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Annemieke Geluk Dept. Infect. Dis. LUMC The Netherlands Immunodiagnostic Tools for Leprosy: Exposure, Infection & Disease Brussels, 19th September 2013 Plenary Session (Facing the) Challenges in Leprosy prevalence still millions patients suffer from leprosy (> MS) NCD: quite static number globally (2012) 232,857 21,349 children Transmission is ongoing Early Diagnosis Transmission M. leprae = killed M. leprae survives 95% 5% 2-6 yrs >10 yrs Early Leprosy diagnosis Leprosy Diagnostic test Transmission Early treatment Diagnosis 2013 Based on clinical signs: # lesions, peripheral neuropathy detection of M. leprae => No early detection Based on immune responses: 1. Anti-PGL-I Ab: HMI: mostly detects MB not PB Positivity does not indicate leprosy 2. Lepromin skin test: CMI: not leprosy-specific Tests covering leprosy spectrum: combined HMI & CMI T-cells/mφ/ neutrophils marker? CMI Infected contacts HMI α-PGL-I IgM Infected contacts Longitudinal studies are lacking TT BT BB BL LL Paucibacillary (PB) - Multibacillary (MB) α-LID-1 IgG α-Ag85 IgG α-LAM IgG M. leprae-specific CMI Pre-genomic era T-ESAT & T-CFP10: TB diagnostics : Quantiferon L-ESAT 63% similarity to T-ESAT6 L-CFP10 40% similarity to T-CFP10 • Extensive recognition of L-ESAT-6 and L-CFP-10 by TB • No application as diagnostic tools in TB endemic countries Ag discovery (2001 – 2010) Identification of M. leprae unique sequences Analyse T cell reactivity in endemic regions Surplus Value of T cell assay using M. leprae unique proteins: 85% Average IFN- (pg/ml) 7% 100% 71% 2500 1500 500 200 100 0 NA PGL-I - PGL-I + students EC PGL-I - PGL-I + PB PGL-I -PGL-I + Rx PGL-I - PGL-I + (H)HC Surplus 1st T cell study IDEAL CSU Fiocruz LSHTM LUMC Selection M. leprae proteins & peptides Brazil Nepal Fiocruz/Goiás TLMN Bangladesh ICDDR,B IDRI Pasteur • Immunogenic in exposed • non-responsive in EC (Rio) Pakistan Aga Khan Ethiopia AHRI IFN- in LST cumulative for 5 sites ML 2283 4000 IFN- (pg/ml) 3000 2000 1000 200 100 0 EC Mlep+ 12/43 EC Mlep- 0/7 BL/LL BT/TT HHC TB 9 /50 10/50 10/46 3/50 IFN- responses in EC • Why IFN- responses in EC & TB to M. leprae unique antigens? • Influence of leprosy endemicity of habitat? IFN- & M.leprae unique protein in WBA allows detection of M. leprae exposure 14000 14000 ML2478 12000 ML0840 14000 10000 8000 8000 6000 6000 6000 4000 4000 4000 500 400 10000 8000 IFN- (pg/ml) 2000 2000 500 400 500 400 300 200 200 200 100 100 100 0 0 0 Bangladesh EC EC TB South-Korea p=0.0232 BT/TT HHC Bangladesh EC EC TB South-Korea p=0.036 2000 300 BT/TT HHC M. leprae 12000 10000 IFN- (pg/ml) IFN- (pg/ml) 12000 300 BT/TT HHC Bangladesh EC EC TB South-Korea Ethiopia/ ML2478 Brazil/ ML2478 5000 p=0.0001 4000 20000 p=0.0021 3000 15000 2000 10000 IFN- (pg/ml) IFN- (pg/ ml) IFN- to M.leprae unique Ag in EClow and EChigh in one city 1000 1000 800 600 400 5000 500 400 300 200 200 100 0 EC low EC high HHC BT/TT 0 EC low EC high Biomarkers for M. leprae Exposure M.leprae unique proteins can be used as tools to identify M.leprae exposed individuals using IFN- as a read out What about tools for M. leprae infection? Stages of M. leprae infection Infection stage: Biomarkers for: 1. Elimination of M.leprae without priming of M. leprae-specific T cells Innate immunity to M. leprae: IGRAneg EChigh 2. M. leprae infection eliminated in association with T cell priming Adaptive immunity Innate immunity to M. leprae HC 3. M. leprae replication maintained at a subclinical level by immune response Early/ Subclinical Innate immunity infection less HC 4. Pathogenic immune response to M. leprae Leprosy disease TT/BT Co-infections Diabetes BL/LL Innate immunity T cell subsets/ Cytokines involved in leprosy Activated T cell: Naive T cell IL-12/IFN- M.leprae IL-4 APC IL-1/IL-6 TGF-, IL-23 Th2 Th17 TGF- Treg IFN- TNF IP-10 