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Background Interferon-gamma (IFN-γ) release assays (IGRAs) were designed to detect latent tuberculosis infection (LTBI). However, discrepancies were found between the tuberculin skin test (TST) and IGRAs results that cannot be attributed to prior Bacille Calmètte Guerin vaccinations. The aim of this study was to evaluate tools for improving LTBI diagnosis by analyzing the IFN-γ response to RD1 proteins in prolonged (long-term response) whole blood tests in those subjects resulting negative to assays such as QuantiFERON-TB Gold In tube (QFT-IT). Methods The study population included 106 healthy TST+ individuals with suspected LTBI (recent contact of smear-positive TB and homeless) consecutively enrolled. As controls, 13 healthy subjects unexposed to M. tuberculosis (TST-, QFT-IT-) and 29 subjects with cured pulmonary TB were enrolled. IFN-γ whole blood response to RD1 proteins and QFT-IT were evaluated at day 1 post-culture. A prolonged test evaluating long-term IFN-γ response (7-day) to RD1 proteins in diluted whole blood was performed. Results Among the enrolled TST+ subjects with suspected LTBI, 70/106 (66.0%) responded to QFT-IT and 64/106 (60.3%) to RD1 proteins at day 1. To evaluate whether a prolonged test could improve the detection of LTBI, we set up the test using cured TB patients (with a microbiologically diagnosed past pulmonary disease) who resulted QFT-IT-negative and healthy controls as comparator groups. Using this assay, a statistically significant difference was found between IFN-γ levels in cured TB patients compared to healthy controls (p < 0.006). Based on these data, we constructed a receiver operating characteristic (ROC) curve and we calculated a cut-off. Based on the cut-off value, we found that among the 36 enrolled TST+ subjects with suspected LTBI not responding to QFT-IT, a long term response to RD1 proteins was detected in 11 subjects (30.6%). Conclusion These results indicate that IFN-γ long-term response to M. tuberculosis RD1 antigens may be used to detect past infection with M. tuberculosis and may help to identify additional individuals with LTBI who resulted negative in the short-term tests. These data may provide useful information for improving immunodiagnostic tests for tuberculosis infection, especially in individuals at high risk for active TB.