Abstract
BackgroundMetagenomic sequencing of respiratory microbial communities for pathogen identification in pneumonia may help overcome the limitations of culture-based methods. We examined the feasibility and clinical validity of rapid-turnaround metagenomics with Nanopore™ sequencing of clinical respiratory specimens.MethodsWe 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 aspirates and applied a microbial DNA enrichment method prior to metagenomic sequencing with the Oxford Nanopore MinION device. For reference, we compared Nanopore results against clinical microbiologic cultures and bacterial 16S rRNA gene sequencing.ResultsHuman DNA depletion enabled in depth sequencing of microbial communities. In culture-positive cases, Nanopore revealed communities with high abundance of the bacterial or fungal species isolated by cultures. In four cases with resistant clinical isolates, Nanopore detected antibiotic resistance genes corresponding to the phenotypic resistance in antibiograms. In culture-negative pneumonia, Nanopore revealed probable bacterial pathogens in 1/5 cases and Candida colonization in 3/5 cases. In controls, Nanopore showed high abundance of oral bacteria in 5/8 subjects, and identified colonizing respiratory pathogens in other subjects. Nanopore and 16S sequencing showed excellent concordance for the most abundant bacterial taxa.ConclusionsWe 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 obtained from sequencing in culture-negative samples. Prospective studies with real-time metagenomics are warranted to examine the impact on antimicrobial decision-making and clinical outcomes.
Highlights
Metagenomic sequencing of respiratory microbial communities for pathogen identification in pneumonia may help overcome the limitations of culture-based methods
With recent technical improvements to overcome the high amounts of contaminating human DNA in clinical respiratory samples [16], Nanopore metagenomics may allow for the development of a novel diagnostic approach for pneumonia
Microbial DNA sequencing approaches We focused our sequencing approach on DNA-based organisms and aimed to perform agnostic profiling for microbes present in the Endotracheal aspirate (ETA) obtained from the patients in the intensive care unit (ICU)
Summary
Metagenomic sequencing of respiratory microbial communities for pathogen identification in pneumonia may help overcome the limitations of culture-based methods. Cultureindependent methods using next-generation sequencing of microbial communities may help overcome the limitations of current diagnostic testing [5,6,7]. Standard 16S sequencing is not clinically applicable due to limited resolution (providing only genus-level bacterial identification) and lengthy sample processing, library preparation and data acquisition timelines [11]. With recent technical improvements to overcome the high amounts of contaminating human DNA in clinical respiratory samples [16], Nanopore metagenomics may allow for the development of a novel diagnostic approach for pneumonia
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