Combined antigen-specific interferon-γ and interleukin-2 release assay (FluoroSpot) for the diagnosis of Mycobacterium tuberculosis infection

PLoS One. 2015 Mar 18;10(3):e0120006. doi: 10.1371/journal.pone.0120006. eCollection 2015.


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 R2 = 0.67 for ESAT-6 and R2 = 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Antigens, Bacterial / immunology*
  • Bacterial Proteins / immunology
  • Case-Control Studies
  • Female
  • Humans
  • Immunoassay / methods*
  • Interferon-gamma / immunology
  • Interferon-gamma / metabolism*
  • Interleukin-2 / immunology
  • Interleukin-2 / metabolism*
  • Male
  • Middle Aged
  • Mycobacterium tuberculosis / immunology*
  • Tuberculosis / diagnosis*
  • Tuberculosis / immunology*
  • Tuberculosis / microbiology


  • Antigens, Bacterial
  • Bacterial Proteins
  • CFP-10 protein, Mycobacterium tuberculosis
  • ESAT-6 protein, Mycobacterium tuberculosis
  • Interleukin-2
  • Interferon-gamma

Grant support

Dumitru Chesov is the recipient of ERS Walther Guerrero CiqueroTuberculosis Fellowship (STRTF 2013-2889). Christoph Lange and Barbara Kalsdorf are supported by the German Center for Infection Research (DZIF). FluoroSpot reagents have been provided by Autoimmun Diagnostika (AID) GmbH, (Straßberg, Germany). The funder provided support in the form of salaries for authors [Rosemarie Preyer], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.