A simple assay to quantify mycobacterial lipid antigen-specific T cell receptors in human tissues and blood.

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.

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

BACKGROUND: Leprosy 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 FINDINGS: A 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. CONCLUSIONS: Quantitative 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.

Epitope mapping from Mycobacterium leprae proteins: Convergent data from in silico and in vitro approaches for serodiagnosis of leprosy.

Knowledge of immunodominant B-cell epitopes is essential to design powerful diagnostic strategies aiming for antibody detection. Outstanding progress in computational prediction has achieved a significant contribution to the biomedical fields, including immunodiagnosis. In silico analysis may have an even more important role when information concerning antigens from etiologic agents of neglected diseases, such as leprosy, is scarce. The aim of this study was to provide mapping of B-cell epitopes from two Mycobacterium leprae-derived antigens (Ag85B and ML2055), confirm their antigenicity, and to assess the ability of in silico immunoinformatics tools to accurately predict them. Linear B-cell epitopes predicted by ABCpred and SVMTrip servers were compared to antigenic regions of synthetic overlapping peptides that exhibited reactivity to antibodies from patients with leprosy. Our in vitro results identified several immunodominant regions that had also been indicated by in silico prediction, providing agreement between experimental and simulated data. After chemical synthesis, we used enzyme-linked immunosorbent assays to determine the effectiveness of the first identified sequence (GTNVPAEFLENFVHG) which had 72 % sensitivity and 78 % specificity (AUC = 0.79) while the second one (PVSSEAQPGDPNAPS) had 72 % sensitivity and 93.8 % specificity (AUC = 0.85). Using dot blotting, an easy-to-read visual test, both peptides could distinguish sera from patients with leprosy from those with tuberculosis and from sera of healthy volunteers. Our findings suggest that these synthetic peptides, with some refinement, may be useful as serological diagnostic antigens for leprosy. In addition, it was displayed that immunoinformatics provides reliable information for mapping potential B-cell epitopes for development of peptide-based diagnostic assays for neglected diseases.

Blood RNA signature RISK4LEP predicts leprosy years before clinical onset.

BACKGROUND: Leprosy, a chronic infectious disease caused by Mycobacterium leprae, is often late- or misdiagnosed leading to irreversible disabilities. Blood transcriptomic biomarkers that prospectively predict those who progress to leprosy (progressors) would allow early diagnosis, better treatment outcomes and facilitate interventions aimed at stopping bacterial transmission. To identify potential risk signatures of leprosy, we collected whole blood of household contacts (HC, n=5,352) of leprosy patients, including individuals who were diagnosed with leprosy 4-61 months after sample collection. METHODS: We investigated differential gene expression (DGE) by RNA-Seq between progressors before presence of symptoms (n=40) and HC (n=40), as well as longitudinal DGE within each progressor. A prospective leprosy signature was identified using a machine learning approach (Random Forest) and validated using reverse transcription quantitative PCR (RT-qPCR). FINDINGS: Although no significant intra-individual longitudinal variation within leprosy progressors was identified, 1,613 genes were differentially expressed in progressors before diagnosis compared to HC. We identified a 13-gene prospective risk signature with an Area Under the Curve (AUC) of 95.2%. Validation of this RNA-Seq signature in an additional set of progressors (n=43) and HC (n=43) by RT-qPCR, resulted in a final 4-gene signature, designated RISK4LEP (MT-ND2, REX1BD, TPGS1, UBC) (AUC=86.4%). INTERPRETATION: This study identifies for the first time a prospective transcriptional risk signature in blood predicting development of leprosy 4 to 61 months before clinical diagnosis. Assessment of this signature in contacts of leprosy patients can function as an adjunct diagnostic tool to target implementation of interventions to restrain leprosy development. FUNDING: This study was supported by R2STOP Research grant, the Order of Malta-Grants-for-Leprosy-Research, the Q.M. Gastmann-Wichers Foundation and the Leprosy Research Initiative (LRI) together with the Turing Foundation (ILEP# 702.02.73 and # 703.15.07).

