Diagnostic value of combined pleural IL-33, ADA and peripheral T-SPOT.TB for tuberculous pleurisy

(cid:0) Objective (cid:0) To investigate the correlation between pleural uid interleukin-33(IL-33) and adenosine deaminase (cid:0) ADA (cid:0) and peripheral blood tuberculosis T cell spot detection (cid:0) T-SPOT.TB (cid:0) ,and the combined value of the three tests for the diagnosis of tuberculous pleurisy. (cid:0) Method (cid:0) 79 patients with pleural effusion admitted from June 2017 to December 2018 were enrolled. They were divided into tuberculous pleural effusion (cid:0) TPE (cid:0) group (57 cases, 72.2%) and malignant pleural effusion group(17 cases,21.5%), pneumonia-like pleural effusion group (5 cases, 6.3%). Correlation between pleural uid IL-33,pleural effusion ADA and peripheral blood T-SPOT.TB was analyzed, comparison of the three separate and combined diagnostic ecacy was also performed. (cid:0) Result (cid:0) The levels of IL-33, ADA and peripheral blood T-SPOT.TB in patients with TPE were signicantly higher than those in non-TPE (P < 0.001). The level of pleural uid IL-33 was positively correlated with pleural effusion ADA and peripheral blood T-SPOT.TB. The Area under the ROC curve (cid:0) AUC (cid:0) of TPE diagnosed by pleural IL-33, ADA and peripheral blood T-SPOT.TB were 0.753, 0.912 and 0.865, respectively. AUC for combined detection of pleural effusion IL-33, ADA and peripheral blood T-SPOT.TB is the largest, with a value of 0.962. Specicity is 100% and sensitivity is 88.5%. (cid:0) Conclusion (cid:0) Combined detection of pleural effusion IL-33, ADA and peripheral blood T-SPOT.TB can improve the diagnostic ecacy of tuberculous pleurisy.


Background
Tuberculous pleurisy is a common form of extrapulmonary tuberculosis. Pleural biopsy and bacteriological testing are the gold standard for diagnosis of tuberculous pleurisy. However, it is di cult to diagnose because of the invasive operation of pleural biopsy and the di culty in cultivating mycobacterium tuberculosis [1,2]. With the advances in enzymology and molecular biology in recent years, enzymes and cytokines have attracted more and more attention in the pathogenesis of various immune diseases [3]. Adenosine deaminase (ADA) is present in various tissues of human body and is mainly involved in the decomposition of purine nucleosides. It has been widely used in clinical diagnosis of tuberculosis in recent years [4]. ADA testing is currently recognized as an ideal indicator for the diagnosis of tuberculous pleurisy [5]. Tuberculosis T cell spot detection (T-SPOT.TB) is a kind of interferon-γ release assay (IGRA). The number of T cells secreting interferon-γ (IFN-γ) can be used as an auxiliary method for early diagnosis of tuberculosis [6,7]. Researches [8][9][10] showed that the level of interleukin-33 (IL-33) in pleural uid was signi cantly higher in patients with tuberculous pleurisy than in other causes. Therefore, we speculate that IL-33 may play an important role in the production of tuberculous pleural effusion (TPE). The combined value of the three tests for the diagnosis of tuberculous pleurisy was investigated to identify the correlation between pleural uid interleukin-33(IL-33),adenosine deaminase ADA and peripheral blood tuberculosis T cell spot detection T-SPOT.TB .It is intended to provide a reference for the clinical diagnosis of tuberculous pleurisy. The results are reported below. Exudate, pleural effusion absorption and clinical symptoms relieved by anti-tuberculosis treatment.

Group of non-tuberculous pleural effusion(non-TPE):
Including malignant pleural effusion group and pneumonia-like pleural effusion group.
Malignant pleural effusion(MPE) group: Imaging results consistent with primary bronchogenic carcinoma complicated with pleural effusion; lung cancer con rmed with pulmonary biopsy obtained by beroptic bronchoscopy or thoracoscopy Metastatic tumor cells detected with Pleural effusion exfoliative cytology.
Pneumonia-like pleural effusion(PPE) group: presence of symptoms of cough, cough, and fever; lung exudation showed by chest imaging and absorption of pleural uid after antibiotic treatment.

Exclusion criteria
Any one of the followings: Patients had chest trauma or received any treatment for invasive pleural examination in the previous year before hospitalization; Patients have received any anti-tumor or antituberculosis treatment before; Patients who have used glucocorticoids, non-steroidal anti-in ammatory drugs, or immunosuppressants Unknown causes of pleural effusion; pleural effusion caused by rheumatic immune diseases.
1.4 Specimen collection method 5 mL of drainage uid was collected by ultrasound-guided thoracentesis from patients with pleural effusion, and centrifuged for 10 minutes at 3000 r/min using heparin with the concentration of 500 U/ml.

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The supernatant was collected and stored in a refrigerator at eighty Celsius degrees below zero.

