Biomarker for evaluating pleural adhesion degree of tuberculous pleural effusion and application

Abstract

The invention belongs to the technical field of biological medicine, and discloses a biomarker for evaluating the pleural adhesion degree of tuberculous pleural effusion (TPE) and an application of the biomarker for evaluating the pleural adhesion degree of the TPE. The invention provides a biomarker for evaluating the pleural adhesion degree of tuberculous pleural effusion. The biomarker comprises dsDNA (double-stranded DNA) in the pleural effusion. The TPE can be more conveniently, economically and practically diagnosed by utilizing the biomarker, and the severity of pleural adhesion of a TPE patient is further evaluated, so that clinical antituberculosis and anti-adhesion treatment is guided.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a biomarker for assessing the degree of pleural adhesion in tuberculous pleural effusion and its application. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by the Mycobacterium tuberculosis complex. It can cause pulmonary tuberculosis or invade other organs such as the liver, kidneys, brain, and lymph nodes, leading to extrapulmonary tuberculosis. TB is a very serious global public health problem. Early diagnosis of TB is crucial for timely and effective treatment and improved prognosis. Tuberculous pleural effusion (TPE) is one of the most common forms of extrapulmonary tuberculosis. Although not uncommon clinically, it is still difficult to distinguish from pleural effusions caused by other diseases (such as malignant tumors, bacterial pneumonia, and heart failure). If not diagnosed and treated promptly, it can easily lead to pleural adhesions, thickening, pleural calcification, chest collapse, and restrictive ventilation, resulting in poor patient prognosis and significantly reduced quality of life.

[0003] Currently, the clinical diagnosis of total pleural effusion (TPE) mainly relies on Ziehl-Neelsen staining, routine pleural effusion analysis, biochemistry, tuberculosis culture, and pleural biopsy. Some studies have also 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 tuberculosis culture in pleural effusion is low, and the culture time is long. Pleural biopsy requires thoracoscopy, which is highly dependent on the hospital and the operator's skill level. Furthermore, thoracoscopy is an invasive procedure with significant surgical risks and low patient acceptance. While using biomarkers such as ADA and IFN-γ in pleural effusion to diagnose TPE is convenient, its sensitivity and specificity are not ideal. In particular, these indicators cannot determine the severity of pleural adhesions in TPE patients, and therefore cannot guide the clinical decision on anti-adhesion therapy. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a biomarker for assessing the degree of pleural adhesion in tuberculous pleural effusion and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a biomarker for assessing the degree of pleural adhesion in tuberculous pleural effusion, the biomarker comprising dsDNA in the pleural effusion.

[0006] For patients with tuberculosis and pleural effusion whose diagnosis is not yet clear, pleural effusion is collected, and the concentration of dsDNA in the pleural effusion is detected. The diagnosis of the disease is determined based on the optimal cutoff value for TPE (441.15 ng / mL). The pleural adhesion of the patient is assessed based on the dsDNA content, and appropriate treatment is then carried out.

[0007] Secondly, the present invention applies the markers described in the first aspect in the preparation of a product for assessing the severity of pleural adhesions in patients with tuberculous pleural effusion.

[0008] Thirdly, the present invention applies the markers described in the first aspect in the preparation of products that guide the treatment of tuberculous pleural effusion.

[0009] In a preferred embodiment of the application described in this invention, when the concentration of dsDNA is >441.15 ng / mL, it suggests that the patient is diagnosed with tuberculous pleural effusion and has pleural adhesions.

[0010] In a preferred embodiment of the application described in this invention, a concentration of dsDNA > 1068.1427 ng / mL indicates that the patient has severe pleural adhesions.

[0011] As a further preferred embodiment of the application described in this invention, the patient should receive anti-adhesion treatment simultaneously while undergoing anti-tuberculosis treatment.

[0012] Fourthly, the present invention utilizes the biomarkers described in the first aspect in products for evaluating the efficacy of treatment for tuberculous pleural effusion.

[0013] Fifthly, the present invention provides a diagnostic reagent for assessing the degree of pleural adhesion in tuberculous pleural effusion, the diagnostic reagent comprising a reagent for detecting the biomarkers described in the first aspect.

