Study design and population
This prospective diagnostic study was conducted in Shanghai Pulmonary Hospital, School of Medicine of Tongji University, between January 2019 and December 2021. The inclusion criteria were 1) aged 18-70 years, 2) healthy volunteers (HC) or patients with ATB, LTBI, non-tuberculosis mycobacterium infection (NTM), or other respiratory diseases (OD). The exclusion criteria were 1) primary or secondary immunodeficiency, including HIV, long-term steroid use, or co-existing autoimmune diseases, 2) diabetes or viral hepatitis, 3) history of anti-tuberculosis treatments. This study complied with the Declaration of Helsinki and was approved by the Ethics Committee of Shanghai Pulmonary Hospital. All participants signed the informed consent forms.
ATB (including active pulmonary TB (PTB) and active EPTB) was diagnosed according to i) no previous history of TB, ii) positive sputum, bronchoalveolar lavage fluid (BALF), or bacteriological tests, including positive acid-fast staining, positive MTB culture (Bectec960), or positive Gene-XPERT test, and iii) no other lung diseases [25-27]. LTBI was defined as household contacts of newly diagnosed TB patients with positive IGRA, with no evidence of clinically manifest active TB. OD was defined as patients with any other respiratory diseases except TB, and with negative IGRA results. Healthy volunteers were those had negative IGRA results and no history of TB exposure. Sputum smear-positive patients with negative sputum MTB culture results were diagnosed as non-tuberculous mycobacteria (NTM).
Demographic information including gender and age of all participants was recorded. For each participant, 0.5 ml whole blood was collected in heparin anticoagulant tubes. Acid fast and Gene-XPERT were used for ATB screening. The results of MTB culture and IGRA were recorded.
ESAT6/CFP10 stimulation and flow cytometry
Within 4 h of blood collection, the whole blood was added into sample tubes containing 10μg/mL ESAT6/CFP10 polypeptide antigen (Beckman Coulter, Brea, CA, USA). Brefeldin A (10μg/mL, Biolegend, San Diego, CA, USA) was added as a Golgi transport inhibitor, and the whole blood was incubated at 37℃ with 5% CO2 for 16 h. After culture, 2.5 mL of erythrocyte lysis solution and fixative solution (3% diethylene glycol, 2% formaldehyde, and 0.75% methanol) were added and incubated at room temperature for 10 min. The cells were centrifuged at 600 ×g for 5 min. The supernatant was discarded, and 375 µL Perfix-NC membrane breaking solution (Beckman Coulter) was added and mixed. IFN-γ-FITC was added. Then, 5μL of CD4-PE, CD3-ECD, CD27-PE-CY5.5, and CD38-PE-CY7 were mixed and incubated for 45 min in the dark. Phosphate-buffered saline (PBS, 3 mL) was added. The cells were centrifuged at 600 ×g for 5 min, and the supernatant was discarded. After adding 300μL PBS, a Beckman Coulter DxFLEX flow cytometer was used for rapid collection (60μL/min). The collection stop condition was set to 100,000 CD4+ cells or 300 s of whole blood. FlowJo V10 (Treestar, Ashland, OR) was used for data analysis. The ratio of CD4+IFN-γ+CD38+/CD4+IFN-γ+, CD4+IFN-γ+CD27-/CD4+IFN-γ+, CD4+IFN-γ+CD38+CD27- /CD4+IFN-γ+, CD4+IFN-γ+CD38+CD27-/CD4+, CD4+IFN-γ+CD38+/CD4+, CD4+IFN-γ+CD27-/CD4+ was measured.
Statistical analyses were performed using SPSS 18 (SPSS, Armonk, NY, USA) and Graph Pad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). Continuous variables are described as means±standard deviation (SD), and categorical variables as n (%). The Kruskal-Wallis test was used for comparison of independent samples between multiple groups, Dunn’s test was used for pairwise comparison between multiple groups, and the Mann-Whitney U-test was used for comparison of independent samples between two groups. Receiver operating characteristic (ROC) analysis was used to assess the diagnostic performance of each biomarker. The areas under the curves (AUCs) were compared by the DeLong test. The optimal cutoff value of variables was identified according to the maximum Youden index. Two-sided P-values <0.05 were considered statistically significant.