Severe COVID-19 pneumonia requiring intensive care unit (ICU) support can be complicated by secondary bacterial or fungal infections. The incidence and impact of secondary pneumonias in COVID-19 are not well-defined because clinical distinction from isolated SARS-CoV-2 infection is challenging and diagnostic practices have been highly variable . Early administration of empiric antibiotics limits the sensitivity of subsequent microbiologic studies, whereas standard invasive workup with bronchoscopy is often avoided due to the risks of healthcare personnel exposure to aerosolized SARS-CoV-2 . To overcome such limitations and comprehensively identify secondary pneumonias in COVID-19, we performed microbial cell-free DNA (mcfDNA) metagenomic sequencing (mcfDNA-Seq) in plasma samples in addition to conventional microbiologic workup.
We enrolled 15 critically-ill patients with COVID-19 (confirmed by nasopharyngeal qPCR for SARS-CoV-2) in a prospective ICU cohort study . Following informed consent, we obtained plasma samples for conducting mcfDNA-Seq with the Karius Test (Karius, Inc. Redwood City, CA). We evaluated detection of mcfDNA in the context of clinical diagnoses and prescribed antimicrobial therapies by the treating physicians, and examined for associations with clinical outcomes.
Of 15 patients analyzed (median age 63, 53% females, 73% mechanically-ventilated), six (40%) died within 30 days from enrollment. Samples were obtained at a median (interquartile range-IQR) of 10 (4–12) days from COVID-19 symptoms onset, and each sample contained a median of 837 (111–4638) total mcfDNA molecules per microliter (MPMs) and 2 (1–4) identified organisms. Of the total 92,791 MPMs reported across 15 samples, 90% belonged to typical pathogenic bacteria (e.g. E.coli and K. Pneumoniae), with the remainder MPMs aligned to commensal bacteria (5%, e.g. oral Streptococcus species), fungi (4%, Candida species) and DNA viruses (1%). Compared to survivors, non-survivors had higher total mcfDNA (p = 0.04), higher pathogenic bacteria MPMs (p = 0.02) and a trend for a higher number of identified organisms per sample (p = 0.06). (Fig. 1).
Secondary pneumonia was clinically suspected or diagnosed by the treating physicians in 11/15 (73%) patients (Group A, Fig. 2), with microbiologic confirmation by positive respiratory cultures in 3/11 subjects (27%); these three patients had high plasma mcfDNA MPMs for common bacterial pathogens, such as E.coli and Ps. aeruginosa. Among the remaining eight patients with clinically-suspected infections and empiric antibiotic treatments, high mcfDNA MPMs of probable bacterial pathogens were detected in 2/8 patients (co-infecting Ps. aeruginosa and K. Pneumoniae; Raoultella ornithinolytica, respectively). In the additional six patients, no evidence of co-infecting bacterial pathogens was present, whereas in one patient (subject 7, Fig. 2) there was high signal for Candida tropicalis (2,490 MPMs) concerning for undiagnosed invasive Candidiasis.
We detected respiratory pathogen MPMs (S. aureus, Ps. aeruginosa and K. Pneumoniae) in 3/4 subjects with low suspicion for secondary infection (Group B, Fig. 2). In these patients, no respiratory specimen cultures were obtained, and antibiotics had not been initiated or had been discontinued based on negative blood cultures by the time of research sampling. Notably, two of these individuals experienced sustained vasodilatory shock and died from multiorgan dysfunction attributed to isolated SARS-CoV-2 infection.
McfDNA-Seq in patients with COVID-19 indicates a higher incidence of probable secondary infections than previously recognized. Despite our small sample size, the significant association between mcfDNA and 30-day mortality suggests that COVID-19 severity may be influenced by circulating bacterial fragments, either from secondary pneumonias or from possible translocation of colonizing microbiota along the disrupted alveolar/epithelial surface of lungs injured by COVID-19 . Integration of mcfDNA detection with clinical data demonstrates opportunity for antibiotic stewardship in patients with suspected infection. On the other hand, the signal for undiagnosed and untreated secondary infections should serve as a call for vigilance and thorough diagnostic workup in patients with severe COVID-19 .
Availability of data and materials
All de-identified data are available from the authors upon request.
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We would like to thank the multiple investigators that contributed to our clinical research efforts for COVID-19, including investigators from the Acute Lung Injury Center of Excellence and the Center for Medicine and the Microbiome at the University of Pittsburgh, and the collaborators from Karius, Inc.
This work was supported by Career Development Award Number IK2 BX004886 from the United States Department of Veterans Affairs Biomedical Laboratory R&D (BLRD) Service (W.B.); the National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Numbers K23 HL129987 (G.D.K); P01HL114453 (B.J.M.); University of Pittsburgh Clinical and Translational Science Institute COVID-19 Pilot Grant Program (G.D.K).
Ethics approval and consent to participate
This study was approved by the University of Pittsburgh Institutional Review Board (Protocol STUDY19050099). Written informed consent was provided by all participants or their surrogates in accordance with the Declaration of Helsinki.
Dr. Bryan J. McVerry has been a consultant for Vapotherm, Inc. and receives research funding from Bayer Pharmaceuticals, Inc. Dr. Georgios Kitsios has received research funding from Karius, Inc. The other authors have no conflicts of interest to declare.
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Kitsios, G.D., Bain, W., Al-Yousif, N. et al. Plasma microbial cell-free DNA load is associated with mortality in patients with COVID-19. Respir Res 22, 24 (2021). https://doi.org/10.1186/s12931-021-01623-0