Epithelioid cell granuloma induction in the guinea pig by haptenated Mycobacterium leprae

Fluorescein isothiocyanate (FITC)-conjugated Mycobacterium leprae (FITC-M. leprae) was injected intradermally into the ears of guinea pigs and granuloma formation in the draining postauricular lymph nodes was studied. At 2 weeks, there was an increase in weights and infiltration of the draining lymph nodes in half of the animals injected with FITC-M. leprae. At 5 weeks, there was a significant increase in the weights and infiltration of these draining lymph nodes in the guinea pigs injected with haptenated M. leprae compared with those injected with unconjugated M. leprae. At 5 weeks, there was also a significant increase in delayed type hypersensitivity responses to 25 μg purified protein derivative. Histologically, epithelioid cell granulomas were seen in these lymph nodes as early as 2 weeks when FITC-M. leprae was used as the source of antigen. Enhancement in the immunogenicity of M. leprae by conjugation with FITC has been postulated.     

Mycobacterium leprae-Infected Macrophages Preferentially Primed Regulatory T Cell Responses and Was Associated with Lepromatous Leprosy

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.     

Profiling of Intracellular Metabolites: An Approach to Understanding the Characteristic Physiology of Mycobacterium leprae

Mycobacterium leprae is the causative agent of leprosy and also known to possess unique features such as inability to proliferate in vitro. Among the cellular components of M. leprae, various glycolipids present on the cell envelope are well characterized and some of them are identified to be pathogenic factors responsible for intracellular survival in host cells, while other intracellular metabolites, assumed to be associated with basic physiological feature, remain largely unknown. In the present study, to elucidate the comprehensive profile of intracellular metabolites, we performed the capillary electrophoresis-mass spectrometry (CE-MS) analysis on M. leprae and compared to that of M. bovis BCG. Interestingly, comparison of these two profiles showed that, in M. leprae, amino acids and their derivatives are significantly accumulated, but most of intermediates related to central carbon metabolism markedly decreased, implying that M. leprae possess unique metabolic features. The present study is the first report demonstrating the unique profiles of M. leprae metabolites and these insights might contribute to understanding undefined metabolism of M. leprae as well as pathogenic characteristics related to the manifestation of the disease.     

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.     

Carbohydrate-Dependent Binding of Langerin to SodC,a Cell Wall Glycoprotein of Mycobacterium leprae

Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [K_D] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (K_D = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells.     

Long-term Survival and Virulence of Mycobacterium leprae in Amoebal Cysts

Leprosy is a curable neglected disease of humans caused by Mycobacterium leprae that affects the skin and peripheral nerves and manifests clinically in various forms ranging from self-resolving, tuberculoid leprosy to lepromatous leprosy having significant pathology with ensuing disfiguration disability and social stigma. Despite the global success of multi-drug therapy (MDT), incidences of clinical leprosy have been observed in individuals with no apparent exposure to other cases, suggestive of possible non-human sources of the bacteria. In this study we show that common free-living amoebae (FLA) can phagocytose M. leprae, and allow the bacillus to remain viable for up to 8 months within amoebic cysts. Viable bacilli were extracted from separate encysted cocultures comprising three common Acanthamoeba spp.: A. lenticulata, A. castellanii, and A. polyphaga and two strains of Hartmannella vermiformis. Trophozoites of these common FLA take up M. leprae by phagocytosis. M. leprae from infected trophozoites induced to encyst for long-term storage of the bacilli emerged viable by assessment of membrane integrity. The majority (80%) of mice that were injected with bacilli extracted from 35 day cocultures of encysted/excysted A. castellanii and A. polyphaga showed lesion development that was similar to mice challenged with fresh M. leprae from passage mice albeit at a slower initial rate. Mice challenged with coculture-extracted bacilli showed evidence of acid-fast bacteria and positive PCR signal for M. leprae. These data support the conclusion that M. leprae can remain viable long-term in environmentally ubiquitous FLA and retain virulence as assessed in the nu/nu mouse model. Additionally, this work supports the idea that M. leprae might be sustained in the environment between hosts in FLA and such residence in FLA may provide a macrophage-like niche contributing to the higher-than-expected rate of leprosy transmission despite a significant decrease in human reservoirs due to MDT.     