[0014] In a sixth aspect, the present invention applies the detection reagent described in the fifth aspect in the preparation of a product for assessing the severity of pleural adhesions in patients with tuberculous pleural effusion.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a biomarker for assessing the degree of pleural adhesion in patients with tuberculous pleural effusion. When assessing the severity of pleural adhesion in patients with tuberculous pleural effusion, only 5-10 ml of pleural fluid needs to be drawn, eliminating the need for thoracoscopy, making it more convenient and safer. After pleural fluid aspiration, dsDNA content can be detected immediately, and the results can be used to differentiate between TPE and non-TPE, resulting in a short waiting time and high efficiency. The sensitivity and specificity of dsDNA in pleural effusion in differentiating between TPE and non-TPE are higher than conventional detection methods (such as ADA and IFN-γ). The dsDNA content in pleural effusion is correlated with the degree of pleural adhesion in patients, which can guide clinical practice early on whether to use anti-adhesion therapy concurrently, avoiding adverse prognoses such as pleural adhesion, thickening, and calcification. Attached Figure Description

[0016] Figure 1 The thoracoscopic findings of patients with different types of pleural effusion.

[0017] Figure 2 The diagnostic value of dsDNA, ADA, and IFN-γ in differentiating TPE from non-TPE is shown in the figure. In the figure, A is the ROC curve of dsDNA in differentiating TPE from non-TPE; B is the ROC curve of ADA in differentiating TPE from non-TPE; C is the ROC curve of IFN-γ in differentiating TPE from non-TPE; D is the ROC curve of dsDNA in differentiating TPE from non-TPE; E is the ROC curve of ADA in differentiating TPE from non-TPE; F is the ROC curve of IFN-γ in differentiating TPE from non-TPE; and G is the ROC curve of dsDNA, ADA, or IFN-γ in differentiating TPE from non-TPE.

[0018] Figure 3 The differences in thoracoscopic findings and dsDNA levels among TPE patients with varying degrees of adhesion were investigated.

[0019] Figure 4 The images show thoracoscopy images of patients 1, 4, 6 and 8. Severe pleural adhesions are visible in patients 1 and 6, while mild pleural adhesions are visible in patients 4 and 8. Detailed Implementation

[0020] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0021] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. The levels of total protein (g / L), glucose (mmol / L), lactate dehydrogenase (LDH, U / L), chloride ions (CI, mmol / L), and adenosine deaminase (ADA, U / L) in the pleural effusion were determined using a Toshiba fully automated biochemical analyzer (TBA40FR); the level of interferon-γ (IFN-γ, pg / mL) in the pleural effusion was determined using a Thermo Fisher IFN-γ assay kit (Thermo Fisher, KHC4021); and the level of double-stranded DNA (dsDNA, ng / mL) in the pleural effusion was determined using a dsDNA assay kit (Thermo Fisher, P7581).

[0022] Example 1 Between March 1, 2021 and November 30, 2023, samples were collected from 254 patients with pleural effusion at Guangzhou First People's Hospital (GZFPH), and thoracoscopy results were also collected. These included 143 patients with tuberculous pleural effusion (TPE), 58 with malignant pleural effusion (MPE), 33 with parapneumonic pleural effusion (PPE), 9 with heart failure-related pleural effusion (HF-PE), and 11 with pleural effusion of unknown cause (Miscellaneous pleural effusion (Misc-PE). The study was approved by the Ethics Committee of Guangzhou First People's Hospital. Written informed consent was obtained from all participants.

[0023] The clinical characteristics of the samples and the contents of total protein (g / L), 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.

[0024] Table 1 Clinical characteristics of the sample Table 2. Content of dsDNA, ADA, and IFN-γ in the samples As shown in Table 1, compared with patients in the non-TPE (pleural effusion of other causes) group, the TPE group had younger patients, higher levels of total protein and LDH in their pleural effusions, and lower levels of glucose and Cl (all P < 0.01). As shown in Table 2, the concentrations of dsDNA, ADA, and IFN-γ in tuberculous pleural effusions were significantly higher than those in non-tuberculous pleural effusions (all P < 0.001), while there were no significant differences among MPE, PPE, HF-PE, and Misc-PE (all P > 0.05).

[0025] Example 2 In Example 1, patients underwent standard thoracentesis within 24 hours of admission to obtain 5-10 mL of pleural effusion. Heparin was added to the collection bottle beforehand to prevent coagulation, and the samples were transported to the laboratory in an ice pack at low temperature. The pleural effusion was aliquoted into 5 mL centrifuge tubes, precisely balanced on a balance, and centrifuged at 1500 rpm for 10 minutes. After centrifugation, the supernatant was aliquoted into 1.5 mL EP tubes and labeled accordingly. These supernatants were then cryopreserved at -80°C for later use in detecting proteins such as cytokines or chemokines.

