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... Show moreLeprosy 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/). Show less
Leprosy has been described in Eurasian red squirrel (Sciurus vulgaris; ERS) carcasses since 2014. Studies of ERS carcasses have not provided information about incubation or disease progression in... Show moreLeprosy has been described in Eurasian red squirrel (Sciurus vulgaris; ERS) carcasses since 2014. Studies of ERS carcasses have not provided information about incubation or disease progression in this host but have provided important insights into pathogen presence and distribution throughout the United Kingdom. Here we present field study data on 31 live ERS from an island population naturally infected with Mycobacterium leprae that were assessed longitudinally over a 2-yr time period. Clinical assessment, serologic (anti-phenolic glycolipid-I antibody [alpha PGL-I] detection) and molecular methods (polymerase chain reaction) were used to diagnose and categorize ERS at each assessment as a leprosy case, a leprosy suspect, colonized by M. leprae, or a contact ERS. Eight ERS (25.8%) were identified as leprosy cases: four at initial assessment, two at 6 mon and two at 24 mon after initial assessment. One ERS was categorized a leprosy suspect when it developed typical lesions 12 mon after initial assessment, despite negative serologic and molecular test results at this time, though M. leprae DNA had been isolated during the initial assessment. Seven ERS (22.6%) were categorized as colonized and of these, six were reassessed but did not develop clinical signs of leprosy within 6 (n = 2), 12 (n = 3), and 18 (n = 1) mon. Most (48.4%, n = 15) were categorized as contact ERS. Progression of leprosy lesions varied between ERS, but always increased in severity over time and was paralleled with increased antibody response. Based on our dataset, we propose the hypotheses: 1) leprosy in ERS is a chronic, slowly progressing disease in this species, similar to that described for other hosts; 2) lesions can undergo repeated ulceration-healing cycles; and 3) in some instances M. leprae DNA and alpha PGL-I antibodies are detectable before the onset of clinical signs of disease. Future studies addressing the progression of leprosy in ERS should follow affected animals over a longer time period and include tissue samples to pair molecular diagnostics with serologic results. Show less
Zhou, Z.J.; Hooij, A. van; Vervenne, R.; Sombroek, C.C.; Fat, E.T.K.M.; Ottenhoff, T.H.M.; ... ; Geluk, A. 2021
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... Show moreSimple 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. Show less
Leprosy is an infectious disease that affects peripheral nerves and can lead to severe lifelong disabilities. Despite the availability of an effective cure, a fairly stable number of about 200,000... Show moreLeprosy is an infectious disease that affects peripheral nerves and can lead to severe lifelong disabilities. Despite the availability of an effective cure, a fairly stable number of about 200,000 new leprosy patients per year has been reported since 2010. This stagnation shows that the transmission of the mycobacteria that cause leprosy, Mycobacterium leprae and Mycobacterium lepromatosis, is still taking place. Timely diagnosis of leprosy patients is therefore vital, so that the time frame in which a person is contagious is shortened, but also irreversible nerve damage and leprosy-associated disabilities can be prevented. However, tools that confirm the diagnosis of leprosy are not yet available. This thesis investigated which factors in blood (the so-called biomarkers) can help to diagnose leprosy. The clinical signs of leprosy have a spectral character and are influenced by the immune response of the host. A combination of biomarkers is described that is able to identify patients with a lot of bacteria (multibacillary) as well as the more difficult to diagnose patients with few bacteria (paucibacillary). Subsequently, these biomarkers have been implemented in user-friendly lateral flow assays, which have been extensively validated in leprosy endemic areas. Show less
Braet, S.M.; Hooij, A. van; Hasker, E.; Fransen, E.; Wirdane, A.; Baco, A.; ... ; Jong, B.C. de 2021
Author summaryLeprosy is the oldest infectious disease known to humankind. We still do not succeed in curbing its transmission, with more than 200,000 new patients detected worldwide each year.... Show moreAuthor summaryLeprosy is the oldest infectious disease known to humankind. We still do not succeed in curbing its transmission, with more than 200,000 new patients detected worldwide each year. Identifying persons with a high burden of bacteria is key to curb transmission. To identify these persons, bacteria are counted in invasive and painful samples like slit skin smears and skin biopsies. We evaluated whether we can use less invasive samples, like fingerstick blood or nasal swabs, to determine the bacterial load. We found that the level of antibodies against M. leprae (alpha PGL-I IgM) in fingerstick blood correlates well with the bacterial load determined in skin biopsies from the same leprosy patient. Therefore, a high level of antibodies against M. leprae in fingerstick blood might identify persons who pose a potential risk for transmission of leprosy and could be prioritized for contact screening, which is essential for control of the disease.The World Health Organization (WHO) endorsed diagnosis of leprosy (also known as Hansen's disease) entirely based on clinical cardinal signs, without microbiological confirmation, which may lead to late or misdiagnosis. The use of slit skin smears is variable, but lacks sensitivity. In 2017-2018 during the ComLep study, on the island of Anjouan (Union of the Comoros; High priority country according to WHO, 310 patients were diagnosed with leprosy (paucibacillary = 159; multibacillary = 151), of whom 263 were sampled for a skin biopsy and fingerstick blood, and 260 for a minimally-invasive nasal swab. In 74.5% of all skin biopsies and in 15.4% of