Ag85B Antigen in Bronchoalveolar Lavage Fluid: A Promising Biomarker for Tuberculosis Diagnosis

Ag85B Antigen in Bronchoalveolar Lavage Fluid: A Promising Biomarker for Tuberculosis Diagnosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ag85B Antigen in Bronchoalveolar Lavage Fluid: A Promising Biomarker for Tuberculosis Diagnosis Guihua Gu, Shiyu Fang, Xinchun Zhou, Jinxiong Jiang, Weiwei Lin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6617609/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: To assess the diagnostic performance of the Mycobacterium tuberculosis (MTB)‑secreted antigen Ag85B in bronchoalveolar lavage fluid (BALF) for pulmonary tuberculosis (PTB) and to explore its utility as a supplemental etiological test. Methods: We prospectively enrolled 104 PTB patients who underwent fibre‑optic bronchoscopy at the Affiliated Hospital of North Sichuan Medical College from May 2021 to July 2023, together with 72 non‑PTB controls who had pulmonary infections of non‑tuberculous origin. Ag85B concentrations in BALF were measured by enzyme‑linked immunosorbent assay (ELISA). Receiver‑operating‑characteristic (ROC) analysis established the optimal diagnostic cut‑off (maximum Youden index). Diagnostic performance indices were compared with acid‑fast bacillus (AFB) smear microscopy and the GeneXpert MTB/RIF assay. Results: BALF‑Ag85B levels were significantly higher in PTB patients than in non‑PTB controls (P 0.05). Sensitivities were 26.92 % for AFB smear, 56.73 % for GeneXpert MTB/RIF, and 64.42 % for Ag85B ELISA; the latter was significantly more sensitive than AFB smear (P < 0.001) and similar to GeneXpert MTB/RIF (P = 0.256). Specificities were 100 % for AFB smear and GeneXpert MTB/RIF versus 79.17 % for Ag85B ELISA (P < 0.001). Ag85B sensitivity was comparable between BC‑PTB (67.80 %) and CD‑PTB (60 %) groups (P = 0.411). Conclusion: ELISA detection of Ag85B in BALF provides a useful adjunct for PTB diagnosis, particularly in bacteriologically negative cases. Pulmonary tuberculosis Tuberculosis antigen Enzyme-linked immunosorbent assay Ag85B AFB smear microscopy GeneXpert MTB/RIF Figures Figure 1 Figure 2 Figure 3 1. INTRODUCTION Tuberculosis remains a major global health threat. China consistently ranks third among the 30 high‑burden nations. Definitive diagnosis of PTB requires integration of epidemiological, clinical, radiological, immunological and bacteriological data. Radiological imaging, while indispensable for delineating lesion extent, cannot reliably distinguish PTB from other pulmonary pathologies. Immunological tests such as the tuberculin skin test and interferon‑γ release assays (IGRAs) are compromised by Bacillus Calmette–Guérin (BCG) vaccination status, host immunity and their inability to differentiate active from latent infection [ 1 ]. Bacteriological confirmation remains the diagnostic cornerstone. Conventional AFB smears have low sensitivity and cannot differentiate MTB from non‑tuberculous mycobacteria, whereas cultures—although definitive—require weeks to yield results and demand resource-intensive biosafety infrastructure [ 2 , 3 ]. The GeneXpert MTB/RIF assay offers rapid detection of MTB DNA and rifampicin resistance with high specificity but suboptimal sensitivity, and it does not distinguish viable from non‑viable organisms [ 4 ]. According to the WHO Global Tuberculosis Report 2024, only 62% of the 6.9 million diagnosed PTB cases were bacteriologically confirmed [ 5 ]. Secreted MTB antigens have emerged as attractive diagnostic targets. Ag85B is a protein abundantly secreted by MTB during early logarithmic growth [ 6 ]. Previous studies documented Ag85B in tuberculosis tissues—including lung [ 7 ], kidney [ 8 ] and lymph node [ 9 ]—and in sputum detected by ELISA [ 14 ]. Because BALF provides superior diagnostic yield for pulmonary infections [ 15 ], we evaluated BALF‑Ag85B levels in PTB patients to determine its diagnostic value. 2. MATERIALS AND METHODS 2.1 Patients From May 2021 to July 2023, 176 consecutive patients undergoing fibre‑optic bronchoscopy at the Affiliated Hospital of North Sichuan Medical College were enrolled. PTB cases (n = 104) comprised BC‑PTB (n = 59; GeneXpert positive, including 28 AFB‑positive) and CD‑PTB (n = 45; GeneXpert‑ and AFB‑negative). All PTB patients responded clinically to anti‑tuberculous therapy. Non‑PTB controls (n = 72) had non‑tuberculous pulmonary infections. Diagnostic criteria followed the Chinese standard WS 288‑2017: Diagnosis of Pulmonary Tuberculosis [16] as follows: BC‑PTB (n = 59) – chest imaging typical of active PTB plus at least one positive bacteriological test (GeneXpert MTB/RIF or AFB smear). All 59 were GeneXpert‑positive; 28 were also AFB‑positive. CD‑PTB (n = 45) – characteristic imaging but negative bacteriology, supported by ≥ 1 of: (i) typical TB symptoms, (ii) positive IGRA, or (iii) histopathology confirming extrapulmonary TB. Non‑PTB controls (n = 72) – other pulmonary infections, negative on GeneXpert and AFB, and no TB history. 2.2 Experimental Methods 2.2.1 BALF Collection BALF was obtained under standard procedures and safety precautions adhering to European guidelines [17]. Three aliquots of sterile saline (10–20 mL each, total 50–60 mL) were instilled and recovered at –13.3 to –19.95 kPa (30–50 % recovery). Combined BALF was homogenised and divided: two aliquots were used for AFB smear and GeneXpert; the remainder was stored at –20 °C in silicone‑coated tubes to avoid freeze–thaw cycles. 2.2.2 Checkerboard Titration Preliminary checkerboard titration on high‑bacillary sputum samples (dilutions 1:1–1:20) identified the undiluted BALF supernatant as optimal (highest positive‑to‑negative OD ratio) for ELISA. 2.2.3 Detection of Ag85B Antigen in BALF by Double Antibody Sandwich ELISA BALF aliquots were thawed overnight at 4 °C, then clarified by centrifugation (3000 rpm, 10 min, 4 °C). The supernatant was analyzed with a commercial double‑antibody sandwich ELISA kit for human MTB‑Ag85B (Camilo Biological, Nanjing; lot 2H‑KMLJh315423) as follows: Preparation of standards: The lyophilized Ag85B standard (20 ng mL⁻¹ after reconstitution in 1 mL of diluent) was serially two‑fold diluted to eight concentrations (100 %, 50 %, 25 %, 12.5 %, 6.3 %, 3.1 %, 1.5 %, 0 %). Plate loading: Duplicate 100 µL aliquots of standards, blank controls and undiluted BALF supernatants were added to antibody‑coated wells. Antigen binding: Plates were sealed and incubated at 37 °C for 90 min, allowing Ag85B to bind the capture antibody. Washing: Each well was washed twice with Tris‑buffered saline to remove unbound material. Detection antibody: Horseradish‑peroxidase (HRP)–labelled avidin (100 µL well⁻¹) was added; plates were resealed and incubated at 37 °C for 30 min in the dark, followed by five washes. Color development TMB substrate (100 µL well⁻¹) was applied and incubated at 37 °C until a clear color gradient appeared in the standard wells. Reaction stop and reading: Stop solution (100 µL well⁻¹) was added; absorbance was read immediately at 450 nm with a microplate reader. Duplicate OD values were averaged, blank‑corrected, and converted to Ag85B concentration from the standard curve. Inter‑duplicate coefficients of variation > 10 % triggered repeat testing. 2.3 Clinical Data Collection Demographic data, clinical features, comorbidities and extrapulmonary tuberculosis were recorded. 2.4 Statistical Analysis All statistical analyses were performed with SPSS Statistics, version 29.0 (IBM Corp., Armonk, NY, USA). Continuous variables were first tested for normality with the Shapiro–Wilk test. · Data presentation and group comparisons o Normally distributed data are reported as mean ± standard deviation (SD) and compared with a two‑tailed Student’s t ‑test. o Non‑normally distributed data are expressed as median (interquartile range) and analyzed with the Mann–Whitney U test. o Categorical variables are summarized as counts and percentages and compared by Pearson’s chi‑square or Fisher’s exact test, as appropriate. · Diagnostic accuracy Receiver‑operating‑characteristic (ROC) curves were constructed to evaluate the ability of BALF Ag85B concentration to discriminate pulmonary tuberculosis (PTB) from non‑PTB. The optimal cutoff value was defined as the point maximizing the Youden index (sensitivity + specificity − 1). