Application of targeted next-generation sequencing in bronchoalveolar lavage fluid testing for pathogen identification in pulmonary infections

Abstract

Pulmonary infections remain a leading cause of morbidity and mortality worldwide. However, conventional microbiological tests (CMTs) frequently fail to identify the causative pathogens, owing to their limited sensitivity, prolonged turnaround times, and inherent inability to detect fastidious or emerging organisms. In this context, targeted next-generation sequencing (tNGS) has emerged as a promising molecular approach, enabling rapid, sensitive, and comprehensive detection of respiratory pathogens directly from clinical specimens. This study evaluated the clinical application of tNGS in the etiological diagnosis of pulmonary infections. This study aimed to systematically characterize the pathogen spectrum, antimicrobial resistance (AMR) gene distribution, and infection-related immune cytokine dynamics in these patients. A retrospective analysis was conducted on patients with pulmonary infections who underwent bronchoalveolar lavage fluid (BALF) testing using tNGS between August 2023 and July 2024. We performed a clinical evaluation of tNGS findings and compared its diagnostic performance with that of CMTs for pathogen identification. The analysis of tNGS results from 733 clinical specimens revealed a pathogen detection positivity rate of 95.63%, with 113 distinct potential pathogens identified among 701 tNGS-positive cases. Bacterial pathogens showed the highest detection rate (70.90%), and bacterial-viral coinfection emerged as the predominant mixed infection pattern (37.84%). Clinical evaluation demonstrated high concordance (91.58%) between tNGS results and initial diagnoses. Notably, tNGS facilitated the identification of mixed infections in 55 patients, leading to subsequent adjustments to antimicrobial therapy. A significant disparity was observed in the overall pathogen detection rates between tNGS and CMTs (95.63% vs. 47.07%, p < 0.001), with tNGS exhibiting superior sensitivity for the identification of bacterial, fungal, viral, and atypical pathogens. Among the 340 specimens that showed concordant positivity in tNGS and CMT results, 268 cases (78.82%) showed complete or partial diagnostic agreement. Additionally, tNGS detected antimicrobial resistance (AMR) genes in 198 samples, with the 23 S ribosomal RNA A2063G mutation being the most prevalent. Analysis of antimicrobial resistance revealed a strong genotype-phenotype correlation, with complete concordance observed in 69.9% of cases. Among 197 tNGS-positive cases with elevated cytokines, the infection types were characterized by a predominance of mixed over single infections. IL-6 was the most commonly elevated cytokine, and 31.98% of samples exhibited synchronous multi-cytokine elevation. tNGS demonstrated superior diagnostic performance over CMTs for the etiological diagnosis of pulmonary infections, enabling high-resolution detection of pathogens, mixed infections, and clinically relevant antimicrobial resistance genes, thereby contributing to more precise antimicrobial therapy.