Noninvasive diagnosis of secondary infections in COVID-19 by sequencing of plasma microbial cell-free DNA.

Abstract

Background: Secondary infection (SI) diagnosis in COVID-19 is challenging, due to overlapping clinical presentations, practical limitations in obtaining samples from the lower respiratory tract (LRT), and low sensitivity of microbiologic cultures. Research Question: Can metagenomic sequencing of plasma microbial cell-free DNA (mcfDNA-Seq) help diagnose SIs complicating COVID-19? Study Design and Methods: We enrolled 42 inpatients with COVID-19 classified as microbiologically-confirmed SI (Micro-SI, n=8), clinically-diagnosed SI (Clinical-SI, n=13, i.e. empiric antimicrobials), or no clinical suspicion for SI (No-Suspected-SI, n=21) at time of enrollment. From baseline and follow-up plasma samples (days 5 and 10 post-enrollment), we quantified mcfDNA for all detected microbes by mcfDNA sequencing and measured nine host-response biomarkers. From LRT samples among intubated subjects, we quantified bacterial burden with 16S rRNA gene quantitative PCR. Results: We performed mcfDNA-Seq in 82 plasma samples. Sequencing was successful in 60/82 (73.2%) samples, which had significantly lower levels of human cfDNA than failed samples (p<0.0001). McfDNA detection was significantly higher in Micro-SI (15/16 [94%]) compared to Clinical-SI samples (8/14 [57%], p=0.03), and unexpectedly common in No-Suspected-SI samples (25/30 [83%]), similar to detection rate in Micro-SI. We detected culture-concordant mcfDNA species in 13/16 Micro-SI samples (81%) and mcfDNA levels tracked with SI outcome (resolution or persistence) under antibiotic therapy. McfDNA levels correlated significantly with LRT bacterial burden (r=0.74, p=0.02) as well as plasma biomarkers of host response (white blood cell count, IL-6, IL-8, and SPD, all p<0.05). Baseline mcfDNA levels were predictive of worse 90-day survival (hazard ratio 1.30 [1.02-1.64] for each log10 mcfDNA, p=0.03). Interpretation: High circulating levels of mcfDNA in a substantial proportion of patients with COVID-19 without clinical suspicion for SI suggest that SIs may often remain undiagnosed. McfDNA-Seq, when clinically available, can offer a non-invasive diagnostic tool for pathogen identification, with prognostic value on host inflammatory response and clinical outcomes.

Competing Interest Statement

Drs. Duttagupta and Ahmed are employed by Karius, Inc. Dr. Kitsios, Dr. Haidar have received research funding from Karius, Inc in 2018-2019. Dr. Mellors is a consultant to Gilead Sciences and owns shares or share options in Co-Crystal Pharmaceuticals, ID Connect, and Abound Bio, all unrelated to the current work. Dr. McVerry has received research funding from Bayer Pharmaceuticals, Inc. and consulting fees from Boehringer Ingelheim, both unrelated to this work. All other authors disclosed no conflict of interest.

Funding Statement

Dr. Kitsios: University of Pittsburgh Clinical and Translational Science Institute, COVID-19 Pilot Award; NIH (K23 HL139987; R03 HL162655). Dr. Bain: Career Development award number IK2 BX004886 from the U.S. Department of Veterans Affairs Biomedical Laboratory R&D (BLRD) Service. Caitlin Schaefer: NIH (P01 HL114453).

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The University of Pittsburgh Institutional Review Board (IRB) approved the protocols and we obtained written or electronic informed consent by all participants or their surrogates in accordance with the Declaration of Helsinki.

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