Background
Leprosy is a chronic, immunoregulatory infectious disease caused by
Mycobacterium leprae that particularly affects the skin and peripheral nerves and often results in severe, life-long disabilities and deformities [
1,
2]. The number of new cases has plateaued at 220,000–250,000 annually, but many linger undetected [
3,
4]. Leprosy remains endemic in Africa, South America and Asia and with increasing migration, new cases are detected in developed countries, where initial misdiagnosis is likely to occur [
5‐
7].
The inter-individual variability in clinical manifestations of leprosy closely parallels the ability of the host to mount an effective immune response to
M. leprae. This is depicted by an immunological and clinical spectrum in those who progress to disease, ranging between two completely different poles i.e. tuberculoid (TT) and lepromatous (LL) leprosy [
8]. Host resistance to
M. leprae is associated with the emergence of a protective Thelper-1 (Th1)-based response characterized by the secretion of the innate and adaptive cytokines IL-12p70, IFN-γ, lymphotoxin-α/β, and (moderate levels of) other pro-inflammatory cytokines such as TNF-α. LL patients secrete predominantly anti-inflammatory mediators such as IL-10, accompanied by the absence of Th1-associated cytokines in response to
M. leprae but characterized by high anti-
M. leprae antibody titers. Conversely, TT patients produce exacerbated levels of pro-inflammatory cytokines, including those produced by Th17 rather than Th1, and frequently driven by strong innate immune activation resulting in the release of IL-1β and/or IL-6, TGF-β and IL-23 [
9,
10].
Although leprosy can be treated effectively with multidrug therapy (MDT), it is complicated by persisters [
11] as well as acute inflammatory episodes called leprosy reactions. These immunological complications, occurring before, during and after MDT treatment in 30–50 % of the patients, represent the major cause of leprosy-related neurological damage [
12,
13]. Two types of reactions are recognized: type 1 or reversal reactions (RRs) and type 2 or erythema nodosum leprosum (ENL). RRs are considered a delayed hypersensitivity reaction with characteristic infiltrations of skin and nerve lesions by CD4
+ T-cells producing IFN-γ and TNF-α [
14‐
16]. Up to 30 % of leprosy patients are affected by RRs, which most commonly occur in borderline forms of leprosy (borderline-tuberculoid (BT), borderline-borderline (BB), borderline-lepromatous (BL)) in which concomitant immunological fluctuations can generate significant neuropathology [
17]. Prompt diagnosis and anti-reactional treatment contributes to recovery significantly thus reducing risks for permanent tissue damage [
18,
19]. Unfortunately, reactions are frequently misdiagnosed due to decreased expertise within integrated health services [
17]. Therefore, reliable tests for early diagnosis of RR could make huge differences in clinical outcomes. A major obstacle to developing such tests is the lack of dependable biomarkers for reactions across endemic populations.
For the complex host immuno-pathogenicity of leprosy [
2,
14], assessment of multiple rather than single biomarkers is more informative of the hosts’ immune status. Therefore, we aimed to identify relevant host immune-biomarkers for early diagnosis of type 1 reactions. We recruited newly diagnosed leprosy patients longitudinally and studied
M. leprae-specific cellular- and humoral immunity in blood of patients 1) in the absence of any clinical signs of reactions at least three months before reactions, 2) very early after clinical presentation of reactions and 3) after completion of treatment. Non-reactional patients (before and after treatment) as well as healthy individuals from the same area were analyzed similarly. To accommodate worldwide applicability, independent of the genetic and environmental background, this study was executed similarly in four distinct, prospective cohorts in Asia, Africa and South-America.
Materials and methods
General study-procedure
Recruitment took place in Bangladesh (International Centre for Diarrhoeal Disease Research Bangladesh, Dhaka), Brazil (National Reference Centre for Sanitary Dermatology and Leprosy, Uberlandia), Ethiopia (ALERT hospital and Health Centre,) and Nepal (Mycobacterial Research Laboratories, Kathmandu). Experiments were performed according to standard operating procedures and each site was provided with identical reagents.
