This study aims to evaluate the diagnostic accuracy of a Mycobacterium tuberculosis (MTB)-specific triple-color FluoroSpot assay (IFN-γ/IL-2/TNF-α) in the differentiation of tuberculosis (TB) infection status in febrile patients. Febrile patients with suspected active TB (ATB) were consecutively enrolled. The frequencies and proportions of MTB-specific T cells secreting IFN-γ, IL-2, and TNF-α were detected at the single-cell level by triple-color FluoroSpot assay. The diagnostic index was fitted with a binary logistic regression model, and the diagnostic accuracy was evaluated according to the receiver operating characteristic (ROC) curve. The sensitivity, specificity, predictive values (PV), and likelihood ratios (LR) were calculated. A total of 210 febrile patients were enrolled, 53 patients were diagnosed with ATB (28 pathogen-confirmed vs. 25 clinically diagnosed) and 157 patients were non-ATB (84 with latent tuberculosis infection (LTBI) vs. 73 uninfected with MTB). Additionally, 30 pathogen-confirmed ATB patients were assembled. When diagnosing ATB, the area under the ROC curve (AUROC) of the MTB-specific triple-color FluoroSpot assay was significantly better than that of T-SPOT.TB (0.882 vs. 0.811, p = 0.017). With the fitted diagnostic index at a cutoff value of 0.378, the sensitivity, specificity, LR+, and LR- were 74.7%, 93.0%, 10.66, and 0.27, respectively. When differentiating ATB from LTBI, the AUROC of the FluoroSpot assay and T-SPOT.TB was 0.878 and 0.692, respectively (p < 0.001). With a diagnostic index of 0.413, the sensitivity, specificity, LR+, and LR were 77.1%, 85.7%, 5.40, and 0.27, respectively. The MTB-specific triple-color FluoroSpot (IFN-γ/IL-2/TNF-α) might be helpful for the differentiation of TB infection status in febrile patients.
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