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated with exact 95 % confidence intervals (CIs) using the Clopper–Pearson method. · Significance threshold P values < 0.05 were considered statistically significant. Graphical outputs—including ROC curves and box‑and‑whisker plots—were generated with GraphPad Prism, version 10.1.2 (GraphPad Software, San Diego, CA, USA). 2.5 Ethical Approval This study received approval from the Ethics Committee of the Affiliated Hospital of North Sichuan Medical College (Approval No. 2023ER224‑1). The research adhered to the principles of non‑maleficence and the protection of patient privacy. 3. RESULTS 3.1 Demographic and Clinical Characteristics of Patients Demographic and clinical data are summarised in Table 1. Age ranged 13–78 years (PTB) and 16–85 years (non‑PTB). Seven PTB patients had concurrent extrapulmonary tuberculosis (tuberculous meningitis = 2; peritonitis = 3; lumbar spinal TB = 2). 3.2 Detection of Ag85B in Bronchoalveolar Lavage Fluid BALF‑Ag85B concentrations were significantly higher in BC‑PTB and CD‑PTB groups than in non‑PTB controls (P < 0.05) with no significant difference between BC‑PTB and CD‑PTB (P = 0.055) (Figure 1). ROC analysis (BC‑PTB vs. non‑PTB) yielded an AUC of 0.783 (95 % CI 0.705–0.860). The maximal Youden index (0.470) corresponded to 4.22 ng/mL, adopted as the diagnostic cut‑off (Figure 2). 3.3 Diagnostic Performance of Antigen Ag85B for Pulmonary Tuberculosis We assessed Ag85B ELISA in 104 PTB patients (BC‑PTB + CD‑PTB) versus 72 non‑PTB controls, using a 4.22 ng/mL cutoff. Of the PTB group, 67/104 (64.4 %) were Ag85B‑positive; among controls, 15/72 (20.8 %) were false‑positive. This yielded a sensitivity of 64.42 % and specificity of 79.17 % for Ag85B. By comparison: · AFB smear microscopy : sensitivity 26.92 %, specificity 100 %. · GeneXpert MTB/RIF : sensitivity 56.73 %, specificity 100 % (Table 2). Ag85B sensitivity exceeded that of AFB smear by 37.5 % (P < 0.001) and was 7.7 % higher than GeneXpert, though this difference was not statistically significant (P = 0.256). Conversely, Ag85B specificity was significantly lower than both AFB smear and GeneXpert (P < 0.001) (Figure 3). 3.4 Comparison of Ag85B Sensitivity Between Bacteriologically Confirmed and Clinically Diagnosed PTB Groups In the BC‑PTB cohort (n = 59), 40 specimens tested positive for Ag85B, yielding a sensitivity of 67.80 % (95 % CI: 54.36 %–79.38 %). In the CD‑PTB cohort (n = 45), 27 specimens were positive, corresponding to a sensitivity of 60.00 % (95 % CI: 44.33 %–74.30 %) (Table 3). A χ² test showed no significant difference between the two groups (χ² = 0.68, P = 0.441). 4. DISCUSSION The Ag85 complex is the most abundant secreted protein family produced by MTB, accounting for approximately 45 % of culture filtrate proteins, and elicits strong humoral and cellular immune responses. Early studies employing indirect ELISA demonstrated that detection of the Ag85 complex in sputum culture filtrates enables rapid tuberculosis diagnosis [18]. Its diagnostic utility extends to extrapulmonary forms: in ascitic fluid, Ag85 achieved 72.22 % sensitivity and 90.48 % specificity for tuberculous peritonitis [19], while in cerebrospinal fluid it reached 92.5 % sensitivity and 97.5 % specificity for tuberculous meningitis [20]. Elevated serum Ag85 levels have also distinguished tuberculosis patients from controls with 82 % sensitivity and 86 % specificity [21]. Collectively, these findings validate the Ag85 complex as a promising biomarker for tuberculosis. Within this complex, Ag85B constitutes ~22 % of the secreted proteins and is the principal antigen recognized by host macrophages [22]. Indirect ELISA assays for Ag85B in sputum report PTB sensitivities of 73.6 %–77 % and specificities of 86.3 %–95.2 % [14, 23]. For extrapulmonary specimens, sensitivities vary by fluid type—62.5 % in pleural effusion, 100 % in joint fluid and lymph node aspirates, and lower in others—while specificity remains high (~96 %) [14]. Immunohistochemical studies detect Ag85B in lung tissue with sensitivities ranging from 50.5 % to 92.7 % and specificities of 83.3 %–100 % [7, 9–13]. Our investigation is the first to quantify Ag85B in BALF, yielding 64.4 % sensitivity and 79.2 % specificity for PTB. The variation in diagnostic performance compared to prior reports likely reflects differences in specimen matrix, antigen concentration, assay format, and control selection. When benchmarked against conventional diagnostics, Ag85B ELISA substantially outperformed AFB smear microscopy (26.9 % sensitivity) and approximated the sensitivity of GeneXpert MTB/RIF (56.7 %), while incurring a specificity trade-off (79.2 % vs. 100 %) (Table 2, Figure 3). Remarkably, Ag85B detected 60 % of clinically diagnosed PTB cases that were smear- and GeneXpert-negative, underscoring its value in paucibacillary or difficult-to-sample presentations. This enhanced detectability may derive from the secretion dynamics of Ag85B. Secreted via classical pathways, extracellular vesicles, or exosome-mediated transport [24–26], Ag85B can disseminate beyond localized bacterial foci. Immunohistochemical analyses have observed diffuse Ag85B distribution around bacillary clusters, contrasting with the more focal pattern of acid-fast bacilli [10]. Previous studies have corroborated this finding: Immunohistochemical comparison with acid-fast staining confirms that Ag85B immunoreactivity extends well beyond visible bacilli, forming a more diffuse halo around bacterial foci and yielding higher sensitivity for antigen detection in lung tissue [7,10–12]. Likewise, in murine models, Ag85B accumulates in lung tissue proportional to bacillary load but declines sharply during late infection stages, reflecting regulated secretion kinetics [24]. These properties may explain the similar Ag85B sensitivities in bacteriologically confirmed and clinically diagnosed PTB cohorts, despite variable bacillary burdens. Although GeneXpert MTB/RIF remains the molecular gold standard for rapid, specific tuberculosis diagnosis, its cost and infrastructural demands limit deployment in resource-constrained settings. In contrast, ELISA for Ag85B is inexpensive, rapid, and requires minimal laboratory infrastructure and technical expertise, making it suitable for peripheral health facilities. Despite sputum’s role as the primary specimen for PTB diagnosis, BALF offers superior sensitivity for lower respiratory infections, albeit requiring bronchoscopy. Our study introduces BALF-based Ag85B ELISA as a supplementary diagnostic modality, particularly beneficial for bacteriologically negative patients. Limitations include the absence of healthy BALF controls due to ethical constraints, which may affect specificity estimates, and a single-center design with a modest sample size, warranting multicenter validation and assessment of Ag85B kinetics during treatment. Conclusion ELISA‑based detection of Ag85B in BALF is a feasible, sensitive and rapid adjunctive test for PTB, particularly valuable when smear and GeneXpert results are negative. Larger studies are required to refine its diagnostic thresholds and evaluate its role in treatment monitoring. Declarations AUTHOR CONTRIBUTIONS Gu Guihua and Fang Shiyu: Conceptualization, Study design, Data acquisition, Formal analysis, and Writing – original draft; Zhou Xinchun: Visualization, Methodology (preparation of figures and tables); Jiang Jinxiong and Lin Weiwei: Investigation (data collection), Validation (statistical analysis); Sun Jie: Writing – review & editing (critical revision for intellectual content); Liu Fengjun: Supervision (ensuring methodological rigor), Project administration (quality control and integrity verification).All authors reviewed and approved the final manuscript. ETHICS APPROVAL AND CONSENT TO PARTICIPATE This study was approved by the Ethics Committee of the Affiliated Hospital of North Sichuan Medical College (Approval No. 2023ER224-1). Written informed consent was obtained from all participants. For participants under the age of 18, consent was provided by their legal guardians. All data were anonymized to protect participant confidentiality. ACKNOWLEDGMENTS The authors greatly thank Yidie Qin for his valuable help. References Carranza C, Pedraza-Sanchez S, de Oyarzabal-Mendez E, Torres M. （2020） Diagnosis for Latent Tuberculosis Infection: New Alternatives. Front Immunol . 