In search of biomarkers for leprosy by unraveling the host immune response to Mycobacterium leprae

Mycobacterium leprae, the causative agent of leprosy, is still actively transmitted in endemic areas reflected by the fairly stable number of new cases detected each year. Recognizing the signs and symptoms of leprosy is challenging, especially at an early stage. Improved diagnostic tools, based on sensitive and specific biomarkers, that facilitate diagnosis of leprosy are therefore urgently needed. In this review, we address the challenges that leprosy biomarker research is facing by reviewing cell types reported to be involved in host immunity to M leprae. These cell types can be associated with different possible fates of M leprae infection being either protective immunity, or pathogenic immune responses inducing nerve damage. Unraveling these responses will facilitate the search for biomarkers. Implications for further studies to disentangle the complex interplay between host responses that lead to leprosy disease are discussed, providing leads for the identification of new biomarkers to improve leprosy diagnostics.Immunogenetics and cellular immunology of bacterial infectious disease

Detection of humoral immunity to mycobacteria causing leprosy in Eurasian red squirrels (Sciurus vulgaris) using a quantitative rapid test

Cancer Signaling networks and Molecular Therapeutic

BCG and adverse events in the context of leprosy

Background: Notwithstanding its beneficial immunoprophylactic outcomes regarding leprosy and childhood TB, BCG vaccination may cause adverse events, particularly of the skin. However, this local hyper-immune reactivity cannot be predicted before vaccination, nor is its association with protection against leprosy known. In this study we investigated the occurrence of adverse events after BCG (re)vaccination in contacts of leprosy patients and analyzed whether the concomitant systemic anti-mycobacterial immunity was associated with these skin manifestations. Methods: Within a randomized controlled BCG vaccination trial in Bangladesh, 14,828 contacts of newly diagnosed leprosy patients received BCG vaccination between 2012 and 2017 and were examined for adverse events 8 to 12 weeks post-vaccination. From a selection of vaccinated contacts, venous blood was obtained at follow-up examination and stimulated with Mycobacterium leprae (M. leprae) antigens in overnight whole-blood assays (WBA). M. leprae phenolic glycolipid-I-specific antibodies and 32 cytokines were determined in WBAs of 13 individuals with and 13 individuals without adverse events after vaccination. Results: Out of the 14,828 contacts who received BCG vaccination, 50 (0.34%) presented with adverse events, mainly (80%) consisting of skin ulcers. Based on the presence of BCG scars, 30 of these contacts (60%) had received BCG in this study as a booster vaccination. Similar to the pathological T-cell immunity observed for tuberculoid leprosy patients, contacts with adverse events at the site of BCG vaccination showed elevated IFN-γ levels in response to M. leprae-specific proteins in WBA. However, decreased levels of sCD40L in serum and GRO (CXCL1) in response to M. leprae simultaneously indicated less T-cell regulation in these individuals, potentially causing uncontrolled T-cell immunity damaging the skin. Conclusion: Skin complications after BCG vaccination present surrogate markers for protective immunity against leprosy, but also indicate a higher risk of developing tuberculoid leprosy

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.Author summary Leprosy, a chronic infectious disease caused by Mycobacterium leprae (M. leprae), targets the skin and nerves and often results in irreversible disabilities as well as social exclusion. Though the disease can be efficiently treated, leprosy elimination is hampered by ongoing transmission of M. leprae. Currently, elimination is monitored by the number of new cases. Since only a small percentage of individuals infected with M. leprae develops disease, this does not accommodate monitoring of transmission. Previous studies have shown that antibody levels against M. leprae in blood correspond to the bacterial load in an individual and can be used as a proxy for infection, although antibodies cannot distinguish between past and present infection. In young children, infection is recent by definition, which allows assessment of more current state of transmission in an area. Thus, this literature review investigated studies on leprosy serology in children without leprosy. Our findings underscore that young children are a fitting group for up-to-date monitoring of M. leprae transmission in an area as seropositivity is inversely related with age. Importantly, a standardized, field-friendly test quantitatively measuring anti-M. leprae antibodies should be applied for population screening to monitor the status of transmission and thereby elimination in an area.Immunogenetics and cellular immunology of bacterial infectious disease

Quantitative rapid test for detection and monitoring of active pulmonary tuberculosis in nonhuman primates

