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This study aimed to compare the accuracy of QuantiFERON-TB Gold Plus (QFT-Plus) and T-SPOT.TB for diagnosing active tuberculosis (ATB) in febrile patients, to explore influencing factors of positive results and to verify the potential value of QFT-Plus in the identification of ATB and latent tuberculosis infection (LTBI).A total of 240 febrile patients with ATB (n = 80) and non-ATB (n = 160) were recruited to assess the accuracy of QFT-Plus and T-SPOT.TB for diagnosing ATB. Multivariable logistic regression was used to analyze the influencing factors of positive results.The proportion of indeterminate results (ITRS) in QFT-Plus and T-SPOT.TB were 3.3% and 0%, respectively. The consistency between the results of the QFT-Plus and T-SPOT.TB was substantial. The area under the receiver operating characteristic curve (AUROC) of the QFT-Plus and T-SPOT.TB for diagnosing ATB was 0.792 and 0.849 (p = 0.070), respectively. The sensitivity of differentiating ATB from non-ATB was 92.2% in QFT-Plus versus 95.0% in T-SPOT.TB. The influencing factors of T-SPOT.TB positive result were male (odds ratio (OR) = 2.33, 95% confidence interval (CI) 1.27-4.26, p = 0.006), evidence of previous TB (OR 11.36, 95% CI 4.62-27.94, p0.001), while male (OR = 3.17, 95% CI 1.73-5.84, p0.001), evidence of previous TB (OR = 7.58, 95% CI 3.60-15.98, p0.001), and use of immunosuppressant (OR = 0.49, 95% CI 0.260.94, p = 0.030) were influencing factors for QFT-Plus positive result. There was no significant difference in QFT-Plus in differentiating ATB from LTBI in febrile patients.There was no significant difference between QFT-Plus and T-SPOT.TB for diagnosing ATB in febrile patients. QFT-Plus is prone to ITRS. The influencing factors including males, evidence of the previous TB, and use of immunosuppressant should be considered when interpreting positive results.

Background To establish the Mycobacterium tuberculosis (MTB) specific IFN-γ/IL-2/TNF-α-FluoroSpot assay, and preliminarily evaluate its accuracy of differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI). Methods Patients with pathologically confirmed and clinically diagnosed ATB in Peking Union Medical College Hospital and Beijing Chest Hospital from April 2020 to May 2021 were enrolled as case group, while patients with LTBI in the same period were enrolled as control group. The FluoroSpot assay was used to simultaneously detect the secretion of IFN-γ, IL-2 and TNF-α in T cells stimulated by the MTB specific antigens ESAT-6 and CFP-10 at the single-cell level. A binary logistic regression model was used to fit the combined diagnostic parameters, and the sensitivity, specificity, predictive value and likelihood ratio of the differential diagnosis of ATB and LTBI were calculated. Figure 1. Schematic diagram of FluoroSpot (IFN-γ/IL-2/TNF-α) detecting cytokine-secreting specific T cells after stimulation with MTB specific antigen. A. The green spots are the total IFN-γ-secreting T cells; B. The red spots are the total IL-2-secreting T cells; C. The blue spots are the total TNF-α-secreting T cells; D. The green spots are the single IFN-γ-secreting T cells; the red spots are the single IL-2-secreting T cells; the blue spots are the single TNF-α-secreting T cells; the yellow spots are the dual IFN-γ/IL-2-secreting T cells; the cyan spots are the dual IFN-γ/TNF-α-secreting T cells; the purple spots are the dual IL-2/TNF-α-secreting T cells; the white spots are the triple IFN-γ/IL-2/TNF-α-secreting T cells. Results 62 patients with ATB (37 pathogen-confirmed ATB, 25 clinical diagnosed ATB), 87 patients with LTBI were included. There was significant correlation of the frequencies of total IFN-γ-secreting T cells detected by IFN-γ/IL-2/TNF-α-FluoroSpot assay compared with T-SPOT.TB after stimulation of MTB-specific antigen (r=0.829 for ESAT-6, P< 0.001, r=0.804 