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.

scholarly journals Diagnosis and Genotyping of Coxiella burnetii Endocarditis in a Patient with Prosthetic Pulmonary Valve Replacement Using Next-Generation Sequencing of Plasma Microbial Cell-Free DNA

2019 ◽  
Vol 6 (6) ◽  
Author(s):  
Maiko Kondo ◽  
Sudeb C Dalai ◽  
Shivkumar Venkatasubrahmanyam ◽  
Nell Eisenberg ◽  
Brian D Robinson ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Valve Replacement ◽  
Coxiella Burnetii ◽  
Pulmonary Valve ◽  
Microbial Cell ◽  
Next Generation ◽  
Cell Free Dna ◽  
Free Dna ◽  
Culture Negative ◽  
Generation Sequencing

Abstract Determining the causative etiology of culture-negative endocarditis can be challenging. We performed next-generation sequencing of plasma microbial cell-free DNA to facilitate rapid diagnosis and genotyping of Coxiella burnetii in a patient with culture-negative endocarditis of a prosthetic pulmonary valve, enabling early targeted treatment prior to valve replacement surgery.

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 1097. Microbial Cell Free DNA Sequencing for Prediction of Culture-Negative Infection Events in Children with Cancer

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S578-S578
Author(s):  
Kathryn Goggin ◽  
Amanda griffen ◽  
Christina Kohler ◽  
Kim J Allison ◽  
Yuki Inaba ◽  
...  
Keyword(s):  
Febrile Neutropenia ◽  
Dna Sequencing ◽  
Bacterial Pathogen ◽  
Microbial Cell ◽  
Research Support ◽  
Convenience Sample ◽  
Cell Free Dna ◽  
Free Dna ◽  
Wide Range ◽  
Culture Negative

Abstract Background Culture-independent diagnostics may help diagnose or predict infection; microbial cell free DNA sequencing (mcfDNA-seq), can detect a wide range of pathogens directly from plasma. Immunocompromised children who develop febrile neutropenia (FN) without documented bloodstream infection (BSI) may have undiagnosed bacterial infection, but identification of this is difficult, and the proportion of such episodes is unknown, as is the relative contribution of non-bacterial etiologies. We analyzed mcfDNA-seq results in a convenience sample of FN cases without known etiology. Methods Participants were &lt; 25 years of age and undergoing treatment for cancer. Remnant plasma was prospectively obtained and stored. Samples from Days 0 and -1 underwent mcfDNA-seq by Karius Inc., reported in molecules per microliter (MPM) of plasma. Samples from participants without impending or recent fever or infection were also tested. Results There were 8 episodes in 7 patients; 4 (50%) had a common bacterial pathogen identified by mcfDNA-seq on Day 0 (Table 1). In 2 (50%) of these cases, the same organism was also identified on Day -1, at a lower concentration. One fungal pathogen was identified prior to and at onset of FN. A common bacterial pathogen was identified in 3/64 (5%) control samples from the population. Culture-negative sepsis was the final diagnosis in one episode; Streptococcus mitis, an important cause of neutropenic sepsis, was found in Day 0 and Day -1 samples. In an episode where E. coli was identified by mcfDNA-seq, FN recurred after antibiotic discontinuation. Table 1. Quantitative mcfDNA-seq Results for Prediction & Diagnosis of Febrile Neutropenia Episodes Conclusion In this sample of culture-negative FN episodes in pediatric patients leukemia, mcfDNA-seq identified a bacterial pathogen in 50% of cases. The same organism was identifiable on the day prior to FN in 50% of cases, suggesting that predictive testing might be feasible. Disclosures Asim A. Ahmed, MD, Karius (Employee) Desiree D. Hollemon, MSN, MPH, Karius inc (Employee) Charles Gawad, MD PhD, Karius inc (Grant/Research Support) Joshua Wolf, MBBS, PhD, FRACP, Karius inc (Grant/Research Support)

scholarly journals 711. Rapid, non-invasive detection and monitoring of Bartonella quintana endocarditis by plasma-based next-generation sequencing of microbial cell-free DNA

