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Mycobacterium leprae, the etiologic agent of leprosy, cannot be cultured on artificial media. This characteristic, coupled with its long generation time, presents a number of unique challenges to studying this pathogen. One of the difficulties facing both researchers and clinicians is the absence of a rapid test to measure the viability of M. leprae in clinical or experimental specimens. The lack of such a tool limits the understanding of M. leprae immunopathogenesis and makes determining the efficacy of drug treatments difficult. With this in mind, we developed a robust two-step molecular viability assay (MVA) that first enumerates the M. leprae in the tissue; then, this data is used to normalize bacterial RNA quantities for the second step, in which the expression of M. leprae esxA and hsp18 are measured. This assay is specific and sensitive enough to be used on most clinical samples. This protocol describes the steps required to extract DNA and RNA from M. leprae-infected tissue, enumerate M. leprae, and measure M. leprae viability based on the normalized expression of two M. leprae-specific genes (hsp18 and esxA). This protocol also outlines an optimal laboratory design and workflow for performing this assay. © 2022 The Leprosy Mission Nepal. Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: DNA and RNA P purification from M. leprae-infected tissue Basic Protocol 2: Enumeration of M. leprae by RLEP qPCR on the DNA fraction Basic Protocol 3: Calculation of M. leprae per tissue and normalization of RNA Basic Protocol 4: Reverse-transcription of normalized RNA to generate cDNA Basic Protocol 5: Determination of M. leprae viability using HSP18 and ESXA qPCR on the cDNA Support Protocol 1: M. leprae qPCR primer/probe stock preparation Support Protocol 2: Preparation of plasmid stocks and standard curves.

Objectives To document serious adverse events (SAE) of prolonged prednisolone use in two double-blind randomized clinical trials (RCT). In the first RCT, treatment effectiveness on restoring nerve function of a 20-week prednisolone regimen was compared with a 32-week course. In the second RCT, patients with subclinical neuropathy received either 20 weeks prednisolone or placebo to assess whether prednisolone could prevent the development of clinical neuropathy. In both trials, patients received a prednisolone starting dose according to weight: high weight (≥50 kg) started at 60 mg and low weight (

Mycobacterium leprae is a slow-growing species of mycobacteria that cannot be cultured in axenic media. This presents a number of challenges for monitoring treatment efficacy and advancing new drugs and regimens for treating leprosy. We previously developed a molecular viability assay (MVA) which measures expression of hsp18 and esxA transcripts to determine viability of M. leprae directly from infected tissue. The objective of the current study was to determine the utility of the MVA for practical use on clinical specimens. Leprosy cases from the Philippines (N = 199), Ethiopia (N = 40), and Nepal (N = 200) were diagnosed by clinical examination, slit-skin smears (SSS) from index sites, and/or histopathology. Biopsy specimens for MVA were collected from an active lesion and stored in 70% ethanol. DNA and RNA were extracted from the tissue, and M. leprae were enumerated on the DNA fraction via RLEP qPCR. Based on this count, DNased RNA was normalized to the equivalent of 3x103M. leprae per reverse transcription reaction, and hsp18 and esxA transcripts were amplified by PCR on the resulting cDNA. There was a strong correlation between RLEP enumeration on the specific biopsy specimen for MVA and the average SSS bacterial index (BI) in all three cohorts (p < 0.001). The MVA could be performed on most biopsies with an average SSS BI ≥ 2 and showed a decrease in M. leprae viability with increasing duration of leprosy multidrug therapy (R2 = 0.81, p < 0.001). The MVA also detected viable M. leprae in relapse patients where it showed significant correlation with the mouse footpad assay (p = 0.018). The MVA is a M. leprae-specific, sensitive, and relatively quick test. Clinically, the MVA would likely be most useful to monitor treatment, confirm suspected relapse cases, and determine efficacy of new leprosy drugs in clinical trials.

T cell receptors (TCRs) encode the history of antigenic challenge within an individual and have the potential to serve as molecular markers of infection. In addition to peptide antigens bound to highly polymorphic MHC molecules, T cells have also evolved to recognize bacterial lipids when bound to non-polymorphic CD1 molecules. One such subset, germline-encoded, mycolyl lipid-reactive (GEM) T cells, recognizes mycobacterial cell wall lipids and expresses a conserved TCR-ɑ chain that is shared among genetically unrelated individuals. We developed a quantitative PCR assay to determine expression of the GEM TCR-ɑ nucleotide sequence in human tissues and blood. This assay was validated on plasmids and T cell lines. We tested blood samples from South African subjects with or without tuberculin reactivity or with active tuberculosis disease. We were able to detect GEM TCR-ɑ above the limit of detection in 92% of donors but found no difference in GEM TCR-ɑ expression among the three groups after normalizing for total TCR-ɑ expression. In a cohort of leprosy patients from Nepal, we successfully detected GEM TCR-ɑ in 100% of skin biopsies with histologically confirmed tuberculoid and lepromatous leprosy. Thus, GEM T cells constitute part of the T cell repertoire in the skin. However, GEM TCR-ɑ expression was not different between leprosy patients and control subjects after normalization. Further, these results reveal the feasibility of developing a simple, field deployable molecular diagnostic based on mycobacterial lipid antigen-specific TCR sequences that are readily detectable in human tissues and blood independent of genetic background., Author summary Our T cell receptor repertoires are unique to each individual and encode the history of our antigenic exposures. Because of this property, they can act as markers of infection. Due to the diversity of T cell receptors, analyzing an individual’s repertoire requires advanced sequencing and machine learning methods. However, there are also subsets of T cells that express conserved T cell receptors shared among genetically unrelated individuals. One such subset, germline-encoded, mycolyl lipid-reactive (GEM) T cells, recognizes mycobacterial cell wall lipids. To determine if we could measure expression of these T cells with a simple assay, we developed a quantitative real-time PCR assay that was validated on plasmids and T cell lines. We then determined whether we could detect the GEM T cell receptor sequence in individuals with or without exposure to Mycobacterium tuberculosis or Mycobacterium leprae. We found that we could detect the GEM T cell receptor in the blood or skin of most individuals, though expression levels did not distinguish between uninfected or infected states. These data show that simple assays to measure mycobacteria-specific T cell receptor sequences are feasible and further advancements focusing on more disease-specific T cell receptors may allow for development of field-friendly diagnostics.