Development of a Loop-mediated isothermal amplification (LAMP) technique for specific and early detection of Mycobacterium leprae in clinical samples.

Leprosy, a progressive, mutilating and highly stigmatized disease caused by Mycobacterium leprae (ML), continues to prevail in the developing world. This is due to the absence of rapid, specific and sensitive diagnostic tools for its early detection since the disease gets notified only with the advent of physical scarring in patients. This study reports the development of a Loop-mediated isothermal amplification (LAMP) technique for fast, sensitive and specific amplification of 16S rRNA gene of ML DNA for early detection of leprosy in resource-limited areas. Various parameters were optimized to obtain robust and reliable amplification of ML DNA. Blind clinical validation studies were performed which showed that this technique had complete concurrence with conventional techniques. Total absence of amplification of negative control DNA confirmed the specificity of this test. Various visual detection methods viz. colorimetric, turbidity differentiation and bridge flocculation were standardized to establish easy-to-read and rapid diagnosis. This technique eliminates the lack of accuracy and sensitivity in skin smear tests in patients and the requirement for expensive lab equipments and trained technicians. The technique holds promise for further expansion and has the potential to cater to the unmet needs of society for a cheap, highly-sensitive and robust rapid diagnosis of ML.

Presence of Senescent and Memory CD8+ Leukocytes as Immunocenescence Markers in Skin Lesions of Elderly Leprosy Patients.

Leprosy is an infectious disease that remains endemic in approximately 100 developing countries, where about 200,000 new cases are diagnosed each year. Moreover, multibacillary leprosy, the most contagious form of the disease, has been detected at continuously higher rates among Brazilian elderly people. Due to the so-called immunosenescence, characterized by several alterations in the quality of the immune response during aging, this group is more susceptible to infectious diseases. In view of such data, the purpose of our work was to investigate if age-related alterations in the immune response could influence the pathogenesis of leprosy. As such, we studied 87 individuals, 62 newly diagnosed and untreated leprosy patients distributed according to the age range and to the clinical forms of the disease and 25 healthy volunteers, who were studied as controls. The frequency of senescent and memory CD8+ leukocytes was assessed by immunofluorescence of biopsies from cutaneous lesions, while the serum levels of IgG anti-CMV antibodies were analyzed by chemiluminescence and the gene expression of T cell receptors' inhibitors by RT-qPCR. We noted an accumulation of memory CD8+ T lymphocytes, as well as reduced CD8+CD28+ cell expression in skin lesions from elderly patients, when compared to younger people. Alterations in LAG3 and PDCD1 gene expression in cutaneous lesions of young MB patients were also observed, when compared to elderly patients. Such data suggest that the age-related alterations of T lymphocyte subsets can facilitate the onset of leprosy in elderly patients, not to mention other chronic inflammatory diseases.

Increased serum levels of interleukin-6 in erythema nodosum leprosum suggest its use as a biomarker.

BACKGROUND: Erythema nodosum leprosum (ENL) is a frequent complication of multibacillary leprosy that can result in significant morbidity, including peripheral nerve damage and physical disability. The identification of possible serum markers could be a valuable tool for the early detection of ENL. AIMS: The purpose of this study was to evaluate selected serum mediators involved in the innate and adaptive immune responses to identify possible immunomarkers for ENL. METHODS: The levels of interleukin-2, interleukin-4, interleukin-6, interleukin-10, interleukin-17, interferon-γ, tumor necrosis factor, nitric oxide and anti-phenolic glycolipid-I antibodies were measured in the sera of leprosy patients with ENL [at the beginning of reaction (M0) and 1 month later (M1)], and then compared with the levels of the same markers in patients with untreated multibacillary leprosy without ENL (controls with leprosy: CTRL) and healthy individuals (healthy controls: CTRH). RESULTS: Significantly higher levels of serum interleukin-6 were observed in M0 than in CTRL. In addition, pairwise comparisons showed higher levels of interleukin-6 in M0 compared to M1. Levels of tumor necrosis factor were higher in M0 than in CTRL, with no significant difference between M0 and M1. There were no differences in the levels of interleukin-2, interleukin-4, interleukin-10, interleukin-17 or interferon-γ between groups. The CTRL group had higher levels of nitric oxide compared to M0 and M1. High levels of anti-phenolic glycolipid-I were observed in M0, M1 and CTRL than in CTRH. LIMITATIONS: Three patients were not assessed at M1, decreasing the number of evaluated patients from 14 to 11. CONCLUSION: High-serum levels of interleukin-6 were observed during ENL, primarily in patients with more severe reactions; levels decreased after specific therapy, suggesting a role for this cytokine in pathogenesis and its utility as an ENL biomarker. Further studies should explore whether interleukin-6 could also be used as a predictive marker for ENL or as a specific target for its treatment.

