A biomarker for evaluating the degree of pleural adhesion of tuberculous pleural effusion and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a biomarker for evaluating the degree of pleural adhesion of tuberculous pleural effusion (TPE) and application. The application provides a biomarker for evaluating the degree of pleural adhesion of tuberculous pleural effusion, wherein the biomarker comprises dsDNA (double-stranded DNA) in the pleural effusion. The biomarker can be used to more conveniently and economically diagnose TPE, and further evaluate the severity of pleural adhesion of TPE patients, thereby guiding anti-tuberculosis and anti-adhesion treatment in the clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a biomarker for evaluating the degree of pleural adhesion of tuberculous pleural effusion and application. BACKGROUND

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis complex. The disease can cause pulmonary tuberculosis and also invade other organs such as liver, kidney, brain and lymph nodes to form extrapulmonary tuberculosis. Tuberculosis is a very serious global public health problem. Early diagnosis of tuberculosis is crucial for timely and effective treatment and improved prognosis. Tuberculous pleural effusion (TPE) is one of the most common extrapulmonary tuberculosis, although it is not uncommon in clinical practice, but it is still difficult to distinguish from other diseases (such as malignant tumors, bacterial pneumonia and heart failure, etc.) caused by pleural effusion. If it cannot be diagnosed and treated in time, it is easy to cause pleural adhesion, thickening, pleural calcification, chest collapse, restrictive ventilation disorder, leading to poor prognosis of patients and significantly reducing the quality of life of patients.

[0003] At present, the diagnosis of TPE in clinical practice mainly relies on Ziehl-Neelsen staining, routine, biochemical, tubercle bacillus culture and pleural biopsy of pleural effusion, and some studies have shown that TPE can be diagnosed by detecting biomarkers such as ADA, IFN-γ, IL-32 and CXCL10 in pleural effusion. However, the detection rate of Ziehl-Neelsen staining and tubercle bacillus culture of pleural effusion is low, and the culture time is long, and pleural biopsy needs to rely on thoracoscopy, which is very dependent on the technical level of the hospital and the operator, and thoracoscopy is an invasive operation, which has a high risk of surgery and a low acceptance of patients. Although it is convenient to diagnose TPE by detecting biomarkers such as ADA and IFN-γ in pleural effusion, the sensitivity and specificity are not ideal, especially these indicators cannot judge the severity of pleural adhesion of TPE patients, and cannot guide whether to use anti-adhesion treatment in clinical practice. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings of the prior art and provide a biomarker for evaluating the degree of pleural adhesion of tuberculous pleural effusion.

[0005] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0006] In a first aspect, the present application provides a biomarker for evaluating the degree of pleural adhesion of tuberculous pleural effusion, wherein the biomarker comprises dsDNA in pleural effusion.

[0007] For diagnosing a patient with pleural effusion tuberculosis whose diagnosis is not clear, collecting the pleural effusion of the patient, detecting the concentration of dsDNA in the pleural effusion, judging the diagnosis of the patient according to the optimal critical value (441.15 ng / mL) of diagnosing TPE, and evaluating the pleural adhesion of the patient according to the content of dsDNA, so as to take corresponding treatment.

[0008] In the second aspect, the marker in the first aspect is applied to prepare a product for evaluating the severity of pleural adhesion of a patient with tuberculous pleural effusion.

[0009] In the third aspect, the marker in the first aspect is applied to prepare a product for guiding the treatment of tuberculous pleural effusion.

[0010] As a preferred embodiment of the application, when the concentration of dsDNA is greater than 441.15 ng / mL, it is suggested that the patient is diagnosed as tuberculous pleural effusion and there is pleural adhesion.

[0011] As a preferred embodiment of the application, when the concentration of dsDNA is greater than 1068.1427 ng / mL, it is suggested that the patient has severe pleural adhesion.

[0012] As a further preferred embodiment of the application, the patient should be treated with anti-adhesion treatment at the same time when treated with anti-tuberculosis treatment.

[0013] In the fourth aspect, the marker in the first aspect is applied to prepare a product for evaluating the therapeutic effect of tuberculous pleural effusion.

[0014] In the fifth aspect, the application provides a detection reagent for evaluating the degree of pleural adhesion of tuberculous pleural effusion, and the detection reagent comprises a reagent for detecting the biomarker in the first aspect.

[0015] In the sixth aspect, the detection reagent in the fifth aspect is applied to prepare a product for evaluating the severity of pleural adhesion of a patient with tuberculous pleural effusion.

