degrees C: Positivity was 45% but the fold increase was less as compared to control and takes were between 80-20%, except one RPMI where take was 100%. CONCLUSION: The viability assessed using the mouse footpad was best and consistent in the inoculas that were injected within 24 hours of harvest from the host tissue (control group). None of the storage temperatures used matched with the controls with respect to bacterial yield or % takes. Among the three storage temperatures, -70 degrees C appeared to be better with 45% of the samples showing growth. There was no significant difference noted between the two preservatives used.

The mouse foot-pad technique for cultivation of Mycobacterium leprae.

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.

Isolation of mycobacterium leprae from untreated borderline tuberculoid, mid-borderline and indeterminate cases using the mouse foot pad technique--a study of 209 cases.

Using the mouse foot pad (MFP) system, isolation of Mycobacterium leprae was attempted in 209 skin biopsies obtained from 114 borderline tuberculoid (BT), 62 mid borderline (BB) and 33 indeterminate (1) untreated cases. Unequivocal growth in the foot pads of mice was seen in 100 (47.8%) cases. Of these 100 cases that showed growth in the mouse foot pad system, in 20 cases acid fast bacilli (AFB) were detected in small numbers (1 + ) in either smear or homogenate. The remaining 80 (42%) cases were negative for AFB in both smear and homogenate. The occurrence of viable bacilli and percentage take at 12 months was highest in BB (76 and 86%) followed by BT (38 and 75%) and I (30% and 52%) cases. In most of the BT (65%) and I (60%) cases, the first peak was seen only at 12 months. These results confirm that viable bacilli can be isolated and expanded from a good proportion of negative BT-BB cases using immunocompetent Swiss White mice.

Diversity of potential short tandem repeats in Mycobacterium leprae and application for molecular typing.

A recent advance in molecular typing for tracing the transmission of leprosy is the discovery of short tandem repeats (STRs) in Mycobacterium leprae. To substantiate polymorphic loci from STR as promising candidates for molecular typing tools in leprosy epidemiology, 44 STR loci including 33 microsatellites and 11 minisatellites were investigated among 27 laboratory strains by sequencing PCR products. Not all STRs were necessarily polymorphic. Thirty-two out of the 44 loci were polymorphic. Nine polymorphic loci were suitable for identifying genotypes according to the discriminatory capacity, stability, and reproducibility. All the strains were classified into independent genotypes by the selected nine loci. Three multi-case households were subjected to molecular typing. M. leprae obtained from household cases showed identical copy numbers by TTC triplet alone, but the isolates from one family contact case were divided into different genotypes by adding eight other polymorphic loci. The combination of information from multiple loci allows increasing levels of discrimination and it is likely that the generation and documentation of data will result in the choice of a potential molecular typing tool for leprosy epidemiology.

Effects of purification and fluorescent staining on viability of Mycobacterium leprae.

Over the years, researchers have carried out experiments with Mycobacterium leprae obtained from either human multibacillary lesions, or infected armadillo tissues, or infected footpad tissues of conventional mice as well as athymic nu/nu mice. In general, these sources of leprosy bacilli are satisfactory for most biochemical and mouse footpad studies, but less than satisfactory for studies in cell biology and immunology where contaminating host tissues pose a serious problem. We examined the utility of a procedure for eliminating mouse footpad tissue from M. leprae suspension using sodium hydroxide solution and its subsequent effect on the viability of the organism by determining the rate of palmitic acid oxidation, bacterial membrane integrity, and growth in the mouse footpad. We found that treating M. leprae suspension, obtained from infected nu/nu mouse footpad, with 0.1N NaOH for 3 min was sufficient to remove the majority of mouse tissue without adversely affecting the viability of the organism. This is a simple and rapid method to get suspensions of nu/nu footpad-derived viable M. leprae essentially free of host tissues, which can be a research reagent for studying the host-pathogen relationship in leprosy. We also report here a method for labeling M. leprae with the fluorescent dye PKH26, without compromising on the viability of the organism. This method may be useful in intracellular trafficking studies of M. leprae or in other cell biology studies that require tracking of the bacteria using fluorescent tag. We observed the staining to be stable in vitro over considerable lengths of time and did not affect the viability of the bacteria.

10-12 years follow-up of highly bacillated BL/LL leprosy patients on combined chemotherapy and immunotherapy.