for CFP-10, P< 0.001). ROC curve was drawn for both T-SPOT.TB and Fluorospot. For T-SPOT.TB, the AUROC was 0.669 (95%CI 0.574-0.765), the sensitivity and specificity of differentiating ATB from LTBI were 70.97% (95%CI 58.05%-81.80%) and 56.32% (95CI 45.26%-66.94%) respectively. While for Fluorospot, the AUROC was 0.906 (95 CI 0.856-0.957), the sensitivity and specificity of differentiating ATB from LTBI were 80.65% (95%CI 68.63% - 89.58%) and 88.51% (95%CI 79.88% - 94.35%) respectively. Figure 2. Correlation between the frequencies of total IFN-γ-secreting T cells detected by FluoroSpot assay and those of T-SPOT.TB. (A) Stimulated by EAST-6. (B) Stimulated by CFP-10. Figure 3. ROC curves and the corresponding AUROC for measurement of frequencies of specific T cells in differentiating ATB and LTBI under stimulation of ESAT-6 or CFP-10. The blue line is drawn with the frequency of IFN-γ-secreting T cells detected by T-SPOT.TB, and the AUC is 0.669 (95%CI, 0.574-0.765). The red line is drawn with combination of the frequencies and proportion of single IFN-γ-'single IL-2-'single TNF-α-'dual IFN-γ/IL-2-'dual IFN-γ/ /TNF-α-'dual IL-2/TNF-α-secreting T cells detected by FluoroSpot, and the AUC is 0.906 (95% CI, 0.856-0.957). Table 1. Diagnostic value of T-SPOT.TB and FluoroSpot for differentiating ATB from LTBI Conclusion Compared with T-SPOT.TB, the IFN-γ/IL-2/TNF-α-Fluorospot assay may be helpful to distinguish ATB from LTBI, and the results need to be verified by large sample prospective cohort study. Disclosures All Authors: No reported disclosures

Background Objective: To compare the accuracy of Quantiferon ®- TB Gold Plus (QFT-Plus) and T-SPOT.TB in the diagnosis of active tuberculosis (ATB). Methods From April 2020 to May 2021, patients with pathologically confirmed or clinically diagnosed ATB in Peking Union Medical College Hospital and Beijing Chest Hospital were enrolled as case group, while patients excluded from ATB in the same period were enrolled as control group, The clinical and laboratory information were collected. QFT-Plus and T-SPOT. TB were tested simultaneously to evaluate the consistency of the results and compare the sensitivity, specificity, predictive values and likelihood ratios for diagnosing ATB. Results Fifty-seven ATB patients and 159 non-ATB patients were included. 33 (57.9%) ATB patients were pathologically confirmed. The proportions of indeterminate results in QFT-Plus and T-SPOT.TB were 3.7% vs 0%, respectively. The agreement between the results of the QFT-Plus and T-SPOT.TB was substantial (kappa=0.644, p＜0.001). The area under the ROC curve of the QFT-Plus and T-SPOT.TB for diagnosing ATB was 0.759 (95%CI 0.689-0.829) vs 0.810 (95%CI 0.742-0.877), respectively, and there was no significant difference (p=0.303). the sensitivity of the QFT-Plus and T-SPOT.TB was 85.2% vs 89.5%, while the specificity was 61.7% vs 52.2%, respectively. In 33 Patients with pathologically confirmed ATB, the sensitivity of QFT-Plus and T-SPOT.TB was 87.9% vs 93.9% (p=0.669), respectively. In 78 patients (36.1%) who received glucocorticoid / immunosuppressive / biological agents, the positive rate of QFT-Plus was 35.9% (28/78), which was significantly lower than that of those who did not receive these agents (77 / 138pm,55.8%) (p=0.005), but there was no significant difference in the positive rate of T-SPOT.TB between the two groups (52.6% vs. 62.3%, p=0.162). The positive rate for both tests was independent of the peripheral blood lymphocyte count (p=0.675 for QFT-Plus vs. P=0.138 for T-SPOT.TB). Conclusion There was no significant difference between the QFT-Plus and T-SPOT.TB in the diagnosis of ATB. QFT-plus might be prone to indeterminate results and influenced by the immunosuppressive status. The results need to be verified by a prospective cohort study with large sample. Disclosures All Authors: No reported disclosures