[0016] Compared with the prior art, the application has the following beneficial effects:

[0017] The biomarker for evaluating the degree of pleural adhesion of tuberculous pleural effusion can only need to extract 5-10 ml of pleural effusion of the patient, without needing to perform thoracoscopy, and is more convenient and safe when evaluating the severity of pleural adhesion of the patient with tuberculous pleural effusion; after extracting the pleural effusion, the dsDNA content can be detected immediately, and TPE or non-TPE can be identified according to the result, the waiting time is short, and the efficiency is high; the sensitivity and specificity of the dsDNA in the pleural effusion for differentiating TPE from non-TPE are higher than those of conventional detection methods (such as ADA and IFN-γ); the dsDNA content in the pleural effusion is related to the degree of pleural adhesion of the patient, can early guide whether the anti-adhesion treatment is used simultaneously in the clinic, and avoids the occurrence of adverse prognosis such as pleural adhesion, thickening and calcification of the patient. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The thoracoscopy performances of patients with different types of pleural effusion.

[0019] Figure 2 The diagnostic values of dsDNA, ADA and IFN-γ for differentiating TPE from non-TPE; in the figure, A is the dsDNA for differentiating TPE from non-TPE; B is the ADA for differentiating TPE from non-TPE; C is the IFN-γ for differentiating TPE from non-TPE; D is the ROC curve of the subject of dsDNA; E is the ROC curve of the subject of ADA; F is the ROC curve of the subject of IFN-γ; and G is the ROC curve of the subject of the combination of dsDNA, ADA or IFN-γ.

[0020] Figure 3 The thoracoscopy performances and dsDNA content differences of TPE patients with different adhesion degrees.

[0021] Figure 4 The thoracoscopy pictures of the patient 1, the patient 4, the patient 6 and the patient 8 under thoracoscopy; it can be seen from the figure that the patient 1 and the patient 6 have severe pleural adhesion, and the patient 4 and the patient 8 have mild pleural adhesion. DETAILED DESCRIPTION

[0022] In order to better illustrate the object, technical scheme and advantages of the present application, the present application will be further described below in combination with specific examples. Those skilled in the art should understand that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0023] The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can be obtained commercially. The total protein (Protein, g / L), glucose (Glucose, mmol / L), lactate dehydrogenase (LDH, U / L), chloride ion (CI, mmol / L), adenosine deaminase (ADA, U / L) in the pleural effusion were determined by the Japanese Toshiba automatic biochemical analyzer (TBA40FR); the content of interferon-γ (IFN-γ, pg / mL) in the pleural effusion was determined by the Thermo Fisher IFN-γ detection kit (Thermo Fisher, KHC4021); the content of double-stranded DNA (dsDNA, ng / mL) in the pleural effusion was determined by the dsDNA detection kit (Thermo Fisher, P7581).

[0024] Example 1

[0025] From March 1, 2021 to November 30, 2023, 254 samples of patients with pleural effusion were collected in Guangzhou First People's Hospital (GZFPH), and the results of thoracoscopy of the patients were also collected. Including 143 cases of tuberculous pleural effusion (TPE), 58 cases of malignant pleural effusion (MPE), 33 cases of parapneumonic pleural effusion (PPE), 9 cases of heart failure-related pleural effusion (HF-PE), and 11 cases of miscellaneous pleural effusion (Misc-PE). Approved by the Ethics Committee of Guangzhou First People's Hospital. All participants obtained written informed consent.

[0026] The clinical characteristics of the samples and the contents of total protein (protein, g / L), glucose (Glucose, mmol / L), lactate dehydrogenase (LDH, U / L), chloride ion (CI, mmol / L), double-stranded DNA (dsDNA, ng / mL), adenosine deaminase (ADA, U / L), and interferon-γ (IFN-γ, pg / mL) in the pleural effusion are shown in Tables 1 and 2.

[0027] Table 1 Clinical characteristics of samples

[0028]

[0029] Table 2 Contents of sample dsDNA, ADA, IFN-γ

[0030]

[0031] As shown in Table 1: compared with non-TPE (pleural effusion of other causes) group patients, TPE group patients were younger, and the total protein and LDH in pleural effusion were higher, and the glucose and Cl contents were lower (all P<0.01). As shown in Table 2: the concentrations of dsDNA, ADA and IFN-γ in tuberculous pleural effusion were significantly higher than those in non-tuberculous pleural effusion (all P<0.001), and there was no significant difference between MPE, PPE, HF-PE and Misc-PE (all P>0.05).

[0032] Example 2

[0033] The patients in Example 1 received standard thoracentesis to extract 5-10 mL of pleural effusion specimens within 24 hours after admission, and heparin was added to the collection bottle in advance to prevent the specimen from coagulating, and the ice box was transported to the laboratory in a low temperature environment. The pleural effusion was divided into 5 mL centrifuge tubes and balanced on the tray balance, and then centrifuged at 1500 rpm for 10 minutes. After centrifugation, the supernatant of the pleural effusion was aspirated and divided into 1.5 mL EP tubes, and the corresponding labels were made, and then the supernatant was transferred to a-80℃ freezer for cryopreservation for subsequent detection of cytokines or chemokines and other proteins.

[0034] As Figure 1 shown: by analyzing the results of thoracoscopy of patients with different disease types, it was found that most TPE patients had severe pleural adhesion, and non-TPE patients had light or basically no adhesion. Figure 1 .

