ARTICLE Synergistic antigen combinations for the development of interferon gamma release assays for paucibacillary leprosy R. M. Oliveira & E. M. Hungria & A. de Araújo Freitas & A. L. O. M. de Sousa & M. B. Costa & S. G. Reed & M. S. Duthie & M. M. A. Stefani Received: 5 December 2013 /Accepted: 31 January 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract The development of immunodiagnostic tests for paucibacillary leprosy (PB) is based on Mycobacterium leprae specific-cell mediated immunity (CMI)/IFN- γ production. Re- cently, novel M. leprae protein antigens that stimulate CMI have been described. This study evaluated different M. leprae antigen combinations in whole blood assay (WBA). Five study groups were tested (20 per group): newly diagnosed, untreated PB patients and multibacillary leprosy patients (MB); household contacts of MB patients (HHC); healthy endemic controls (EC); pulmonary tuberculosis patients (TB). WBA (heparinized, 24 h 37 °C 5 % CO 2) were stimulated with: 10 μg/ml of each individual M. leprae recombinant protein (rML) and five com- binations of rML (46f+LID-1, ML0276+LID-1, ML2055+ ML1632+ML2044, ML0276+46f, ML2055+LID-1) — M. leprae cell sonicate (MLCS, 10 μg/ml), PHA (1 μg/ml), and PBS alone. Human IFN-γ ELISA (QuantiFERON®-TB Gold/QFT-G, Cellestis) was performed using stimulated plasma (arbitrary cut-off=50 pg/ml). Three out of five antigen combina- tions (46f+LID-1, ML0276+LID-1, ML2055+ML1632+ ML2044) were able to increase the levels of IFN-γ production in WBA in a larger number of responders among both PB leprosy and contacts. However, the magnitude of IFN-γ re- sponses was higher among contacts. The antigen combination (46f+ML0276) stimulated IFN-γ only in symptomatic PB lep- rosy patients and not in asymptomatic contacts. Few controls (EC, TB) responded to combinations (0–15 %), indicating the specificity of the response in an endemic area with high BCG coverage. The synergistic effect of new combinations of M. leprae proteins upon IFN-γ production in WBA indicates their potential use for the development of an interferon gamma release assay/IGRA for the diagnosis of PB leprosy. Abbreviations BB Borderline borderline BI Bacilloscopic index BL Borderline lepromatous BSA Bovine serum albumin BT Borderline tuberculoid CMI Cell-mediated immunity ELISA Enzyme-linked immunosorbent assay HHC Healthy household contact IFN-γ Interferon gamma IGRA IFN-γ release assays LL Lepromatous MB Multibacillary MDT Multi-drug therapy M. leprae Mycobacterium leprae M. tuberculosis Mycobacterium tuberculosis PB Paucibacillary PBS Phosphate buffered solution PGL-I Phenolic glycolipid-I rML M. leprae recombinant proteins TB Tuberculosis TT Tuberculoid leprosy WBA Whole blood assay Introduction Leprosy is a chronic infectious disease that can result in significant impairment of nerve function and permanent MMAS and MSD share the senior authorship R. M. Oliveira : E. M. Hungria : A. de Araújo Freitas : A. L. O. M. de Sousa : M. B. Costa : M. M. A. Stefani (*) Tropical Pathology and Public Health Institute, Federal University of Goiás, Goiânia, GO, Brazil e-mail: mmastefani@gmail.com S. G. Reed : M. S. Duthie Infectious Disease Research Institute, 1124 Columbia Street, Suite 400, Seattle, WA 98104, USA Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-014-2077-z