Background Leprosy is curable with multidrug therapy and treatment in the early stages can prevent disability. However, local nerve damage can lead to injury and consequently recurring and disfiguring ulcers. The aim of this study is to evaluate the treatment of leprosy ulcers using an autologous blood product; leukocyte and platelet-rich fibrin (L-PRF) to promote healing. Methods This is a single-centre study in the Anandaban Hospital, The Leprosy Mission Nepal, Kathmandu, Nepal. Consenting patients (n=130) will be individually randomised in a single-blinded, controlled trial. Participants will be 18 years of age or older, admitted to the hospital with a clean, dry and infection-free chronic foot ulcer between 2 and 20 cm2 in size. If the ulcer is infected, it will be treated before enrolment into the study. The intervention involves the application of leukocyte and platelet-rich fibrin (L-PRF) matrix on the ulcer beds during twice-weekly dressing changes. Controls receive usual care in the form of saline dressings only during their twice-weekly dressing changes. Primary outcomes are the rate of healing assessed using standardised photographs by observers blind to allocated treatment, and time to complete re-epithelialization. Follow-up is at 6 months from randomisation. Discussion This research will provide valuable information on the clinical and cost-effectiveness of L-PRF in the treatment of leprosy ulcers. An additional benefit is the evaluation of the effects of treatment on quality of life for people living with leprosy ulcers. The results will improve our understanding of the scalability of this treatment across low-income countries for ulcer healing in leprosy and potentially other conditions such as diabetic ulcers. Trial registration ISRCTN14933421. Registered on 16 June 2020

Background Subclinical infection with Mycobacterium leprae is one potential source of leprosy transmission, and post-exposure prophylaxis (PEP) regimens have been proposed to control this source. Because PEP trials require considerable investment, we applied a sensitive variation of the kinetic mouse footpad (MFP) screening assay to aid in the choice of drugs and regimens for clinical trials. Methodology/Principal findings Athymic nude mice were inoculated in the footpad (FP) with 6 x 103 viable M. leprae and treated by gastric gavage with a single dose of Rifampin (SDR), Rifampin + Ofloxacin + Minocycline (SD-ROM), or Rifapentine + Minocycline + Moxifloxacin (SD-PMM) or with the proposed PEP++ regimen of three once-monthly doses of Rifampin + Moxifloxacin (RM), Rifampin + Clarithromycin (RC), Rifapentine + Moxifloxacin (PM), or Rifapentine + Clarithromycin (PC). At various times post-treatment, DNA was purified from the FP, and M. leprae were enumerated by RLEP quantitative PCR. A regression analysis was calculated to determine the expected RLEP value if 99.9% of the bacilli were killed after the administration of each regimen. SDR and SD-ROM induced little growth delay in this highly susceptible murine model of subclinical infection. In contrast, SD-PMM delayed measurable M. leprae growth above the inoculum by 8 months. The four multi-dose regimens delayed bacterial growth for >9months post-treatment cessation. Conclusions/Significance The delay in discernable M. leprae growth post-treatment was an excellent indicator of drug efficacy for both early (3–4 months) and late (8–9 months) drug efficacy. Our data indicates that multi-dose PEP may be required to control infection in highly susceptible individuals with subclinical leprosy to prevent disease and decrease transmission., Author summary While multi-drug therapy (MDT) has been successful in decreasing the worldwide prevalence of leprosy, the new case detection rate, or incidence, remains consistent. These circumstances indicate that leprosy transmission is still occurring. Subclinical asymptomatic leprosy infections are considered a leading cause of ongoing transmission. One means to control this source is an effective post-exposure prophylaxis (PEP) regimen that would prevent both subsequent progression to clinical leprosy for the individual and transmission of the disease to others. Therefore, in this study, we used a modified kinetic mouse footpad screening assay and sensitive molecular bacterial enumeration in a susceptible-subclinical mouse model to identify effective potential PEP drug regimens for leprosy. Using these methods, we showed that a single dose PEP regimen is not effective in a susceptible host, and multiple intermittent doses of combination therapies are required. This model could provide useful pre-clinical information for the development of PEP regimens for leprosy.

Early diagnosis of leprosy is challenging, particularly its inflammatory reactions, the major cause of irreversible neuropathy in leprosy. Current diagnostics cannot identify which patients are at risk of developing reactions. This study assessed blood RNA expression levels as potential biomarkers for leprosy. Prospective cohorts of newly diagnosed leprosy patients, including reactions, and healthy controls were recruited in Bangladesh, Brazil, Ethiopia and Nepal. RNA expression in 1,090 whole blood samples was determined for 103 target genes for innate and adaptive immune profiling by dual color Reverse-Transcription Multiplex Ligation-dependent Probe Amplification (dcRT-MLPA) followed by cluster analysis. We identified transcriptomic biomarkers associated with leprosy disease, different leprosy phenotypes as well as high exposure to Mycobacterium leprae which respectively allow improved diagnosis and classification of leprosy patients and detection of infection. Importantly, a transcriptomic signature of risk for reversal reactions consisting of five genes (CCL2, CD8A, IL2, IL15 and MARCO) was identified based on cross-sectional comparison of RNA expression. In addition, intra-individual longitudinal analyses of leprosy patients before, during and after treatment of reversal reactions, indicated that several IFN-induced genes increased significantly at onset of reaction whereas IL15 decreased. This multi-site study, situated in four leprosy endemic areas, demonstrates the potential of host transcriptomic biomarkers as correlates of risk for leprosy. Importantly, a prospective five-gene signature for reversal reactions could predict reversal reactions at least 2 weeks before onset. Thus, transcriptomic biomarkers provide promise for early detection of these acute inflammatory episodes and thereby help prevent permanent neuropathy and disability in leprosy patients.

