Combined Antigen-Specific Interferon-γ and Interleukin-2 Release Assay (FluoroSpot) for the Diagnosis of Mycobacterium tuberculosis Infection

BACKGROUND: To evaluate interleukin (IL)-2 and interferon (IFN)-γ secreting T-cells in parallel for the differentiation of latent infection with Mycobacterium tuberculosis infection (LTBI) from active tuberculosis. METHODS: Following ex-vivo stimulation of peripheral blood mononuclear cells (PBMC) with M. tuberculosis-specific antigens early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10, immune responses were assessed by enzyme-linked immunospot IFN-γ release assay (EliSpot-IGRA) and a novel dual cytokine detecting fluorescence-linked immunospot (FluoroSpot) in 18 patients with pulmonary tuberculosis, 10 persons with previously cured tuberculosis, 25 individuals with LTBI and 16 healthy controls. RESULTS: Correlation of IFN- γ(+) spot-forming cells in EliSpot-IGRA and FluoroSpot were R(2) = 0.67 for ESAT-6 and R(2) = 0.73 for CFP-10. The number of IL-2(-) IFN- γ(+) producing cells was higher in patients with tuberculosis compared with past tuberculosis (CFP-10-induced p = 0.0068) or individuals with LTBI (ESAT-6-induced p = 0.0136). A cutoff value of >16 CFP-10-induced IFN-γ(+) secreting cells/200.000 PBMC in the EliSpot-IGRA discriminated with highest sensitivity and specificity (89% and 76%, respectively). However, overlap in cytokine responses precludes distinction between the cohorts on an individual basis. CONCLUSIONS: Combined analysis of IFN-γ and IL-2 secretion by antigen specific T-cells does not allow a reliable differentiation between different states of M. tuberculosis infection in clinical practice.