Single Nucleotide Polymorphism Analysis of European Archaeological M. leprae DNA

Background

Leprosy was common in Europe eight to twelve centuries ago but molecular confirmation of this has been lacking. We have extracted M. leprae ancient DNA (aDNA) from medieval bones and single nucleotide polymorphism (SNP) typed the DNA, this provides insight into the pattern of leprosy transmission in Europe and may assist in the understanding of M. leprae evolution.

Methods and Findings

Skeletons have been exhumed from 3 European countries (the United Kingdom, Denmark and Croatia) and are dated around the medieval period (476 to 1350 A.D.). we tested for the presence of 3 previously identified single nucleotide polymorphisms (SNPs) in 10 aDNA extractions. M. leprae aDNA was extracted from 6 of the 10 bone samples. SNP analysis of these 6 extractions were compared to previously analysed European SNP data using the same PCR assays and were found to be the same. Testing for the presence of SNPs in M. leprae DNA extracted from ancient bone samples is a novel approach to analysing European M. leprae DNA and the findings concur with the previously published data that European M. leprae strains fall in to one group (SNP group 3).

Conclusions

These findings support the suggestion that the M. leprae genome is extremely stable and show that archaeological M. leprae DNA can be analysed to gain detailed information about the genotypic make-up of European leprosy, which may assist in the understanding of leprosy transmission worldwide.     

Contribution of GM-CSF on the enhancement of the T cell-stimulating activity of macrophages

Mycobacterium leprae is an intracellular parasitic organism that multiplies in macrophages (MØ). It inhibits the fusion of mycobacterial phagosome with lysosome and induces interleukin (IL)-10 production from macrophages. However, macrophages are heterogenous in various aspects. We examined macrophages that differentiated from monocytes using either recombinant (r) granulocyte-MØ colony-stimulating factor (GM-CSF) (these MØ are named as GM-MØ) or rMØ colony-stimulating factor (M-CSF) (cells named as M-MØ) in terms of the T cell-stimulating activity. Although both macrophages phagocytosed the mycobacteria equally, GM-MØ infected with M. leprae and subsequently treated with IFN-γ- and CD40 ligand (L) stimulated T cells to produce interferon-gamma (IFN-γ), but M-MØ lacked the ability to stimulate T cells. While M-MØ mounted a massive IL-10 production, GM-MØ did not produce the cytokine on infection with M. leprae. M. leprae-infected, IFN-γ- and CD40L-treated GM-MØ expressed a higher level of HLA-DR and CD86 Ags than those of M-MØ, and expressed one of the dominant antigenic molecules of M. leprae, Major Membrane Protein-II on their surface. These results indicate that GM-CSF, but not M-CSF, contributes to the up-regulation of the T cell-stimulating activity of M. leprae-infected macrophages.     

Detection of anti-M. leprae antibodies in children in leprosy-endemic areas: A systematic review

BackgroundLeprosy elimination primarily targets transmission of Mycobacterium leprae which is not restricted to patients’ households. As interruption of transmission is imminent in many countries, a test to detect infected asymptomatic individuals who can perpetuate transmission is required. Antibodies directed against M. leprae antigens are indicative of M. leprae infection but cannot discriminate between active and past infection. Seroprevalence in young children, however, reflects recent M. leprae infection and may thus be used to monitor transmission in an area. Therefore, this literature review aimed to evaluate what has been reported on serological tests measuring anti-M. leprae antibodies in children without leprosy below the age of 15 in leprosy-endemic areas.Methods and findingsA literature search was performed in the databases Pubmed, Infolep, Web of Science and The Virtual Health Library. From the 724 articles identified through the search criteria, 28 full-text articles fulfilled all inclusion criteria. Two additional papers were identified through snowballing, resulting in a total of 30 articles reporting data from ten countries. All serological tests measured antibodies against phenolic glycolipid-I or synthetic derivatives thereof, either quantitatively (ELISA or UCP-LFA) or qualitatively (ML-flow or NDO-LID rapid test). The median seroprevalence in children in endemic areas was 14.9% and was stable over time if disease incidence remained unchanged. Importantly, seroprevalence decreased with age, indicating that children are a suitable group for sensitive assessment of recent M. leprae infection. However, direct comparison between areas, solely based on the data reported in these studies, was impeded by the use of different tests and variable cut-off levels.ConclusionsQuantitative anti-PGL-I serology in young children holds promise as a screening test to assess M. leprae infection and may be applied as a proxy for transmission and thereby as a means to monitor the effect of (prophylactic) interventions on the route to leprosy elimination.     

