Optimisation and clinical validation of a metagenomic third-generation sequencing approach for aetiological diagnosis in bronchoalveolar lavage fluid of patients with pneumonia

作者：Shanshan Zhang, Xi Li, Xia Li, Yingkun Fu, Long Chen, Wei Wang, Qinqing Lin, Hui Lou, Yake Yao, Wenyu Chen, Cheng Zhong, Jian Ye, Yan Yao, Huimin Guo, Yunsong Yu, Hua Zhou · 发表于：EBioMedicine · 年份：2025 · DOI：10.1016/j.ebiom.2025.105752 · 被引用次数：10 · 研究领域：Genomics and Phylogenetic Studies、Bacterial Identification and Susceptibility Testing、Pneumocystis jirovecii pneumonia detection and treatment

BACKGROUND: Metagenomic Third Generation Sequencing (mTGS), based on nanopore technology, has emerged as a promising tool for the rapid diagnosis of pneumonia pathogens. However, this technology currently lacks standardised technical protocols, quality control measures, and comprehensive performance evaluations for the simultaneous detection of bacteria, fungi, and viruses in clinical settings. METHODS: We optimised the mTGS workflow by refining key parameters (cell wall lysis, fragment size selection, host DNA depletion, and sequencing depth) using reference samples and bronchoalveolar lavage fluid (BALF) from eight patients with pneumonia. These optimisations formed the basis for a standardised mTGS protocol. To assess the clinical diagnostic value of the optimised mTGS, a multicentre prospective cohort study involving 313 pneumonia-suspected patients was conducted. Each BALF sample was tested using conventional microbiological testing (CMTs), metagenomic next-generation sequencing (mNGS), pre-optimised mTGS, and optimised mTGS. FINDINGS: The optimised mTGS protocol, based on the refined cell wall lysis, fragment size selection, no host DNA depletion, and 800 MB sequencing depth, achieved a tenfold increase in sensitivity compared with pre-optimised mTGS for detecting the species of Bacillus subtilis, Mycobacterium tuberculosis, Mycobacterium avium, Cryptococcus neoformans, and Human papillomavirus in reference samples. In the prospective cohort, 274 patients with a confirm...