Supplementary Material for: Bifunctional T-Cell-Derived Cytokines for the Diagnosis of Tuberculosis and Treatment Monitoring

<b><i>Background:</i></b> Diagnosis and treatment monitoring of patients with tuberculosis remain challenging. <b><i>Objective:</i></b> We have evaluated whether <i>Mycobacterium</i>-specific interferon (IFN)-γ and interleukin (IL)-2 bifunctional cytokine immune response assays improve the diagnosis of and correlate to treatment response in pulmonary tuberculosis. <b><i>Methods:</i></b> Early secretory antigenic target (ESAT)6/culture filtrate protein 10 (CFP10), microsomal triglyceride transfer protein 65 (MTP65) and the purified protein derivative (PPD) tuberculin-specific immune profiles were investigated in peripheral blood mononuclear cells from 19 patients with culture-confirmed tuberculosis and 23 healthy community controls (HCCs; 82.6% with latent <i>M. tuberculosis</i> infection) using a novel fluorescence-based dual-colour enzyme-linked immunospot (EliSpot) technology (FluoroSpot). <b><i>Results:</i></b> The frequency of ESAT6/CFP10-induced IFN-γ+IL-2- producing cells was elevated (p < 0.001), whereas the percentages of specific IFN-γ-IL-2+ (p = 0.002) and IFN-γ+IL-2+ double producing cells (p = 0.037) were diminished in tuberculosis patients in comparison to HCCs. A 3-host marker model using a combination of those IFN-γ and IL-2 single-cell responses showed 93.8% sensitivity and 77.8% specificity for tuberculosis. During tuberculosis treatment, the PPD-induced immune responses shifted from an IFN-γ+IL-2- dominated profile towards a balance of IFN-γ-IL-2+ and IFN-γ+IL-2+ double producing cells (all p ≤ 0.05). <b><i>Conclusions:</i></b> The addition of antigen-specific IL-2 production to IFN-γ responses by EliSpot in IFN-γ release assays increases diagnostic sensitivity for active tuberculosis.