Reversal reactions (RRs) in leprosy are characterized by a reduction in the number of bacilli in lesions associated with an increase in cell-mediated immunity against the intracellular bacterium Mycobacterium leprae, the causative pathogen of leprosy. To identify the mechanisms that contribute to cell-mediated immunity in leprosy, we measured changes in the whole blood-derived transcriptome of patients with leprosy before, during and after RR. We identified an ‘RR signature’ of 1017 genes that were upregulated at the time of the clinical diagnosis of RR. Using weighted gene correlated network analysis (WGCNA), we detected a module of 794 genes, bisque4, that was significantly correlated with RR, of which 434 genes were part of the RR signature. An enrichment for both IFN-γ and IFN-β downstream gene pathways was present in the RR signature as well as the RR upregulated genes in the bisque4 module, including those encoding proteins of the guanylate binding protein (GBP) family that contributes to antimicrobial responses against mycobacteria. Specifically, GBP1, GBP2, GBP3 and GBP5 mRNAs were upregulated in the RR peripheral blood transcriptome, with GBP1, GBP2 and GBP5 mRNAs also upregulated in the RR disease lesion transcriptome. These data indicate that RRs involve a systemic upregulation of IFN-γ downstream genes including GBP family members as part of the host antimicrobial response against mycobacteria., Author summary Reversal reaction (RR) is a major cause of tissue injury and disabilities in leprosy, resulting from the rapid onset of cell-mediated immune responses to the intracellular bacterium Mycobacterium leprae. To identify the mechanisms related to the increase in cell-mediated immunity in RR, we measured changes in the whole blood-derived transcriptome of patients with leprosy before, during and after RR. We identified that RRs are associated with an IFN-γ induced inflammatory response including an antimicrobial gene network containing the GBP1, GBP2, GBP3 and GBP5 mRNAs in the peripheral blood. Furthermore, we show that GBP1, GBP2 and GBP5 mRNAs are also upregulated at the site of disease in RR patients. In summary, our study suggests that RR involves a systemic induction of IFN-γ regulated genes that contributes to an antimicrobial response against the pathogen, releasing ligands and antigens that can further amplify the inflammatory response.

IntroductionErythema nodosum leprosum (ENL) is an immunological complication of leprosy. ENL results in morbidity and disability and if it is not treated can lead to death. The current treatment consists of thalidomide or high doses of oral corticosteroids for prolonged periods. Thalidomide is not available in many leprosy endemic countries. The use of corticosteroids is associated with morbidity and mortality. Identifying treatment regimens that reduce the use of corticosteroids in ENL is essential. Methotrexate (MTX) is used to treat many inflammatory diseases and has been used successfully to treat patients with ENL not controlled by other drugs, including prednisolone and thalidomide. We present the protocol of the ‘MTX and prednisolone study in ENL’ (MaPs in ENL) a randomised controlled trial (RCT) designed to test the efficacy of MTX in the management of ENL.Methods and analysisMaPs in ENL is an international multicentre RCT, which will be conducted in leprosy referral centres in Bangladesh, Brazil, Ethiopia, India, Indonesia and Nepal. Patients diagnosed with ENL who consent to participate will be randomly allocated to receive 48 weeks of weekly oral MTX plus 20 weeks of prednisolone or 48 weeks of placebo plus 20 weeks of prednisolone. Participants will be stratified by type of ENL into those with acute ENL and those with chronic and recurrent ENL. The primary objective is to determine whether MTX reduces the requirement for additional prednisolone. Patients’ reported outcome measures will be used to assess the efficacy of MTX. Participants will be closely monitored for adverse events.Ethics and disseminationResults will be submitted for publication in peer-reviewed journals. Ethical approval was obtained from the Observational/Interventions Research Ethics Committee of the London School of Hygiene & Tropical Medicine (15762); The Leprosy Mission International Bangladesh Institutional Research Board (in process); AHRI-ALERT Ethical Review Committee, Ethiopia; Ethics Committee of the Managing Committee of the Bombay Leprosy Project; and The Leprosy Mission Trust India Ethics Committee; the Nepal Health and Research Council and Health Research Ethics Committee Dr. Soetomo, Indonesia. This study is registered at www.clinicaltrials.gov. This is the first RCT of MTX for ENL and will contribute to the evidence for the management of ENL.Trial registration numberNCT 03775460.

Objectives Neuropathic pain (NP) can occur as a chronic complication of leprosy neuropathy. NP epidemiology and its impact on patients have not been well documented. This study investigates NP prevalence and impact in the years after patients are declared “released from treatment” (RFT) following multidrug therapy (MDT) completion. Methods In this cross-sectional study, 85 RFT patients were recruited within leprosy referral services in Nepal. The Douleur Neuropathique 4 Questionnaire (DN4) was used to screen for NP. Pain severity, impacts on patients’ daily activities and mental health were measured by using the Brief Pain Inventory (BPI), Screening of Activity Limitation and Safety Awareness (SALSA), and General Health Questionnaire-12 (GHQ-12) respectively. Results 96% surveyed had been treated for multibacillary leprosy. 44 (52%) complained of pain of which 30 (68%) were diagnosed with NP. NP was not associated with age, gender, or presence of skin lesions or nerve symptoms at leprosy diagnosis. 70% of patients with NP had either history of or ongoing reactions and 47% had grade 2 disability. Nerve tenderness (p = 0.023) and current reactions (p = 0.018) were significant risk factors for NP. Patients with NP suffered significantly higher intensity pain (p = 0.023) and daily life interference (p = 0.003) and were more likely to have moderate to extreme daily activity limitations (p = 0.005). 13 (43%) exhibited psychological distress, and medications only reduced moderate degree (50–60%) of pain. Conclusions In our study, 35% of RFT patients had ongoing NP. Risk factors include nerve tenderness and reaction. They suffer from more daily life interference and psychological distress. Leprosy patient care should include recognition and management of NP., Author summary Leprosy could present with chronic neuropathic pain, which might severely disturb daily life and mental health of leprosy-infected patients. This complication could also occur even years after antibiotics therapy completion, and we noticed that it is associated with recent leprosy reactions and nerve tenderness on physical examination. This is the first study that describes the epidemiology of neuropathic pain in leprosy patients in Nepal. More than one-third of leprosy patients who had completed multidrug therapy, are still suffering from neuropathic pain, and this study demonstrated that it has caused a great impact to their daily activities and psychological status. Therefore, prompt recognition and management of neuropathic pain are mandatory while taking care of the patients with leprosy.