Increased serum levels of interleukin-6 in erythema nodosum leprosum suggest its use as a biomarker

Background: Erythema nodosum leprosum (ENL) is a frequent complication of multibacillary leprosy that can result in significant morbidity, including peripheral nerve damage and physical disability. The identification of possible serum markers could be a valuable tool for the early detection of ENL. Aims: The purpose of this study was to evaluate selected serum mediators involved in the innate and adaptive immune responses to identify possible immunomarkers for ENL.Methods: The levels of interleukin-2, interleukin-4, interleukin-6, interleukin-10, interleukin-17, interferon-γ, tumor necrosis factor, nitric oxide and anti-phenolic glycolipid-I antibodies were measured in the sera of leprosy patients with ENL [at the beginning of reaction (M0) and 1 month later (M1)], and then compared with the levels of the same markers in patients with untreated multibacillary leprosy without ENL (controls with leprosy: CTRL) and healthy individuals (healthy controls: CTRH).Results: Significantly higher levels of serum interleukin­6 were observed in M0 than in CTRL. In addition, pairwise comparisons showed higher levels of interleukin-6 in M0 compared to M1. Levels of tumor necrosis factor were higher in M0 than in CTRL, with no significant difference between M0 and M1. There were no differences in the levels of interleukin-2, interleukin-4, interleukin-10, interleukin-17 or interferon-γ between groups. The CTRL group had higher levels of nitric oxide compared to M0 and M1. High levels of anti-phenolic glycolipid-I were observed in M0, M1 and CTRL than in CTRH.Limitations: Three patients were not assessed at M1, decreasing the number of evaluated patients from 14 to 11. Conclusion: High-serum levels of interleukin-6 were observed during ENL, primarily in patients with more severe reactions; levels decreased after specific therapy, suggesting a role for this cytokine in pathogenesis and its utility as an ENL biomarker. Further studies should explore whether interleukin-6 could also be used as a predictive marker for ENL or as a specific target for its treatment.

Cardiovascular disease risk factors and markers of oxidative stress and DNA damage in leprosy patients in Southern Nigeria.

