Leprae Infection Research Articles

Field-evaluation of a new lateral flow assay for detection of cellular and humoral immunity against Mycobacterium leprae.

BackgroundField-applicable tests detecting asymptomatic Mycobacterium leprae (M. leprae) infection or predicting progression to leprosy, are urgently required. Since the outcome of M. leprae infection is determined by cellular- and humoral immunity, we aim to develop diagnostic tests detecting pro-/anti-inflammatory and regulatory cytokines as well as antibodies against M. leprae. Previously, we developed lateral flow assays (LFA) for detection of cytokines and anti-PGL-I antibodies. Here we evaluate progress of newly developed LFAs for applications in resource-poor settings.MethodsThe combined diagnostic value of IP-10, IL-10 and anti-PGL-I antibodies was tested using M. leprae-stimulated blood of leprosy patients and endemic controls (EC). For reduction of the overall test-to-result time the minimal whole blood assay time required to detect distinctive responses was investigated. To accommodate LFAs for field settings, dry-format LFAs for IP-10 and anti-PGL-I antibodies were developed allowing storage and shipment at ambient temperatures. Additionally, a multiplex LFA-format was applied for simultaneous detection of anti-PGL-I antibodies and IP-10. For improved sensitivity and quantitation upconverting phosphor (UCP) reporter technology was applied in all LFAs.ResultsSingle and multiplex UCP-LFAs correlated well with ELISAs. The performance of dry reagent assays and portable, lightweight UCP-LF strip readers indicated excellent field-robustness. Notably, detection of IP-10 levels in stimulated samples allowed a reduction of the whole blood assay time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, indicating the feasibility to identify M. leprae infection in endemic areas.ConclusionsDry-format UCP-LFAs are low-tech, robust assays allowing detection of relevant cytokines and antibodies in response to M. leprae in the field. The high levels of IP-10 and the required shorter whole blood assay time, render this cytokine useful to discriminate between leprosy patients and EC.

Role of contact tracing and prevention strategies in the interruption of leprosy transmission

The global prevalence of leprosy has declined from 5.2 million in the 1980 s to 200,000 today. However, the new case detection rate remains high: over the last 8 years, around 220,000-250,000 people have been diagnosed with leprosy each year. In June 2013, an international meeting was organised by the Novartis Foundation for Sustainable Development in Geneva, Switzerland,2 with the objective of discussing the feasibility of interrupting the transmission of leprosy. The group of physicians, epidemiologists and public health professionals concluded that a successful programme would require early diagnosis and prompt multidrug therapy (MDT) for all patients, tracing and post-exposure prophylaxis (PEP) for contacts of patients newly diagnosed with leprosy, improvements in diagnostic tools, as well as strict epidemiological surveillance and response systems to monitor progress. As a follow-up, a second expert group meeting was convened by the Novartis Foundation in January 2014 in Zurich, Switzerland, with the objective of reviewing the evidence for chemoprophylaxis in contacts and high-risk communities. The meeting also considered the definitions of 'contacts' and 'contact tracing', discussed alternative prophylaxis regimens, preliminary findings of operational pilot projects on PEP in Indonesia, as well as the development of diagnostic tools, and identified the priority questions for operational research in leprosy transmission. The meeting outlined how contact tracing and chemoprophylaxis programmes can be implemented to interrupt leprosy transmission. The expert panel reached the following conclusions: Chemoprophylaxis with single-dose rifampicin (SDR) is efficacious in reducing the risk of developing leprosy, although the protective effect appears to be smaller in contacts closer to the index patient than in more distant contacts.3 SDR can be targeted to contacts or implemented as community mass prophylaxis in certain circumstances; the preferred approach depends on local factors, such as the case detection rate, the level of community stigma against leprosy, and the degree of access to healthcare for patients and contacts. Alternative prophylaxis regimens and the role of post-exposure immunoprophylaxis need to be further investigated. Contact tracing combined with PEP across very diverse settings offers protection rates similar to those reported in controlled trials. For high-incidence pockets ('hotspots') or remote or confined high-incidence populations ('hotpops'), blanket administration of PEP may be a better option. Implementation of contact-tracing programmes is feasible and cost-effective, particularly in high-risk groups, but it should be integrated into local healthcare services to ensure their long-term sustainability. Funding and support must be maintained after an initial pilot has finished. New programmes for contact tracing need effective surveillance systems to enable appropriate follow-up and outcome evaluation. The Novartis Foundation and Netherlands Leprosy Relief (NLR) are currently developing and implementing a large international programme to demonstrate the feasibility, acceptability, cost-effectiveness and real-world efficacy of PEP as a strategy to interrupt leprosy transmission, in six pilot projects in Asia, Africa and South America. These new pilot projects will be developed together with the local health authorities, healthcare workers, communities and patients, in order to create local ownership from the outset. The pilots should aim to be scalable and sustainable, and should therefore include an objective outcome assessment. Local ownership ensures that locally appropriate language and definitions of contacts are used in each of the pilots. A test to identify subclinical disease and distinguish M. leprae exposure from infection would facilitate early and appropriate therapy (with PEP or MDT). The identification and validation of new, sensitive biomarkers for M. leprae infection and exposure may allow better targeting of PEP to those contacts at highest risk of developing leprosy.