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S407-S407
Author(s):  
Dipesh Solanky ◽  
Asim A Ahmed ◽  
Joshua Fierer ◽  
Sanjay Mehta
Keyword(s):  
Next Generation Sequencing ◽  
Aortic Valve ◽  
Valve Replacement ◽  
Microbial Cell ◽  
Next Generation ◽  
Cell Free Dna ◽  
Bartonella Quintana ◽  
Free Dna ◽  
Culture Negative ◽  
Generation Sequencing

Abstract Background There are up to 50,000 new cases of infective endocarditis each year in the United States, of which approximately 20% are culture negative endocarditis (CNE). In-hospital mortality remains high at 20 to 30%. Despite advances in diagnostic testing, determining the timing of surgery and duration of treatment in CNE are significant challenges for clinicians. Plasma next-generation sequencing (NGS) for circulating microbial cell-free DNA (mcfDNA) has shown utility in diagnosing and monitoring the response to treatment in endocarditis. Methods Serial blood samples were obtained prior to and after aortic valve replacement in a patient with culture negative endocarditis. Microbial cfDNA was extracted from plasma and NGS was performed by Karius, Inc. (Redwood City, California). Human sequences were removed and remaining sequences were aligned to a curated database of over 1,400 pathogens. Organisms present above a predefined statistical significance threshold were reported and quantified in DNA molecules per microliter (MPM). Chart review was performed for clinical correlation. Results A 53-year old man with history of homelessness, well-controlled HIV infection and a bioprosthetic aortic valve presented with symptomatic severe aortic stenosis and elevated inflammatory markers 3 years following valve surgery. Transesophageal echocardiography showed a paravalvular leak. Bartonella quintana was detected by Karius NGS (in parallel Bartonella henselae serologies were positive). After 4 weeks of parenteral antibiotics, repeat Karius testing demonstrated a 94% (16-fold) decrease in the Bartonella quintana mcfDNA signal to 8813 MPM. He underwent surgical valve replacement; twenty-four hours after removal of the infected valve repeat Karius testing showed a rapid decay of the Bartonella quintana mcfDNA signal to 103 MPM. The patient completed 3 months of oral antibiotics post-operatively, ultimately returning to his former performance status. Conclusion Plasma-based next-generation sequencing assays for circulating microbial cell-free DNA offer a unique means of pathogen detection, assessment of infection burden and monitoring of response to both medical treatment and surgical debridement/definitive source control in a case of Bartonella quintana endocarditis. Disclosures Asim A. Ahmed, MD, Karius (Employee)

scholarly journals 1030. Chasing the Long Tail of Infectious Diseases: Detecting Capnocytophaga canimorsus and Pasteurella multocida Infections with A Plasma-based Microbial Cell-Free DNA Next Generation Sequencing Test

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S605-S606
Author(s):  
Nicholas R Degner ◽  
Ricardo Galvan-Castillo ◽  
Jose Alexander ◽  
Aparna Arun ◽  
Christiaan R de Vries ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pasteurella Multocida ◽  
Graft Infection ◽  
Microbial Cell ◽  
Next Generation ◽  
Capnocytophaga Canimorsus ◽  
Cell Free Dna ◽  
Free Dna ◽  
Culture Negative ◽  
Generation Sequencing