Leprosy reduces quality of life of affected persons. Oxidative stress caused by reactive oxygen species may play a vital role in the pathogenesis of leprosy. This study evaluated anthropometric indices, fasting plasma glucose (FPG), lipid profile, total antioxidant capacity (TAC), total plasma peroxide (TPP), oxidative stress index (OSI), malondialdehyde (MDA), glutathione (GSH) and 8-hydroxy-2-deoxyguanosine (8-OHdg) in leprosy patients. Sixty test participants of both genders, aged 18-65years and diagnosed of multibacillary leprosy and 30 apparently healthy controls were consecutively recruited for this study. The test participants comprised of 30 patients on multidrug therapy (MDT) and 30 patients relieved from therapy (RFT). Body mass index (BMI), Waist-hip ratio (WHR), FPG, lipid profile, TAC, TPP, OSI, MDA, GSH and 8-OHdg were determined using appropriate methods. Data were analyzed using Analysis of variance; p<0.05 was considered statistically significant. The MDT group had significantly lower BMI (p = 0.0001), Total cholesterol (p = 0.001), HDL-C (p = 0.019), LDL-C (p = 0.005), TAC (p = 0.0001) and higher TPP (p = 0.001), MDA (p = 0.0001), OSI (p = 0.005) and 8-OHdg (p = 0.035) compared to the controls. The RFT group had significantly lower BMI (p = 0.001) Total cholesterol (0.0001), HDL-C (p = 0.006) LDL-C (p = 0.0001), TAC (p = 0.001) and higher WHR (p = 0.010), VLDL-C (p = 0.035), TG (p = 0.023) Atherogenic index of plasma (p = 0.0001) and TPP (p = 0.001), MDA (p = 0.0001) compared to the control group. GSH levels correlated negatively with duration of treatment (r = -0.401, p = 0.028). This study has shown that there is oxidative stress in multibacillary leprosy patients irrespective of drug treatment status. This study also shows that leprosy patients relieved from treatment may be susceptible to cardiovascular events. Antioxidants supplementation may be beneficial in the treatment of leprosy and clinical follow up on patients relieved from treatment may also be necessary to monitor health status and prevent development of cardiovascular events.

Leprosy surveillance study in a highly endemic Brazilian area using leprosy specific serologic tests and IFNγ whole blood assay.

This surveillance study evaluated leprosy-serologic tests and the IFNγ whole-blood-assay/WBA as adjunct diagnostic tools. Previously diagnosed leprosy index cases, intradomiciliary, peridomiciliary contacts from a Brazilian endemic area were enrolled during domiciliary visits. Physical evaluation was performed by trained nurses and leprosy diagnosis confirmed by expert dermatologist. ELISA detected IgM anti-PGL-I, IgG anti-LID-1, and IgM/IgG anti-ND-O-LID antibodies. Heparinized WBA plasma stimulated with LID-1, 46f + LID-1, ML0276 + LID-1 (24 h, 37 °C, 5% CO2) was tested for human IFNγ (QuantiFERON®-TB Gold/QFT-G; Qiagen). The survey included 1731 participants: 44 leprosy index cases, 64 intradomiciliary, 1623 peridomiciliary contacts. Women represented 57.7%, median age was 32 years, 72.2% had BCG scar. Leprosy prevalence was higher in intradomiciliary (8.57%) versus peridomiciliary contacts (0.67%), p < 0.001. Among 23 suspects, five leprosy cases were confirmed: 4 multibacillary/MB and 1 paucibacillary/PB. Leprosy incidence was 0.30%: 1.56% in intradomiciliary versus 0.25% in peridomiciliary (p = 0.028). Seropositivity rates were 1.9% to PGL-I, 4.9% to LID-1, and 1.0% to ND-O-LID. LID-1 positivity was higher in all groups; incident cases were LID-1 seropositive. ND-O-LID positivity was higher in intra- versus peridomiciliary contacts (p = 0.022). IFNγ WBA (40 index cases, 19 suspects, 35 intradomiciliary, 74 peridomiciliary contacts) showed higher LID-1/WBA positivity in peridomiciliary contacts (p > 0.05); significant differences among groups were seen with 46f + LID-1 but 0276 + LID-1 induced higher IFNγ levels. Incident cases were LID-1 seropositive, while IFNγ-WBA had marginal diagnostic application. As seropositivity indicates exposed individuals at higher risk of disease development, the utility of serologic screening for surveillance and prophylactic measures remains to be demonstrated.

Cytokine gene polymorphisms in type I and type II reactions in Hansen's disease.