Study participants
Patients and endemic controls (EC) were recruited on a voluntary basis between February 2008-March 2013 (Table
1
). Leprosy was diagnosed based on clinical, bacteriological and histological observations and classified by skin biopsies according to Ridley and Jopling [
1]. Leprosy patients were treated according to WHO standards. Clinical monitoring for reactions was performed during monthly clinic visits. Clinical and demographic data was collected in clinical research forms (Additional file
1) and subsequently transferred in databases with special emphasis on standardizing data collection and definition of reaction between all cohorts [
20,
21]. For patients who presented with reactions the type, severity, skin- and/or nerve involvement, number of lesions and relapse were noted, according to state-of-the-art clinical expertise and international consensus scoring [
21,
22]. EC were assessed for the absence of clinical signs and symptoms of leprosy and TB. Staff of leprosy- or TB clinics were excluded.
Table 1
Participating study sites and study groups
Bangladesh | EC | nad | 0.9 | 20–40 | 20 |
BL/LL | 2.20 | 5 | 18–61 | 31 |
RR | 1.68 | 2.5 | 21–63 | 20 |
Brazil | EC | nad | 1.3 | 24–76 | 23 |
BL/LL | 1.51 | 1 | 22–26 | 25 |
RR | 1.95 | 3.3 | 25–68 | 20 |
Ethiopia | EC | nad | 1.8 | 18–45 | 11 |
BL/LL | 1.25 | 1.7 | 18–52 | 25 |
RR | 0.46 | 2.8 | 18–60 | 15 |
Nepal | EC | nad | 3.6 | 19–28 | 20 |
BL/LL | 2.96 | 2 | 35–58 | 13 |
RR | 1.45 | 2.5 | 27–50 | 20 |
Leprosy prevalence
Dhaka, prevalence: 2.45/10,000, new case detection rate (NCDR): 0.31/10,000 (Annual Reports of Leprosy Control Institute & Hospital, Dhaka); Uberlandia, prevalence: 0.96/10,000, NCDR: 1,12/10,000 (National Disease Surveillance System, Secretariat of Health Surveillance, Ministry of Health Brazil); Addis Ababa, prevalence: 0.6/10,000 in 2010–2011, 0.4/10,000 in 2012, NCDR: 0.35/10,000 (FMOH reports); Kathmandu, prevalence: 1.1-0.79/10,000, NCDR: 1.67- 1.15/10,000 (Annual Report 2012–2013, Leprosy Control Division, Department of Health Services, Kathmandu).
Recruitment
Newly diagnosed, untreated leprosy patients without clinical reactions were enrolled and blood was drawn before MDT (t = 0). Patients who presented reactions within three months of the start of therapy were excluded to avoid profile analyses of patients with latent reactions. If patients presented with reactions after more than three months of MDT, blood was drawn before initiation of anti-reactional therapy (t = x). Newly diagnosed leprosy patients who visited clinics with RR were recruited (t = x) but consequently lacked t = 0 samples. From all patients, blood was collected after MDT and/or steroid therapy (t = end). For patients with RR this was done at least one month after completion of steroid therapy to avoid assessment of the effect of steroids. All patients were assessed for the absence of reactions three months after t = end. For patients showing clinical signs of reactions within three months after t = end, this time point was excluded. In case patients died, moved or withdrew from the study, preventing follow-up, their samples were excluded. Blood was used for isolation of peripheral blood mononuclear cells (PBMC). Supernatants and sera were stored at −20 °C.
Antigens
M. leprae recombinant proteins were produced as described [
23].
M. leprae whole cell sonicate was provided through the NIH/NIAID “Leprosy Research Support” Contract N01 AI-25469 (
http://www.beiresources.org).
Cytokine/chemokine analysis
PBMC, freshly isolated from venous blood, were cultured for 6 days with antigens as described [
23]. IFN-γ was determined by ELISA (U-CyTech, Utrecht, The Netherlands) [
24]. A positive, reference supernatant was provided to all laboratories. IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IFN-γ, IP-10, G-CSF, GM-CSF, MCP-1, MIG, MIP-1β and TNF in supernatants or sera were measured using the Bio-Plex-suspension-array-system (Bio-Rad, Veenendaal, NL) [
23]. IFN-β was determined in undiluted sera (25ul) using Procartaplex IFN-β simplex-kit (eBioscience, Hatfield, UK) and CCL18 was determined (1:10 dilutions; 100 μl) by ELISA (DY394 DuoSet, R & D Systems, Minneapolis, MN) according to manufacturers’ instructions.
