Metagenomic identification of severe pneumonia pathogens with rapid Nanopore sequencing in mechanically-ventilated patients

BackgroundMetagenomic sequencing of respiratory microbial communities for etiologic pathogen identification in pneumonia may help overcome the limitations of current culture-based methods. We examined the feasibility and clinical validity of rapid-turnaround metagenomics with Nanopore™ sequencing of respiratory samples for severe pneumonia diagnosis.Methods and FindingsWe conducted a case-control study of mechanically-ventilated patients with pneumonia (nine culture-positive and five culture-negative) and without pneumonia (eight controls). We collected endotracheal aspirate samples (ETAs) and applied a microbial DNA enrichment method prior to performing metagenomic sequencing with the Oxford Nanopore MinION device. We compared Nanopore results against clinical microbiologic cultures and bacterial 16S rRNA gene sequencing. In nine culture-positive cases, Nanopore revealed communities with low alpha diversity and high abundance of the bacterial (n=8) or fungal (n=1) species isolated by clinical cultures. In four culture-positive cases with resistant organisms, Nanopore detected antibiotic resistance genes corresponding to the phenotypic resistance identified by clinical antibiograms. In culture-negative pneumonia, Nanopore revealed probable bacterial pathogens in 1/5 cases and airway colonization by Candida species in 3/5 cases. In controls, Nanopore showed high abundance of oral bacteria in 5/8 subjects, and identified colonizing respiratory pathogens in the three other subjects. Nanopore and 16S sequencing showed excellent concordance for the most abundant bacterial taxa.ConclusionWe demonstrated technical feasibility and proof-of-concept clinical validity of Nanopore metagenomics for severe pneumonia diagnosis, with striking concordance with positive microbiologic cultures and clinically actionable information offered from the sequencing profiles of culture-negative samples. Prospective studies with real-time metagenomics are warranted to examine the impact on antimicrobial decision-making and clinical outcomes.