INTRODUCTION: Leprosy or Hansen's disease is a chronic debilitating disease caused by Mycobacterium leprae. Host genetics are believed to strongly influence the course of the disease. It is known that cytokines play an important role in leprosy and cytokine gene polymorphisms probably influence the course of the disease. METHODS: In the present study, we evaluated 70 patients with leprosy and 243 controls. DNA was extracted from the peripheral blood and genotyping was done for the following polymorphisms: IL-1 RA intron 2, IL-1ß-511 C/T and TNF-α A/G. RESULTS: A strong association of TNF-α-308 G/A polymorphism with Hansen's disease with both genotypes and alleles was found. However, no correlation was identified between the other two polymorphisms and Hansen's disease. A strong association between the IL-1ß gene polymorphisms and the type of reactions seen in leprosy was found. In contrast, the other two polymorphisms did not show any such association. LIMITATIONS: Genetic polymorphisms are association studies. They are not a direct reflection of the transcriptome or proteome and this is a major limitation of this study. CONCLUSION: In conclusion, cytokine gene polymorphisms appear to influence the susceptibility and course of Hansen's disease. An evaluation of the cytokine levels in the skin during lepra reactions would confirm this observation. Possibly, in future, this would be a guide to therapeutic decisions in cases of lepra reactions.

Metabolism and interactions of antileprosy drugs.

Leprosy is a chronic infectious disease caused my Mycobacterium leprae that primarily affects peripheral nervous system and extremities and is prevalent in tropical countries. Treatment for leprosy with multidrug regimens is very effective compared to monotherapy especially in multibacillary cases. The three major antileprosy drugs currently in use are 4, 4'-diaminodiphenyl sulfone (DDS, dapsone), rifampicin, and clofazimine. During multidrug therapy, the potent antibiotic rifampicin induces the metabolism of dapsone, which results in decreased plasma half-life of dapsone and its metabolites. Furthermore, rifampicin induces its own metabolism and decreases its half-life during monotherapy. Rifampicin upregulates several hepatic microsomal drug-metabolizing enzymes, especially cytochrome P450 (CYP) family that in turn induce the metabolism of dapsone. Clofazimine lacks significant induction of any drug-metabolizing enzyme including CYP family and does not interact with dapsone metabolism. Rifampicin does not induce clofazimine metabolism during combination treatment. Administration of dapsone in the acetylated form (acedapsone) can release the drug slowly into circulation up to 75 days and could be useful for the effective treatment of paucibacillary cases along with rifampicin. This review summarizes the major aspects of antileprosy drug metabolism and drug interactions and the role of cytochrome P450 family of drug metabolizing enzymes, especially CYP3A4 during multidrug regimens for the treatment of leprosy.

[The use of synthetic glycoconjugates as components of the immunochromatographic test for rapid serological diagnosis of leprosy.]

The glycoconjugates with BSA (bovine serum albumin) were synthesized using a next saccharide: disaccharide derivative M.leprae PGL-1 (phenolic glycolipid-1); a complex of the disaccharide fragment and the branched hexasaccharide fragment LAM (lipoarabinomannan); diarabinofuranose fragment LAM. These glycoconjugates were used as antigenic components for leprosy rapid serotest construction in immunochromatographic format (leprosy LF serotest). The data obtained with sera of leprosy patients, patients who have been in contact with leprosy, and healthy donors indicate that the most promising antigenic component is a BSA conjugate with two synthetic epitopes - a disaccharide derivative of PGL-1 and a branched hexasaccharide fragment of LAM. The leprosy LF serotest with such glycoconjugate demonstrated the greatest diagnostic sensitivity for main forms of leprosy - paucibacillary (PB) and multibacillary (MB).

Cytokine Levels in Neural Pain in Leprosy.

