Plasma microbial cell-free DNA load is associated with mortality in patients with COVID-19

© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Introduction 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 welldefined because clinical distinction from isolated SARSCoV-2 infection is challenging and diagnostic practices have been highly variable [1]. 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 SARSCoV-2 [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.


Introduction
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 welldefined because clinical distinction from isolated SARS-CoV-2 infection is challenging and diagnostic practices have been highly variable [1]. 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 [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.

Methods
We enrolled 15 critically-ill patients with COVID-19 (confirmed by nasopharyngeal qPCR for SARS-CoV-2) in a prospective ICU cohort study [3]. Following informed consent, we obtained plasma samples for conducting mcfDNA-Seq with the Karius Test (Karius, Inc. Redwood City, CA) [4]. 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.
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 Open Access 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.
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.

Discussion
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 [5]. Integration of mcfDNA detection with clinical data demonstrates opportunity for antibiotic stewardship in   Fig. 2 Case-based analysis of 15 critically ill patients with COVID-19 with depicted clinical diagnoses, plasma microbial cell-free DNA metagenomics and survival outcomes. The Y-axis margin indicates two groups of clinical diagnoses: Group A includes 11 patients who received antibiotics for either microbiologically-confirmed (n = 3) or clinically-suspected infections despite negative microbiologic workup (n = 8), whereas Group B includes four patients with low clinical suspicion for secondary infection and no antibiotic therapies at time of sampling. The Y-axis ticks denote each patient sample and the x-height of each stacked bar represents the number of microbial cell-free DNA molecules per plasma microliter (MPMs) by metagenomic sequencing, with different colors for the top 10 microbes by ranked abundance. The "other" category (shown in grey) represents the sum of lower abundance taxa of commensal origin. Five out of 11 subjects of Group A (45%, Subjects 1-5) had high MPM signal for probable respiratory pathogens, whereas in the remaining 6/11 subjects there was no evidence of co-infecting bacterial pathogens. Subject 7 was clinically-diagnosed with culture-negative sepsis and treated with prolonged course of empiric broad-spectrum antibiotics while on extracorporeal membrane oxygenation support for refractory hypoxemic respiratory failure from COVID-19; the high mcfDNA signal for C.tropicalis (2,490 MPMs) is concerning for undiagnosed invasive Candidiasis, corroborated by persistent growth of yeast organisms (not further speciated) from clinical bronchoalveolar lavage samples obtained on days 5, 9 and 14 after the research sample acquisition. Two out of four patients of Group B (subjects 12 and 13) who did not survive and had not received empiric antimicrobials were found to have high mcfDNA signal (> 4000 total MPMs) of probable respiratory pathogens, indicative of undiagnosed (and untreated) secondary infections 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 [6].