Simple Summary Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is the most lethal infectious disease from a single pathogen for which there is no effective vaccine available. Rhesus macaques are extremely susceptible to MTB and therefore represent relevant models to study the pathogenesis of TB and assess the potential of TB drugs and vaccines. However, there are no diagnostic tools currently available that allow rapid, user-friendly detection of TB for either TB research purposes or monitoring nonhuman primate colonies. To develop a rapid diagnostic test, we investigated whether low complexity lateral flow assays (LFAs) that we recently developed for rapid and quantitative detection of human serum proteins are applicable to detect and monitor active pulmonary TB in NHPs. We found that serum levels of SAA1, IP-10, and IL-6 detected by LFAs were significantly increased after MTB infection in rhesus macaques. Moreover, levels of these biomarkers correlated with disease severity as determined by pathology scoring and allowed detection of the effect of vaccination and drug treatment in experimentally MTB infected macaques. These UCP-LFAs thus offer a low-cost, convenient, and minimally invasive diagnostic tool that can be used to assess new therapeutic and prophylactic treatment methods in macaques to tackle TB. Nonhuman primates (NHPs) are relevant models to study the pathogenesis of tuberculosis (TB) and evaluate the potential of TB therapies, but rapid tools allowing diagnosis of active pulmonary TB in NHPs are lacking. This study investigates whether low complexity lateral flow assays utilizing upconverting reporter particles (UCP-LFAs) developed for rapid detection of human serum proteins can be applied to detect and monitor active pulmonary TB in NHPs. UCP-LFAs were used to assess serum proteins levels and changes in relation to the MTB challenge dosage, lung pathology, treatment, and disease outcome in experimentally MTB-infected macaques. Serum levels of SAA1, IP-10, and IL-6 showed a significant increase after MTB infection in rhesus macaques and correlated with disease severity as determined by pathology scoring. Moreover, these biomarkers could sensitively detect the reduction of bacterial levels in the lungs of macaques due to BCG vaccination or drug treatment. Quantitative measurements by rapid UCP-LFAs specific for SAA1, IP-10, and IL-6 in serum can be utilized to detect active progressive pulmonary TB in macaques. The UCP-LFAs thus offer a low-cost, convenient, and minimally invasive diagnostic tool that can be applied in studies on TB vaccine and drug development involving macaques.Immunogenetics and cellular immunology of bacterial infectious disease

Field-friendly serological tests for determination of M. leprae-specific antibodies

Microscopic imaging and technolog

Detection and monitoring of Mycobacterium leprae infection in nine banded armadillos (Dasypus novemcinctus) using a quantitative rapid test

Leprosy is an infectious disease caused by Mycobacterium leprae with tropism for skin and peripheral nerves. Incessant transmission in endemic areas is still impeding elimination of leprosy. Although detection of M. leprae infection remains a challenge in asymptomatic individuals, the presence of antibodies specific for phenolglycolipid-I (PGL-I) correlate with bacterial load. Therefore, serosurveillance utilizing field-friendly tests detecting anti-PGL-I antibodies, can be applied to identify those who may transmit bacteria and to study (reduction of) M. leprae transmission. However, serology based on antibody detection cannot discriminate between past and present M. leprae infection in humans, nor can it detect individuals carrying low bacillary loads. In humans, anti-PGL-I IgM levels are long-lasting and usually detected in more individuals than anti-PGL-I IgG levels. Inherent to the characteristically long incubation time of leprosy, IgM/IgG relations (antibody kinetics) in leprosy patients and infected individuals are not completely clear. To investigate the antibody response directly after infection, we have measured antibody levels by ELISA, in longitudinal samples of experimentally M. leprae infected, susceptible nine-banded armadillos (Dasypus novemcinctus). In addition, we assessed the user- and field-friendly, low-cost lateral flow assay (LFA) utilizing upconverting reporter particles (UCP), developed for quantitative detection of human anti-PGL-I IgM (UCP-LFA), to detect treatment- or vaccination-induced changes in viable bacterial load. Our results show that serum levels of anti-PGL-I IgM, and to a lesser extent IgG, significantly increase soon after experimental M. leprae infection in armadillos. In view of leprosy phenotypes in armadillos, this animal model can provide useful insight into antibody kinetics in early infection in the various spectral forms of human leprosy. The UCP-LFA for quantitative detection of anti-PGL-I IgM allows monitoring the efficacy of vaccination and rifampin-treatment in the armadillo leprosy model, thereby providing a convenient tool to evaluate the effects of drugs and vaccines and new diagnostics.Cancer Signaling networks and Molecular Therapeutic