Pain is a frequent symptom in leprosy patients. It may be predominantly nociceptive, as in neuritis, or neuropathic, due to injury or nerve dysfunction. The differential diagnosis of these two forms of pain is a challenge in clinical practice, especially because it is quite common for a patient to suffer from both types of pain. A better understanding of cytokine profile may serve as a tool in assessing patients and also help to comprehend pathophysiology of leprosy pain. Patients with leprosy and neural pain (n = 22), neuropathic pain (n = 18), neuritis (nociceptive pain) (n = 4), or no pain (n = 17), further to those with diabetic neuropathy and neuropathic pain (n = 17) were recruited at Souza Araujo Out-Patient Unit (Fiocruz, Rio de Janeiro, RJ, Brazil). Serum levels of IL1ß, IL-6, IL-10, IL-17, TNF, CCL-2/MCP-1, IFN-γ, CXCL-10/IP-10, and TGF-ß were evaluated in the different Groups. Impairment in thermal or pain sensitivity was the most frequent clinical finding (95.5%) in leprosy neuropathy patients with and without pain, but less frequent in Diabetic Group (88.2%). Previous reactional episodes have occurred in patients in the leprosy and Pain Group (p = 0.027) more often. Analysis of cytokine levels have demonstrated that the concentrations of IL-1ß, TNF, TGF-ß, and IL-17 in serum samples of patients having leprosy neuropathy in combination with neuropathic or nociceptive pain were higher when compared to the samples of leprosy neuropathy patients without pain. In addition, these cytokine levels were significantly augmented in leprosy patients with neuropathic pain in relation to those with neuropathic pain due to diabetes. IL-1ß levels are an independent variable associated with both types of pain in patients with leprosy neuropathy. IL-6 concentration was increased in both groups with pain. Moreover, CCL-2/MCP-1 and CXCL-10/IP-10 levels were higher in patients with diabetic neuropathy over those with leprosy neuropathy. In brief, IL-1ß is an independent variable related to neuropathic and nociceptive pain in patients with leprosy, and could be an important biomarker for patient follow-up. IL-6 was higher in both groups with pain (leprosy and diabetic patients), and could be a therapeutic target in pain control.

Novel PCR primers for improved detection of Mycobacterium leprae and diagnosis of leprosy.

AIMS: Diagnosis of leprosy, a chronic infection caused by Mycobacterium leprae, predominantly depends on clinical manifestations and histopathological analysis, hampering rapid and accurate diagnostics. Our aim was to increase accuracy of leprosy diagnosis by improving M. leprae's DNA detection based on polymerase chain reaction (PCR) technique using new specific primers for the RLEP repetitive sequence. METHODS AND RESULTS: The specific target region, RLEP, of M. leprae's genome was selected based on comparative genomics. After confirming the specificity of this region, using blastn analysis, primers were designed and tested for their in silico specificity. To evaluate the specificity and sensitivity of these primers in vitro, 184 blood samples from patients were used in qPCR. The new primer pair LYON1/LYON2 produced 91% positive samples, whereas the current primer pair LP1/LP2 produced 46%. Specificity and DNA detection limit test were carried out to compare the efficiency of the developed primer pair. The LYON1/LYON2 primer showed 100% specificity, whereas LP1/LP2 showed 64%. The DNA detection limit of LYON1/LYON2 was 10 copies of bacterial genomes per millilitre, whereas LP1/LP2 was 1000 copies of bacterial genomes per millilitre. CONCLUSIONS: In conclusion, the developed LYON1/LYON2 primer pair presented to be a specific and sensitive new molecular marker for the diagnosis of leprosy. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of a specific primer pair for the detection of the M. leprae genome through qPCR technique contributes to a fast, sensitive and specific diagnosis, which is essential to prevent spreading and progression of this disease.

Expansion and suppressive capacity of regulatory T cells isolated from patients across the leprosy spectrum: a pilot study.

Knowledge of the role of Tregs in the immunopathogenesis of the different clinical outcomes within the leprosy spectrum remains limited due to the lack of studies directly assessing their suppression capacity. We thus tested a protocol to expand Tregs from the peripheral blood of patients across the leprosy spectrum and analyzed their suppressive capacity in autologous TCD4+ responses. Results of these pilot assays show that Tregs can be expanded and exert suppressive capacity, but also that their rate of expansion and suppressive capacity are influenced by the patient's clinical classification, suggesting that they possibly retain some in vivo characteristics.

Whole blood RNA signatures in leprosy patients identify reversal reactions before clinical onset: a prospective, multicenter study.

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.

Epidemiological, clinical and immune factors that influence the persistence of antiphospholipid antibodies in leprosy.