11:2006. Published 2020 Sep 10. doi:10.3389/fimmu.2020.02006 Consolidated guidance on tuberculosis data generation and use. Module 1. Tuberculosis surveillance. Geneva: World Health Organization; 2024. Licence: CC BY-NC-SA 3.0 IGO. https://www.who.int/publications/i/item/9789240075290 Rageade F, Picot N, Blanc-Michaud A, et al. （2014） Performance of solid and liquid culture media for the detection of Mycobacterium tuberculosis in clinical materials: meta-analysis of recent studies. Eur J Clin Microbiol Infect Dis . 33(6):867-870. doi:10.1007/s10096-014-2105-z Garg A, Agarwal L, Mathur R. （2022） Role of GeneXpert or CBNAAT in diagnosing tuberculosis: Present scenario. Med J DY Patil Vidyapeeth . 15:14-9. doi: 10.4103/mjdrdypu.mjdrdypu_182_20 Global tuberculosis report 2024. Geneva: World Health Organization; 2024. Licence: CC BY-NC-SA 3.0 IGO. https://www.who.int/publications/i/item/9789240101531 Wiker HG, Harboe M. （1992） The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. Microbiol Rev . 56(4):648-661. doi:10.1128/mr.56.4.648-661.1992 Sun J, Zhou X, Yu J, Fang S, Duan S, Liu F. （2024） Diagnostic value of tuberculosis-Specific antigens Ag85B, ESAT-6 and CFP10 in pulmonary tuberculosis. J Clin Tuberc Other Mycobact Dis . 37:100486. Published 2024 Oct 19. doi:10.1016/j.jctube.2024.100486 Man CJ, Dong XY, Wang X, et al. （2014） Application of Mycobacterium tuberculosis secretory antigens Ag85 and ESAT-6 in the diagnosis of renal tuberculosis infection. Chinese Journal of Nosocomiology , 24(18): 4660-4662. doi: 10.11816/cn.ni.2014-134242 Che N, QU Y, Zhang C, et al. （2014） Expression characteristics of Mycobacterium tuberculosis Ag85B protein and its diagnostic value in pathology. Chinese Journal of Pathology, 43(9): 600-603. doi:10.3760/cm8.j.issn.0529-5807.2014.09.006 Che N, Qu Y, Zhang C, Zhang L, Zhang H. （2016） Double staining of bacilli and antigen Ag85B improves the accuracy of the pathological diagnosis of pulmonary tuberculosis. J Clin Pathol . 69(7):600-606. doi:10.1136/jclinpath-2015-203244 Duan, S. Q. (2024). Significance of Immunohistochemical Detection of Mycobacterium tuberculosis Secreted Antigens Ag85B, ESAT-6, and CFP10 in the Diagnosis of Pulmonary Tuberculosis [Master's thesis, North Sichuan Medical College]. CNKI. (in Chinese) Retrieved from https://kns.cnki.net/kcms2/article/abstract?v=-Y4qPNLDuvYoYW9wNeBTiUp8JUkWCt8jrA_D-1Wz1GvtYE-IBykTiOwpWPf7nWFSwHG761CdLtUg5Dw91YNw2NtnJgy4_rB4pATV9kO98NAHieZUAm4ju1-yv1sSWrAXzKfn3V4AgXHda7YOhtsgMBHgZ2fdyoaiz5MQl26ONvcUp3BMA4Xo5eGeYERC46p9&uniplatform=NZKPT&language=CHS Fang SY, Duan SQ, Chen XQ, et al. （2023） Immunohistochemical staining for detection of Ag85B in pulmonary tuberculosis tissues. Journal of North Sichuan Medical College . 38(5): 1165-1168. doi: 10. 3969 / j. issn. 1005⁃3697. 2023. 09. 003 Dong Y, Zhou L, Zhang C, et al. （2023） Detection of antigen Ag85B expression is useful for the diagnosis of tuberculosis, especially for those with an antituberculosis treatment history. Am J Clin Pathol . 160(1):62-71. doi:10.1093/ajcp/aqad012 Singh N, Sreenivas V, Gupta KB, et al. （2015） Diagnosis of pulmonary and extrapulmonary tuberculosis based on detection of mycobacterial antigen 85B by immuno-PCR. Diagn Microbiol Infect Dis . 83(4):359-364. doi:10.1016/j.diagmicrobio.2015.08.015 Liu X, Hou XF, Gao L, et al. （2018） Indicators for prediction of Mycobacterium tuberculosis positivity detected with bronchoalveolar lavage fluid. Infect Dis Poverty . 7(1):22. Published 2018 Mar 24. doi:10.1186/s40249-018-0403-x National Health Commission of China. (2017). WS 288—2017: Diagnostic criteria for pulmonary tuberculosis . Beijing: Standards Press of China. (in Chinese) Report of the European Society of Pneumology Task Group. （1989） Technical recommendations and guidelines for bronchoalveolar lavage (BAL). Eur Respir J . 2(6):561-585. PMID: 2663535. Phunpae P, Chanwong S, Tayapiwatana C, Apiratmateekul N, Makeudom A, Kasinrerk W. （2014）Rapid diagnosis of tuberculosis by identification of Antigen 85 in mycobacterial culture system. Diagn Microbiol Infect Dis . 78(3):242-248. doi:10.1016/j.diagmicrobio.2013.11.028 Kashyap RS, Saha SM, Nagdev KJ, et al. （2010） Diagnostic markers for tuberculosis ascites: a preliminary study. Biomark Insights . 5:87-94. Published 2010 Aug 25. doi:10.4137/bmi.s5196 Kashyap RS, Dobos KM, Belisle JT, et al. （2005） Demonstration of components of antigen 85 complex in cerebrospinal fluid of tuberculous meningitis patients. Clin Diagn Lab Immunol . 12(6):752-758. doi:10.1128/CDLI.12.6.752-758.2005 Kashyap RS, Rajan AN, Ramteke SS, et al. （2007） Diagnosis of tuberculosis in an Indian population by an indirect ELISA protocol based on detection of Antigen 85 complex: a prospective cohort study. BMC Infect Dis . 7:74. Published 2007 Jul 10. doi:10.1186/1471-2334-7-74 Xu Z, Hu T, Xia A, et al. （2017） Generation of Monoclonal Antibodies against Ag85A Antigen of Mycobacterium tuberculosis and Application in a Competitive ELISA for Serodiagnosis of Bovine Tuberculosis. Front Vet Sci . 4:107. Published 2017 Jun 30. doi:10.3389/fvets.2017.00107 Mehta PK, Singh N, Dharra R, et al. （2016） Diagnosis of tuberculosis based on the detection of a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor by immuno-PCR. J Microbiol Methods . 127:24-27. doi:10.1016/j.mimet.2016.05.003 Ernst JD, Cornelius A, Bolz M. （2019） Dynamics of Mycobacterium tuberculosis Ag85B Revealed by a Sensitive Enzyme-Linked Immunosorbent Assay. mBio . 10(2):e00611-19. Published 2019 Apr 23. doi:10.1128/mBio.00611-19 Giri PK, Kruh NA, Dobos KM, Schorey JS. （2010） Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis-infected and culture filtrate protein-treated macrophages. Proteomics . 10(17):3190-3202. doi:10.1002/pmic.200900840 Srivastava S, Grace PS, Ernst JD. （2016） Antigen Export Reduces Antigen Presentation and Limits T Cell Control of M. tuberculosis. Cell Host Microbe . 19(1):44-54. doi:10.1016/j.chom.2015.12.003 Table Table 1 : Demographic and clinical characteristics of patients. Characteristics PTB group Non-PTB group n=104(%) n=72(%) Age（Median, year） 44 58.5 Gender Male 75(72.1) 48(66.7) Female 29(27.9) 24(33.3) Symptoms cough 68(65.3) 54(75.0) Fever 14(13.4) 23(31.9) Weight loss 19(18.2) 11(15.2) Night sweat 27(25.9) 5(6.9) No symptoms 18(17.3) 12(16.6) Comorbidities diabetes mellitus 12(11.5) 9(12.5) Hypertension 2(1.9) 9(12.5) extrapulmonary tuberculosis 7(6.7) 0 Note : The number of cases is expressed as frequency and percentage (n(%)). Table 2 Diagnostic Value of Antigen Ag85B, Acid-Fast Bacilli (AFB) Staining, and GeneXpert MTB/RIF for Active Pulmonary Tuberculosis (n=176) PTB (n=104) Non-PTB (n=72) Sensitivity （%） Specificity （%） PPV （%） NPV （%） AFB + 28 0 26.92 (18.69~36.51) 100.00 (95.01~100) 100.00 (87.66~100) 48.65 (40.36~56.99) - 76 72 GeneXpert MTB/RIF + 59 0 56.73 (46.65~66.41) 100.00 (95.01~100) 100.00 (93.94~100) 61.54 (52.09~70.38) - 45 72 Ag85B + 67 15 64.42 (54.43~73.57) 79.17 (67.98~87.84) 81.71 (71.63~89.38) 60.64 (50.02~70.56) - 37 57 Note : PPV: Positive Predictive Value; NPV: Negative Predictive Value. Sensitivity, specificity, PPV, and NPV are expressed as percentages with 95% confidence intervals (95% CI). Table 3 Sensitivity of Ag85B in the