Mycobacterium leprae Upregulates IRGM Expression in Monocytes and Monocyte-Derived Macrophages

Leprosy is caused by the infection of Mycobacterium leprae, which evokes a strong inflammatory response and leads to nerve damage. Immunity-related GTPase family M protein (IRGM) plays critical roles in controlling inflammation. The objective of the study was to investigate whether IRGM is involved in the infection of M. leprae. Levels of IRGM were assessed in M. leprae-infected CD4(+) T cells, monocytes, and monocyte-derived macrophages. Data revealed that both protein and mRNA levels of IRGM were increased in monocytes after M. leprae infection. Interestingly, monocyte-derived macrophages showed more prominent IRGM expression with M. leprae infection, whereas the bacteria did not affect IRGM in CD4(+) T cells. Furthermore, we assessed levels of IRGM in CD4(+) T cells and monocytes from 78 leprosy patients and 40 healthy controls, and observed upregulated protein level of IRGM in the monocytes from leprosy patients. Also, IRGM expression was inversely correlated with the severity of the disease. These findings suggested a close involvement of IRGM in M. leprae infection and indicated a potential mechanism of defending M. leprae infection.

Mycobacterium leprae and Mycobacterium haemophilum co-infection in an iatrogenically immunosuppressed patient

We present the case of a native Texan who was diagnosed with tuberculoid leprosy and later developed a cutaneous infection with M. haemophilum following iatrogenic immunosuppression. To our knowledge, there are no such reports of M. haemophilum and M. leprae infection occurring simultaneously in the same host.

Evaluation of novel tools to facilitate the detection and characterization of leprosy patients in China.

Leprosy is the disabling outcome of chronic infection with Mycobacterium leprae. The disease often evades early detection, particularly now that fewer clinicians are able to confidently diagnose the disease following the integration of leprosy control measures within general health services in many countries. Although leprosy is officially eliminated in China, endemic regions remain in some difficult-to-reach, underdeveloped areas in Southwest China. In order to better understand the extent of M. leprae infection and identify new leprosy cases in a timely manner, simple tools that can detect infection and the early disease are required. In this report we evaluated the performance of antigen-specific ELISA, the NDO-LID rapid diagnostic test, and antigen-specific whole blood assays (WBA) as potential diagnostic tools. Our data support the use of antibody detection tests and WBA to facilitate the diagnosis of multibacillary and paucibacillary leprosy, respectively. These tools could be invaluable for increased, but simplified, monitoring of individuals in order to provide referrals for clinical exam and early leprosy diagnosis.