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: Leprosy is complicated by immunological reactions which can occur before, during and after successful completion of multidrug therapy. Genetic studies have suggested that polymorphisms in toll-like receptors (TLRs) may affect the susceptibility of an individual with leprosy to developing Type 1 reactions. OBJECTIVES: To examine the gene and protein expression of TLRs in the cutaneous lesions of leprosy Type 1 reactions at the onset of reaction and during systemic corticosteroid therapy. METHODS: Patients who were being treated for leprosy type 1 reactions with corticosteroids as part of a randomized controlled trial of corticosteroid treatment had skin biopsies performed before, during and at the end of treatment. The gene and protein expression of TLR2 and TLR4 were measured. RESULTS: We have demonstrated that the gene hARP-P0 is a suitable control gene for TLR gene expression studies in this population. The gene and protein expression of TLR2 and TLR4 were both reduced significantly during corticosteroid treatment. CONCLUSIONS: This is the first study to examine the expression of TLR2 and TLR4 in vivo in individuals experiencing leprosy Type 1 reactions. The data support the possibility of an important role for TLR2 and TLR4 in the pathogenesis of this important complication of leprosy.

Objectives To determine whether the measured change in score of a validated clinical severity scale reflected physician assessed improvement in individuals who had received corticosteroid therapy for leprosy associated nerve damage. Design Patients with nerve function impairment who participated in a randomised controlled trial of corticosteroids were classified into two groups using a retrospectively determined physician assessment of improvement. One group consisted of patients who had recovered or improved the other of patients who were unchanged or had deteriorated. The change in the clinical severity scale scores of these two groups was compared. Results The change in the clinical severity scale scores of the 34 eligible individuals in the two groups were significantly different (P = 0.003). Individuals in the group who recovered or improved had a greater change in severity score than those whose nerve function was unchanged or deteriorated. Conclusion The scale for measuring the severity of leprosy Type 1 reactions (T1Rs) and/or nerve function impairment reflects the clinical improvement of individuals with leprosy associated nerve damage.

Ongoing transmission of leprosy is evident from the stable disease incidence in high burden areas. Tools for early detection of Mycobacterium leprae (M. leprae) infection, particularly in sub-clinically infected individuals, are urgently required to reduce transmission. Following the sequencing of the M. leprae genome, many M. leprae-unique candidate proteins have been identified, several of which have been tested for induction of M. leprae specific T cell responses in different leprosy endemic areas. In this study, 21 M. leprae-unique proteins and 10 peptide pools covering the complete sequence of five M. leprae-unique proteins (ML0576, ML1989, ML1990, ML2283, and ML2567) were evaluated in 160 individuals in Nepal and Ethiopia. These included: tuberculoid and borderline tuberculoid (TT/BT), borderline borderline and borderline lepromatous (BB/BL) leprosy patients; healthy household contacts (HHC); tuberculosis (TB) patients and endemic controls (EC). Immunogenicity of the proteins was determined by IFN-gamma secretion via stimulation of PBMC in 6 days lymphocyte stimulation tests (LST) or in whole blood assays (WBA). In LST, BB/BL patients (40%) responded to ML0573 and ML1601 whereas ML1604 was most immunogenic in TT/BT (35%) and HHC (36%). Additionally, significant numbers of EC displayed IFN-gamma production in response to ML0573 (54%), ML1601 (50%) and ML1604 (54%). TB patients on the other hand, hardly responded to any of the proteins except for ML1989. Comparison of IFN-gamma responses to ML0121, ML0141 and ML0188 for TT/BT patients showed specific increase in diluted 6 days WBA compared to the undiluted 24 hours WBA, whereas EC showed a reduced response in the diluted WBA, which may indicate detection of disease-specific responses in the 6 days WBA. In summary, identification of multiple M. leprae proteins inducing M. leprae-specific T cell responses in groups at high risk of developing leprosy may contribute to improve early detection for M. leprae infection.

Nepal is geographically located between India and China, a region containing significant Tuberculosis (TB) and Multi-Drug Resistance (MDR-TB) burdens. However, limited information is available on the phylogenetic diversity of Mycobacterium tuberculosis (Mtb) in Nepal. To gain further insight into the diversity of Mtb in Nepal, consecutive clinical samples from 176 newly diagnosed pulmonary tuberculosis patients were collected from two hospitals in Nepal. Insertion Site IS6110 Fluorescent Amplified Fragment Length Polymorphism (FAFLP) PCR and rpoB sequence analysis were carried out on genomic DNA extracts of cultured strains to assign them to accepted genetic lineages and identify MDR-TB. In this study, the IS6110 based characterisation showed a prevalence of 36.36% Central Asian Strain (CAS), 18.75% Beijing, 7.95 % Haarlem, 3.97% X, 2.2% each of Latin American Mediterranean (LAM), T-Uganda and T, 1.7% S and 24.4% were unassigned. Further, 3.9% of total M. tuberculosis isolates were of rifampicin resistant genotypes thus indicating that the prevalence of MDR could be higher than the country wide prevalence of MDR among new TB cases (2.2%) as reported by the national drug resistance survey carried out in 2011/2012.

