Discordant results between the interferon-gamma release assays (IGRAs) and tuberculin skin test (TST) are common in latent tuberculosis infection (LTBI). We evaluated whether the measurement of IFN-γ and interleukin (IL)-2T-cell responses, after prolonged Mycobacterium tuberculosis-specific antigen stimulation, can be used as adjunctive biomarker for LTBI detection in subjects with discordant results between TST and QuantiFERON-Gold In-Tube (QFT). 196 healthcare workers were screened for LTBI and in 90 of those participants, the QFT was repeated after 18 h, and IFN-γ/IL-2 immune response was measured after 72 h long-term stimulation. Of the 196 patients, 34 had positive, 155 negative, and 7 indeterminate QFT results. Discordant TST +/QFT - results were found in 29 (14.7%) patients, of whom 6 (20.6%) were Bacillus Calmette-Guerin (BCG) vaccinated. None of 23 non-BCG vaccinated subjects showed a specific IFN-γ immune response after 18 h nor 72 h of incubation, whereas 3/23 (13.04%) discordant subjects produced a specific long-term IL-2 response, which might reflect a LTBI status. In LTBI group (TST +/QFT +) both cytokine levels were increased after long-term in comparison to short-term stimulation. No significant long-term IFN-γ/IL-2 secretion was detected in control group (TST -/QFT -). Taken together, our data showed that the 87% of discordant patients who did not respond to the long-term assay, as controls subjects, were judged LTBI negative. The use of classic QFT and long-term IL-2 response may have a potential role to clarify the LTBI status in individuals in whom the diagnosis of LTBI is uncertain due to the discordance of the available diagnostic tests, such as TST and IGRA. © 2014 Elsevier B.V. All rights reserved.