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A two-step procedure using a tuberculin skin test (TST) followed by an interferon (IFN)-γ assay in cases in which the TST result is positive has been advocated to screen for latent tuberculosis infection. However, TST could also boost the in vitro immune response. In this study, we evaluated the effect of TST on the results of the IFN-γ assay.Our study included 84 health-care workers who had been working in the department of pulmonary medicine for > 1 year. First, a whole-blood IFN-γ assay was performed, and then the TST was applied. Two to 4 weeks later, a follow-up IFN-γ assay was performed. A commercially available IFN-γ assay (QuantiFERON-TB GOLD; Cellestis Ltd; Carnegie, VIC, Australia) was used.Valid TST results were available in 82 individuals because 2 participants refused to undergo the TST after the IFN-γ assay. The TST result was positive in 36 of 82 participants (42.7%), and the IFN-γ assay was positive in 16 of 82 participants (19.5%). The overall agreement between the two tests was 67.5% (κ = 0.31; 95% confidence interval, 0.22 to 0.40). The IFN-γ levels increased significantly from 0.05 to 0.19 (p = 0.011), and 3 of 18 participants (16.7%) had conversion of their IFN-γ assay results in the TST-positive group. However, in the TST-negative group, the IFN-γ levels did not change after the TST.The agreement between the results of the TST and the IFN-γ assay was low, and IFN-γ level could be influenced by the TST, in the TST-positive population, when a follow-up IFN-γ assay is performed 2 to 4 weeks later.