[0035] In order to explore the diagnostic value of dsDNA, ADA and IFN-γ for pleural effusion, the concentrations of dsDNA, ADA and IFN-γ in the supernatant of pleural effusion were detected, and according to the receiver operating characteristic (ROC) curve, the corresponding sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), and negative likelihood ratio (NLR) were calculated. The diagnostic value of dsDNA concentration in pleural effusion was analyzed to determine the best critical value for differentiating TPE from non-TPE. As Figure 2The ROC curves of the subjects were plotted to evaluate the sensitivity, specificity, PPV, NPV, PLR and NLR of these biomarkers. In the Guangzhou cohort, when the cut-off value was 441.15 ng / mL, the AUC of dsDNA in distinguishing TPE from non-TPE was 0.947 (95% CI, 0.931-0.963), which was higher than that of ADA (0.904) and IFN-γ (0.945) (see Figure 2 ). In addition, the diagnostic accuracy parameters of dsDNA combined with ADA, dsDNA combined with IFN-γ, ADA combined with IFN-γ, and dsDNA combined with ADA combined with IFN-γ are also shown in Figure 2 . Among them, their AUCs were 0.970 (95% CI, 0.956-0.989), 0.976 (95% CI, 0.967-0.994), 0.957 (95% CI, 0.940-0.982), and 0.979 (95% CI, 0.970-0.995), respectively. These results show that immune cells release extracellular traps in the environment of bacterial and other pathogen infections, especially under the stimulation of Mycobacterium tuberculosis and virulence molecules, and immune cells such as neutrophils and macrophages release more extracellular traps. Therefore, the content of dsDNA in pleural effusion has high value for the diagnosis of tuberculous pleural effusion.

[0036] Under thoracoscopy, it was found that not all TPE patients had severe pleural adhesion. The dsDNA in the pleural effusion was detected, and it was found to be consistent with the thoracoscopy results. The content of dsDNA in patients with severe adhesion was higher than that in patients with light adhesion, especially when the dsDNA concentration was > 1068.1427 ng / mL, which indicated that the patient had severe pleural adhesion (see Figure 3 ).

[0037] The above results show that dsDNA has potential value for evaluating the severity of pleural adhesion in TPE patients. The content of dsDNA in pleural effusion can significantly distinguish TPE from non-TPE. When the content of dsDNA in the pleural effusion of patients is greater than the cut-off value of 441.15 ng / mL, it is suggested that for TPE patients rich in dsDNA, anti-tuberculosis treatment should be performed at the same time, and anti-adhesion treatment should be performed at the same time, which will help to avoid complications such as pleural thickening in patients, thereby better improving the prognosis of patients.

[0038] Example 3

[0039] The samples of 10 patients with pleural effusion in Guangzhou First People's Hospital (GZFPH) from January 1, 2024 to January 31, 2024 were collected, including 3 patients with malignant pleural effusion, 4 patients with tuberculous pleural effusion, and 3 patients with pneumonia parapleural effusion. The clinical characteristics and the content of dsDNA in the pleural effusion of the patients were detected as shown in Table 3.

[0040] Table 3 Clinical data of patients and the content of dsDNA in pleural effusion

[0041]

[0042] Figure 4 The thoracoscope pictures of patient 1, patient 4, patient 6 and patient 8 showed that patient 1 and patient 6 had severe pleural adhesion, and patient 4 and patient 8 had mild pleural adhesion.

[0043] The content of dsDNA in the pleural effusion of the 4 patients with TPE was significantly higher than the critical value of 441.15 ng / mL. At the same time, the content of dsDNA in the pleural effusion of patient 1 and patient 6 was 1302.09 ng / mL and 1948.38 ng / mL, respectively, which was higher than the critical value of 1068.1427 ng / mL for severe pleural adhesion, indicating that they had severe pleural adhesion, which was consistent with the results of the thoracoscope pictures of Figure 4

[0044] The above results further prove that the dsDNA in the pleural effusion is a new reliable biomarker for diagnosing TPE, and can be used as a detection and evaluation marker for judging the severity of pleural adhesion in patients with TPE.

[0045] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and do not limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.​

Claims

1. The application of the test reagent in the preparation of products guiding the treatment of tuberculous pleural effusion, characterized in that, The detection reagents include reagents for detecting biomarkers; the biomarker is dsDNA in pleural effusion; a dsDNA concentration > 441.15 ng / mL indicates pleural adhesions in the patient; a dsDNA concentration > 1068.1427 ng / mL indicates severe pleural adhesions in the patient; the patient is receiving anti-adhesion treatment concurrently with anti-tuberculosis treatment.

2. The application of the test reagent in the preparation of a product for assessing the severity of pleural adhesions in patients with tuberculous pleural effusion, characterized in that, The detection reagents include reagents for detecting biomarkers; the biomarker is dsDNA in pleural effusion; when the concentration of dsDNA is >441.15 ng / mL, it indicates that the patient has pleural adhesions; when the concentration of dsDNA is >1068.1427 ng / mL, it indicates that the patient has severe pleural adhesions.

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