Fundamental research

Leprosy research

New, rapid diagnostics for a centennial, slow disease

Leprosy is a slow, chronic disease with a long incubation period, which still represents a considerable health treat in developing countries: worldwide there are 2 to 3 million people with leprosy-related disabilities and associated social stigma . Also in The Netherlands there are about 400 leprosy patients. The causative agent of leprosy, Mycobacterium leprae (M.leprae) , that is closely related to the bacterium causing tuberculosis, was the first bacterium shown to cause human disease. Although hardly fatal, the chronic symptoms often afflict individuals in their most productive stage of life and therefore impose a significant social and economic burden on society.

Most individuals who are exposed to M.leprae, develop protective immunity and are able to kill the bacterium without any complications. Those who are not able to mount an efficient immune response, develop leprosy. The disease can be manifested in several forms making it a clinical spectrum. On one pole of this spectrum the diffuse form, lepromatous leprosy, is found which is characterized by large numbers of bacteria located in tissue . The other polar form is called tuberculoid leprosy and is characterized by strong, almost unrestrained immune response against the bacterium, causing secondary damage to skin and nerve tissue.

Transmission

Transmission of M.leprae is clearly unabated as evidenced by the number of new cases, 10% of whom are children, that plateaued at an annual rate of more than 200,000 annually since 2005. It is still not exactly known how and when transmission takes place nor what are the most important risk factors for transmission. Continued transmission in endemic areas is likely caused by the large reservoir of individuals who are infected but who do not have any symptoms yet. The incubation time for leprosy is typically between 2-5 years (but cases of 10-20 years are observed as well). During this period, the infected individuals can transmit bacteria to others. Prevention of M.leprae transmission therefore requires early detection of infected individuals to allow preventive treatment before symptoms occur and to prevent further transmission in the community.

Immune reactions

Although leprosy can be treated effectively with multidrug therapy (MDT), it is complicated by acute episodes of exacerbated inflammation, called leprosy reactions, occurring before, during but also after completion of MDT. These immunological complications occur in up to 50% of leprosy patients and represent the major cause of irreversible, neurological damage and consequent anatomical deformities. Prompt diagnosis and treatment, will aid recovery from inflammatory nerve damage and reduce risks for permanent disability considerably. However, prevention of irreversible tissue damage and consequent lifelong handicaps, requires more detailed knowledge on the mechanisms underlying leprosy reactions.

Biomarkers: a combination of bacterium- and host-derived tools

Currently, leprosy is diagnosed based on clinical symptoms and detection of  bacteria in slit skin smears. However, no laboratory test exists for early detection of  leprosy or leprosy reactions, or for asymptomatic infection. Such diagnostic tests would contribute considerably to detection of leprosy, allowing reduction of transmission as well as the occurrence of nerve damage.

Since the publication of its genome sequence, several M.leprae-specific proteins (antigens) have been identified. Analysis of the activation of immune cells in response to these unique proteins has led to the identification of antigens that can be used to detect infection with M.leprae and who form the basis of newly developed tests that can be used to measure anti-mycobacterial immune responses in blood .

The remaining challenge in leprosy research is to determine which compounds formed by the immune system (called immune-biomarkers) are characteristic for the development of leprosy, leprosy reactions or that indicate a protective response against M.leprae. This involves a combination of biomarkers and their ratio, a so-called biomarker profile. In addition to immune biomarkers, we study genetic and metabolic biomarkers in blood and urine, respectively.

Besides cross-sectional analysis at one time point, investments in l arge-scale, follow-up studies, allowing intra-individual comparison of immune profiles of individuals in leprosy-endemic areas worldwide, are essential to evaluate which biomarkers correlate with progression to disease and may be used as predictive biomarkers. Therefore, we have initiated longitudinal studies in Bangladesh, Brazil, and China to follow contacts of leprosy  for several years. This will enable detection  of biomarkers discriminating between patients and healthy individuals, but also of the variation in biomarkers within one individual at different points in time.

Diagnostic tests

Leprosy endemic areasare often short of sophisticated laboratories which makes it imperative to develop diagnostic tests that are suitable for field settings. In collaboration with the LUMC Department of Molecular Cell Biology, we have developed a first generation, field-friendly diagnostic test for leprosy (reactions), based on immune biomarkers in combination with M. leprae-specific proteins, which is at present brought further to the field in extended, longitudinal studies in three leprosy endemic continents. The format of this test is now also investigated for application in TB diagnosis.  Current research focusses on the development of version 2.0 in which several biomarkers are detected simultaneously.