Abstract Background Capnocytophaga canimorsus (Cc) and Pasteurella multocida (Pm) are gram negative bacterial commensal pathogens typically from dogs or cats that can cause severe infection in humans when spread through licks, scratches or bites. The diagnosis of these infections can be limited by: (1) their fastidious nature and difficulty to culture; (2) the nonspecific manifestations of the infections; and (3) the unreliability of dog or cat exposure history. Open-ended microbial cell free DNA (mcfDNA) next-generation sequencing (NGS) offers a potential solution to overcome these limitations. Methods The Karius TestTM (KT) developed and validated in Karius’s CLIA certified/CAP accredited lab in Redwood City, CA detects mcfDNA in plasma. After mcfDNA is extracted and NGS performed, human reads are removed, and remaining sequences are aligned to a curated database of &gt; 1500 organisms. McfDNA from organisms present above a statistical threshold are reported and quantified in molecules/µL (MPM). KT detections of Cc and Pm were reviewed from August 2017 - present; clinical information was obtained with test requisition or consultation upon result reporting. Results KT detected 5 cases of Cc (25,039 MPM +/- 41,062) and 8 cases of Pm (33,264 MPM +/- 69,301) (Table 1). All detections of Cc were in adults (60% male) and included 2 cases of culture-negative endocarditis (one with known liver disease) and one case of sepsis with diffuse rash. Pm detections occurred in 6 adults and 2 children (75% male) and included 2 cases of culture-negative endocarditis, and single cases each of endovascular graft infection, pneumonia, fever of unknown origin, and a cranial dog bite complicated by an abscess. Two patients had immunocompromising conditions including neuroblastoma and aplastic anemia. Conclusion Unbiased, plasma-based mcfDNA NGS provides a rapid, non-invasive test to diagnose diverse clinical infections by Cc and Pm. These cases highlight the potential of the KT to diagnose infections caused by fastidious/unculturable pathogens with non-specific clinical manifestations and broad differential diagnoses. Disclosures Nicholas R. Degner, MD, MPH, MS, Karius Inc. (Employee, Shareholder) Ricardo Galvan-Castillo, MD, Karius Inc. (Employee, Shareholder) Jose Alexander, MD, D(ABMM), FCCM, CIC, SM, MB(ASCP), BCMAS, Karius (Employee) Aparna Arun, MD, Karius (Employee) Ann Macintyre, DO, Karius, Inc. (Employee) Bradley Perkins, MD, Karius, Inc. (Employee) Asim A. Ahmed, MD, Karius, Inc. (Employee) Matthew Smollin, PharmD, Karius, Inc. (Employee)

scholarly journals 395. Rapid, Non-invasive Detection of Infection Using Plasma-based Next-Generation Sequencing for Microbial Cell-free DNA in Individuals Testing Negative for SARS-CoV-2 in a Pandemic Setting

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S266-S266
Author(s):  
William V La Via ◽  
Sudeb Dalai ◽  
Christiaan R de Vries ◽  
Ann Macintyre ◽  
Asim A Ahmed
Keyword(s):  
Next Generation Sequencing ◽  
Severe Pneumonia ◽  
Microbial Cell ◽  
Next Generation ◽  
Independent Contractor ◽  
Cell Free Dna ◽  
Non Invasive ◽  
Free Dna ◽  
Wide Range ◽  
Generation Sequencing

Abstract Background The clinical presentation of patients with severe COVID-19 infection can be protracted and deteriorate to ARDS and multi-organ dysfunction with prolonged fever. As such, there is clinical overlap with many infectious diseases especially those that cause pneumonia. Due to of the prevalence of COVID-19 illness amidst the pandemic, concerns about testing sensitivity and the attendant risk to health care personnel (HCP) delivering care, patients are frequently tested multiple times to ascertain that they are SARS-CoV-2 free. Often, alternative diagnoses are not considered because some diagnostic modalities—such as bronchoalveolar lavage (BAL)—pose an unacceptable risk to the patient and/or HCP. Methods We interrogated plasma for microbial cell-free DNA from 58 patients who were known to be SARS-CoV-2 negative. Clinical information is taken from information submitted with the test requisition or obtained at the time of result reporting from clinical consultations with the ordering provider. In each case, a plasma sample was analyzed with the Karius Test (KT) which is a CLIA certified/CAP-accredited next-generation sequencing (NGS) plasma test designed to detect and quantify circulating microbial cell-free DNA (mcfDNA), which can assist with the diagnosis of deep-seated infections. After mcfDNA is extracted and NGS performed, human reads are removed and remaining sequences are aligned to a curated database of &gt;1400 organisms. Organisms present above a statistical threshold are reported. The time to result is on average 24 hours from sample receipt. Results In a subset of 20 samples, we found a broad range of pathogens. Pneumocystis jirovecii was the most common. These detections were unexpected in the majority of these patients. (see Table) Broad range of Karius detected pathogens (including fastidious bacteria, mycobacteria, fungi and viruses) Conclusion Open-ended, plasma-based NGS for mcfDNA with the KT provides a rapid, non-invasive method to diagnose deep-seated infection like pneumonia. This broad-based test detected a wide range of pathogens – many unsuspected – in patients with severe pneumonia and other invasive infections during the COVID-19 pandemic. These detections highlight the utility of the tool; which allowed better management including de-escalation of SARS-CoV-2 testing and selection of appropriate antibiotic therapy for the unexpected diagnoses. Disclosures William V. La Via, MD, Karius (Employee) Sudeb Dalai, MD, Karius (Employee) Christiaan R. de Vries, MD, PhD, Karius (Consultant, Independent Contractor)Stanford University (Employee) Ann Macintyre, DO, Karius (Employee) Asim A. Ahmed, MD, Karius (Employee)