Autophagy Gene Polymorphism is Associated with Susceptibility to Leprosy by Affecting Inflammatory Cytokines

Autophagy and inflammation closely interact with each other, and together, they play critical roles in bacterial infection. Leprosy is caused by the infection of Mycobacterium leprae (M. leprae). The objective of the study was to investigate the association between polymorphisms in IRGM, an autophagy gene, and susceptibility to leprosy, and identify possible functions of the polymorphism in the infection of M. leprae. Two polymorphisms in IRGM, rs4958842 and rs13361189, were tested in 412 leprosy cases and 432 healthy controls. Levels of inflammatory cytokines including interleukin 1 beta, IL-4, IL-6, and interferon gamma (INF-γ) were measured after the infection of M. leprae in the peripheral blood mononuclear cell (PBMC) of subjects with different genotypes of rs13361189. Data showed that prevalence of rs13361189TC and CC genotypes were significantly higher in leprosy patients than in healthy controls (odds ratio (OR) = 1.49, 95 % confidence interval (CI) 1.09-2.04, P = 0.012; OR = 2.58, 95 % CI 1.65-4.05, P < 0.001; respectively). Furthermore, the frequency of rs13361189CC genotype was increased in patients with complications than those without complications (P = 0.011). When analyzing the effect of rs13361189 polymorphism on M. leprae infection, we identified that M. leprae-infected PBMC with rs13361189CC genotype expressed significantly elevated levels of INF-γ and IL-4 than those with TT genotype. Our results suggested autophagy gene polymorphism was associated with the increased risk of leprosy by affecting inflammatory cytokines.

Increased Frequency of CD4 and CD8 Regulatory T Cells in Individuals under 15 Years with Multibacillary Leprosy

BackgroundLeprosy is a chronic disease, caused by Mycobacterium leprae, which poses a serious public health problem worldwide. Its high incidence in people under 15 years old in Ceará state, Brazil, reflects the difficulty of its control. The spectrum of clinical manifestations is associated with the immune response developed, with the Th1 and Th2 responses being related to the paucibacillary and multibacillary forms, respectively. Regulatory T cells (Treg), which can suppress Th1 and Th2 response, have received special attention in the literature and have been associated with development of chronic infections. However, their role in leprosy in individuals under 15 years old has not yet been elucidated. We evaluated the frequency of CD4+/CD8+CD25highFOXP3+ and CD4+/CD8+CD25highFOXP3high cells in leprosy patients and household contacts, in both cases under 15 years old.Methodology/Principal FindingsPBMC from 12 patients and 17 contacts were cultured for 72 hours with anti-CD3 and anti-CD28 (activators) or with activators associated with total sonicated fraction of M. leprae. After culture, the frequency of CD4+/CD8+ Treg was identified by flow cytometry. Cells stimulated by activators and antigen from multibacillary patients showed Treg frequencies almost two times that of the contacts: CD4+FOXP3+ (21.93±8.43 vs. 13.79±8.19%, p = 0.0500), CD4+FOXP3high (10.33±5.69 vs. 5.57±4.03%, p = 0.0362), CD8+FOXP3+ (13.88±9.19 vs. 6.18±5.56%, p = 0.0230) and CD8+FOXP3high (5.36±4.17 vs. 2.23±2.68%, p = 0.0461). Furthermore, the mean fluorescence intensity of FOXP3 in Treg was higher in multibacillary patients than in the contacts. Interestingly, there was a positive correlation of the bacillary index and number of lesions with the frequency of all Treg evaluated in patients.Conclusions/SignificanceWe have demonstrated for the first time that multibacillary leprosy patients under 15 years old have greater CD4+ and CD8+ Treg frequencies and these correlate with clinical and laboratorial aspects of disease. These findings suggest the involvement of these cells in the perpetuation of M. leprae infection.

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

AbstractEarlier studies indicate that genotyping of Mycobaterium leprae based on single-nucleotide polymorphisms (SNPs) is useful for analysis of the global spread of leprosy. In the present study, we investigated the diversity of M. leprae at eight SNP loci using 180 clinical isolates obtained from patients with leprosy residing mainly in Delhi and Purulia (West Bengal) regions. It was observed that the frequency of SNP type 1 and subtype D was most predominant in the Indian population. Further, the SNP type 2 subtype E was noted only from East Delhi region and SNP type 2 subtype G was noted only from the nearby areas of Hoogly district of West Bengal. These results indicate the occurrence of focal transmission of M. leprae infection and demonstrate that analysis by SNP typing has great potential to help researchers in understanding the transmission of M. leprae infection in the community.