BC-PTB and CD-PTB Groups Positive Negative Sensitivity BC-PTB (n=59) 40 19 67.80% (95% CI: 54.36%-79.38%) CD-PTB (n=45) 27 18 60% (95% CI: 44.33%-74.30%) χ² - - 0.677 P - - 0.441 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6617609","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":466920595,"identity":"3b7dced3-004f-49e9-939f-41fcabcd5189","order_by":0,"name":"Guihua Gu","email":"","orcid":"","institution":"Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Guihua","middleName":"","lastName":"Gu","suffix":""},{"id":466920596,"identity":"d3f64332-b8d2-4feb-a862-a6bc65b4559f","order_by":1,"name":"Shiyu Fang","email":"","orcid":"","institution":"Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Shiyu","middleName":"","lastName":"Fang","suffix":""},{"id":466920597,"identity":"f06544f8-f9b3-40da-a062-66b1d169ed78","order_by":2,"name":"Xinchun Zhou","email":"","orcid":"","institution":"Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xinchun","middleName":"","lastName":"Zhou","suffix":""},{"id":466920598,"identity":"e0b38c4a-0fa0-4245-a4d8-3b1022fc5328","order_by":3,"name":"Jinxiong Jiang","email":"","orcid":"","institution":"Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jinxiong","middleName":"","lastName":"Jiang","suffix":""},{"id":466920599,"identity":"fed44d51-e7c7-4405-9195-43d750031ae0","order_by":4,"name":"Weiwei Lin","email":"","orcid":"","institution":"Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Lin","suffix":""},{"id":466920600,"identity":"91f02010-b0da-481f-9db7-c8f3607d4eb2","order_by":5,"name":"Sun Jie","email":"","orcid":"","institution":"Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Sun","middleName":"","lastName":"Jie","suffix":""},{"id":466920601,"identity":"9dc13fa4-0321-44fe-a6ac-434a594efe0a","order_by":6,"name":"Fengjun Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYBACPuYDCQcSKmx4+NkbiNTCxpaQeODBmTQZyZ4DxGthPviw5bCNwQ0HorUwPDiQ2HCeh+EGA+OHjznEaUk4kLjjNg/j7AZmyZnbiNEi3wDUcuY2D7PMATZmXqK0gG1pO8fDJpFAmpYDPDykaUk4k8wjwXOwmTi/8LPxJH/8UWFnb3+8+eCHj8RoYWDgSYAyGBuIUg8E7AeIVTkKRsEoGAUjFQAABUU3XdqLHLIAAAAASUVORK5CYII=","orcid":"","institution":"Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":true,"prefix":"","firstName":"Fengjun","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-05-08 07:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6617609/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6617609/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84276813,"identity":"5a2b7ed9-47cf-4636-9036-cbf307f8d10a","added_by":"auto","created_at":"2025-06-10 05:43:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40689,"visible":true,"origin":"","legend":"\u003cp\u003eBox‑and‑whisker plots of Ag85B concentrations in BALF for BC‑PTB, CD‑PTB, and non‑PTB groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e: Boxes represent the 25th to 75th percentiles, horizontal lines the medians; whiskers show the minimum and maximum values; the dashed line indicates the diagnostic cutoff (4.22 ng/mL).\u003cbr\u003e\nAbbreviations: BC‑PTB, bacteriologically confirmed pulmonary tuberculosis; CD‑PTB, clinically diagnosed pulmonary tuberculosis; non‑PTB, non‑pulmonary tuberculosis controls\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6617609/v1/fb07868bb5ae5b3988315bc6.png"},{"id":84278028,"identity":"3a568829-3475-4021-b87a-6e4d26616880","added_by":"auto","created_at":"2025-06-10 06:04:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12808,"visible":true,"origin":"","legend":"\u003cp\u003eReceiver operating characteristic (ROC) curve of BALF Ag85B concentrations distinguishing BC‑PTB from non‑PTB subjects. The area under the ROC curve (AUC) indicates diagnostic performance.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6617609/v1/113a90ee9b5d519339ab7762.png"},{"id":84277674,"identity":"04c5cb75-0065-47eb-8b82-cb7fd50af790","added_by":"auto","created_at":"2025-06-10 06:03:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":22273,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of Sensitivity and Specificity of Three Detection Methods for Diagnosing Pulmonary Tuberculosis (n=176)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6617609/v1/70a5742e6f978f0d04585fbb.png"},{"id":86349517,"identity":"caf98e29-cdd4-4f65-98a6-f13e25ca4536","added_by":"auto","created_at":"2025-07-09 15:39:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":795977,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6617609/v1/8f70f28b-3987-43c6-a417-eb11c23e4640.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ag85B Antigen in Bronchoalveolar Lavage Fluid: A Promising Biomarker for Tuberculosis Diagnosis","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eTuberculosis remains a major global health threat. China consistently ranks third among the 30 high‑burden nations. Definitive diagnosis of PTB requires integration of epidemiological, clinical, radiological, immunological and bacteriological data. Radiological imaging, while indispensable for delineating lesion extent, cannot reliably distinguish PTB from other pulmonary pathologies. Immunological tests such as the tuberculin skin test and interferon‑γ release assays (IGRAs) are compromised by Bacillus Calmette\u0026ndash;Gu\u0026eacute;rin (BCG) vaccination status, host immunity and their inability to differentiate active from latent infection [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBacteriological confirmation remains the diagnostic cornerstone. Conventional AFB smears have low sensitivity and cannot differentiate MTB from non‑tuberculous mycobacteria, whereas cultures\u0026mdash;although definitive\u0026mdash;require weeks to yield results and demand resource-intensive biosafety infrastructure [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The GeneXpert MTB/RIF assay offers rapid detection of MTB DNA and rifampicin resistance with high specificity but suboptimal sensitivity, and it does not distinguish viable from non‑viable organisms [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. According to the WHO Global Tuberculosis Report 2024, only 62% of the 6.9\u0026nbsp;million diagnosed PTB cases were bacteriologically confirmed [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSecreted MTB antigens have emerged as attractive diagnostic targets. Ag85B is a protein abundantly secreted by MTB during early logarithmic growth [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Previous studies documented Ag85B in tuberculosis tissues\u0026mdash;including lung [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], kidney [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and lymph node [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u0026mdash;and in sputum detected by ELISA [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Because BALF provides superior diagnostic yield for pulmonary infections [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], we evaluated BALF‑Ag85B levels in PTB patients to determine its diagnostic value.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e2.1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePatients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom May 2021 to July 2023, 176 consecutive patients undergoing fibre‑optic bronchoscopy at the Affiliated Hospital of North Sichuan Medical College were enrolled. PTB cases (n = 104) comprised BC‑PTB (n = 59; GeneXpert positive, including 28 AFB‑positive) and CD‑PTB (n = 45; GeneXpert‑ and AFB‑negative). All PTB patients responded clinically to anti‑tuberculous therapy. Non‑PTB controls (n = 72) had non‑tuberculous pulmonary infections. Diagnostic criteria followed the Chinese standard \u003cem\u003eWS\u0026nbsp;288‑2017: Diagnosis of Pulmonary Tuberculosis\u003c/em\u003e [16] as follows:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBC‑PTB (n\u0026nbsp;=\u0026nbsp;59)\u003c/em\u003e\u003c/strong\u003e \u0026ndash; chest imaging typical of active PTB \u003cstrong\u003eplus\u003c/strong\u003e at least one positive bacteriological test (GeneXpert MTB/RIF or AFB smear). All 59 were GeneXpert‑positive; 28 were also AFB‑positive.