Detection method
The IL-33 in pleural effusion was measured by enzyme linked immunosorbent assay (ELISA). The kit was provided by abcam company (UK). The procedure was carried out strictly in accordance with the operating instructions. The levels of serum ADA and serum lactate dehydrogenase (LDH) peripheral blood T-SPOT.TB were measured in the Second A liated Hospital of Wenzhou Medical University in accordance with the instructions.

Statistical analysis
Data were analyzed with the statistical software SPSS(version 22.0). It was expressed as mean ± standard deviation (x ± s) for normal distributed date.Non-normally distributed data was expressed as median and interquartile range(IQR). Measurement data were compared using the independent sample T test. Pearson correlation was employed to analysis the correlations between pleural uid IL-33, pleural effusion ADA and peripheral blood T-SPOT. The receiver operating characteristic curve (ROC curve) was plotted with the sensitivity as the Y-axis and 1-speci city as the x-axis. The optimal threshold was determined according to the Yoden index (sensitivity + speci city − 1). P < 0.05 was taken as a statistically signi cant difference.

Ethics statement
This study was approved by the institutional review board of the second a liated hospital of WenZhou medical university (Approval No L-2020-1).Written informed consent was provided by all patients.

Demographic characteristics
A total of 79 patients were collected, and demographic characteristics are shown in Table 1.

Levels of IL-33, ADA, LDH and T-SPOT.TB in peripheral blood
The levels of pleural uid IL-33, ADA and peripheral blood T-SPOT.TB in patients with TPE were signi cantly higher than those in non-tuberculous pleural effusions, and the difference was statistically signi cant (P<0.001). As shown in Table 2. 2.3 Correlations.
The level of IL-33 in pleural uid was positively correlated with pleural effusion ADA and peripheral blood T-SPOT.TB r=0.343 0.450 P 0.05 . As shown in Figure1, Figure2. 2.4. Diagnostic value of ADA, IL-33 and peripheral blood T-SPOT.TB for tuberculous pleurisy in pleural effusion. Table 3 shows the diagnostic value of pleural IL-33, ADA and peripheral blood T-SPOT.TB. ROC curve is shown in Figure 3.
2.5 Diagnostic value of combined detection of pleural effusion IL-33, ADA and peripheral blood T-SPOT.TB for tuberculous pleurisy.
ROC curve analysis was performed to de ne the diagnostic pro le of combined detection of pleural effusion IL-33, ADA and peripheral blood T-SPOT.TB in identifying tuberculous pleurisy. The area under the ROC curve for combined detection the pleural effusion IL-33, ADA and peripheral blood T-SPOT.TB was the largest at 0.962, with speci city 100%, sensitivity 88.5%. As shown in Table 4.