Evolutionary history of Mycobacterium leprae in the Pacific Islands

As one of the oldest known human diseases, leprosy or Hansen''s disease remains a public health concern around the world with over 200 000 new cases in 2018. Most human leprosy cases are caused by Mycobacterium leprae, but a small number of cases are now known to be caused by Mycobacterium lepromatosis, a sister taxon of M. leprae. The global pattern of genomic variation in M. leprae is not well defined. Particularly, in the Pacific Islands, the origins of leprosy are disputed. Historically, it has been argued that leprosy arrived on the islands during nineteenth century colonialism, but some oral traditions and palaeopathological evidence suggest an older introduction. To address this, as well as investigate patterns of pathogen exchange across the Pacific Islands, we extracted DNA from 39 formalin-fixed paraffin-embedded biopsy blocks dating to 1992–2016. Using whole-genome enrichment and next-generation sequencing, we produced nine M. leprae genomes dating to 1998–2015 and ranging from 4-63× depth of coverage. Phylogenetic analyses indicate that these strains belong to basal lineages within the M. leprae phylogeny, specifically falling in branches 0 and 5. The phylogeographical patterning and evolutionary dating analysis of these strains support a pre-modern introduction of M. leprae into the Pacific Islands.This article is part of the theme issue ‘Insights into health and disease from ancient biomolecules’.     

Comparative Sequence Analysis of Mycobacterium leprae and the New Leprosy-Causing Mycobacterium lepromatosis

