The application of nanopore targeted sequencing for pathogen diagnosis in bronchoalveolar lavage fluid of patients with pneumonia: a prospective multicenter study

To evaluate the value of nanopore targeted sequencing in diagnosing pneumonia pathogens. This large-scale multicentre prospective study performed in 8 hospitals across China from April to October 2022. Hospitalised patients with a diagnosis of pneumonia at admission were included. Complete clinical data were collected, and bronchoalveolar lavage fluid were obtained from each patient. These samples underwent simultaneous testing using conventional microbial testing, metagenomic next-generation sequencing, and nanopore targeted sequencing. A total of 218 patients were included. Among the 168 cases of pulmonary infection, 246 strains of pathogens were confirmed. Nanopore targeted sequencing outperformed conventional microbial testing, identifying more pathogens with a sensitivity increase of 47.9% (77.2% vs. 29.3%). Metagenomic next-generation sequencing had a sensitivity of 82.9%. Total of 70.1% patients had consistent results in both metagenomic next-generation sequencing and nanopore targeted sequencing. Nanopore targeted sequencing exhibited significantly higher sensitivity in detecting Pneumocystis jiroveci, cytomegalovirus, Mycobacterium tuberculosis, Nontuberculous mycobacteria, Streptococcus pneumoniae, and Mycoplasma pneumoniae compared to conventional microbial testing. However, metagenomic next-generation sequencing demonstrated higher sensitivity than nanopore targeted sequencing for Aspergillus (88.5% vs. 53.8%). Regarding the detection of co-infections, nanopore targeted sequencing displayed significantly higher sensitivity than conventional microbial testing (76.7% vs. 28.7%) and was on par with metagenomic next-generation sequencing (76.7% vs. 82.9%). Nanopore targeted sequencing performs equally well as metagenomic next-generation sequencing in bronchoalveolar lavage fluid for pathogen diagnosis in pneumonia, both methods showing higher sensitivity than conventional microbial testing. Nanopore targeted sequencing can be considered a reliable method for diagnosing pathogens in pneumonia.

本研究旨在评估靶向纳米孔测序（nanopore targeted sequencing）在肺炎病原体诊断中的应用价值。本研究为一项大规模多中心前瞻性研究，于2022年4月至10月在中国8家医院开展。研究纳入入院时确诊为肺炎的住院患者，收集所有患者的完整临床资料，并采集每位患者的支气管肺泡灌洗液（bronchoalveolar lavage fluid）。对上述样本同步开展传统微生物检测、宏基因组下一代测序（metagenomic next-generation sequencing）以及靶向纳米孔测序检测。本研究共纳入218例患者，其中168例确诊为肺部感染，共确认246株病原体。靶向纳米孔测序的检测性能优于传统微生物检测，可检出更多病原体，灵敏度提升47.9%（77.2% vs. 29.3%）。宏基因组下一代测序的灵敏度为82.9%。70.1%的患者在宏基因组下一代测序与靶向纳米孔测序中得到一致的检测结果。相较于传统微生物检测，靶向纳米孔测序对耶氏肺孢子菌（Pneumocystis jiroveci）、巨细胞病毒（cytomegalovirus）、结核分枝杆菌（Mycobacterium tuberculosis）、非结核分枝杆菌（Nontuberculous mycobacteria）、肺炎链球菌（Streptococcus pneumoniae）及肺炎支原体（Mycoplasma pneumoniae）的检测灵敏度显著更高。但在曲霉属（Aspergillus）的检测中，宏基因组下一代测序的灵敏度（88.5%）高于靶向纳米孔测序（53.8%）。在混合感染检测方面，靶向纳米孔测序的灵敏度显著高于传统微生物检测（76.7% vs. 28.7%），且与宏基因组下一代测序结果相当（76.7% vs. 82.9%）。在支气管肺泡灌洗液的肺炎病原体诊断中，靶向纳米孔测序的表现与宏基因组下一代测序相当，且两种方法的灵敏度均高于传统微生物检测。综上，靶向纳米孔测序可作为肺炎病原体诊断的可靠手段。