[0026] like Figure 1 As shown: Analysis of thoracoscopic results in patients with different disease types revealed that most patients with total pulmonary embolism (TPE) had severe pleural adhesions, while non-TPE patients had milder or virtually no pleural adhesions. Figure 1 ).

[0027] To investigate the diagnostic value of dsDNA, ADA, and IFN-γ in pleural effusion, the concentrations of dsDNA, ADA, and IFN-γ in the supernatant of pleural effusion were measured. Based on receiver operating characteristic (ROC) curves, 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 optimal cutoff value for differentiating between TPE and non-TPE. Figure 2 As shown: Subject ROC curves were plotted to assess the sensitivity, specificity, PPV, NPV, PLR, and NLR of these biomarkers. In the Guangzhou cohort, at a cutoff value of 441.15 ng / mL, the AUC of dsDNA distinguishing between TPE and non-TPE was 0.947 (95% CI, 0.931–0.963), which was higher than the AUC of ADA (0.904) and IFN-γ (0.945) (see [reference needed]). Figure 2Furthermore, the diagnostic accuracy parameters for 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 [reference needed]. Figure 2 The AUCs of these samples 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 indicate that immune cells release extracellular traps in the environment of bacterial and other pathogen infection. In particular, under the stimulation of Mycobacterium tuberculosis and virulence molecules, immune cells such as neutrophils and macrophages release more extracellular traps. Therefore, the dsDNA content in pleural effusion has high diagnostic value for tuberculous pleural effusion.

[0028] Thoracoscopic examination revealed that not all patients with total pleural effusion (TPE) had severe pleural adhesions. Analysis of dsDNA in the pleural effusion showed results consistent with thoracoscopy: patients with severe adhesions had higher dsDNA levels than those with milder adhesions, especially when the dsDNA concentration was >1068.1427 ng / mL, indicating severe pleural adhesions (see...). Figure 3 ).

[0029] The above results indicate that dsDNA has the potential value in assessing the severity of pleural adhesions in patients with total pulmonary embolism (TPE). The dsDNA content in pleural effusion can significantly distinguish between TPE and non-TPE. When the dsDNA content in the pleural effusion of patients is greater than the critical value of 441.15 ng / mL, it suggests that for TPE patients rich in dsDNA, anti-adhesion therapy should be carried out simultaneously with anti-tuberculosis treatment. This will help avoid complications such as pleural thickening and thus improve the prognosis of patients.

[0030] Example 3 Samples were collected from 10 patients with pleural effusion at Guangzhou First People's Hospital (GZFPH) between January 1 and January 31, 2024, including 3 patients with malignant pleural effusion, 4 patients with tuberculous pleural effusion, and 3 patients with parapneumonic pleural effusion. The clinical characteristics and dsDNA content in the pleural effusions of the patients are shown in Table 3.

[0031] Table 3. Patient clinical data and dsDNA content in pleural effusion. Figure 4 The images show thoracoscopy images of patients 1, 4, 6 and 8. Patients 1 and 6 have severe pleural adhesions, while patients 4 and 8 have mild pleural adhesions.

[0032] The dsDNA levels in the pleural effusions of all four patients with TPE were significantly higher than the critical value of 441.15 ng / mL. Furthermore, the dsDNA levels in the pleural effusions of patients 1 and 6 were 1302.09 ng / mL and 1948.38 ng / mL, respectively, both exceeding the critical value of 1068.1427 ng / mL for severe pleural adhesions, suggesting the presence of severe pleural adhesions. Figure 4 The results of the thoracoscopy images were consistent.

[0033] The above results further demonstrate that dsDNA in pleural effusion is a novel and reliable biomarker for diagnosing TPE, and can be used as a marker for the detection and assessment of the severity of pleural adhesions in TPE patients.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of dsDNA in pleural effusion in the preparation of a product for assessing the severity of pleural adhesions in patients with tuberculous pleural effusion.

2. Application of dsDNA in pleural effusion in the preparation of products to guide the treatment of tuberculous pleural effusion.

3. The application according to claim 2, characterized in that, A concentration of dsDNA > 441.15 ng / mL indicates the presence of pleural adhesions in the patient.

4. The application according to claim 2, characterized in that, When the concentration of dsDNA is >1068.1427 ng / mL, it indicates that the patient has severe pleural adhesions.

5. The application according to claim 3 or 4, characterized in that, The patient was undergoing anti-tuberculosis treatment and anti-adhesion treatment simultaneously.

6. 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 biomarkers include dsDNA in pleural effusion.

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