New biomarkers for the diagnosis of tuberculous pleural effusion.

• Grant number: 25/19347-8
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2025
• End date: October 31, 2026
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Roberta Karla Barbosa de Sales
• Grantee: Pedro Augusto Bento de Sousa
• Host Institution: Instituto do Coração Professor Euryclides de Jesus Zerbini (INCOR). Hospital das Clínicas da Faculdade de Medicina da USP (HCFMUSP). Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

This is a prospective study analyzing pleural fluid from patients with pleural effusions caused by tuberculosis, parapneumonic effusion, empyema, malignancies, and transudates, all with a clinical indication for thoracentesis and/or pleural drainage. Patients will be recruited between April 2025 and March 2027. The study will be conducted at the Heart Institute of the Hospital das Clínicas, University of São Paulo Medical School (InCor-HCFMUSP). Adults over 18 years of age who agree to participate and sign an informed consent form authorizing the collection of blood and pleural fluid samples for research purposes will be included, in accordance with approval by the institutional Research Ethics Committee. The following diagnostic criteria will be used to define the etiology of pleural effusions: tuberculosis will be diagnosed when at least one of the following is present-detection of Mycobacterium tuberculosis (MTB) or granuloma with caseous necrosis on pleural biopsy, positive culture for MTB from pleural fluid or pleural tissue, positive sputum culture for MTB, or a lymphocytic pleural effusion with high adenosine deaminase (ADA), negative cytology for malignant cells, and clinical response to specific treatment. Malignant pleural effusion will be confirmed by the presence of neoplastic cells on pleural fluid cytology or pleural biopsy. Parapneumonic effusion will be defined by a positive pleural fluid culture without purulent characteristics, or by a neutrophilic effusion associated with community-acquired pneumonia. Empyema will be defined as purulent pleural fluid with positive Gram stain and bacterial culture. Transudative effusions will be defined according to Light's criteria, with a pleural fluid/serum LDH ratio < 0.6, total protein ratio < 0.5, or pleural fluid LDH less than two-thirds of the upper reference limit for serum LDH. Blood samples will be collected by venipuncture, and pleural fluid samples will be obtained via thoracentesis. Samples will be placed in dry or anticoagulant-containing tubes, according to the requirements of each laboratory test. Pleural fluid will be analyzed for total and differential cell counts (using a Neubauer chamber and Leishman stain), pH, and biochemical markers such as glucose, total protein, lactate dehydrogenase (LDH), and ADA. Microbiological tests will include Gram staining and culture. Blood will be analyzed for LDH and total protein to support classification by Light's criteria. Pleural fluid samples will be centrifuged and analyzed for cytokine levels-specifically IFN-¿, IL-6, and IL-27-using enzyme-linked immunosorbent assay (ELISA) with capture kits from R&D Systems (Minnesota, USA). The GeneXpert MTB/RIF Ultra assay, an automated molecular test designed for rapid and sensitive detection of Mycobacterium tuberculosis and rifampicin resistance, will also be performed. Statistical analysis will be performed using SigmaStat software (San Rafael, CA, USA). ANOVA will be used to compare values among groups, followed by Tukey's test for multiple comparisons. When data are not normally distributed, the Kruskal-Wallis test and Dunn's multiple comparison test will be applied. Pearson or Spearman correlation coefficients will be used to assess associations. Diagnostic performance will be evaluated using receiver operating characteristic (ROC) curve analysis, with subsequent assessment of sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy. A p-value of less than 0.05 will be considered statistically significant. (AU)