Serology
Antibodies against ML2028 (Ag85B) and ND-O-BSA, a synthetic analogue of phenolic glycolipid I (PGL-I), were determined as described [
25].
Ethics
This study was performed according to the Helsinki Declaration (2008 revision). Participants were informed about the study-objectives, the samples and their right to refuse to take part/withdraw from the study without consequences for their treatment. Written informed consent was obtained before enrolment. All patients received treatment according to national guidelines. Ethical approval of the study-protocol was obtained through appropriate ethics committees: Ethical Review Committee of ICDDR, B (#PR-10032; #PR-2007-069); Brazilian National Council of Ethics in Research (CONEP) and UFU Research Ethics Committee (#499/2008); National Health Research Ethical Review committee Ethiopia (NERC # RDHE/127-83/08); Nepal Health Research Council (NHR #751).
Statistical analysis
Differences in cytokine concentrations were analysed with two-tailed Mann–Whitney U tests (unpaired samples) for non-parametric distribution and Wilcoxon matched-pairs signed rank test or paired t test for longitudinal analyses using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, CA, USA;
www.graphpad.com). The statistical significance level used was
p < 0.05.
Discussion
Biomarkers as reliable correlates of disease complications and response to therapy are essential tools for early diagnosis of disease states in chronic infections. Generally, the performance of one biomarker can be significantly enhanced by using instead a custom-made grouping of independent biomarkers, called a profile or signature. In the current situation of leprosy elimination, the availability of sensitive and specific biomarkers that aid early diagnosis of leprosy reactions as well as monitor therapy, would be a strategic advantage enabling health care workers to identify, treat and possibly prevent these episodes at early stages, thereby reducing nerve damage. Since the immunopathology of leprosy, particularly in reactional states, is linked to temporal changes in the immune response to M. leprae, leprosy represents a uniquely suitable model to study immune-biomarker changes in relation to clinical disease manifestations.
This is the first study in which cellular- and humoral immunity specific for
M. leprae in leprosy patients within the three main continents reporting leprosy were monitored longitudinally during treatment. Although previous studies have analyzed circulating cytokines and chemokines [
29] around the time of leprosy reactions. The addition of an
M. leprae antigen-specific component, as utilized in this study, provides more specificity to this approach.
The data demonstrate translational importance since similar intra-individual trends were observed for development of RR in different endemic areas, allowing global application of these biomarkers in tests for early diagnosis of RR. In this respect, the importance of the combined effect of M. leprae-induced cytokine production (IFN-γ, IL-17, IP-10, IL-1β, VEGF), determined by their ratios versus IL-10, was highlighted, providing valuable tools for diagnosis of reactional states.
The biomarker profiles identified in this study for RR can be used in blood-based diagnostic tests [
28] to detect (intra-individual) changes during these acute inflammatory periods but also provide an approach for other chronic diseases with acute inflammatory states such as tuberculosis [
34] and buruli ulcer [
35] (paradoxical reactions) and Crohn’s disease [
36,
37], to help early diagnose such episodes thereby contributing to timely treatment and prevention of disease-specific tissue damage.
The acknowledged immunosuppressive role of IL-10 in lepromatous leprosy [
38] as well as in
M. leprae infected mice [
39,
40] was also evident from its reduction at RR-onset [
41]. Thus, during RR the breakdown of regulation, in favour of inflammation, seems to underlie the aetiology of reactional tissue damage, whereas balanced ratios of these immune responses, as present in nonreactional leprosy patients, are protective against RR [
42]. This is in line with the associations of IL-10 genetic variants with development of leprosy and leprosy reactions [
6,
43‐
46]. Suppression of IL-10 in a borderline tuberculoid-like murine model significantly augmented CD4/44
+ and CD8/44
+ longitudinal infiltrative responses specific to
M. leprae antigens and permitted CD4
+ T-cells to penetrate and fragment nerve [
47], in line with our current field findings and supporting monitoring patient IL-10 levels in ratio to cytokines proven to escalate during RR as a potential early indicator of impending clinical RR.