INTRODUCTION: Antiphospholipid antibodies (aPL) are described in individuals with leprosy without the clinical features of antiphospholipid antibody syndrome (APS), a condition involving thromboembolic phenomena. We have described the persistence of these antibodies for over 5 years in patients with leprosy after specific treatment. OBJECTIVES: To determine whether epidemiological, clinical and immunological factors played a role in the long-term persistence of aPL antibodies in leprosy patients after multidrug therapy (MDT) had finished. METHODS: The study sample consisted of 38 patients with a diagnosis of leprosy being followed up at the Dermatology and Venereology Outpatient Department at the Alfredo da Matta Foundation (FUAM) in Manaus, AM. ELISA was used to detect anticardiolipin (aCL) and anti-ß2 glycoprotein I (anti-ß2GPI) antibodies. Patients were reassessed on average of 5 years after specific treatment for the disease (MDT) had been completed. RESULTS: Persistence of aPL antibodies among the 38 leprosy patients was 84% (32/38), and all had the IgM isotype. Mean age was 48.1 ± 15.9 years, and 23 (72.0%) were male. The lepromatous form (LL) of leprosy was the most common (n = 16, 50%). Reactional episodes were observed in three patients (9.4%). Eighteen (47.37%) were still taking medication (prednisone and/or thalidomide). Mean IgM levels were 64 U/mL for aCL and 62 U/mL for anti-ß2GPI. In the multivariate binary logistic regression the following variables showed a significant association: age (p = 0.045, OR = 0.91 and CI 95% 0.82-0.98), LL clinical presention (p = 0.034; OR = 0.02 and CI 95% = 0.0-0.76) and bacterial index (p = 0.044; OR = 2.74 and CI 95% = 1.03-7.33). We did not find association between prednisone or thalidomide doses and positivity for aPL (p = 0.504 and p = 0.670, respectively). No differences in the variables vascular thrombosis, pregnancy morbidity, diabetes, smoking and alcoholism were found between aPL-positive and aPL-negative patients. CONCLUSION: Persistence of positivity for aPL antibodies was influenced by age, clinical presentation and bacterial index. However, further studies are needed to elucidate the reason for this persistence, the role played by aPL antibodies in the disease and the B cell lineages responsible for generation of these antibodies.

Application of new host biomarker profiles in quantitative point-of-care tests facilitates leprosy diagnosis in the field.

BACKGROUND: Transmission of Mycobacterium leprae, the pathogen causing leprosy, is still persistent. To facilitate timely (prophylactic) treatment and reduce transmission it is vital to both early diagnose leprosy, and identify infected individuals lacking clinical symptoms. However, leprosy-specific biomarkers are limited, particularly for paucibacillary disease. Therefore, our objective was to identify new biomarkers for leprosy and assess their applicability in point-of-care (POC) tests. METHODS: Using multiplex-bead-arrays, 60 host-proteins were measured in a cross-sectional approach in 24-h whole blood assays (WBAs) collected in Bangladesh (79 patients; 54 contacts; 51 endemic controls (EC)). Next, 17 promising biomarkers were validated in WBAs of a separate cohort (55 patients; 27 EC). Finally, in a third cohort (36 patients; 20 EC), five candidate markers detectable in plasma were assessed for application in POC tests. FINDINGS: This study identified three new biomarkers for leprosy (ApoA1, IL-1Ra, S100A12), and confirmed five previously described biomarkers (CCL4, CRP, IL-10, IP-10, αPGL-I IgM). Overnight stimulation in WBAs provided increased specificity for leprosy and was required for IL-10, IL-1Ra and CCL4. The remaining five biomarkers were directly detectable in plasma, hence suitable for rapid POC tests. Indeed, lateral flow assays (LFAs) utilizing this five-marker profile detected both multi- and paucibacillary leprosy patients with variable immune responses. INTERPRETATION: Application of novel host-biomarker profiles to rapid, quantitative LFAs improves leprosy diagnosis and allows POC testing in low-resource settings. This platform can thus aid diagnosis and classification of leprosy and also provides a tool to detect M.leprae infection in large-scale contact screening in the field.