Genetic association of G896A polymorphism of TLR4 gene in leprosy through family-based and case-control study designs

Polymorphisms in TLR4 may change the function of the protein and alter the efficiency of immune response of host to infection. The high relevance of host gene polymorphisms with outcome of Mycobacterium leprae infection led us to study the genetic association of TLR4 G896A polymorphism in order to identify its risk among contacts of affected leprosy patients. For case-control study design a total of 628 individuals were recruited; 17 multicase leprosy families which included 32 case-parent trios were considered for family-based study. Genotyping was done using PCR-RFLP method. In case-control study AA genotype was positively associated while GA genotype was negatively associated with leprosy. In family based transmission disequilibrium test (TDT) analysis allele G was found to be over transmitted to the affected individuals. Case-control study suggests that homozygous AA genotype may confer susceptibility and heterozygous GA genotype may confer resistance to leprosy, while allele A was observed to increase risk and that of allele G may confer resistance to leprosy. No strong transmission disequilibrium was detected in family-based TDT analysis, possibly due to lower number of trios. In contrast to case-control data allele G was over transmitted to the affected ones in TDT analysis. To conclude, the frequencies of genotypes in household contacts were almost the same as in leprosy patients, suggesting that contacts with AA genotype may be at higher risk of leprosy and may therefore require prophylactic inputs.

Assessment of serum level of interleukin-1β and interleukin-12 in leprosy: impact of previous Bacillus Calmitte Guerin vaccination

Macrophages play an important role in attempt to eliminate mycobacteria, via production of cytokines, including interleukin-1, and interleukin-12. Bacillus Calmitte Guerin (BCG) vaccination, known to induce interleukin-1β in tuberculosis, was originally aimed at tuberculosis control, but it showed efficacy against leprosy. Our aim was to estimate serum levels of interleukin-1β and interleukin-12, in leprosy, and to assess the impact of previous BCG vaccination on their levels. Serum interleukin-1β and interleukin-12 p70 were estimated in 43 leprotic patients and 43 controls by enzyme-linked immunosorbent assay. Patients were grouped according to presence or absence of reactions, as well as bacillary load. Serum interleukin-1β was significantly higher in patients as compared to controls (p = 0.047), and was significantly different in patients' groups (p = 0.036); with significantly higher level in multibacillary patients, both non reactional and with erythema nodosum leprosum, compared with paucibacillary/non reactional patients (p = 0.012 and 0.049 respectively). A statistically significant higher interleukin-1β was found in BCG vaccinated paucibacillary patients as compared to unvaccinated patients (p = 0.031). Significantly elevated interleukin-12 was present in patients as compared to controls (p < 0.001), with no statistically significant difference comparing patients' groups. BCG vaccination showed stimulatory effect on monocytes only in the immunocompetent paucibacillary leprosy patients, as evidenced by higher Interleukin-1β in this group. Interleukin-1β was shown to have a pro-inflammatory role in multibacillary patients with or without erythema nodosum leprosum. Targeting interleukin-1β may be promising to control episodic refractory erythema nodosum leprosum. Interleukin-12 may be a general marker of active Mycobacterium leprae infection.

Anti-PGL1 salivary IgA/IgM, serum IgG/IgM, and nasal Mycobacterium leprae DNA in individuals with household contact with leprosy