Leprosy is a chronic disease characterized by skin and peripheral nerve pathology and immune responses that fail to control Mycobacterium leprae. Toll-interacting protein (TOLLIP) regulates Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) signaling against mycobacteria. We analyzed messenger RNA (mRNA) expression of candidate immune genes in skin biopsy specimens from 85 individuals with leprosy. TOLLIP mRNA was highly and specifically correlated with IL-1R antagonist (IL-1Ra). In a case-control gene-association study with 477 cases and 1021 controls in Nepal, TOLLIP single-nucleotide polymorphism rs3793964 TT genotype was associated with increased susceptibility to leprosy (recessive, P = 1.4 × 10(-3)) and with increased skin expression of TOLLIP and IL-1Ra. Stimulation of TOLLIP-deficient monocytes with M. leprae produced significantly less IL-1Ra (P < .001), compared with control. These data suggest that M. leprae upregulates IL-1Ra by a TOLLIP-dependent mechanism. Inhibition of TOLLIP may decrease an individual's susceptibility to leprosy and offer a novel therapeutic target for IL-1-dependent diseases.

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.

Regulatory T (Treg) 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+ Treg cells play a role in M. leprae-Th1 unresponsiveness in LL., Author Summary Leprosy is a curable infectious disease caused by Mycobacterium leprae (M. leprae) that affects the skin and peripheral nerves. It is manifested in different forms ranging from self-healing, tuberculoid leprosy (TT) with low bacillary load and high cellular immunity against M. leprae, to lepromatous leprosy (LL) with high bacillary load and high antibody titers to M. leprae antigens. However, LL patients have poor cell mediated response against M. leprae leading to delayed clearance of the bacilli. A possible explanation for this bacterial persistence could lie in the presence of more regulatory cells at infection sites and in peripheral blood. This study shows the recovery of the cell mediated response by depletion of CD25+ cells in a subset of LL patients, while another patient subset was not affected similarly. Moreover, an increased frequency of FoxP3+ T cells together with anti-inflammatory macrophages was observed in LL patients' skin biopsies. Thus, these data show that CD25+ Treg cells play a role in M. leprae-unresponsiveness in leprosy patients.

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.

A variety of host immunogenetic factors appear to influence both an individual's susceptibility to infection with Mycobacterium leprae and the pathologic course of the disease. Animal models can contribute to a better understanding of the role of immunogenetics in leprosy through comparative studies helping to confirm the significance of various identified traits and in deciphering the underlying mechanisms that may be involved in expression of different disease related phenotypes. Genetically engineered mice, with specific immune or biochemical pathway defects, are particularly useful for investigating granuloma formation and resistance to infection and are shedding new light on borderline areas of the leprosy spectrum which are clinically unstable and have a tendency toward immunological complications. Though armadillos are less developed in this regard, these animals are the only other natural hosts of M. leprae and they present a unique opportunity for comparative study of genetic markers and mechanisms associable with disease susceptibility or resistance, especially the neurological aspects of leprosy. In this paper, we review the recent contributions of genetically engineered mice and armadillos toward our understanding of the immunogenetics of leprosy.

The diagnosis of leprosy continues to be based on clinical symptoms and early diagnosis and treatment are critical to preventing disability and transmission. Sensitive and specific laboratory tests are not available for diagnosing leprosy. Despite the limited applicability of anti-phenolic glycolipid-I (PGL-I) serology for diagnosis, it has been suggested as an additional tool to classify leprosy patients (LPs) for treatment purposes. Two formats of rapid tests to detect anti-PGL-I antibodies [ML immunochromatography assay (ICA) and ML Flow] were compared in different groups, multibacillary patients, paucibacillary patients, household contacts and healthy controls in Brazil and Nepal. High ML Flow intra-test concordance was observed and low to moderate agreement between the results of ML ICA and ML Flow tests on the serum of LPs was observed. LPs were "seroclassified" according to the results of these tests and the seroclassification was compared to other currently used classification systems: the World Health Organization operational classification, the bacilloscopic index and the Ridley-Jopling classification. When analysing the usefulness of these tests in the operational classification of PB and MB leprosy for treatment and follow-up purposes, the ML Flow test was the best point-of-care test for subjects in Nepal and despite the need for sample dilution, the ML ICA test yielded better performance among Brazilian subjects. Our results identified possible ways to improve the performance of both tests.

To determine whether the measured change in score of a validated clinical severity scale reflected physician assessed improvement in individuals who had received corticosteroid therapy for leprosy associated nerve damage.Patients with nerve function impairment who participated in a randomised controlled trial of corticosteroids were classified into two groups using a retrospectively determined physician assessment of improvement. One group consisted of patients who had recovered or improved the other of patients who were unchanged or had deteriorated. The change in the clinical severity scale scores of these two groups was compared.The change in the clinical severity scale scores of the 34 eligible individuals in the two groups were significantly different (P = 0.003). Individuals in the group who recovered or improved had a greater change in severity score than those whose nerve function was unchanged or deteriorated.The scale for measuring the severity of leprosy Type 1 reactions (T1Rs) and/or nerve function impairment reflects the clinical improvement of individuals with leprosy associated nerve damage.