The LUMC Department Infectious Diseases is the national reference centre for routine serological diagnosis of leprosy, and provides this service also to European Institutes.

Model disease

LUMC leprosy research is not restricted to infectious diseases. Since its inter-individual variability in clinical manifestations closely parallels the ability of the host to establish effective immunity to M. leprae, leprosy represents an intriguing model of human immunoregulatory disease. This has resulted in leprosy being the first disease for which researchers identified HLA disease association, human regulatory cells and the Th1/Th2 concept for human T cells.

Moreover, leprosy shares genetic susceptibility genes with chronic/ autoimmune diseases such as rheumatoid arthritis, psoriasis and Crohn's disease where acute inflammation related to leprosy reactions occur as well. Current research therefore focusses on “personalized diagnostics”, consisting of conscientious monitoring of patients to early detect destructive immune responses.

LUMC leprosy research is conducted in collaboration with numerous foreign research institutes mainly in developing countries (Brazil, Bangladesh, China, Ethiopia, Nepal, India). The LUMC has a leading role within the international IDEAL (Initiative for Diagnostic and Epidemiological Assays for Leprosy) consortium.

Leprosy research at the LUMC has been supported and funded for many years by external funding organisations such as The Netherlands Leprosy Relief (NLR), The Turing Foundation, The Leprosy Research Initiative (LRI), Die Deutsche Lepra- und Tuberkulosehilfe (DAHW), The Order of Malta-Grants-for-Leprosy-Research (MALTALEP), The Heiser Program for Research in Leprosy in The New York Community Trust and The Q.M. Gastmann Wichers Foundation.

Staffmembers involved at the Department of Infectious Diseases: Prof. dr. A Geluk and Prof. dr. THM Ottenhoff.

Application form for routine serological leprosy diagnosis

Publications

Geluk, A. and PLAM Corstjens. 2017. CRP: unique tell-tale biomarker or common denominator. Lancet Infectious Diseases. DOI: http://dx.doi.org/10.1016/S1473-3099(17)30472-3.

van Hooij A, Tjon Kon Fat EM. van den Eeden SJF, Wilson L, Batista da Silva M, Salgado CB, Spencer JS, Corstjents PLAM, and A Geluk. 2017. Field-friendly serological tests for determination of M. leprae-specific antibodies. Scientific reports 7(1):88688. DOI: http://dx.doi.org/10.1038/s41598-017-07803-7.

van Hooij, A, DM Boeters, EM Tjon Kon Fat, SJF van den Eeden, PLAM Corstjens, AHM van der Helm-van Mil, and A Geluk. 2017. Longitudinal IP-10 serum levels are associated with the course of disease activity and remission in rheumatoid arthritis. Clin. Vaccina Immunol. DOI: http://dx.doi.org/10.1128/CVI.00060-17.

Hagge, DA, Parajuli P, CB Kunwar, DRSJB Rana, R Thapa, KD Neupane, P Nicholls, LB Adams, A Geluk, M Shah, and IB Napit. 2017. Opening a can of worms: leprosy reactions and complicit soil-transmitted helminths. Ebioscience 23:119-124.

van Hooij A et al. Quantitative lateral flow strip assays as User-Friendly Tools To Detect Biomarker Profiles For Leprosy. Scientific Reports. 2016. 6:34260.

Inkeles MS, Teles RM, Pouldar D, et al. Cell-type deconvolution with immune pathways identifies gene networks of host defense and immunopathology in leprosy. JCI Insight.2016. 1(15):e88843.

Corstjens, P. L., H. A. van, E. M. Tjon Kon Fat, S. J. van den Eeden, L. Wilson, and A. Geluk. 2016. Field-friendly test for monitoring multiple immune response markers during onset and treatment of exacerbated immunity in leprosy. Clin. Vaccine Immunol. Mar 30. pii: CVI.00033-16

Roset Bahmanyar, E, C Smith, P Brennan, R Cummings, M Duthie, JH Richardus, P Saunderson, T Shwe, S Rosen and A Geluk. 2016. Leprosy diagnostic test development as a prerequisite towards elimination: requirements from the user’s perspective. PLoS Negl Trop Dis doi:10.1371/journal.pntd.0004331.

Mayboroda, OA, A van Hooij, SJF van den Eeden, K Dijkman, S Khadge, P Thapa, CB Kunwar, DA Hagge, and A Geluk. 2016. Exploratory urinary metabolomics of Type 1 Leprosy Reactions. Int. J. Infect. Dis. DOI : 10.1016/j.ijid.2016.02.012.