Discussion
Tuberculous pleurisy is the most common cause of pleural effusion, accounting for 49.5 to 54.5% of the cause of hospitalized pleural effusion in China [11].A rapid and effective detection method for early diagnosis and treatment of patients with tuberculous pleural effusion is needed to reduce complications such as tuberculous empyema and lung damage caused by tuberculous pleural effusion [12].
IL-33 was discovered by Schmitz in 2005 and belongs to the IL-1 class cytokine superfamily. It has a homologous clover-like structures [13]. The results of this study showed that the level of IL-33 in pleural effusion was signi cantly higher in patients with tuberculous pleurisy than in non-tuberculous pleural effusion. The area under the ROC curve for IL-33 in TPE was 0.753, and the sensitivity was 49.1%, speci city is 100% at the best cutoff value of 155.96 ng/L. The level of pleural uid IL-33 was positively correlated with pleural effusion ADA and peripheral blood T-SPOT.TB. Lee KS and Xuan WX and other scholars [9,10] found that the level of IL-33 in pleural effusion of patients with tuberculous pleurisy was signi cantly higher than other causes of pleural effusion and serum IL-33 levels, the sensitivity was 78% and 86.96%, speci city was 65% and 90.48% respectively. It was also suggested by Lee KS and other scholars that pleural uid IL-33 level and pleural ADA are signi cantly positively correlated [9]. Li D and other scholars [8] also showed that the sensitivity of IL-33 in the diagnosis of tuberculous pleurisy was 83.9%, the speci city was 87.3%, and the area under the ROC curve was 0.823. Therefore, the above evidence shows that IL-33 is related to the pathophysiology of TPE. Although we did not probe the speci c mechanism of IL-33 in the pathogenesis of TPE, the signi cant relationship between IL-33 and tuberculous pleurisy observed in this study can be explained by the following hypothesis: IL-33 is shown to exhibit an immunomodulatory effect to some extent, such as the induction of cytokines and responsive cells. A growing number of basic studies [14][15][16] have shown that IL-33 can mediate and even enhance Th1 cellular immune responses by increasing interferon-gamma (Interferon-γ, IFN-γ). On the other hand, some studies also found that IL-33 expression is up-regulated by IFN-γ and tumor necrosis factor-α (TNF-α) [17,18]. Therefore, IFN-γ is not only an upstream regulator of IL-33, but also a downstream product of IL-33 signaling [15,16,19]. Therefore, in tuberculous pleurisy, IL-33 and IFN-γ may form a coupled positive feedback loop [15][16][17][18][19][20]. Therefore, IL-33 may be involved in the pathogenesis and development of tuberculous pleurisy, and its elevated level may play a role of in the stimulation of in ammation by mycobacterium tuberculosis. IL-33 has an important diagnostic value in the diagnosis of tuberculous pleurisy.
ADA is an important enzyme in the metabolism of purine nucleosides in various tissues of human body, especially lymphocytes. The pathogenesis of tuberculous pleurisy is delayed-type hypersensitivity caused by MTB infection. The tuberculosis protein of MTB enters the pleural cavity and causes pleural in ammatory reaction, which leads to lymphocyte differentiation and proliferation, resulting in increased ADA content [4]. ADA is one of the most widely studied and recommended biomarkers and has been found to have a good performance in diagnosing TPE. A meta-analysis of 63 studies [21] evaluated the value of pleural ADA activity in identifying TPE and non-TPE, demonstrating its high sensitivity and speci city (92% and 90%, respectively). ADA is one of the highly recommended biomarkers and has been found to have good performance in diagnosing TPE. The results of this study showed that the ADA level in pleural effusion was signi cantly higher in patients with tuberculous pleurisy than in non-tuberculous pleural effusion, the sensitivity of diagnosis of tuberculous pleurisy was 93.0%, the speci city was 90.9%, which was similar to the previous study [21].
Tuberculous pleurisy is mainly mediated by cellular immunity. After stimulation by MTB antigen, T cells get activated to secrete cytokine IFN-γ to participate in the immune response. There are corresponding speci c T cells in the peripheral blood of patients [22]. The principle of T-SPOT.TB detection is to isolate MTB-speci c T cells in peripheral blood, which can secret IFN-γ after in vitro culture and antigen restimulation, so we can diagnose the presence of MTB infection by examining the IFN-γ concentration with the corresponding antibody. Its diagnostic value is not affected by the patient's gender, age, tumor, immunosuppression, etc. It can be used not only for the diagnosis of extrapulmonary tuberculosis, but also as a tool for therapeutic effect evaluation, which has a high practical value [23]. The results of this study showed that the level of T-SPOT.TB in peripheral blood of patients with tuberculous pleural effusion was signi cantly higher than that of patients with non-tuberculous pleural effusion. The sensitivity of peripheral blood T-SPOT. TB for diagnose of TPE was 92.3% and the speci city was 71.4%. However, studies have shown that peripheral blood T-SPOT.TB has its own defects because it is affected by the number of peripheral blood T lymphocytes [24], especially in immunode cient patients [25], which may cause false negative results. Moreover, peripheral blood T-SPOT.TB cannot differential MTB latent infection from active tuberculosis, which further limits its use.
This study found that the combined detection of pleural effusion IL-33, ADA and peripheral blood T-SPOT.TB could further improve the sensitivity and speci city of TPE diagnosis. The area under the ROC curve was the largest at 0.962 when combined those three, with 100% speci city, 88.5% sensitivity.
In summary, this study compared the AUC, sensitivity and speci city of pleural effusion IL-33, ADA and blood TSPOT for the diagnosis of tuberculous pleurisy. We found that IL-33 has the highest speci city of 100%. It can be used for the exclusion of tuberculous pleurisy, but the sensitivity of IL-33 is not high, only at 49.1%. However, the diagnostic sensitivity was increased to 76.9% when combined with TSPOT.TB. Combined IL-33, ADA, TSPOT.TB, the diagnostic speci city was 100%, and the sensitivity was 88.5%, suggesting that the combined detection has a higher diagnostic value for tuberculous pleurisy. It can prevent missed diagnosis, which is helpful for early detection and early treatment, so that it has important auxiliary diagnostic value for tuberculous pleurisy.

Conclusion
Combined detection of pleural effusion IL-33, ADA and peripheral blood T-SPOT.TB can improve the diagnostic e cacy of tuberculous pleurisy.

Declarations Ethical Approval and Consent to participate
This study was approved by the institutional review board of the second a liated hospital of WenZhou medical university (Approval No L-2020-1).Written informed consent was provided by all patients. See the attached le named Ethics Committee Consent form1, Ethics Committee Consent form2 and patients' informed consent form.

Consent for publication
Written informed consent was obtained from all subjects before the study. See the le of patients' informed consent form. Sign below: Availability of data and materials See the le named date and materials.

Competing interests
The Authors declare that there is no con ict of interest.

Funding
The authors disclosed receipt of the following nancial support for the research, and publication of this article: This paper was supported by grants Wenzhou science and Technology Bureau    Diagnostic value of combined detection of pleural effusion IL-33, ADA and peripheral blood T-SPOT.TB for tuberculous pleurisy. Combined IL-33, ADA, TSPOT.TB, the diagnostic specificity was 100%, and the sensitivity was 88.5%. Figure 1 Correlation between pleural uid IL-33 level and pleural ADA level