This study reports the follow-up results of 36 highly bacillated untreated BL/LL cases who were serially allocated to three treatment groups. Group I patients received a modified WHO regimen (Rifampicin 600 mg once a month supervised, 50 mg of Clofazimine and 100 mg of Dapsone daily unsupervised) and BCG 0.1 mg per dose 6 monthly; group II patients received the same multi-drug treatment (MDT) and Mw (2 x 10(8) killed bacilli per dose) 6 monthly: group III patients received the same MDT with 0.1 ml of distilled water 6 monthly and acted as a control. Treatment was continued till smear negativity. All these three groups were comparable by their initial clinical score, bacteriological index (BI), viable bacilli as assessed by the mouse foot pad (MFP), bacillary adenosine triphosphate (ATP) content and also histologically at the time of starting treatment. All these parameters were evaluated every 6 months. The vaccines were well tolerated. All the patients in group I became smear negative by 3.5 years, in group II in 3 years whereas those in group III took 5 years. The incidence of reactions was the same in all the groups during the first 2 years, however, patients of group III (MDT + placebo) continued to have reactions up to 3 years. No viable bacilli could be detected in the local and distal sites as estimated by MFP and bacillary ATP after 12 months in both the immunotherapy groups. These could be detected in patients on MDT alone up to 24 months of therapy. Histologically patients in both the immunotherapy groups (groups I and II) showed accelerated granuloma clearance, histological upgrading and non-specific healing without granuloma formation both at the local and distal sites and this was achieved much earlier compared to the MDT + placebo group. Thus, by the addition of immunotherapy the effective treatment period of achieving bacteriological negativity could be reduced by about 40%, time period of reactions reduced by 33% and there were no reactions and/or relapses in the 10-12 years post-treatment follow-up.

Viability and drug susceptibility testing of M. leprae using mouse footpad in 37 relapse cases of leprosy.

Mycobacteria leprae isolates obtained from 37 referral relapse cases of leprosy (37 skin and 10 nerve biopsy samples) received during the years 1994-2001, were tested for viability and drug sensitivity in the mouse footpad. A significant M. leprae yield in the footpads of control mice was obtained, with 32/47 (68%) isolates (from 26 cases) thus confirming viability. Of the 28 isolates successfully drug tested, 6 (21%) were resistant to one or more drugs. All except one, were multidrug treated cases (5/24 = 21%). One of the isolates was resistant to all three drugs, i.e., dapsone (di-aminodiphenyl sulphone, DDS), rifampin (RFP), and clofazimine (CLF). Two were resistant to two drugs, i.e., DDS and RFP, and each of the others were mono resistant to DDS, RFP, or CLF. Notably, one of the isolates that showed combined resistance to DDS and RFP was derived from a borderline tuberculoid case. Also, in one case skin and nerve showed that discordance viz: M. leprae derived from skin were resistant to RFP, while those derived from nerve tested sensitive to all three drugs, indicating tissue related difference.

Electron microscope appearance of lepromatous footpads of nude mice [corrected].

Footpad lesions of 3 nude mice infected by Mycobacterium leprae were studied at 9, 12, and 14 months after inoculation with light and electron microscope. The lesions were somewhat similar to those found in nodules in polar lepromatous leprosy. Striated muscles rather than nerves were the preferred site of the growth of M. leprae. Yet, M. leprae were identified in Schwann cells and endothelial cells, singly and in clumps. M. leprae filled macrophages, and free M. leprae were found in large numbers in the endoneurium without producing any significant demyelination.

A second case of multidrug-resistant Mycobacterium leprae isolated from a Japanese patient with relapsed lepromatous leprosy.

Emergence of drug resistant strains of Mycobacterium leprae was reported soon after the introduction of dapsone (diamino-diphenyl sulphone, DDS) for leprosy treatment (6, 10, 11). Three cases of multidrug-resistant strains of M. leprae have been reported recently (2, 8, 9, 13). In order to prevent multiple drug resistant strains of M. leprae from developing, current leprosy control strategies are based on early detection of cases and treatment with multidrug therapy (MDT) as recommended by the World Health Organization (WHO). We report here the identification of a multidrug-resistant strain of M. leprae from a patient who received inadequate therapy for leprosy. The drug resistant profile of the isolated strain was confirmed by the mouse footpad method and the identification of mutations in genes previously shown to be associated with resistance to each drug was made.

The study of Mycobacterium leprae infection in interferon-gamma gene--disrupted mice as a model to explore the immunopathologic spectrum of leprosy.

Mycobacterium leprae infection was evaluated in interferon-gamma knockout (GKO) mice. At 4 months, growth of the bacilli in the footpads of GKO mice plateaued a log(10) 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-1alpha 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.

Dosage and site of entry influence growth and dissemination of Mycobacterium leprae in T900r mice.

The role of dosage of Mycobacterium leprae and the environment of the inoculated site, in producing leprosy lesions in immunologically-suppressed, highly-susceptible T900r mice, was investigated. Various doses of M. leprae, i.e., 10(7), 10(6), 10(5), 10(4), were inoculated into both flanks and footpads of two different groups of mice. The sites of inoculation were biopsied for histopathological examination and for M. leprae counts at the end of 6, 8 and 12 months. M. leprae multiplied at the infected site and disseminated [figure: see text] to other parts of the body at all concentrations in the mice that were infected in the footpad with a temperature of 31 degrees C. In animals inoculated at the flanks with a temperature of 37 degrees C, multiplication was recorded only when the dosage of M. leprae was high and there was no dissemination of the organism in any of them. The temperature at the site of entry and the dose of infecting M. leprae may play an important role in the development of leprosy in susceptible individuals exposed to M. leprae.