Khadge, S., S. Banu, K. Bobosha, van der Ploeg-van Schip JJ, I. M. Goulart, P. Thapa, C. B. Kunwar, K. E. van Meijgaarden, S. J. van den Eeden, L. Wilson, S. Kabir, H. Dey, L. R. Goulart, J. Lobato, W. Carvalho, Y. Bekele, K. L. Franken, A. Aseffa, J. S. Spencer, L. Oskam, T. H. Otttenhoff, D. A. Hagge, and A. Geluk. 2015. Longitudinal immune profiles in type 1 leprosy reactions in Bangladesh, Brazil, Ethiopia and Nepal. BMC. Infect. Dis. 15: 477.

Corstjens, PLAM, EM Tjon Kon Fat, CJ de Dood, JJ van der Ploeg-van Schip, KLMC Franken, NN Chegou, JS Sutherland, R Howe, A Mihret, D Kassa, M van der Vyver, J Sheehama, A Crampin, H Mayanja-Kizza, THM Ottenhoff, G Walzl and A Geluk. 2015. Multi-center evaluation of a user-friendly lateral flow assay to determine IP-10 and CCL4 levels in blood of TB and non-TB cases in Africa.Clinical Biochemistry. S0009-9120(15)00393-8 [pii];10.1016/ j.clinbiochem.2015.08.013 [doi].

Bobosha, K, EM Tjon Kon Fat, SJF van den Eeden, Y Bekele, JJ van der Ploeg-van Schip, CJ de Dood, K Dijkman, KLMC Franken, L Wilson, A Aseffa , JS Spencer, THM Ottenhoff, PLAM Corstjens and A Geluk. 2014. Field-Evaluation of a New Lateral Flow Assay for Detection of Cellular and Humoral Immunity against Mycobacterium leprae. PLoS Negl Trop Dis 8(5): e2845. doi:10.1371/journal.pntd.0002845.

Geluk A, KE van Meijgaarden, L Wilson, K Bobosha, JJ van der Ploeg- van Schip, Susan JF van den Eeden, E Quinten, K Dijkman, KLMC Franken, I Haisma, MC Haks, CLM van Hees, and THM Ottenhoff. 2014. Longitudinal immune responses and gene expression profiles associated with type 1 leprosy reactions. J. Clinical Immunology. 34: 245-255.

Geluk A 2013.  Challenges in immunodiagnostic tests for leprosy. Expert Opinion on Medical Diagnostics. 7:265-274.

Geluk, A, K Bobosha, JJ van der Ploeg-van Schip, JS Spencer, S Banu, M Brandao Martins, S Cho, KLMC Franken, HJ Kim, Y Bekele, MKM Uddin, SA Hadi, A Aseffa, MCV Pessolani, GMB Pereira, HM Dockrell, and THM Ottenhoff. 2012. New biomarkers for Mycobacterium leprae infection applicable in areas highly endemic for leprosy. J.Immunol. 188: 4782-4791.

Geluk, A, MS Duthie, and JS Spencer. 2011. Postgenomic Mycobacterium leprae antigens for cellular and serological diagnosis of M. leprae exposure, infection and leprosy disease. Leprosy Review 82: 398-417.

Corstjens, PLAM, CJ de Dood, JJ Ploeg-van Schip, CC Wiesmeijer, T Riuttamäki-Rantanen, KE van Meijgaarden, JS Spencer, HJ Tanke, THM Ottenhoff, A Geluk. 2011. Lateral flow assay for simultaneous detection of cellular- and humoral immune responses. Clinical Biochemistry 44: 1241-1246.

Geluk, A, SJF van den Eeden, K Dijkman, L Wilson, HJ Kim, K Franken, MCV Pessolani, JS Spencer, GMB Pereira, and THM Ottenhoff. 2011. In vivo cytotoxic function of the ML1419c HLA-A*0201-restricted T cell epitope of Mycobacterium leprae. J. Immunology 187: 1393–1402.

Geluk, A, KE van Meijgaarden, KLMC Franken, Y W Subronto, B. Wieles, SM Arend, EP Sampaio, T de Boer, WR Faber, B Naafs, and TH M Ottenhoff. 2002. Identification and characterization of the ESAT-6 homologue of Mycobacterium leprae and T cell cross-reactivity with Mycobacterium tuberculosis. Infect. Immun. 70: 2544-2548.

Ottenhoff, THM, PR Klatser, J Ivanyi, DG Elferink, MY de Wit, RRP de Vries. 1986. Mycobacterium leprae-specific protein antigens defined by cloned human helper T cells. Nature 319: 66-68.