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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.

Background Acute inflammatory reactions are a frequently occurring, tissue destructing phenomenon in infectious- as well as autoimmune diseases, providing clinical challenges for early diagnosis. In leprosy, an infectious disease initiated by Mycobacterium leprae (M. leprae), these reactions represent the major cause of permanent neuropathy. However, laboratory tests for early diagnosis of reactional episodes which would significantly contribute to prevention of tissue damage are not yet available. Although classical diagnostics involve a variety of tests, current research utilizes limited approaches for biomarker identification. In this study, we therefore studied leprosy as a model to identify biomarkers specific for inflammatory reactional episodes. Methods To identify host biomarker profiles associated with early onset of type 1 leprosy reactions, prospective cohorts including leprosy patients with and without reactions were recruited in Bangladesh, Brazil, Ethiopia and Nepal. The presence of multiple cyto-/chemokines induced by M. leprae antigen stimulation of peripheral blood mononuclear cells as well as the levels of antibodies directed against M. leprae-specific antigens in sera, were measured longitudinally in patients. Results At all sites, longitudinal analyses showed that IFN-γ-, IP-10-, IL-17- and VEGF-production by M. leprae (antigen)-stimulated PBMC peaked at diagnosis of type 1 reactions, compared to when reactions were absent. In contrast, IL-10 production decreased during type 1 reaction while increasing after treatment. Thus, ratios of these pro-inflammatory cytokines versus IL-10 provide useful tools for early diagnosing type 1 reactions and evaluating treatment. Of further importance for rapid diagnosis, circulating IP-10 in sera were significantly increased during type 1 reactions. On the other hand, humoral immunity, characterized by M. leprae-specific antibody detection, did not identify onset of type 1 reactions, but allowed treatment monitoring instead. Conclusions This study identifies immune-profiles as promising host biomarkers for detecting intra-individual changes during acute inflammation in leprosy, also providing an approach for other chronic (infectious) diseases to help early diagnose these episodes and contribute to timely treatment and prevention of tissue damage. Electronic supplementary material The online version of this article (doi:10.1186/s12879-015-1128-0) contains supplementary material, which is available to authorized users.

Background New tools are required for the diagnosis of pre-symptomatic leprosy towards further reduction of disease burden and its associated reactions. To address this need, two new skin test antigens were developed to assess safety and efficacy in human trials. Methods A Phase I safety trial was first conducted in a non-endemic region for leprosy (U.S.A.). Healthy non-exposed subjects (n = 10) received three titrated doses (2.5 µg, 1.0 µg and 0.1 µg) of MLSA-LAM (n = 5) or MLCwA (n = 5) and control antigens [Rees MLSA (1.0 µg) and saline]. A randomized double blind Phase II safety and efficacy trial followed in an endemic region for leprosy (Nepal), but involved only the 1.0 µg (high dose) and 0.1 µg (low dose) of each antigen; Tuberculin PPD served as a control antigen. This Phase II safety and efficacy trial consisted of three Stages: Stage A and B studies were an expansion of Phase I involving 10 and 90 subjects respectively, and Stage C was then conducted in two parts (high dose and low dose), each enrolling 80 participants: 20 borderline lepromatous/lepromatous (BL/LL) leprosy patients, 20 borderline tuberculoid/tuberculoid (BT/TT) leprosy patients, 20 household contacts of leprosy patients (HC), and 20 tuberculosis (TB) patients. The primary outcome measure for the skin test was delayed type hypersensitivity induration. Findings In the small Phase I safety trial, reactions were primarily against the 2.5 µg dose of both antigens and Rees control antigen, which were then excluded from subsequent studies. In the Phase II, Stage A/B ramped-up safety study, 26% of subjects (13 of 50) showed induration against the high dose of each antigen, and 4% (2 of 50) reacted to the low dose of MLSA-LAM. Phase II, Stage C safety and initial efficacy trial showed that both antigens at the low dose exhibited low sensitivity at 20% and 25% in BT/TT leprosy patients, but high specificity at 100% and 95% compared to TB patients. The high dose of both antigens showed lower specificity (70% and 60%) and sensitivity (10% and 15%). BL/LL leprosy patients were anergic to the leprosy antigens. Interpretation MLSA-LAM and MLCwA at both high (1.0 µg) and low (0.1 µg) doses were found to be safe for use in humans without known exposure to leprosy and in target populations. At a sensitivity rate of 20–25% these antigens are not suitable as a skin test for the detection of the early stages of leprosy infection; however, the degree of specificity is impressive given the presence of cross-reactive antigens in these complex native M. leprae preparations. Trial Registration ClinicalTrails.gov NCT01920750 (Phase I), NCT00128193 (Phase II), Author Summary Clinically useful skin test reagents should be safe and sufficiently sensitive to detect infection prior to physical manifestations of leprosy disease. While in these small scale human studies, leprosy reagents were safe for use in humans, they failed in respect of sensitivity at a rate of 20–25% in the key indicator group, BT/TT leprosy patients. Specificity in terms of leprosy vs. tuberculosis at a rate of 95–100% was surprisingly high in light of the extensive presence of cross-reactive antigens in the complex native M. leprae preparations. These results could justify a further trial at lower dosages.

