Objectives: Distinguishing between active tuberculosis (ATB) and latent tuberculosis infection (LTBI) remains challenging. Methods: The modified T-SPOT.TB assay was performed in 499 participants (243 ATB and 256 LTBI) and another 322 participants (162 ATB and 160 LTBI) who were diagnosed in Qiaokou (training) and Caidian (validation) cohort respectively. Results: The mean spot sizes (MSS) of early secreted antigenic target 6 (ESAT-6) spot-forming cells (SFC) of T-SPOT.TB assay in ATB patients was significantly higher than that in LTBI individuals. 1.0 x 10(5) was the optimal number of cells added to phytohaemagglutinin (PHA) well for obtaining more accurate TBspecific antigen to phytohaemagglutinin (TBAg/PHA) ratio. The area under the curve of the diagnostic model by combination of ESAT-6 SFC MSS and modified TBAg/PHA ratio in distinguishing ATB from LTBI was 0.959 in training cohort, with a sensitivity of 90.12% and a specificity of 91.02% when a cutoff value of 0.46 was used. This diagnostic model showed similar performance in the validation cohort. The area under the curve, sensitivity, and specificity were 0.962, 93.21%, and 90.00%, respectively. Further flow cytometry analysis showed that ESAT-6 stimulation induced a significantly higher mean fluorescence intensity of IFN-gamma(+) cells in lymphocytes compared with culture filtrate protein 10 (CFP-10) stimulation. In contrast, CFP-10 stimulation induced a significantly higher percentage of IFN-gamma(+) cells in lymphocytes compared with ESAT-6 stimulation. Conclusions: The combination of the MSS of ESAT-6 SFC and the modified TBAg/PHA ratio of T-SPOT.TB assay showed great value in discriminating ATB from LTBI. (C) 2020 The Author(s). Published by Elsevier Ltd on behalf of The British Infection Association.