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCD‑PTB (n\u0026nbsp;=\u0026nbsp;45)\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026ndash; characteristic imaging \u003cstrong\u003ebut\u003c/strong\u003e negative bacteriology, supported by \u0026ge; 1 of: (i) typical TB symptoms, (ii) positive IGRA, or (iii) histopathology confirming extrapulmonary TB.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNon‑PTB controls (n\u0026nbsp;=\u0026nbsp;72)\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026ndash; other pulmonary infections, negative on GeneXpert and AFB, and no TB history.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Experimental Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1 BALF Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBALF was obtained under standard procedures and safety precautions adhering to European guidelines\u0026nbsp;[17]. Three aliquots of sterile saline (10\u0026ndash;20\u0026nbsp;mL each, total 50\u0026ndash;60\u0026nbsp;mL) were instilled and recovered at \u0026ndash;13.3 to \u0026ndash;19.95\u0026nbsp;kPa (30\u0026ndash;50\u0026nbsp;% recovery). Combined BALF was homogenised and divided: two aliquots were used for AFB smear and GeneXpert; the remainder was stored at \u0026ndash;20\u0026nbsp;\u0026deg;C in silicone‑coated tubes to avoid freeze\u0026ndash;thaw cycles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2 Checkerboard Titration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePreliminary checkerboard titration on high‑bacillary sputum samples (dilutions 1:1\u0026ndash;1:20) identified the undiluted BALF supernatant as optimal (highest positive‑to‑negative OD ratio) for ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.3 Detection of Ag85B Antigen in BALF by Double Antibody Sandwich ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBALF aliquots were thawed overnight at\u0026nbsp;4\u0026nbsp;\u0026deg;C, then clarified by centrifugation (3000\u0026nbsp;rpm, 10\u0026nbsp;min, 4\u0026nbsp;\u0026deg;C). The supernatant was analyzed with a commercial double‑antibody sandwich ELISA kit for human MTB‑Ag85B (Camilo Biological, Nanjing; lot\u0026nbsp;2H‑KMLJh315423) as follows:\u003c/p\u003e\n\u003cp\u003ePreparation of standards:\u0026ensp;The lyophilized Ag85B standard (20\u0026nbsp;ng\u0026nbsp;mL⁻\u0026sup1; after reconstitution in 1\u0026nbsp;mL of diluent) was serially two‑fold diluted to eight concentrations (100\u0026nbsp;%,\u0026nbsp;50\u0026nbsp;%,\u0026nbsp;25\u0026nbsp;%,\u0026nbsp;12.5\u0026nbsp;%,\u0026nbsp;6.3\u0026nbsp;%,\u0026nbsp;3.1\u0026nbsp;%,\u0026nbsp;1.5\u0026nbsp;%,\u0026nbsp;0\u0026nbsp;%).\u003c/p\u003e\n\u003cp\u003ePlate loading:\u0026ensp;Duplicate 100\u0026nbsp;\u0026micro;L aliquots of standards, blank controls and undiluted BALF supernatants were added to antibody‑coated wells.\u003c/p\u003e\n\u003cp\u003eAntigen binding:\u0026ensp;Plates were sealed and incubated at\u0026nbsp;37\u0026nbsp;\u0026deg;C for 90\u0026nbsp;min, allowing Ag85B to bind the capture antibody.\u003c/p\u003e\n\u003cp\u003eWashing:\u0026ensp;Each well was washed twice with Tris‑buffered saline to remove unbound material.\u003c/p\u003e\n\u003cp\u003eDetection antibody:\u0026ensp;Horseradish‑peroxidase (HRP)\u0026ndash;labelled avidin (100\u0026nbsp;\u0026micro;L\u0026nbsp;well⁻\u0026sup1;) was added; plates were resealed and incubated at\u0026nbsp;37\u0026nbsp;\u0026deg;C for 30\u0026nbsp;min in the dark, followed by five washes. Color development\u0026ensp;TMB substrate (100\u0026nbsp;\u0026micro;L\u0026nbsp;well⁻\u0026sup1;) was applied and incubated at\u0026nbsp;37\u0026nbsp;\u0026deg;C until a clear color gradient appeared in the standard wells.\u003c/p\u003e\n\u003cp\u003eReaction stop and reading:\u0026ensp;Stop solution (100\u0026nbsp;\u0026micro;L\u0026nbsp;well⁻\u0026sup1;) was added; absorbance was read immediately at 450\u0026nbsp;nm with a microplate reader.\u003c/p\u003e\n\u003cp\u003eDuplicate OD values were averaged, blank‑corrected, and converted to Ag85B concentration from the standard curve. Inter‑duplicate coefficients of variation \u0026gt;\u0026nbsp;10\u0026nbsp;% triggered repeat testing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Clinical Data Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDemographic data, clinical features, comorbidities and extrapulmonary tuberculosis were recorded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4\u0026nbsp;\u0026nbsp; Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed with SPSS\u0026nbsp;Statistics, version\u0026nbsp;29.0 (IBM Corp., Armonk, NY, USA). Continuous variables were first tested for normality with the Shapiro\u0026ndash;Wilk test.\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eData presentation and group comparisons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eo Normally distributed data are reported as mean \u0026plusmn; standard deviation (SD) and compared with a two‑tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e‑test.\u003c/p\u003e\n\u003cp\u003eo Non‑normally distributed data are expressed as median (interquartile range) and analyzed with the Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e test.\u003c/p\u003e\n\u003cp\u003eo Categorical variables are summarized as counts and percentages and compared by Pearson\u0026rsquo;s chi‑square or Fisher\u0026rsquo;s exact test, as appropriate.\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eDiagnostic accuracy\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Receiver‑operating‑characteristic (ROC) curves were constructed to evaluate the ability of BALF Ag85B concentration to discriminate pulmonary tuberculosis (PTB) from non‑PTB. The optimal cutoff value was defined as the point maximizing the Youden index (sensitivity + specificity \u0026minus; 1). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated with exact 95 % confidence intervals (CIs) using the Clopper\u0026ndash;Pearson method.\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003eSignificance threshold\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;P values \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e\n\u003cp\u003eGraphical outputs\u0026mdash;including ROC curves and box‑and‑whisker plots\u0026mdash;were generated with GraphPad\u0026nbsp;Prism, version\u0026nbsp;10.1.2 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5\u0026nbsp;\u0026nbsp; Ethical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received approval from the Ethics Committee of the Affiliated Hospital of North Sichuan Medical College (Approval No. 2023ER224‑1). The research adhered to the principles of non‑maleficence and the protection of patient privacy.\u003c/p\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e\u003cstrong\u003e3.1 Demographic and Clinical Characteristics of Patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDemographic and clinical data are summarised in Table\u0026nbsp;1. Age ranged 13–78\u0026nbsp;years (PTB) and 16–85\u0026nbsp;years (non‑PTB). Seven PTB patients had concurrent extrapulmonary tuberculosis (tuberculous meningitis\u0026nbsp;=\u0026nbsp;2; peritonitis\u0026nbsp;=\u0026nbsp;3; lumbar spinal TB\u0026nbsp;=\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Detection of Ag85B in Bronchoalveolar Lavage Fluid\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBALF‑Ag85B concentrations were significantly higher in BC‑PTB and CD‑PTB groups than in non‑PTB controls (P\u0026nbsp;\u0026lt;\u0026nbsp;0.05) with no significant difference between BC‑PTB and CD‑PTB (P\u0026nbsp;=\u0026nbsp;0.055) (Figure\u0026nbsp;1). ROC analysis (BC‑PTB vs. non‑PTB) yielded an AUC of\u0026nbsp;0.783 (95\u0026nbsp;%\u0026nbsp;CI 0.705–0.860). The maximal Youden index (0.470) corresponded to 4.22\u0026nbsp;ng/mL, adopted as the diagnostic cut‑off (Figure\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Diagnostic Performance of Antigen Ag85B for Pulmonary Tuberculosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe assessed Ag85B ELISA in 104 PTB patients (BC‑PTB\u0026nbsp;+\u0026nbsp;CD‑PTB) versus 72 non‑PTB controls, using a 4.22\u0026nbsp;ng/mL cutoff. Of the PTB group, 67/104 (64.4\u0026nbsp;%) were Ag85B‑positive; among controls, 15/72 (20.8\u0026nbsp;%) were false‑positive. This yielded a sensitivity of 64.42\u0026nbsp;% and specificity of 79.17\u0026nbsp;% for Ag85B.