Biomarkers Th1 (Possible) Role in : • Protection • TT/BT • RR IL-4 IL5 IL-13 α-PGL-I • LL/BL • ENL IL17A IL-17F IL-22 IL21 • TT/BT • RR • ENL IFN- IL-10 • LL/BL Biomarkers for M. leprae infection endemic vs not endemic (anymore) areas MIP-1 MIP-1/ M. leprae IP-10/IP-10 M. leprae ns 16000 p=0.0007 p=0.0039 ns 10000 ns p=0.029 14000 12000 8000 10000 8000 ns 4000 4000 pg/ ml pg/ ml 6000 ns 6000 4000 3000 2000 2000 1000 0 0 TT/BT HHC Bangladesh EC EC TB South-Korea TT/BT HHC Bangladesh EC EC TB South-Korea Biomarkers for M. leprae infection endemic vs not endemic (anymore) areas MCP-1/ M.leprae MCP-1 IL-1 IL-1/ M.leprae p=0.0006 14000 p=0.0021 ns p=0.0001 p=0.001 4000 12000 10000 3000 ns 8000 pg/ ml pg/ ml p=0.066 6000 p=0.044 2000 4000 1000 2000 0 0 TT/BT HHC Bangladesh EC EC TB South-Korea TT/BT HHC Bangladesh EC EC TB South-Korea Biomarkers for M. leprae infection (mycobacterial disease) ROC curve TT/BT vs EC MCP-1, MIP-1, IL-1 sensitivity 150 100 50 0 0 20 40 60 80 100% - specificity% Area Std. Error 95% confidence interval P value 0.9900 0.01616 0.9583 to 1.022 0.0002145 100 Biomarkers for M. leprae infection (mycobacterial disease) ROC curve TT/BT vs EC MCP-1, MIP-1, IL-1 sensitivity 150 100 50 0 0 20 40 60 80 100% - specificity% Area Std. Error 95% confidence interval P value 0.9900 0.01616 0.9583 to 1.022 0.0002145 100 Summary I • M.leprae unique Ag can identify M. leprae exposed individuals using IFN-; importance of proper reference group as EC (same socio-economic background, same part of town) • Combinations of additional cytokines & chemokines can discriminate between between M. leprae infected vs. uninfected (but exposed) healthy individuals in highly endemic areas • Longitudinal follow-up studies in HC from leprosy-endemic areas will be essential for evaluation (intra-individual comparison) Early Diagnosis 2 Transmission M. leprae = killed M. leprae survives 95% 5% 2-6 yrs >10 yrs 1. Early Leprosy diagnosis 2. Early diagnosis leprosy reactions M. leprae infection Diagnostic test Transmission Early treatment Biomarkers for Leprosy Reactions Biomarker for T1R 2007 2008- 2010- 1. Nepal Anandaban Hospital 2. Bangladesh ICDDR,B 3. Brazil UFU, Uberlandia 4. Brazil Fiocruz, Rio 5. Ethiopia AHRI Biomarkers for T1R Overall objective: • Identify novel biomarker signatures for early diagnosis of leprosy reactions Working hypothesis: • The development of leprosy reactions coincides with changes in the: - immunological profile and/ or - gene expression profile Biomarker Study Set-up: prospective cohort t = 0: • at recruitment • before MDT • no signs of T1R > 3months t = x: • onset of T1R t = end: • end of therapy 1. CMI 2. Serum 3. RNA 1. CMI 2. Serum 3. RNA 1. CMI 2. Serum 3. RNA T1R MB T1R T1R MB EC Cellular Biomarkers: Cytokine 4 Bangladesh Nepal 4000 20000 3000 2000 1000 pg/ ml pg/ ml 2000 1500 1000 200 500 500 150 400 100 300 50 200 100 0 0 RR RR RR t=0 t=x t=end RR t=0 RR t=x RR t=end Longitudinal trial Nilphamari Biobanking : 1. WBA samples 2. RNA samples 24h Test development: UCP-LF • A field-friendly, LF is developed for cellular (cytokines) and humoral (PGL-I) IR to M. leprae. • Th1 (IFN-) & Th2 cytokines (IL-10) • More cytokines under development • Evaluated in Africa (AE-TBC/ EDCTP) IFN IL10 PGL-I UCP-LF Diagnostic Test Strip 1 Strip 2 flow flow samples value LUMC, NL Jolien vd Ploeg-Schip Elisa Tjon Kon Fat Kees Franken Louis Wilson Marielle Haks Paul Corstjens Tom Ottenhoff LSHTM Hazel Dockrel UFU, Brazil Luiz & Isabela Goulart Janaina Lobato AHRI Kidist Bobosha ICDDR,B Sayera Banu Senjuti Kabir Fiocruz, Brazil Euzenir Sarno Roberta Olmo Pinheiro Cristina Pessolani Geraldo Pereira Marcia Brandao Milton Moraes KIT: Linda Oskam CSU: John Spencer Yonsei: Ray Cho Anandaban Hospital, Nepal Saraswoti Khadge Prathiba Thapa Chhatra B. Kunwar Murdo McDonald Deanna Hagge Q.M. GastmannWichers Foundation