Leprosy household contacts represent a group at high risk of developing the disease. The aim of this study was to detect Mycobacterium leprae subclinical infection in this group through serological and molecular parameters. Serum anti-PGL1 IgG/IgM and salivary anti-PGL1 IgA/IgM was investigated using an ELISA, and nasal carriage of M. leprae DNA was detected by PCR, in leprosy household contacts of paucibacillary (PB) and multibacillary (MB) household leprosy patients (n=135), their index cases (n=30), and in persons living in a low endemic city (n=17). Salivary anti-PGL1 IgA and IgM and serum anti-PGL1 IgG showed good correlation comparing contacts and index cases (p<0.01, p<0.005, and p<0.0001, respectively). This was not observed for serum anti-PGL1 IgM (p>0.05). A high frequency of anti-PGL1 IgM positivity was found in IgG-negative samples (p<0.0001). For IgG-positive samples, IgM antibodies were also positive in most of the samples. None of the 17 volunteers living in a low endemic city presented seropositivity for IgG; however, two of them showed positivity for anti-PGL1 IgM. M. leprae DNA was found in the nasal swabs of nine out of the 85 MB household leprosy contacts (10.6%) and in three out of the 50 PB household leprosy contacts (6.0%). We strongly suggest that serum IgG/IgM and salivary anti-PGL1 IgA/IgM measurements are used to follow leprosy household contacts.

Gene Expression Profiling Specifies Chemokine, Mitochondrial and Lipid Metabolism Signatures in Leprosy

Herein, we performed microarray experiments in Schwann cells infected with live M. leprae and identified novel differentially expressed genes (DEG) in M. leprae infected cells. Also, we selected candidate genes associated or implicated with leprosy in genetic studies and biological experiments. Forty-seven genes were selected for validation in two independent types of samples by multiplex qPCR. First, an in vitro model using THP-1 cells was infected with live Mycobacterium leprae and M. bovis bacillus Calmette-Guérin (BCG). In a second situation, mRNA obtained from nerve biopsies from patients with leprosy or other peripheral neuropathies was tested. We detected DEGs that discriminate M. bovis BCG from M. leprae infection. Specific signatures of susceptible responses after M. leprae infection when compared to BCG lead to repression of genes, including CCL2, CCL3, IL8 and SOD2. The same 47-gene set was screened in nerve biopsies, which corroborated the down-regulation of CCL2 and CCL3 in leprosy, but also evidenced the down-regulation of genes involved in mitochondrial metabolism, and the up-regulation of genes involved in lipid metabolism and ubiquitination. Finally, a gene expression signature from DEG was identified in patients confirmed of having leprosy. A classification tree was able to ascertain 80% of the cases as leprosy or non-leprous peripheral neuropathy based on the expression of only LDLR and CCL4. A general immune and mitochondrial hypo-responsive state occurs in response to M. leprae infection. Also, the most important genes and pathways have been highlighted providing new tools for early diagnosis and treatment of leprosy.

NLRP1 haplotypes associated with leprosy in Brazilian patients

Polymorphisms in innate immunity genes are known to be involved in the multifactorial susceptibility to Mycobacterium leprae infection. M. leprae can downregulate IL-1β secretion escaping monocyte digestion. The intracellular receptors NLRPs (NACHT, LRR and PYD domains-containing proteins) sense pathogen associated molecular patterns (PAMPs) activating caspase-1 and IL-1β secretion in the context of inflammasome. Considering the possible role of inflammasome in the immune response against M. leprae, known single nucleotide polymorphisms (SNPs) in two NLRP genes, NLRP1 and NLRP3, were analyzed in Brazilian leprosy patients.Disease-associated SNPs (5 in NLRP1 and 2 in NLRP3), previously associated to infections and to immunologic disorders, were genotyped in 467 leprosy patients (327 multibacillary, MB; 96 paucibacillary, PB) and in 380 healthy controls (HC) from the state of Sao Paulo (Brazil), and in 183 patients (147MB; 64PB) and 186 HC from Mato Grosso (Brazil). Logistic regression analysis was performed considering susceptibility to leprosy di per se (leprosy versus HC) and clinical form (MB versus PB), adjusting for gender and ethnicity.Whereas none of the considered SNPs were statistically associated with leprosy, the NLRP1 combined haplotype rs2137722/G-rs12150220/T-rs2670660/G resulted significantly more frequent in patients than in HC as well as in PB than in MB. While both associations were lost after correction for gender and ethnicity, the NLRP1 combined haplotype rs2137722/G-rs12150220/A-rs2670660/G resulted strongly associated to PB.NLRP1 might be involved in the susceptibility to leprosy with particular emphasis for PB clinical form. Although preliminary, this is the first report linking NLRPs and inflammasome with leprosy: replication studies as well as functional assays are envisaged to deeper investigate the role of NLRP1 in M. leprae infection. Interestingly, NLRP1 SNPs were previously associated to susceptibility to Crohn disease, suggesting that NLRP1 could be a new modifier gene in common between leprosy and Crohn disease.