\u003c/p\u003e\n\u003cp\u003eBy comparison:\u003c/p\u003e\n\u003cp\u003e· \u003cstrong\u003e\u003cem\u003eAFB smear microscopy\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e:\u003c/em\u003e sensitivity 26.92 %, specificity 100 %.\u003c/p\u003e\n\u003cp\u003e· \u003cstrong\u003e\u003cem\u003eGeneXpert\u0026nbsp;MTB/RIF\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e:\u003c/em\u003e sensitivity 56.73 %, specificity 100 % (Table 2).\u003c/p\u003e\n\u003cp\u003eAg85B sensitivity exceeded that of AFB smear by 37.5\u0026nbsp;% (P\u0026nbsp;\u0026lt;\u0026nbsp;0.001) and was 7.7\u0026nbsp;% higher than GeneXpert, though this difference was not statistically significant (P\u0026nbsp;=\u0026nbsp;0.256). Conversely, Ag85B specificity was significantly lower than both AFB smear and GeneXpert (P\u0026nbsp;\u0026lt;\u0026nbsp;0.001) (Figure\u0026nbsp;3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Comparison of Ag85B Sensitivity Between Bacteriologically Confirmed and Clinically Diagnosed PTB Groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the BC‑PTB cohort (n = 59), 40 specimens tested positive for Ag85B, yielding a sensitivity of 67.80 % (95 % CI: 54.36 %–79.38 %). In the CD‑PTB cohort (n = 45), 27 specimens were positive, corresponding to a sensitivity of 60.00 % (95 % CI: 44.33 %–74.30 %) (Table 3). A χ² test showed no significant difference between the two groups (χ² = 0.68, P = 0.441).\u003c/p\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThe Ag85 complex is the most abundant secreted protein family produced by MTB, accounting for approximately 45 % of culture filtrate proteins, and elicits strong humoral and cellular immune responses. Early studies employing indirect ELISA demonstrated that detection of the Ag85 complex in sputum culture filtrates enables rapid tuberculosis diagnosis [18]. Its diagnostic utility extends to extrapulmonary forms: in ascitic fluid, Ag85 achieved 72.22 % sensitivity and 90.48 % specificity for tuberculous peritonitis [19], while in cerebrospinal fluid it reached 92.5 % sensitivity and 97.5 % specificity for tuberculous meningitis [20]. Elevated serum Ag85 levels have also distinguished tuberculosis patients from controls with 82 % sensitivity and 86 % specificity [21]. Collectively, these findings validate the Ag85 complex as a promising biomarker for tuberculosis.\u003c/p\u003e\n\u003cp\u003eWithin this complex, Ag85B constitutes ~22 % of the secreted proteins and is the principal antigen recognized by host macrophages [22]. Indirect ELISA assays for Ag85B in sputum report PTB sensitivities of 73.6 %–77 % and specificities of 86.3 %–95.2 % [14, 23]. For extrapulmonary specimens, sensitivities vary by fluid type—62.5 % in pleural effusion, 100 % in joint fluid and lymph node aspirates, and lower in others—while specificity remains high (~96 %) [14]. Immunohistochemical studies detect Ag85B in lung tissue with sensitivities ranging from 50.5 % to 92.7 % and specificities of 83.3 %–100 % [7, 9–13]. Our investigation is the first to quantify Ag85B in BALF, yielding 64.4 % sensitivity and 79.2 % specificity for PTB. The variation in diagnostic performance compared to prior reports likely reflects differences in specimen matrix, antigen concentration, assay format, and control selection.\u003c/p\u003e\n\u003cp\u003eWhen benchmarked against conventional diagnostics, Ag85B ELISA substantially outperformed AFB smear microscopy (26.9 % sensitivity) and approximated the sensitivity of GeneXpert MTB/RIF (56.7 %), while incurring a specificity trade-off (79.2 % vs. 100 %) (Table 2, Figure 3). Remarkably, Ag85B detected 60 % of clinically diagnosed PTB cases that were smear- and GeneXpert-negative, underscoring its value in paucibacillary or difficult-to-sample presentations.\u003c/p\u003e\n\u003cp\u003eThis enhanced detectability may derive from the secretion dynamics of Ag85B. Secreted via classical pathways, extracellular vesicles, or exosome-mediated transport [24–26], Ag85B can disseminate beyond localized bacterial foci. Immunohistochemical analyses have observed diffuse Ag85B distribution around bacillary clusters, contrasting with the more focal pattern of acid-fast bacilli [10]. Previous studies have corroborated this finding: Immunohistochemical comparison with acid-fast staining confirms that Ag85B immunoreactivity extends well beyond visible bacilli, forming a more diffuse halo around bacterial foci and yielding higher sensitivity for antigen detection in lung tissue [7,10–12]. Likewise, in murine models, Ag85B accumulates in lung tissue proportional to bacillary load but declines sharply during late infection stages, reflecting regulated secretion kinetics [24]. These properties may explain the similar Ag85B sensitivities in bacteriologically confirmed and clinically diagnosed PTB cohorts, despite variable bacillary burdens.\u003c/p\u003e\n\u003cp\u003eAlthough GeneXpert MTB/RIF remains the molecular gold standard for rapid, specific tuberculosis diagnosis, its cost and infrastructural demands limit deployment in resource-constrained settings. In contrast, ELISA for Ag85B is inexpensive, rapid, and requires minimal laboratory infrastructure and technical expertise, making it suitable for peripheral health facilities.\u003c/p\u003e\n\u003cp\u003eDespite sputum’s role as the primary specimen for PTB diagnosis, BALF offers superior sensitivity for lower respiratory infections, albeit requiring bronchoscopy. Our study introduces BALF-based Ag85B ELISA as a supplementary diagnostic modality, particularly beneficial for bacteriologically negative patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations\u003c/strong\u003e include the absence of healthy BALF controls due to ethical constraints, which may affect specificity estimates, and a single-center design with a modest sample size, warranting multicenter validation and assessment of Ag85B kinetics during treatment.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eELISA‑based detection of Ag85B in BALF is a feasible, sensitive and rapid adjunctive test for PTB, particularly valuable when smear and GeneXpert results are negative. Larger studies are required to refine its diagnostic thresholds and evaluate its role in treatment monitoring.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGu Guihua and Fang Shiyu: Conceptualization, Study design, Data acquisition, Formal analysis, and Writing\u0026nbsp;–\u0026nbsp;original draft; Zhou Xinchun: Visualization, Methodology (preparation of figures and tables); Jiang Jinxiong and Lin Weiwei: Investigation (data collection), Validation (statistical analysis); Sun Jie: Writing\u0026nbsp;–\u0026nbsp;review \u0026amp; editing (critical revision for intellectual content); Liu Fengjun: Supervision (ensuring methodological rigor), Project administration (quality control and integrity verification).All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of the Affiliated Hospital of North Sichuan Medical College (Approval No. 2023ER224-1). Written informed consent was obtained from all participants. For participants under the age of 18, consent was provided by their legal guardians. All data were anonymized to protect participant confidentiality.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors greatly thank Yidie Qin for his valuable help.