scholarly journals 245. Plasma and Respiratory Specimen Metagenomic Sequencing for the Diagnosis of Severe Pneumonia in Mechanically-Ventilated Patients

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S138-S138
Author(s):  
Georgios Kitsios ◽  
Libing Yang ◽  
Rachel Nettles ◽  
Zia Haris ◽  
Daniel Dunlap ◽  
...  
Keyword(s):  
Severe Pneumonia ◽  
Oral Bacteria ◽  
Uninfected Control ◽  
Metagenomic Sequencing ◽  
Mechanically Ventilated ◽  
Mechanically Ventilated Patients ◽  
Culture Positive ◽  
Culture Negative ◽  
Polymicrobial Infections ◽  
Ventilated Patients

Abstract Background Metagenomic sequencing of respiratory microbial communities may overcome the limitations of culture-based pneumonia diagnostics. Nonetheless, respiratory metagenomics requires high-quality specimens, may miss deep-seated infections and cannot distinguish colonization from infection. Plasma microbial cell-free DNA (mcfDNA) sequencing may offer a noninvasive alternative for culture-independent diagnosis and help refine interpretations of respiratory metagenomics. Methods We obtained concurrent plasma and endo-tracheal aspirate (ETA) samples from 29 mechanically-ventilated patients (15 culture-positive, 11 culture-negative pneumonia, 3 uninfected control patients). We performed plasma mcfDNA sequencing (Karius®Test, Redwood City, CA) and ETA metagenomics (MiNION, Oxford Nanopore Technologies). We compared sequencing results with clinical microbiologic cultures for identified DNA pathogens. Results Uninfected control patients had a negative signal for mcfDNA in plasma and oral bacteria in ETA specimens. In culture-positive pneumonia samples, Karius testing identified a median of 2 pathogens per sample (range 0–10), which were concordant with clinical isolates in 11/15 (73%) cases (figure). In 5/11 (45%) of concordant cases, Karius and MiNION suggested polymicrobial infections with additional pathogens not identified by cultures. In culture-negative cases, Karius detected potential pathogens in 8/11 (73% cases), which matched the species identified in ETA specimens by MiNION. In cases of clinical aspiration, Karius detected more organisms (median 8, range 0–14) per sample mainly consisting of oral origin bacteria compared with cases without history of aspiration (median 1, range 0–6, P = 0.04). Conclusion Metagenomic sequencing in plasma and ETA samples showed good concordance between the blood and lung compartments as well as with culture results in pneumonia patients. Metagenomics revealed potential pathogens missed by cultures in ~75% of culture-negative pneumonias and suggested polymicrobial infections especially in cases with aspiration. Further research is needed to evaluate the clinical utility of real-time metagenomics for pneumonia diagnosis in mechanically ventilated patients. Disclosures All authors: No reported disclosures.

scholarly journals Metagenomic identification of severe pneumonia pathogens in mechanically-ventilated patients: a feasibility and clinical validity study

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Libing Yang ◽  
Ghady Haidar ◽  
Haris Zia ◽  
Rachel Nettles ◽  
Shulin Qin ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Severe Pneumonia ◽  
High Abundance ◽  
Metagenomic Sequencing ◽  
Clinical Validity ◽  
Mechanically Ventilated ◽  
Mechanically Ventilated Patients ◽  
Culture Positive ◽  
Culture Negative ◽  
Ventilated Patients

Abstract Background Metagenomic 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. Methods We 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. Results Human 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. Conclusions We 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.

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

2019 ◽  
Author(s):  
Libing Yang ◽  
Ghady Haidar ◽  
Haris Zia ◽  
Rachel Nettles ◽  
Shulin Qin ◽  
...  
Keyword(s):  
Severe Pneumonia ◽  
High Abundance ◽  
Metagenomic Sequencing ◽  
Clinical Validity ◽  
Mechanically Ventilated ◽  
Mechanically Ventilated Patients ◽  
Culture Positive ◽  
Culture Negative ◽  
Pneumonia Diagnosis ◽  
Ventilated Patients

AbstractBackgroundMetagenomic 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.

Circulating microbial cell-free DNA is associated with inflammatory host-responses in severe pneumonia

Thorax ◽  
2021 ◽  
pp. thoraxjnl-2020-216013
Author(s):  
Haopu Yang ◽  
Ghady Haidar ◽  
Nameer S Al-Yousif ◽  
Haris Zia ◽  
Daniel Kotok ◽  
...  
Keyword(s):  
Large Scale ◽  
Inflammatory Responses ◽  
Severe Pneumonia ◽  
Systemic Circulation ◽  
Microbial Cell ◽  
Metagenomic Sequencing ◽  
Cell Free Dna ◽  
Free Dna ◽  
Culture Positive ◽  
Culture Negative

Host inflammatory responses predict worse outcome in severe pneumonia, yet little is known about what drives dysregulated inflammation. We performed metagenomic sequencing of microbial cell-free DNA (mcfDNA) in 83 mechanically ventilated patients (26 culture-positive, 41 culture-negative pneumonia, 16 uninfected controls). Culture-positive patients had higher levels of mcfDNA than those with culture-negative pneumonia and uninfected controls (p<0.005). Plasma levels of inflammatory biomarkers (fractalkine, procalcitonin, pentraxin-3 and suppression of tumorigenicity-2) were independently associated with mcfDNA levels (adjusted p<0.05) among all patients with pneumonia. Such host–microbe interactions in the systemic circulation of patients with severe pneumonia warrant further large-scale clinical and mechanistic investigations.

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