Influence of Intron II microsatellite polymorphism in human toll-like receptor 2 gene in leprosy

Leprosy is a chronic granulomatous infection caused by the obligate intracellular organism Mycobacterium leprae. TLR2 plays a key role when activated by M. leprae lipoproteins initiating protective responses which induce bacterial killing and therefore control of disease spread. Microsatellite polymorphisms in intron2 of TLR2 gene have been reported to be associated with development of clinical features of several infectious diseases. The study aims to evaluate the influence of GT microsatellite on the expression of TLR2 which could make humans prone to M. leprae infections. A total of 279 individuals were enrolled in the study, 88 were leprosy patients, 95 were house hold contacts (HHC) and 96 were healthy controls (HC). Genotyping was done using PCR-Sequencing method. TLR2 mRNA expression was analyzed by RT-PCR. IL-10 and IFN-γ levels were measured using ELISA in MLSA stimulated cell culture supernatants. Statistical analysis was performed using Chi-Square (χ(2)) test and t-tests. Allele/genotype of TLR2 microsatellite which includes longer GT repeats was associated with low TLR2 mRNA expression and high IL-10 production while that including shorter GT repeats was associated with high TLR2 mRNA expression and low IL-10 production. High IL10 producing allele of TLR2 microsatellite might predispose house hold contacts to leprosy.

Challenges in immunodiagnostic tests for leprosy

Despite the effectiveness of multidrug therapy, leprosy still represents a significant global health problem: transmission of Mycobacterium leprae (M. leprae) is not sufficiently reduced as witnessed by unwavering new case rates in leprosy-endemic countries. Early detection of M. leprae infection (before clinical manifestations occur) is vital to reduction of transmission. Current diagnosis relies on detection of clinical signs since there are no tests available to detect asymptomatic M. leprae infection or predict progression to leprosy. Identification of risk factors (immunological or genetic biomarkers) for disease development and/or onset of leprosy reactions is imperative for efficient diagnosis. Tests simultaneously detecting biomarkers specific for cellular and humoral immunity are well suited for diagnosis of different clinical outcomes of leprosy. This review describes the challenges of discovery of biomarkers for M. leprae infection and their implementation in field-friendly tests. In view of the complicated nature of M. leprae infections, it is essential to invest in longitudinal studies allowing intra-individual comparison of immune and genetic biomarkers in various leprosy-endemic areas. Furthermore, the effect of co-infections on biomarker profiles should also be taken into account. Diagnostic tests based on such biomarkers can contribute significantly to early detection of leprosy (reactions) thus helping reduce nerve damage.

Biomarkers for Leprosy: would you prefer T (cells)?

The need for rapid diagnostic tests that can be applied in non-expert settings may now be greater than ever before, due to changes in leprosy control programmes and the decrease in special expertise required for (early) diagnosis of leprosy. However, there is no test available that can detect asymptomatic Mycobacterium leprae infection or predict progression of infection to clinical disease. The characteristics of the leprosy disease spectrum, in which both cellular and humoral immunity against M. leprae determine the outcome of infection, are pre-eminently suitable for development of tests that simultaneously detect biomarkers specific for both types of immune responses, thereby covering the complete immunological leprosy spectrum. Since publication of the M. leprae genome, many research groups have investigated the potential of M. leprae-unique antigens in serologic as well as T cell assays. Despite the early promise of the genome towards identifying antigens that induce M. leprae-specific T cells, it took more than a decade before promising candidates were identified. This review describes recent studies on M. leprae-specific T cell responses and discusses the application of measuring specific cellular mediated immune responses possibly combined with serologic responses in field-friendly tests for early diagnosis of M. leprae exposure and infection.