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 (

Background Leprosy, a chronic granulomatous disease affecting the skin and nerves, is caused by Mycobacterium leprae (M. leprae). The type of leprosy developed depends upon the host immune response. Type 1 reactions (T1Rs), that complicate borderline and lepromatous leprosy, are due to an increase in cell-mediated immunity and manifest as nerve damage and skin inflammation. Owing to the increase in inflammation in the skin of patients with T1Rs, we sought to investigate the activation of the innate immune system during reactionary events. Specifically, we investigated the expression levels of human beta-defensins (hBDs) 2 and 3 in the skin of patients with T1Rs, in keratinocytes, and in macrophages stimulated with M. leprae and corticosteroids. Results Skin biopsies from twenty-three patients with Type 1 reactions were found to have higher transcript levels of hBD3 as compared to fifteen leprosy patients without Type 1 reactions, as measured by qPCR. Moreover, we observed that keratinocytes but not macrophages up-regulated hBD2 and hBD3 in response to M. leprae stimulation in vitro. Corticosteroid treatment of patients with T1Rs caused a suppression of hBD2 and hBD3 in skin biopsies, as measured by qPCR. In vitro, corticosteroids suppressed M. leprae-dependent induction of hBD2 and hBD3 in keratinocytes. Conclusions This study demonstrates that hBD3 is induced in leprosy Type 1 Reactions and suppressed by corticosteroids. Furthermore, our findings demonstrate that keratinocytes are responsive to M. leprae and lend support for additional studies on keratinocyte innate immunity in leprosy and T1Rs. Trial Registration Controlled-Trials.com ISRCTN31894035, Author Summary Leprosy, caused by Mycobacterium leprae (M. leprae), is a chronic infection leading to potentially debilitating nerve damage. Although the infection is curable with multi-drug therapy, many patients continue to suffer from episodes of inflammation, called Type 1 reactions. These reactions may lead to nerve damage and deformation. Patients with Type 1 reactions are treated with corticosteroids to reduce the inflammation. Elucidating the immune factors contributing to leprosy Type 1 reactions will further clarify why many leprosy patients develop nerve and skin inflammation. We conducted a study to understand whether the antimicrobial peptides, human beta-defensins (hBD) 2 and 3, are increased in the skin of leprosy patients with Type 1 reactions. We also investigated keratinocyte expression of hBD2 and hBD3 in response to M. leprae. In this study, we found that hBD3 was significantly increased in the skin of leprosy patients with Type 1 reactions. Moreover, we demonstrated that corticosteroid treatment reduced the expression of hBD3 in patients with Type 1 reactions. We also found that keratinocytes incubated with M. leprae upregulated hBD2 and hBD3 in vitro; corticosteroids were found to suppress this response. These studies suggest a role for the innate immune system in leprosy Type 1 reactions.

Background Leprosy Type 1 reactions are a major cause of nerve damage and the preventable disability that results. Type 1 reactions are treated with oral corticosteroids and there are few data to support the optimal dose and duration of treatment. Type 1 reactions have a Th1 immune profile: cells in cutaneous and neural lesions expressing interferon-γ and interleukin-12. Methylprednisolone has been used in other Th1 mediated diseases such as rheumatoid arthritis in an attempt to switch off the immune response and so we investigated the efficacy of three days of high dose (1 g) intravenous methylprednisolone at the start of prednisolone therapy in leprosy Type 1 reactions and nerve function impairment. Results Forty-two individuals were randomised to receive methylprednisolone followed by oral prednisolone (n = 20) or oral prednisolone alone (n = 22). There were no significant differences in the rate of adverse events or clinical improvement at the completion of the study. However individuals treated with methylprednisolone were less likely than those treated with prednisolone alone to experience deterioration in sensory function between day 29 and day 113 of the study. The study also demonstrated that 50% of individuals with Type 1 reactions and/or nerve function impairment required additional prednisolone despite treatment with 16 weeks of corticosteroids. Conclusions The study lends further support to the use of more prolonged courses of corticosteroid to treat Type 1 reactions and the investigation of risk factors for the recurrence of Type 1 reaction and nerve function impairment during and after a corticosteroid treatment. Trial Registration Controlled-Trials.comISRCTN31894035, Author Summary Leprosy is caused by a bacterium and is curable with a combination of antibiotics known as multi-drug therapy that patients take for six or 12 months. However a significant proportion of leprosy patients experience inflammation in their skin and/or nerves, which may occur even after successful completion of multi-drug therapy. These episodes of inflammation are called leprosy Type 1 reactions. Type 1 reactions are an important complication of leprosy because they may result in nerve damage that leads to disability and deformity. Type 1 reactions require treatment with immunosuppressive agents such as corticosteroids. The optimum dose and duration of corticosteroid therapy remains unclear. We conducted a study to see if it would be safe to use a large dose of a corticosteroid called methylprednisolone for three days at the start of a 16 week corticosteroid treatment regime of prednisolone in patients with leprosy Type 1 reactions and leprosy patients with nerve damage present for less than six months. We did this by comparing individuals who were given methylprednisolone followed by prednisolone and those who received just prednisolone. In this small study we did not see any significant difference in the frequency of adverse events due to corticosteroid treatment in the two groups. We did not demonstrate a significant difference in improvement in individuals in the methylprednisolone group (who received a larger dose of corticosteroids) than those in the prednisolone treated group. Overall approximately 50% of individuals required more prednisolone in addition to the 16 week course of treatment to prevent further nerve damage or reactions. This suggests that it would be worthwhile investigating longer treatment courses with corticosteroids and other immunosuppressive drugs.

Background. Because of its wide spectrum of clinical manifestations and its well-defined immunological complications, leprosy is a useful disease for studying genetic regulation of the host response to infection. We hypothesized that polymorphisms in the nucleotide-binding oligomerization domain containing 2 (NOD2) gene, for a cytosolic receptor known to detect mycobacteria, are associated with susceptibility to leprosy and its clinical outcomes. Methods. We used a case-control study design with 933 patients in Nepal. Our study included 240 patients with type 1 (reversal) reactions and 124 patients with type 2 (erythema nodosum leprosum) reactions. We compared the frequencies of 32 common polymorphisms in the NOD2 gene region between patients with the different clinical types of leprosy as well as between the patients and 101 control participants without leprosy. Results. Four polymorphisms were associated with susceptibility to leprosy when comparing allele frequencies, and 8 were associated when comparing genotype frequencies with a dominant model. Five polymorphisms were associated with protection from reversal reaction in an allelic analysis, and 7 were associated with reversal reaction with a dominant model. Four polymorphisms were associated with increased susceptibility to erythema nodosum leprosum in an allelic analysis, whereas 7 of 32 polymorphisms were associated with a dominant model. Conclusion. These data suggest that NOD2 genetic variants are associated with susceptibility to leprosy and the development of leprosy reactive states.