As a second biomarker for RR in multiple ethnic backgrounds, increased serum IP-10 levels were identified, whereas CCL18, which is elevated in lepromatous leprosy [
30], decreased at early RR in 6/10 patients who developed RR. Since CCL18 is secreted by dendritic cells upon recognition of
M. tuberculosis [
48] and has been implicated in differentiation of macrophages into an alternative phenotype [
49] this suggests that decreased CCL18 levels lead to fewer alternatively activated macrophages and less T-cell regulation [
6,
50]. These data therefore indicate that new biomarker discovery approaches for RR also contribute to our understanding of the RR-associated immunopathologic mechanisms, suggesting new opportunities for therapeutic interventions.
Since RRs are considered delayed hypersensitivity reactions caused by overreaction and/or dysregulation of host defence mechanisms, conscientious (personalized) treatment monitoring is vital similar to other diseases with acute inflammatory states such as psoriasis and Crohn’s disease which share specific susceptibility genes with leprosy [
36,
51]. Our data showed that pro-inflammatory cytokine/IL-10 ratios, serum IP-10 can be used for monitoring treatment while not on steroids. Therefore, besides for early diagnosis of reactions, tests to monitor efficacy of treatment are useful as well, especially in the light of the reoccurrence of these episodes.
To allow access to diagnostic test at resource-poor field settings, we recently developed low-tech, robust lateral flow assays (LFAs) for (simultaneous) detection of inflammatory (IP-10) and regulatory (IL-10) immune responses together with anti-PGL-I IgM antibodies in short term whole blood assays [
28,
52]. In the light of the currently identified immune markers for RR, field-friendly LFAs measuring these cytokines for leprosy patients on MDT at each clinic-visit may be helpful to early detect RR if used for intra-individual testing. Thus, to provide a rapid test, the diagnostic potential of the cytokine ratios defined here, need to be determined in future studies using whole blood assays as well.
Conclusions
Type 1 or reversal reactions (RRs) are a major cause of leprosy-related nerve impairment and bear similarities with acute inflammation induced episodes in other (infectious) diseases. Since there is no laboratory test for the early diagnosis of these episodes, this multi-continental, longitudinal study on the occurrence of RRs in leprosy patients, showed for the first time that both M. leprae-specific cellular- as well as humoral host immune-profiles, correlating with early onset of these inflammatory episodes, can be identified. Biomarkers associated with diagnosis or efficiency of treatment of type 1 reactions were identified, based on intra-individual changes rather than single values. In particular, ratios of cytokines secreted by M. leprae stimulated blood cells as well as circulating cytokines in sera, contributed to these biomarker profiles. Thus, these profiles can be applied for the early diagnosis and to monitor reactional episodes and contribute to timely treatment and reduction/prevention of tissue damage.
Acknowledgements
The authors gratefully acknowledge all patients and blood donors. AHRI, CSU, ICDDR, B, KIT, LUMC and MRL Anandaban are part of the IDEAL (Initiative for Diagnostic and Epidemiological Assays for Leprosy) Consortium. We are indebted to Kapil Dev Neupane (Anandaban), Dr. Sheikh Abdul Hadi (Dhaka), Genet Amare, Haregewoin Yetesha, Alemayehu Kifle, Dr. Saba M. Lambert and Martha Zewdie (AHRI/ALERT, Addis Ababa) for recruitment of study participants and sample collection.
Competing interest
The authors declare that they have no competing interests.
Author contributions
Designed research: AG, LO.(Facilitation of) patient recruitment and laboratory analysis: SKh, SB, KB, IG, PT, CK, SKa, YB, DH, LG, AA Performed research: SKh, KB, JP, PT, CK, KF, KM, SE, LW, HD, JL, WC, YB, JS. Contributed reagents/ tools: JS. Analyzed the data: AG, SKh, KB, JP, SE, JS. Wrote the paper: AG, DH, TO. All authors read and approved of the final manuscript.