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCarranza C, Pedraza-Sanchez S, de Oyarzabal-Mendez E, Torres M. （2020） Diagnosis for Latent Tuberculosis Infection: New Alternatives. \u003cem\u003eFront Immunol\u003c/em\u003e. 11:2006. Published 2020 Sep 10. doi:10.3389/fimmu.2020.02006\u003c/li\u003e\n\u003cli\u003eConsolidated guidance on tuberculosis data generation and use. Module 1. Tuberculosis surveillance. Geneva: World Health Organization; 2024. Licence: CC BY-NC-SA 3.0 IGO. https://www.who.int/publications/i/item/9789240075290\u003c/li\u003e\n\u003cli\u003eRageade F, Picot N, Blanc-Michaud A, et al. （2014） Performance of solid and liquid culture media for the detection of Mycobacterium tuberculosis in clinical materials: meta-analysis of recent studies. \u003cem\u003eEur J Clin Microbiol Infect Dis\u003c/em\u003e. 33(6):867-870. doi:10.1007/s10096-014-2105-z\u003c/li\u003e\n\u003cli\u003eGarg A, Agarwal L, Mathur R. （2022） Role of GeneXpert or CBNAAT in diagnosing tuberculosis: Present scenario. \u003cem\u003eMed J DY Patil Vidyapeeth\u003c/em\u003e. 15:14-9. doi: 10.4103/mjdrdypu.mjdrdypu_182_20\u003c/li\u003e\n\u003cli\u003eGlobal tuberculosis report 2024. Geneva: World Health Organization; 2024. Licence: CC BY-NC-SA 3.0 IGO. https://www.who.int/publications/i/item/9789240101531\u003c/li\u003e\n\u003cli\u003eWiker HG, Harboe M. （1992） The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. \u003cem\u003eMicrobiol Rev\u003c/em\u003e. 56(4):648-661. doi:10.1128/mr.56.4.648-661.1992\u003c/li\u003e\n\u003cli\u003eSun J, Zhou X, Yu J, Fang S, Duan S, Liu F. （2024） Diagnostic value of tuberculosis-Specific antigens Ag85B, ESAT-6 and CFP10 in pulmonary tuberculosis. \u003cem\u003eJ Clin Tuberc Other Mycobact Dis\u003c/em\u003e. 37:100486. Published 2024 Oct 19. doi:10.1016/j.jctube.2024.100486\u003c/li\u003e\n\u003cli\u003eMan CJ, Dong XY, Wang X, et al. （2014） Application of Mycobacterium tuberculosis secretory antigens Ag85 and ESAT-6 in the diagnosis of renal tuberculosis infection. \u003cem\u003eChinese Journal of Nosocomiology\u003c/em\u003e, 24(18): 4660-4662. doi: 10.11816/cn.ni.2014-134242\u003c/li\u003e\n\u003cli\u003eChe N, QU Y, Zhang C, et al. （2014） Expression characteristics of Mycobacterium tuberculosis Ag85B protein and its diagnostic value in pathology. \u003cem\u003eChinese Journal of Pathology,\u003c/em\u003e 43(9): 600-603. doi:10.3760/cm8.j.issn.0529-5807.2014.09.006\u003c/li\u003e\n\u003cli\u003eChe N, Qu Y, Zhang C, Zhang L, Zhang H. （2016） Double staining of bacilli and antigen Ag85B improves the accuracy of the pathological diagnosis of pulmonary tuberculosis. \u003cem\u003eJ Clin Pathol\u003c/em\u003e. 69(7):600-606. doi:10.1136/jclinpath-2015-203244\u003c/li\u003e\n\u003cli\u003eDuan, S. Q. (2024). Significance of Immunohistochemical Detection of Mycobacterium tuberculosis Secreted Antigens Ag85B, ESAT-6, and CFP10 in the Diagnosis of Pulmonary Tuberculosis [Master\u0026apos;s thesis, North Sichuan Medical College]. CNKI. (in Chinese) Retrieved from https://kns.cnki.net/kcms2/article/abstract?v=-Y4qPNLDuvYoYW9wNeBTiUp8JUkWCt8jrA_D-1Wz1GvtYE-IBykTiOwpWPf7nWFSwHG761CdLtUg5Dw91YNw2NtnJgy4_rB4pATV9kO98NAHieZUAm4ju1-yv1sSWrAXzKfn3V4AgXHda7YOhtsgMBHgZ2fdyoaiz5MQl26ONvcUp3BMA4Xo5eGeYERC46p9\u0026amp;uniplatform=NZKPT\u0026amp;language=CHS\u003c/li\u003e\n\u003cli\u003eFang SY, Duan SQ, Chen XQ, et al. （2023） Immunohistochemical staining for detection of Ag85B in pulmonary tuberculosis tissues. \u003cem\u003eJournal of North Sichuan Medical College\u003c/em\u003e. 38(5): 1165-1168. doi: 10. 3969 / j. issn. 1005⁃3697. 2023. 09. 003\u003c/li\u003e\n\u003cli\u003eDong Y, Zhou L, Zhang C, et al. （2023） Detection of antigen Ag85B expression is useful for the diagnosis of tuberculosis, especially for those with an antituberculosis treatment history. \u003cem\u003eAm J Clin Pathol\u003c/em\u003e. 160(1):62-71. doi:10.1093/ajcp/aqad012\u003c/li\u003e\n\u003cli\u003eSingh N, Sreenivas V, Gupta KB, et al. （2015） Diagnosis of pulmonary and extrapulmonary tuberculosis based on detection of mycobacterial antigen 85B by immuno-PCR. \u003cem\u003eDiagn Microbiol Infect Dis\u003c/em\u003e. 83(4):359-364. doi:10.1016/j.diagmicrobio.2015.08.015 \u003c/li\u003e\n\u003cli\u003eLiu X, Hou XF, Gao L, et al. （2018） Indicators for prediction of Mycobacterium tuberculosis positivity detected with bronchoalveolar lavage fluid. \u003cem\u003eInfect Dis Poverty\u003c/em\u003e. 7(1):22. Published 2018 Mar 24. doi:10.1186/s40249-018-0403-x\u003c/li\u003e\n\u003cli\u003eNational Health Commission of China. (2017). \u003cem\u003eWS 288\u0026mdash;2017: Diagnostic criteria for pulmonary tuberculosis\u003c/em\u003e. Beijing: Standards Press of China. (in Chinese)\u003c/li\u003e\n\u003cli\u003eReport of the European Society of Pneumology Task Group. （1989） Technical recommendations and guidelines for bronchoalveolar lavage (BAL). \u003cem\u003eEur Respir J\u003c/em\u003e. 2(6):561-585. PMID: 2663535.\u003c/li\u003e\n\u003cli\u003ePhunpae P, Chanwong S, Tayapiwatana C, Apiratmateekul N, Makeudom A, Kasinrerk W. （2014）Rapid diagnosis of tuberculosis by identification of Antigen 85 in mycobacterial culture system. \u003cem\u003eDiagn Microbiol Infect Dis\u003c/em\u003e. 78(3):242-248. doi:10.1016/j.diagmicrobio.2013.11.028 \u003c/li\u003e\n\u003cli\u003eKashyap RS, Saha SM, Nagdev KJ, et al. （2010） Diagnostic markers for tuberculosis ascites: a preliminary study. \u003cem\u003eBiomark Insights\u003c/em\u003e. 5:87-94. Published 2010 Aug 25. doi:10.4137/bmi.s5196\u003c/li\u003e\n\u003cli\u003eKashyap RS, Dobos KM, Belisle JT, et al. （2005） Demonstration of components of antigen 85 complex in cerebrospinal fluid of tuberculous meningitis patients. \u003cem\u003eClin Diagn Lab Immunol\u003c/em\u003e. 12(6):752-758. doi:10.1128/CDLI.12.6.752-758.2005\u003c/li\u003e\n\u003cli\u003eKashyap RS, Rajan AN, Ramteke SS, et al. （2007） Diagnosis of tuberculosis in an Indian population by an indirect ELISA protocol based on detection of Antigen 85 complex: a prospective cohort study. \u003cem\u003eBMC Infect Dis\u003c/em\u003e. 7:74. Published 2007 Jul 10. doi:10.1186/1471-2334-7-74\u003c/li\u003e\n\u003cli\u003eXu Z, Hu T, Xia A, et al. （2017） Generation of Monoclonal Antibodies against Ag85A Antigen of \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e and Application in a Competitive ELISA for Serodiagnosis of Bovine Tuberculosis. \u003cem\u003eFront Vet Sci\u003c/em\u003e. 4:107. Published 2017 Jun 30. doi:10.3389/fvets.2017.00107\u003c/li\u003e\n\u003cli\u003eMehta PK, Singh N, Dharra R, et al. （2016） Diagnosis of tuberculosis based on the detection of a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor by immuno-PCR. \u003cem\u003eJ Microbiol Methods\u003c/em\u003e. 127:24-27. doi:10.1016/j.mimet.2016.05.003\u003c/li\u003e\n\u003cli\u003eErnst JD, Cornelius A, Bolz M. （2019） Dynamics of Mycobacterium tuberculosis Ag85B Revealed by a Sensitive Enzyme-Linked Immunosorbent Assay. \u003cem\u003emBio\u003c/em\u003e. 10(2):e00611-19. Published 2019 Apr 23. doi:10.1128/mBio.00611-19\u003c/li\u003e\n\u003cli\u003eGiri PK, Kruh NA, Dobos KM, Schorey JS. （2010） Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis-infected and culture filtrate protein-treated macrophages. \u003cem\u003eProteomics\u003c/em\u003e. 10(17):3190-3202. doi:10.1002/pmic.200900840\u003c/li\u003e\n\u003cli\u003eSrivastava S, Grace PS, Ernst JD. （2016） Antigen Export Reduces Antigen Presentation and Limits T Cell Control of M. tuberculosis. \u003cem\u003eCell Host Microbe\u003c/em\u003e. 19(1):44-54. doi:10.1016/j.chom.2015.12.003\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e: Demographic and clinical characteristics of patients.\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003ePTB group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003eNon-PTB group\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003en=104(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003en=72(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eAge（Median, year）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e58.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eGender\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e75(72.