Targeted next-generation sequencing of bronchoalveolar lavage fluid for pathogen detection in connective tissue disease-related pulmonary infections

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ABSTRACT The study aims to assess the clinical value of targeted next-generation sequencing of bronchoalveolar lavage fluid (BALF-tNGS) in pulmonary infection associated with connective tissue diseases (CTDs) and characterize the infection profiles in these patients. CTD patients undergoing bronchoscopy with BALF-tNGS between January 2022 to July 2025 were enrolled. tNGS results were compared with conventional microbiological tests (CMTs). Logistic regression identified risk factors for monomicrobial infection and polymicrobial infection. Among 124 CTD patients enrolled, 45 (36.3%) were male with the mean age of 52.6 ± 15.4 years, and the mean disease duration was 4.1 ± 6.1 years. CMTs detected pathogens in 43 patients, yielding 55 isolates: 23 bacteria, 13 viruses, 12 fungi, and 9 Mycoplasma pneumoniae . tNGS identified pathogens in 105 patients, yielding 168 isolates: 70 bacteria, 48 viruses, 43 fungi, and 7 M. pneumoniae . The sensitivity of tNGS was significantly higher than that of CMTs ( P = 0.02). Treatment regimens were adjusted in 54 patients (49.5%) based on tNGS results. Polymicrobial infection was observed in 44 patients (40.37%), whereas 65 patients (59.63%) had monomicrobial infection. Multivariable analysis identified interstitial lung disease (ILD; odds ratio [OR] 3.90, 95% confidence interval [CI] 1.42–10.72), chronic kidney disease (CKD; OR 6.75, 95% CI 1.09–41.81), and decreased T-cell count (OR 0.30, 95% CI 0.13–0.69) as independent risk factors for polymicrobial infection. BALF-tNGS substantially increases the sensitivity of pathogen detection in CTD-related pulmonary infection and guides pathogen-directed antimicrobial therapy. Polymicrobial infection is common in CTD patients; ILD, CKD, and reduced T-cell counts are independent risk factors for its development. IMPORTANCE The study delineates the infection landscape of connective tissue disease-related pneumonia and demonstrates that targeted next-generation sequencing of bronchoalveolar lavage fluid (BALF-tNGS) doubles the yield of identified pathogens, frequently revealing polymicrobial disease. This molecular insight prompted a change in antimicrobial therapy in nearly half of the cohort. Patients with concurrent interstitial lung disease, renal impairment, or lymphopenia were particularly susceptible to mixed infections. By enabling pathogen-directed treatment, BALF-tNGS reduces unnecessary antibiotic exposure, shortens recovery, and supports early, precision therapy.

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