Diagnostic accuracy of Mycobacterium tuberculosis-specific triple-color FluoroSpot assay in differentiating tuberculosis infection status in febrile patients with suspected tuberculosis

Lifan Zhang; Yuanchun Li; Xiaoqing Zou; Huimin Ma; Mengqiu Gao; Qiping Ge; Yueqiu Zhang; Zhengrong Yang; Xinuo Song; Qiwen Yang; Xiaoqing Liu

doi:10.3389/fimmu.2024.1462222

Diagnostic accuracy of Mycobacterium tuberculosis-specific triple-color FluoroSpot assay in differentiating tuberculosis infection status in febrile patients with suspected tuberculosis

Front Immunol. 2025 Jan 8:15:1462222. doi: 10.3389/fimmu.2024.1462222. eCollection 2024.

Authors

Lifan Zhang^#^{1

2

3}, Yuanchun Li^#¹, Xiaoqing Zou^#^{1

4}, Huimin Ma¹, Mengqiu Gao⁵, Qiping Ge⁵, Yueqiu Zhang¹, Zhengrong Yang¹, Xinuo Song⁶, Qiwen Yang⁶, Xiaoqing Liu^{1

2

3

4}

Affiliations

¹ Division of Infectious Diseases, Department of Internal Medicine, State Key Laboratory of Complex Severe and Rare Disease, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
² Clinical Epidemiology Unit, Peking Union Medical College, International Clinical Epidemiology Network, Beijing, China.
³ Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
⁴ Department of Scientific Research, Xiangya Hospital, Central South University, Changsha, China.
⁵ Department of Tuberculosis, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, China.
⁶ Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, China.

^# Contributed equally.

Abstract

Objective: 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.

Method: 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.

Result: 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.

Conclusion: 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.

Keywords: FluoroSpot; T-SPOT.TB; active tuberculosis; latent tuberculosis infection; triple-color.

MeSH terms

Adult
Aged
Diagnosis, Differential
Female
Fever* / diagnosis
Fever* / immunology
Fever* / microbiology
Humans
Interferon-gamma / metabolism
Interleukin-2
Latent Tuberculosis / diagnosis
Latent Tuberculosis / immunology
Male
Middle Aged
Mycobacterium tuberculosis* / immunology
ROC Curve
Sensitivity and Specificity
Tuberculosis* / diagnosis
Tuberculosis* / immunology
Tuberculosis* / microbiology
Tumor Necrosis Factor-alpha / analysis

Substances

Interferon-gamma
Tumor Necrosis Factor-alpha
Interleukin-2

Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by Capital’s Funds for Health Improvement and Research (2024-2-4016), the National High Level Hospital Clinical Research Funding (2022 PUMCH-A-119, 2022-PUMCH-C-013), and, National Natural Science Foundation of China (82373648).