IL12B SNPs and copy number variation in IL23R gene associated with susceptibility to leprosy

BackgroundGenome-wide studies have identified both human leucocyte antigen (HLA) and non-HLA regions in association with leprosy. Involvement of novel functional loci within these regions has been proposed by us earlier.MethodsWe...

Clofazimine Modulates the Expression of Lipid Metabolism Proteins in Mycobacterium leprae-Infected Macrophages

Mycobacterium leprae (M. leprae) lives and replicates within macrophages in a foamy, lipid-laden phagosome. The lipids provide essential nutrition for the mycobacteria, and M. leprae infection modulates expression of important host proteins related to lipid metabolism. Thus, M. leprae infection increases the expression of adipophilin/adipose differentiation-related protein (ADRP) and decreases hormone-sensitive lipase (HSL), facilitating the accumulation and maintenance of lipid-rich environments suitable for the intracellular survival of M. leprae. HSL levels are not detectable in skin smear specimens taken from leprosy patients, but re-appear shortly after multidrug therapy (MDT). This study examined the effect of MDT components on host lipid metabolism in vitro, and the outcome of rifampicin, dapsone and clofazimine treatment on ADRP and HSL expression in THP-1 cells. Clofazimine attenuated the mRNA and protein levels of ADRP in M. leprae-infected cells, while those of HSL were increased. Rifampicin and dapsone did not show any significant effects on ADRP and HSL expression levels. A transient increase of interferon (IFN)-β and IFN-γ mRNA was also observed in cells infected with M. leprae and treated with clofazimine. Lipid droplets accumulated by M. leprae-infection were significantly decreased 48 h after clofazimine treatment. Such effects were not evident in cells without M. leprae infection. In clinical samples, ADRP expression was decreased and HSL expression was increased after treatment. These results suggest that clofazimine modulates lipid metabolism in M. leprae-infected macrophages by modulating the expression of ADRP and HSL. It also induces IFN production in M. leprae-infected cells. The resultant decrease in lipid accumulation, increase in lipolysis, and activation of innate immunity may be some of the key actions of clofazimine.

Mycobacterium leprae virulence-associated peptides are indicators of exposure to M. leprae in Brazil, Ethiopia and Nepal

Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.

Susceptibility to Leprosy is Associated with M-ficolin Polymorphisms

Mycobacterium leprae exploits complement activation and opsonophagocytosis to infect phagocytes. M-ficolin is encoded by the FCN1 gene and initiates the lectin pathway on monocyte surfaces. We investigated FCN1 promoter polymorphisms that could be responsible for the high interindividual variability of M-ficolin levels and for modulating leprosy susceptibility. We genotyped rs2989727 (-1981 G > A), rs28909068 (-791 G > A), rs10120023 (-542 G > A), rs17039495 (-399 G > A), rs28909976 (-271IndelT), rs10117466 (-144C > A) and rs10858293 (+33 T > G) in 400 controls and 315 leprosy patients from Southern Brazil, and in 296 Danish healthy individuals with known M-ficolin levels. Ten haplotypes were identified with sequence-specific PCR and/or haplotype-specific sequencing. We found evidence for a protective codominant additive effect of FCN1*-542A-144C with leprosy in Euro-Brazilians (P=0.003, PBf =0.021, OR=0.243 [CI95% =0.083-0.71]), which was independent of age, ethnic group and gender effects (P=0.029). There was a trend for a positive association of the -399A variant in Afro-Brazilians (P=0.022, PBf =0.154, OR=4.151 [CI95% =1.115-15.454], as well as for a negative association of the FCN1*3A haplotype with lepromatous leprosy, compared with less severe forms of the disease (P=0.016, PBf =0.112, OR=0.324 [CI95% =0.123-0.858]). Danish individuals with this haplotype presented M-ficolin levels higher than the population average of circa 1,000 ng/ml, and -542A-144C, which is able to modify the recognition of transcription factors in silico, occurred in individuals with levels under the 25 percentil (P=0.031). Our data provide the first evidence that FCN1 polymorphisms are associated with leprosy. M-ficolin may represent a novel key to understand the immunopathogenesis of M. leprae infection.