Background: Infective endocarditis (IE) is a rare and life-threatening condition with considerable mortality rates. Diagnosis is often complicated by negative blood culture results, limiting the accurate identification of causative pathogens. This study aimed to evaluate the effectiveness of metagenomic next-generation sequencing (mNGS) of cardiac valve specimens compared to conventional clinical laboratory methods for identifying pathogens in IE.

Methods: Nineteen patients with suspected IE who were scheduled for surgical valve removal were prospectively enrolled. The metagenomic workflow included bacterial DNA enrichment from resected valves using the Molzym Ultra-Deep Microbiome Prep, sequencing of metagenomic libraries using the Illumina MiSeq platform, and Kraken 2 taxonomic assignments based on read data.

Results: Valve mNGS achieved a sensitivity of 82.4% and a specificity of 100% relative to the final adjudicated pathogen diagnosis. Blood culture, considered the reference standard, exhibited slightly higher sensitivity (88.2%) with comparable specificity (100%). In comparison, valve culture (sensitivity: 29.4%, specificity: 50.0%) and microscopy (sensitivity: 35.3%, specificity: 100%) showed lower diagnostic performance. Delays between blood culture negativization and valve resection impacted mNGS sensitivity, likely due to pathogen clearance. Notably, valves resected within 12 days from blood culture negativization achieved 100% diagnostic accuracy, emphasizing the importance of timing for optimal mNGS results.

Conclusion: This study underscores mNGS as a valuable diagnostic tool for detecting IE pathogens, complementing traditional diagnostic methods. The detection of antibiotic resistance genes and multi-locus sequence typing profiles in some samples further demonstrated its utility.