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.

Recent studies identified an association between genetic variants in the lymphotoxin-alpha (LTalpha) gene and leprosy. To study the influence of LTalpha on the control of experimental leprosy, both low- and high-dose Mycobacterium leprae foot pad (FP) infections were evaluated in LTalpha-deficient chimeric (cLTalpha(-/-)) and control chimeric (cB6) mice. Cellular responses to low-dose infection in cLTalpha(-/-) mice were dramatically different, with reduced accumulation of CD4(+) and CD8(+) lymphocytes and macrophages and failure to form granulomas. Growth of M. leprae was contained for 6 months, but augmented late in infection. In contrast, tumor necrosis factor knockout and tumor necrosis factor receptor 1 knockout FPs exhibited extensive inflammatory infiltration with an increase in M. leprae growth throughout infection. Following high-dose infection, cB6 FP induration peaked at 4 weeks and was maintained for 12 weeks. Induration was not sustained in cLTalpha(-/-) FPs that contained few lymphocytes and no granulomas. There was a reduction in the expression levels of inflammatory cytokines, chemokines, and chemokine receptors, including nitric oxide synthase 2, vascular cell adhesion molecule, and intercellular cell adhesion molecule. Furthermore, cLTalpha(-/-) popliteal lymph nodes contained a higher proportion of naïve CD44(lo)CD62L(hi) T cells than cB6 mice, suggestive of reduced T cell activation. Therefore, both LTalpha and tumor necrosis factor are essential for the regulation of the granuloma, but they have distinctive roles in the recruitment of lymphocytes and maintenance of the granulomatous response during chronic M. leprae infection.

Cytokine-activated macrophages (MPhi) employ reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to combat pathogens. The requirement for ROI for an effective host response to experimental leprosy using mice which have a disruption in the 91-kD subunit of the NAPDH oxidase cytochrome b (phox91-/-) was examined. Mycobacterium leprae multiplication in phox91-/- foot pads (FP) was elevated early in infection but subsequently arrested similarly to control mice within a noninvasive granuloma. Using a modified lepromin test model, a similar cellular composition in the M. leprae-induced FP granuloma in both strains with lymphocyte infiltration consisting primarily of CD4+CD44(hi)CD62L(lo) effector cells was found. Of great interest was the disparity in the T cell population between the granuloma and the draining lymph node which contained predominantly naïve CD4+CD44(lo)CD62L(hi) cells and was, therefore, not representative of the infection site. TH1 cytokines, chemokines and inducible nitric oxide synthase were comparably expressed in the FP of both strains. When infected in vitro, normal MPhi from B6 and phox91-/- mice supported bacterial viability, whereas IFNgamma-activated MPhi killed M. leprae in a RNI-dependent manner, emphasizing that ROI was dispensable. These data show that phox91-/- mice generate a strong adaptive immune response and control long-term infection with M. leprae.

BACKGROUND Dengue fever (DF) is an emerging public health problem in Nepal, and yet neither the magnitude of the DF burden or its epidemiological trends are well understood. METHODS We conducted a sero-epidemiological, seasonal trend and demographic analysis of the trends in DF in the Terai region of Nepal, from 2007 to 2013. In that period, 2002 serum samples were collected from febrile patients suspected of dengue virus infection. Samples were screened by IgM antibody ELISA analysis for the presence of anti-dengue IgM antibodies. RESULTS The overall prevalence of DF cases was found to range from 26.1 to 55.4%. Infection was found to be more common among adults and males. Seasonal trends revealed that cases peaked in October and November. CONCLUSIONS The study offers perspective on the dengue fever burden before, during and after a major epidemic and can now be used as a basis for formulating strategies by policy makers that will enhance and develop relevant control and preventive measures against dengue fever. The findings of this study reinforce the perceived need for urgent dengue virus surveillance to enhance dengue control strategies that need to be developed for future preparedness. In the light of the recent earthquakes, future major outbreaks of vector borne disease are likely to recur.

Erythema nodosum leprosum (ENL) is a severe multisystem immune mediated complication of borderline lepromatous leprosy and lepromatous leprosy. ENL is associated with skin lesions, neuritis, arthritis, dactylitis, eye inflammation, osteitis, orchitis, lymphadenitis and nephritis. The treatment of ENL requires immunosuppression, which is often required for prolonged periods of time and may lead to serious adverse effects. ENL and its treatment is associated with increased mortality and economic hardship. Improved, evidence-based treatments for ENL are needed; however, defining the severity of ENL and outcome measures for treatment studies is difficult because of the multiple organ systems involved. A cross-sectional study was performed, by the members of the Erythema Nodosum Leprosum International STudy (ENLIST) Group, of patients with ENL attending seven leprosy referral centres in Brazil, Ethiopia, India, Nepal, the Philippines and the United Kingdom. We systematically documented the clinical features and type of ENL, its severity and the drugs used to treat it. Patients with chronic ENL were more likely to be assessed as having severe ENL. Pain, the most frequent symptom, assessed using a semi-quantitative scale was significantly worse in individuals with “severe” ENL. Our findings will determine the items to be included in a severity scale of ENL which we are developing and validating. The study also provides data on the clinical features of ENL, which can be incorporated into a definition of ENL and used for outcome measures in treatment studies., Author Summary Leprosy is caused by a bacterium and is curable with a combination of antibiotics known as multi-drug therapy, which patients take for six or 12 months. However, a significant proportion of leprosy patients experience severe inflammation in their skin, nerves and other organs known as erythema nodosum leprosum (ENL). ENL is a severe complication of leprosy which can occur before, during, or after completion of antibacterial treatment. ENL causes severe morbidity affecting not only the skin, but also bones, joints, eyes, nerves, testes and kidneys. ENL may be fatal and it has been shown that the adverse effects of corticosteroid treatment also contribute to mortality. An effective treatment for ENL is thalidomide but this is not available in many leprosy endemic countries or is severely constrained by availability, cost and justified concerns about teratogenicity. Thalidomide, despite its effectiveness, is rarely used as a first line agent. Other effective drugs need to be identified to reduce the burden of this condition. In order to establish which treatments will be effective in ENL it is necessary to have a clear picture of the clinical features of the disease and how these influence severity. This is the first systematic study of ENL to define these and will help in developing tools to facilitate treatment studies of this severe, debilitating complication of leprosy.