Leprosy care has been integrated with peripheral health services, away from vertical programmes. This includes the diagnosis and management of leprosy reactions, which cause significant morbidity. We surveyed patients with leprosy reactions at two leprosy hospitals in Nepal to assess their experience of leprosy reaction management following integration to identify any gaps in service delivery. Methods Direct and referral patients with leprosy reactions were interviewed in two of Nepal's leprosy hospitals. We also collected quantitative and qualitative data from clinical examination and case-note review to document the patient pathway. Results Seventy-five patients were interviewed. On development of reaction symptoms 39% presented directly to specialist services, 23% to a private doctor, 17% to a district hospital, 10% to a traditional healer, 7% to a health post and 4% elsewhere. Those who presented directly to specialist services were 6.6 times more likely to start appropriate treatment than those presenting elsewhere (95% CI: 3.01 to 14.45). The average delay between symptom onset to commencing corticosteroids was 2.9 months (range 0–24 months). Obstacles to early presentation and treatment included diagnostic challenge, patients' lack of knowledge and the patients' view of health as a low priority. 40% received corticosteroids for longer than 12 weeks and 72% required an inpatient stay. Treatment follow-up was conducted at locations ranging from health posts to specialist hospitals. Inconsistency in the availability of corticosteroids peripherally was identified and 41% of patients treated for leprosy and a reaction on an outpatient basis attended multiple sites for follow-up treatment. Conclusion This study demonstrates that specialist services are necessary and continue to provide significant critical support within an integrated health system approach towards the diagnosis and management of leprosy reactions., Author Summary The global strategy for leprosy has moved patient care to general health services with the aim of improving access to treatment. We suspected that leprosy patients with a common complication, leprosy reactions, are not being diagnosed and treated promptly in integrated services. Leprosy reactions cause nerve damage and, if not treated early, can cause significant disability. We interviewed 75 patients with a leprosy reaction in Nepal, a country with a fully-integrated leprosy service. Patients were experiencing average delays of 2.9 months between developing leprosy reactions and starting treatment. Many required extended courses of treatment, an inpatient stay or experienced a recurrence of their reaction. Patients also continue to attend specialist services for both diagnosis and follow-up. Patient care could be improved by utilising specialist knowledge for training and the management of complex cases. Health care workers and patients need to be educated about leprosy reactions. A wider implication of the study is that health policy-makers need to be cautious not to over-simplify medical conditions when restructuring services.

Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR–high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1 , rpoB , and gyrA , targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1 , two rpoB , and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (

Exposure to Mycobacterium tuberculosis produces varied early outcomes, ranging from resistance to infection to progressive disease. Here we report results from a forward genetic screen in zebrafish larvae that identify multiple mutant classes with distinct patterns of innate susceptibility to Mycobacterium marinum. A hypersusceptible mutant maps to the lta4h locus encoding leukotriene A(4) hydrolase, which catalyzes the final step in the synthesis of leukotriene B(4) (LTB(4)), a potent chemoattractant and proinflammatory eicosanoid. lta4h mutations confer hypersusceptibility independent of LTB(4) reduction, by redirecting eicosanoid substrates to anti-inflammatory lipoxins. The resultant anti-inflammatory state permits increased mycobacterial proliferation by limiting production of tumor necrosis factor. In humans, we find that protection from both tuberculosis and multibacillary leprosy is associated with heterozygosity for LTA4H polymorphisms that have previously been correlated with differential LTB(4) production. Our results suggest conserved roles for balanced eicosanoid production in vertebrate resistance to mycobacterial infection.

Meeting abstracts High-fat diets and chronic alcohol consumption (CAC) can both induce a low-grade inflammation. We report that when CAC (16.6% of total calories) is administered in combination with the Lieber-DeCarli high-fat diet an additive myeloid response is induced. This increase in