Mycobacterium lepromatosis is a newly discovered leprosy-causing organism. Preliminary phylogenetic analysis of its 16S rRNA gene and a few other gene segments revealed significant divergence from Mycobacterium leprae, a well-known cause of leprosy, that justifies the status of M. lepromatosis as a new species. In this study we analyzed the sequences of 20 genes and pseudogenes (22,814 nucleotides). Overall, the level of matching of these sequences with M. leprae sequences was 90.9%, which substantiated the species-level difference; the levels of matching for the 16S rRNA genes and 14 protein-encoding genes were 98.0% and 93.1%, respectively, but the level of matching for five pseudogenes was only 79.1%. Five conserved protein-encoding genes were selected to construct phylogenetic trees and to calculate the numbers of synonymous substitutions (dS values) and nonsynonymous substitutions (dN values) in the two species. Robust phylogenetic trees constructed using concatenated alignment of these genes placed M. lepromatosis and M. leprae in a tight cluster with long terminal branches, implying that the divergence occurred long ago. The dS and dN values were also much higher than those for other closest pairs of mycobacteria. The dS values were 14 to 28% of the dS values for M. leprae and Mycobacterium tuberculosis, a more divergent pair of species. These results thus indicate that M. lepromatosis and M. leprae diverged ∼10 million years ago. The M. lepromatosis pseudogenes analyzed that were also pseudogenes in M. leprae showed nearly neutral evolution, and their relative ages were similar to those of M. leprae pseudogenes, suggesting that they were pseudogenes before divergence. Taken together, the results described above indicate that M. lepromatosis and M. leprae diverged from a common ancestor after the massive gene inactivation event described previously for M. leprae.Leprosy, one of the oldest human diseases, remains a significant public health problem in many developing countries (8). Mycobacterium leprae was the only known cause of leprosy until recently, when a new mycobacterium, Mycobacterium lepromatosis, was found to be the cause of diffuse lepromatous leprosy (DLL), a unique form of leprosy endemic in Mexico and the Caribbean (17). The discovery of this new species may provide an explanation for the clinical and geographic variability of leprosy.The initial phylogenetic analysis of M. lepromatosis was carried out using the sequences of the 16S rRNA gene and segments of groEL, rpoB, and other genes (total, 4.99 kb) (17). This study revealed significant sequence differences between M. lepromatosis and all known Mycobacterium species and placed M. lepromatosis closest to M. leprae. However, the sequence variation justified assigning a new species for the new organism instead of classifying it as a variant of M. leprae. All M. leprae strains collected worldwide have been found to be clonal and to differ by only single-nucleotide polymorphism or variable numbers of tandem repeats (24). Also, the genomes of two M. leprae strains, strain TN from India (GenBank accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"AL583917 to AL583926","start_term":"AL583917","end_term":"AL583926","start_term_id":"13092412","end_term_id":"13093786"}}AL583917 to AL583926) (4) and strain Br4923 from Brazil (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"FM211192","term_id":"219932450","term_text":"FM211192"}}FM211192) (N. Honore et al., unpublished data) share 99.98% identity.Like M. leprae, M. lepromatosis has not been cultivated on artificial media. In addition, our previous study also showed other similarities between these organisms, such as degeneration of mmaA3 into a pseudogene, the presence of unique AT-rich inserted sequences in the 16S rRNA gene, identical six-base tandem repeats in rpoT, similar G+C contents, and great evolutionary distance from other mycobacteria (17).The M. leprae genome (3.3 Mb) is much smaller than the Mycobacterium tuberculosis genome (4.4 Mb) (3, 4). More intriguingly, the M. leprae genome has undergone reductive evolution; ∼40% of the genes are inactivated (4), and ∼50% of the genes of the last common ancestor of M. leprae and M. tuberculosis have been lost (13). On the other hand, the M. leprae genome has been far more stable than the M. tuberculosis genome, and the worldwide clonality of the M. leprae strains paralleled the global spread of M. leprae strains that occurred via human activity and migration during the last ∼100,000 years (24). Recently, by comparing the genomes of M. leprae and M. tuberculosis and by analyzing the ages of the M. leprae pseudogenes, Gomez-Valero et al. (13) estimated that a massive gene inactivation event took place in the M. leprae genome in the last 20 million years.The discovery of M. lepromatosis and its differences from M. leprae make it relevant for further study for diagnosis, treatment, and prevention of DLL. Likewise, the many similarities between these two organisms prompted questions about their evolutionary histories and about how M. lepromatosis became endemic mainly in Mexico, while M. leprae occurs worldwide. In this study, we extended and refined our previous phylogenetic study by determining and analyzing the sequences of 20 genes and pseudogenes of M. lepromatosis. Our findings solidified the phylogeny of this new organism and provided new insights into the history of pseudogenes.     

Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs

Zoonotic pathogens that cause leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts.     

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.     