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e48(66.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e29(27.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e24(33.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eSymptoms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003ecough\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e68(65.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e54(75.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eFever\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e14(13.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e23(31.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eWeight loss\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e19(18.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e11(15.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eNight sweat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e27(25.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e5(6.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eNo symptoms\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e18(17.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e12(16.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eComorbidities\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003ediabetes mellitus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e12(11.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e9(12.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eHypertension\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e2(1.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e9(12.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 36px;\"\u003e\n \u003cp\u003eextrapulmonary tuberculosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 31px;\"\u003e\n \u003cp\u003e7(6.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 32px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e: The number of cases is expressed as frequency and percentage (n(%)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Diagnostic Value of Antigen Ag85B, Acid-Fast Bacilli (AFB) Staining, and GeneXpert MTB/RIF for Active Pulmonary Tuberculosis (n=176)\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePTB\u003c/p\u003e\n \u003cp\u003e(n=104)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNon-PTB\u003c/p\u003e\n \u003cp\u003e(n=72)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSensitivity\u003c/p\u003e\n \u003cp\u003e（%）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSpecificity\u003c/p\u003e\n \u003cp\u003e（%）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePPV\u003c/p\u003e\n \u003cp\u003e（%）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNPV\u003c/p\u003e\n \u003cp\u003e（%）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eAFB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e26.92\u003c/p\u003e\n \u003cp\u003e(18.69~36.51)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e100.00\u003c/p\u003e\n \u003cp\u003e(95.01~100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e100.00\u003c/p\u003e\n \u003cp\u003e(87.66~100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"bottom\"\u003e\n \u003cp\u003e48.65\u003c/p\u003e\n \u003cp\u003e(40.36~56.99)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eGeneXpert MTB/RIF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e56.73\u003c/p\u003e\n \u003cp\u003e(46.65~66.41)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e100.00\u003c/p\u003e\n \u003cp\u003e(95.01~100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e100.00\u003c/p\u003e\n \u003cp\u003e(93.94~100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e61.54\u003c/p\u003e\n \u003cp\u003e(52.09~70.38)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eAg85B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e64.42\u003c/p\u003e\n \u003cp\u003e(54.43~73.57)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e79.17\u003c/p\u003e\n \u003cp\u003e(67.98~87.84)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e81.71\u003c/p\u003e\n \u003cp\u003e(71.63~89.38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e60.64\u003c/p\u003e\n \u003cp\u003e(50.02~70.56)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e: PPV: Positive Predictive Value; NPV: Negative Predictive Value. Sensitivity, specificity, PPV, and NPV are expressed as percentages with 95% confidence intervals (95% CI).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Sensitivity of Ag85B in the BC-PTB and CD-PTB Groups\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 252px;\"\u003e\n \u003cp\u003eSensitivity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eBC-PTB (n=59)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 252px;\"\u003e\n \u003cp\u003e67.80% (95% CI: 54.36%-79.38%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003eCD-PTB (n=45)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 252px;\"\u003e\n \u003cp\u003e60% (95% CI: 44.33%-74.30%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026chi;\u0026sup2;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 252px;\"\u003e\n \u003cp\u003e0.677\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 252px;\"\u003e\n \u003cp\u003e0.441\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pulmonary tuberculosis, Tuberculosis antigen, Enzyme-linked immunosorbent assay, Ag85B, AFB smear microscopy, GeneXpert MTB/RIF","lastPublishedDoi":"10.21203/rs.3.rs-6617609/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6617609/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e To assess the diagnostic performance of the Mycobacterium tuberculosis (MTB)‑secreted antigen Ag85B in bronchoalveolar lavage fluid (BALF) for pulmonary tuberculosis (PTB) and to explore its utility as a supplemental etiological test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe prospectively enrolled 104 PTB patients who underwent fibre‑optic bronchoscopy at the Affiliated Hospital of North Sichuan Medical College from May 2021 to July 2023, together with 72 non‑PTB controls who had pulmonary infections of non‑tuberculous origin. Ag85B concentrations in BALF were measured by enzyme‑linked immunosorbent assay (ELISA). Receiver‑operating‑characteristic (ROC) analysis established the optimal diagnostic cut‑off (maximum Youden index). Diagnostic performance indices were compared with acid‑fast bacillus (AFB) smear microscopy and the GeneXpert MTB/RIF assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eBALF‑Ag85B levels were significantly higher in PTB patients than in non‑PTB controls (P \u0026lt; 0.001) and did not differ between bacteriologically confirmed (BC‑PTB) and clinically diagnosed (CD‑PTB) cases (P \u0026gt; 0.05). Sensitivities were 26.92 % for AFB smear, 56.73 % for GeneXpert MTB/RIF, and 64.42 % for Ag85B ELISA; the latter was significantly more sensitive than AFB smear (P \u0026lt; 0.001) and similar to GeneXpert MTB/RIF (P = 0.256). Specificities were 100 % for AFB smear and GeneXpert MTB/RIF versus 79.17 % for Ag85B ELISA (P \u0026lt; 0.001). Ag85B sensitivity was comparable between BC‑PTB (67.80 %) and CD‑PTB (60 %) groups (P = 0.411).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eELISA detection of Ag85B in BALF provides a useful adjunct for PTB diagnosis, particularly in bacteriologically negative cases.\u003c/p\u003e","manuscriptTitle":"Ag85B Antigen in Bronchoalveolar Lavage Fluid: A Promising Biomarker for Tuberculosis Diagnosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-10 05:43:37","doi":"10.21203/rs.3.rs-6617609/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a81a4aa4-eba1-4af3-ba0d-4a9889ff3584","owner":[],"postedDate":"June 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-09T15:38:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-10 05:43:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6617609","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6617609","identity":"rs-6617609","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}