Millions of patients with leprosy suffer from nerve damage resulting in disabilities as a consequence of Mycobacterium leprae infection. However, mechanisms of nerve damage have not been elucidated because of the lack of a model that maintains M. leprae viability and mimics disease conditions. A model was developed using viable M. leprae, rat Schwann cells, and Schwann cell-neuron cocultures incubated at 33 degrees C. M. leprae retained 56% viability in Schwann cells for 3 weeks after infection at 33 degrees C, compared with 3.6% viability at 37 degrees C. Infected Schwann cells had altered morphology and expression of genes encoding cellular adhesion molecules at 33 degrees C but were capable of interacting with and myelinating neurons. Cocultures, infected after myelination occurred, showed no morphological changes in myelin architecture after 1 month of incubation at 33 degrees C, and M. leprae retained 53% viability. This article describes a new model for studying the effects of M. leprae on Schwann cells.

Background Leprosy is characterized by polar clinical, histologic and immunological presentations. Previous immunologic studies of leprosy polarity were limited by the repertoire of cytokines known at the time. Methodology We used a candidate gene approach to measure mRNA levels in skin biopsies from leprosy lesions. mRNA from 24 chemokines and cytokines, and 6 immune cell type markers were measured from 85 Nepalese leprosy subjects. Selected findings were confirmed with immunohistochemistry. Principal Results Expression of three soluble mediators (CCL18, CCL17 and IL-10) and one macrophage cell type marker (CD14) was significantly elevated in lepromatous (CCL18, IL-10 and CD14) or tuberculoid (CCL17) lesions. Higher CCL18 protein expression by immunohistochemistry and a trend in increased serum CCL18 in lepromatous lesions was observed. No cytokines were associated with erythema nodosum leprosum or Type I reversal reaction following multiple comparison correction. Hierarchical clustering suggested that CCL18 was correlated with cell markers CD209 and CD14, while neither CCL17 nor CCL18 were highly correlated with classical TH1 and TH2 cytokines. Conclusions Our findings suggest that CCL17 and CCL18 dermal expression is associated with leprosy polarity., Author Summary Leprosy presents with a polarized spectrum, with lepromatous leprosy having high bacillary numbers and TH2 dermal cytokines, versus tuberculoid leprosy showing very few bacilli and TH1 cytokines. The mechanism underlying this polarized presentation is largely unknown. In the following study, we isolated mRNA from skin biopsies from 85 individuals with leprosy and measured the expression of a panel of 24 cytokines and 6 cell markers. We found that three soluble mediators (CCL17, CCL18 and IL10) and one cell marker (CD14) were differentially expressed in leprosy dermal lesions. CCL18 and IL10 were more highly expressed within lepromatous lesions, and CCL17 and CD14 were more highly expressed within tuberculoid lesions. In addition, CCL18 protein expression was confirmed by immunostaining. CCL17 and CCL18, were more strongly associated with leprosy polarity than traditional TH1 and TH2 cytokines. These data suggest that newer soluble chemokines may be important in leprosy pathogenesis and uncover a molecular signature of the two polar phenotypes of leprosy, which may be useful in future diagnostics.

Background Although immunopathology dictates clinical outcome in leprosy, the dynamics of early and chronic infection are poorly defined. In the tuberculoid region of the spectrum, Mycobacterium leprae growth is restricted yet a severe granulomatous lesion can occur. The evolution and maintenance of chronic inflammatory processes like those observed in the leprosy granuloma involve an ongoing network of communications via cytokines. IL-10 has immunosuppressive properties and IL-10 genetic variants have been associated with leprosy development and reactions. Methodology/Principal Findings The role of IL-10 in resistance and inflammation in leprosy was investigated using Mycobacterium leprae infection of mice deficient in IL-10 (IL-10−/−), as well as mice deficient in both inducible nitric oxide synthase (NOS2−/−) and IL-10 (10NOS2−/−). Although a lack of IL-10 did not affect M. leprae multiplication in the footpads (FP), inflammation increased from C57Bl/6 (B6), Author Summary Despite effective antimicrobial therapy, 30–50% of leprosy patients develop immunological complications called leprosy reactions before, during or even years after being cured. Leprosy reactions are a major risk for neuritis that leads to peripheral nerve damage, disfigurement and disability. Unfortunately, why and how leprosy reactions occur is not well understood. Based on the latest human genetic leprosy susceptibility research and mouse infection models, we generated a double knockout mouse strain (10NOS2−/−) which has deficiencies in two key immune factors, interleukin-10 (IL-10) and inducible nitric oxide synthase (NOS2). We investigated the dynamics of the immune response to Mycobacterium leprae infection and chronicled the types of immune cells recruited to the site of infection. 10NOS2−/− mice developed a substantial induration in response to infection, as well as an increased interferon-gamma response to components of the leprosy bacillus. Interestingly, these animals also exhibited CD4+ T cell infiltration into the nerves, a phenomenon which has not been previously reported in leprosy mouse models. This new model provides insight into potential mechanisms whereby immune modulators may regulate leprosy reactions and neuritis and could aid the development of tests for monitoring and treatment of leprosy patients.