T-Cell Regulation in Lepromatous Leprosy

Regulatory T (T_reg) cells are known for their role in maintaining self-tolerance and balancing immune reactions in autoimmune diseases and chronic infections. However, regulatory mechanisms can also lead to prolonged survival of pathogens in chronic infections like leprosy and tuberculosis (TB). Despite high humoral responses against Mycobacterium leprae (M. leprae), lepromatous leprosy (LL) patients have the characteristic inability to generate T helper 1 (Th1) responses against the bacterium. In this study, we investigated the unresponsiveness to M. leprae in peripheral blood mononuclear cells (PBMC) of LL patients by analysis of IFN-γ responses to M. leprae before and after depletion of CD25⁺ cells, by cell subsets analysis of PBMC and by immunohistochemistry of patients'' skin lesions. Depletion of CD25⁺ cells from total PBMC identified two groups of LL patients: 7/18 (38.8%) gained in vitro responsiveness towards M. leprae after depletion of CD25⁺ cells, which was reversed to M. leprae-specific T-cell unresponsiveness by addition of autologous CD25⁺ cells. In contrast, 11/18 (61.1%) remained anergic in the absence of CD25⁺ T-cells. For both groups mitogen-induced IFN-γ was, however, not affected by depletion of CD25⁺ cells. In M. leprae responding healthy controls, treated lepromatous leprosy (LL) and borderline tuberculoid leprosy (BT) patients, depletion of CD25⁺ cells only slightly increased the IFN-γ response. Furthermore, cell subset analysis showed significantly higher (p = 0.02) numbers of FoxP3⁺ CD8⁺CD25⁺ T-cells in LL compared to BT patients, whereas confocal microscopy of skin biopsies revealed increased numbers of CD68⁺CD163⁺ as well as FoxP3⁺ cells in lesions of LL compared to tuberculoid and borderline tuberculoid leprosy (TT/BT) lesions. Thus, these data show that CD25⁺ T_reg cells play a role in M. leprae-Th1 unresponsiveness in LL.     

Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis

Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator C_p variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.     

Field-Evaluation of a New Lateral Flow Assay for Detection of Cellular and Humoral Immunity against Mycobacterium leprae

Background

Field-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.

Methods

The 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.

Results

Single 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.

Conclusions

Dry-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.     

The armadillo: a model for the neuropathy of leprosy and potentially other neurodegenerative diseases

Leprosy (also known as Hansen’s disease) is an infectious peripheral neurological disorder caused by Mycobacterium leprae that even today leaves millions of individuals worldwide with life-long disabilities. The specific mechanisms by which this bacterium induces nerve injury remain largely unknown, mainly owing to ethical and practical limitations in obtaining affected human nerve samples. In addition to humans, nine-banded armadillos (Dasypus novemcinctus) are the only other natural host of M. leprae, and they develop a systemically disseminated disease with extensive neurological involvement. M. leprae is an obligate intracellular parasite that cannot be cultivated in vitro. Because of the heavy burdens of bacilli they harbor, nine-banded armadillos have become the organism of choice for propagating large quantities of M. leprae, and they are now advancing as models of leprosy pathogenesis and nerve damage. Although armadillos are exotic laboratory animals, the recently completed whole genome sequence for this animal is enabling researchers to undertake more sophisticated molecular studies and to develop armadillo-specific reagents. These advances will facilitate the use of armadillos in piloting new therapies and diagnostic regimens, and will provide new insights into the oldest known infectious neurodegenerative disorder.     

Human Genetic Ancestral Composition Correlates with the Origin of Mycobacterium leprae Strains in a Leprosy Endemic Population

Recent reports have suggested that leprosy originated in Africa, extended to Asia and Europe, and arrived in the Americas during European colonization and the African slave trade. Due to colonization, the contemporary Colombian population is an admixture of Native-American, European and African ancestries. Because microorganisms are known to accompany humans during migrations, patterns of human migration can be traced by examining genomic changes in associated microbes. The current study analyzed 118 leprosy cases and 116 unrelated controls from two Colombian regions endemic for leprosy (Atlantic and Andean) in order to determine possible associations of leprosy with patient ancestral background (determined using 36 ancestry informative markers), Mycobacterium leprae genotype and/or patient geographical origin. We found significant differences between ancestral genetic composition. European components were predominant in Andean populations. In contrast, African components were higher in the Atlantic region. M. leprae genotypes were then analyzed for cluster associations and compared with the ancestral composition of leprosy patients. Two M. leprae principal clusters were found: haplotypes C54 and T45. Haplotype C54 associated with African origin and was more frequent in patients from the Atlantic region with a high African component. In contrast, haplotype T45 associated with European origin and was more frequent in Andean patients with a higher European component. These results suggest that the human and M. leprae genomes have co-existed since the African and European origins of the disease, with leprosy ultimately arriving in Colombia during colonization. Distinct M. leprae strains followed European and African settlement in the country and can be detected in contemporary Colombian populations.