Comparative evaluation of probe-capture and conventional metagenomic sequencing across multiple clinical sample types, with analysis of paired bronchoalveolar lavage fluid and blood samples.

Abstract

Conventional metagenomic next-generation sequencing (mNGS) suffers from host nucleic acid interference and poor performance in low-biomass samples. Probe-capture metagenomic sequencing (PC-mNGS), which enriches microbial targets via hybridization probes, shows superior sensitivity but lacks systematic multi-sample evaluations. This study compared PC-mNGS and mNGS across diverse clinical specimens (bronchoalveolar lavage fluid [BALF], blood, cerebrospinal fluid [CSF]) and assessed the clinical utility of pathogen co-detection in paired BALF-blood samples from sepsis patients. A total of 282 samples (81 BALF, 141 blood, 25 CSF, 35 others) sequenced by both PC-mNGS and mNGS were analyzed. Additionally, 621 paired BALF-blood samples from sepsis patients with pulmonary infections were evaluated. PC-mNGS achieved higher pathogen detection rates (66.67% vs 57.10%, P = 0.000198) than mNGS, particularly in blood (66.67% vs 47.52%, P = 2.5 × 10⁻⁵). PC-mNGS detected more bacteria (19 species exclusive) and fungi (11 species exclusive) than mNGS. Viruses showed comparable detection. BALF and CSF exhibited high overall agreement (OPA: 96.30% and 88%, respectively), while blood had lower concordance (NPA: 54.05%, OPA: 70.92%). A total of 60.55% of BALF-positive samples (PC-mNGS) had co-detected pathogens in blood. Gram-negative bacteria (e.g., Klebsiella pneumoniae) and fungi (e.g., Candida albicans) showed higher blood co-detection rates than viruses. In this study, PC-mNGS detected more pathogens and showed a higher positivity rate than mNGS in blood samples. BALF sequencing data, particularly bacterial reads per million (RPM), may predict bloodstream co-detection, aiding in sepsis management. However, clinical validation and integration with traditional diagnostics are needed to confirm utility. This study highlights PC-mNGS as a promising tool for complex infections but underscores the need for rigorous multi-context validation.IMPORTANCEAccurate and rapid identification of pathogens is critical for effective treatment of severe infectious diseases, such as sepsis. This study demonstrates that probe-capture metagenomic sequencing (PC-mNGS) detected more pathogens in blood samples compared to conventional metagenomic sequencing, especially for bacterial and fungal infections. By analyzing paired lung and blood samples, we show that high pathogen levels in lung fluid may predict bloodstream infection, offering a potential early warning for clinicians. These findings support the use of PC-mNGS as a more sensitive diagnostic tool, which could lead to faster, more targeted therapies and better outcomes for patients with complex infections.