scholarly journals #75: Microbial Cell-free DNA Sequencing for Prediction of Culture-Negative Infection Events in Children with Cancer

2021 ◽  
Vol 10 (Supplement_1) ◽  
pp. S8-S9
Author(s):  
K P Goggin ◽  
A Griffen ◽  
C Kohler ◽  
K J Allison ◽  
Y Inaba ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Pediatric Patients ◽  
Bacterial Pathogen ◽  
Antifungal Prophylaxis ◽  
Microbial Cell ◽  
Antibacterial Prophylaxis ◽  
Cell Free Dna ◽  
Free Dna ◽  
Culture Negative ◽  
Control Samples

Abstract Background New culture-independent diagnostics are being investigated for diagnosis and prediction of infection. One method is microbial cell-free DNA sequencing (mcfDNA-seq), which can detect a wide range of pathogens directly from plasma. Immunocompromised children who develop febrile neutropenia (FN) without documented bloodstream infection (BSI) may have an undiagnosed bacterial infection, but identification of this subset is difficult. The percentage of episodes of FN caused by bacterial pathogens that did not grow in culture is unknown, as is the relative contribution of other specific etiologies such as viruses, fungi, immunotherapies, and immune reconstitution or graft vs. host disease. The value of mcfDNA-seq in FN patients is also unknown. As part of a larger study evaluating the ability of mcfDNA-seq to predict BSI in a cohort of pediatric patients with relapsed or refractory leukemia, we analyzed mcfDNA-seq results in a sample of cases of FN for which the definitive etiology was unknown. Methods Eligible participants were &lt;25 years of age and undergoing treatment for cancer. Remnant plasma from clinical testing was prospectively obtained and stored. Samples collected on the day of onset of FN (Day 0) and the day prior (Day –1) underwent mcfDNA-seq by Karius Inc. in a CLIA and CAP-accredited laboratory. Pathogen detection results were reported in molecules per microliter (MPM) of plasma. Negative control samples from study participants without impending or recent fever or infection were also obtained. Testing was batched and blinded, so results were not clinically available. Results mcfDNA-seq results were obtained from eight episodes of FN in seven patients. Five episodes occurred in participants awaiting engraftment after HCT and three were receiving chemotherapy only. All participants receiving chemotherapy were receiving antibacterial prophylaxis with vancomycin and ciprofloxacin, antifungal prophylaxis with micafungin or voriconazole, and PJP prophylaxis with TMP-SMX or pentamidine. No HCT recipients were receiving antibacterial prophylaxis, but all received PJP prophylaxis, antifungal prophylaxis or treatment, and antiviral prophylaxis or treatment. Of 8 FN episodes, 4 (50%) had a common bacterial pathogen identified by mcfDNA-seq on Day 0 (Table 1). In 2 (50%) of these cases, the same organism was also identified on Day –1, at a lower concentration. One fungal pathogen was identified prior to and at the onset of FN. A common bacterial pathogen was identified in 3/64 (5%) control samples from the study population. Culture-negative sepsis was the final diagnosis in one episode; in this case, Streptococcus mitis, an important cause of sepsis in neutropenic patients, was identified in both Day 0 and Day –1 samples. In another episode where E. coli was identified, antibiotics were discontinued after 48 hours, but the patient was re-admitted within 24 hours for recurrent FN. Conclusions In this sample of culture-negative FN episodes in pediatric patients with relapsed or refractory leukemia, mcfDNA-seq identified a common bacterial pathogen in 50% of cases. The same organism was identifiable on the day prior to FN in 50% of cases, suggesting that predictive testing might be feasible. More data regarding sensitivity and specificity of mcfDNA-seq to diagnose and predict FN are needed.

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.

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