all nasal swabs, M. leprae DNA was detected. In 63.1% of fingerstick blood samples, M. leprae specific antibodies were detected with the quantitative alpha PGL-I test. Results show a strong correlation of alpha PGL-I IgM levels in fingerstick blood and RLEP-qPCR positivity of nasal swabs, with the M. leprae bacterial load measured by RLEP-qPCR of skin biopsies. Patients with a high bacterial load (>= 50,000 bacilli in a skin biopsy) can be identified with combination of counting lesions and the alpha PGL-I test. To our knowledge, this is the first study that compared alpha PGL-I IgM levels in fingerstick blood with the bacterial load determined by RLEP-qPCR in skin biopsies of leprosy patients. The demonstrated potential of minimally invasive sampling such as fingerstick blood samples to identify high bacterial load persons likely to be accountable for the ongoing transmission, merits further evaluation in follow-up studies. Show less
Zhou, Z.J.; Pena, M.; Hooij, A. van; Pierneef, L.; Jong, D. de; Stevenson, R.; ... ; Geluk, A. 2021
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.... Show moreLeprosy 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. Show less
Pierneef, L.; Hooij, A. van; Taal, A.; Rumbaut, R.; Nobre, M.L.; Brakel, W. van; Geluk, A. 2021
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,... Show moreBackground 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. Show less
The presence of Mycobacterium lepromatosis and Mycobacterium leprae in Eurasian red squirrel (Sciurus vulgar's, ERS) carcasses throughout the British Isles, and leprosy as a disease, have recently... Show moreThe presence of Mycobacterium lepromatosis and Mycobacterium leprae in Eurasian red squirrel (Sciurus vulgar's, ERS) carcasses throughout the British Isles, and leprosy as a disease, have recently been reported using histological and molecular diagnostic methods. In 2016, the first longitudinal study of ERS affected by leprosy was initiated. One of the main challenges was the reliable diagnosis of leprosy in live ERS, which is important for (a) welfare and case management and (b) surveillance or pretranslocation screening efforts. We explored diagnostic methods ranging from detailed clinical assessment and informative categorization of observed lesions, thermal imaging, serology (antiphenolic glycolipid-I antibody [alpha PGL-I] detection) to molecular methods (polymerase chain reaction [PCR). For PCR the ear was established as the optimal sampling site. Based on the experiences from this 2-yr study we propose an objective categorization system for clinical lesions and a diagnostic framework for the combination of the diagnostic tools we found to be effective in live ERS: clinical assessment, alpha PGL-I serology, and PCR. Thermal imaging did not offer additional information for leprosy diagnostics in ERS. We propose an amended definition of leprosy lesions in ERS as "skin areas of local hair loss, in which a firm-rubbery, glossy swelling develops, that may ulcerate" and standardized terminology for describing ERS leprosy status. The information presented forms the basis of a consistent, reliable diagnostic and reporting system for leprosy cases in ERS. Show less
Tio-Coma, M.; Kielbasa, S.M.; Eeden, S.J.F. van den; Mei, H.L.; Roy, J.C.; Wallinga, J.; ... ; Geluk, A. 2021
Background: Leprosy, a chronic infectious disease caused by Mycobacterium leprae, is often late-or misdiag-nosed leading to irreversible disabilities. Blood transcriptomic biomarkers that... Show moreBackground: Leprosy, a chronic infectious disease caused by Mycobacterium leprae, is often late-or misdiag-nosed 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 pres-ence 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 iden-tified, 1,613 genes were differentially expressed in progressors before diagnosis compared to HC. We identi-fied 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 ina 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 pre-dicting development of leprosy 4 to 61 months before clinical diagnosis. Assessment of this signature in con-tacts of leprosy patients can function as an adjunct diagnostic tool to target implementation of interventions to restrain leprosy development. (C) 2021 The Author(s). Published by Elsevier B.V. Show less
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... Show moreMycobacterium 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. Show less
Dijk, J.H.M. van; Hooij, A. van; Groot, L.M.; Geboers, J.; Moretti, R.; Verhard-Seymonsbergen, E.; ... ; Geluk, A. 2021
Point-of-care (POC) diagnostic tests for the rapid detection of individuals infected with Mycobacterium leprae, the causative pathogen of leprosy, represent efficient tools to guide therapeutic and... Show morePoint-of-care (POC) diagnostic tests for the rapid detection of individuals infected with Mycobacterium leprae, the causative pathogen of leprosy, represent efficient tools to guide therapeutic and prophylactic treatment strategies in leprosy control programs, thus positively contributing to clinical outcome and reducing transmission of this infectious disease. Levels of antibodies directed against the M. leprae-specific phenolic glycolipid I (PGL-I) closely correlate with an individual's bacterial load and a higher risk of developing leprosy. We describe herein the assembly of a set of PGL glycans carrying the characteristic phenol aglycon and featuring different methylation patterns. The PGL trisaccharides were applied to construct neoglycoproteins that were used to detect anti-PGL IgM antibodies in leprosy patients. ELISAs and quantitative lateral-flow assays based on up-converting nanoparticles (UCP-LFAs) showed that the generated PGL-I and PGL-II trisaccharide neoglycoconjugates can be applied for the detection of anti M. leprae IgM antibodies in POC tests. Show less
Hooij, A. van; Fat, E.M.T.K.; Jong, D. de; Khatun, M.; Soren, S.; Chowdhury, A.; ... ; Corstjens, P.L.A.M. 2021
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... Show moreTo 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. Show less