Prototype multi-biomarker test for point-of-care leprosy diagnostics

To end the decade-long, obstinately stagnant number of new leprosy cases, there is an urgent need for field-applicable diagnostic tools that detect infection with Mycobacterium leprae, leprosy's etiologic agent. Since immunity against M. leprae is characterized by humoral and cellular markers, we developed a lateral flow test measuring multiple host proteins based on six previously identified biomarkers for various leprosy phenotypes. This multi-biomarker test (MBT) demonstrated feasibility of quantitative detection of six host serum proteins simultaneously, jointly allowing discrimination of patients with multibacillary and paucibacillary leprosy from control individuals in high and low leprosy endemic areas. Pilot testing of fingerstick blood showed similar MBT performance in point-of-care (POC) settings as observed for plasma and serum. Thus, this newly developed prototype MBT measures six biomarkers covering immunity against M. leprae across the leprosy spectrum. The MBT thereby provides the basis for immunodiagnostic POC tests for leprosy with potential for other (infectious) diseases as well.Cancer Signaling networks and Molecular Therapeutic

BCG-induced immunity profiles in household contacts of leprosy patients differentiate between protection and disease

Leprosy is an infectious disease caused by Mycobacterium leprae leading to irreversible disabilities along with social exclusion. Leprosy is a spectral disease for which the clinical outcome after M. leprae infection is determined by host factors. The spectrum spans from anti-inflammatory T helper-2 (Th2) immunity concomitant with large numbers of bacteria as well as antibodies against M. leprae antigens in multibacil-lary (MB) leprosy, to paucibacillary (PB) leprosy characterised by strong pro-inflammatory, Th1 as well as Th17 immunity. Despite decades of availability of adequate antibiotic treatment, transmission of M. leprae is unabated. Since individuals with close and frequent contact with untreated leprosy patients are particularly at risk to develop the disease themselves, prophylactic strategies currently focus on household contacts of newly diagnosed patients. It has been shown that BCG (re)vaccination can reduce the risk of leprosy. However, BCG immunopro-phylaxis in contacts of leprosy patients has also been reported to induce PB leprosy, indicating that BCG (re)vaccination may tip the balance between protective immunity and overactivation immunity causing skin/nerve tissue damage. In order to identify who is at risk of developing PB leprosy after BCG vaccination, amongst individuals who are chronically exposed to M. leprae, we analyzed innate and adaptive immune markers in whole blood of household contacts of newly diagnosed leprosy patients in Bangladesh, some of which received BCG vaccination. As controls, individuals from the same area without known contact with leprosy patients were similarly assessed. Our data show the added effect of BCG vaccination on immune markers on top of the effect already induced by M. leprae exposure. Moreover, we identified BCG-induced markers that differentiate between protective and disease prone immunity in those contacts. (c) 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).Immunogenetics and cellular immunology of bacterial infectious disease

Fingerstick test quantifying humoral and cellular biomarkers indicative for M-leprae infection

Objectives: New user-friendly diagnostic tests for detection of individuals infected by Mycobacterium leprae (M. leprae), the causative pathogen of leprosy, can help guide therapeutic and prophylactic treatment, thus positively contributing to clinical outcome and reduction of transmission. To facilitate point-of-care testing without the presence of phlebotomists, the use of fingerstick blood (FSB) rather than whole blood-derived serum is preferred. This study is a first proof-of-principle validating that previously described rapid serum tests detecting antibodies and cytokines can also be used with FSB. Methods: Quantitative detection of previously identified biomarkers for leprosy and M. leprae infection, anti-M. leprae PGL-I IgM antibodies (αPGL-I), IP-10 and CRP, was performed with lateral flow (LF) strips utilizing luminescent up-converting reporter particles (UCP) and a portable reader generating unbiased read-outs. Precise amounts of FSB samples were collected using disposable heparinized capillaries. Biomarker levels in paired FSB and serum samples were determined using UCP-LF test strips for leprosy patients and controls in Bangladesh, Brazil, South-Africa and the Netherlands. Results: Correlations between serum and FSB from the same individuals for αPGL-I, CRP and IP-10 were highly significant (p < .0001) even after FSB samples had been frozen. The αPGL-I FSB test was able to correctly identify all multibacillary leprosy patients presenting a good quantitative correlation with the bacterial index. Conclusions: Reader-assisted, quantitative UCP-LF tests for the detection of humoral and cellular biomarkers for M. leprae infection, are compatible with FSB. This allows near-patient testing for M. leprae infection and immunomonitoring of treatment without highly trained staff. On site availability of test-result concedes immediate initiation of appropriate counselling and treatment. Alternatively, the UCP-LF format allows frozen storage of FSB samples compatible with deferred testing in central laboratories