Inflammatory syndromes, including those caused by infection, are a major cause of hospital admissions among children and are often misdiagnosed because of a lack of advanced molecular diagnostic tools. In this study, we explored the utility of circulating cell-free RNA (cfRNA) in plasma as an analyte for the differential diagnosis and characterization of pediatric inflammatory syndromes. We profiled cfRNA in 370 plasma samples from pediatric patients with a range of inflammatory conditions, including Kawasaki disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), viral infections and bacterial infections. We developed machine learning models based on these cfRNA profiles, which effectively differentiated KD from MIS-C - two conditions presenting with overlapping symptoms - with high performance (Test Area Under the Curve (AUC) = 0.97). We further extended this methodology into a multiclass machine learning framework that achieved 81% accuracy in distinguishing among KD, MIS-C, viral, and bacterial infections. We further demonstrated that cfRNA profiles can be used to quantify injury to specific tissues and organs, including the liver, heart, endothelium, nervous system, and the upper respiratory tract. Overall, this study identified cfRNA as a versatile analyte for the differential diagnosis and characterization of a wide range of pediatric inflammatory syndromes.

C.J.L and I.D.V are inventors on submitted patents pertaining to cell-free nucleic acids (US patent applications 63/237,367 and 63/429,733). I.D.V. is a member of the Scientific Advisory Board of Karius Inc., Kanvas Biosciences and GenDX. I.D.V. is listed as an inventor on submitted patents pertaining to cell-free nucleic acids (US patent applications 63/237,367, 63/056,249, 63/015,095, 16/500,929, 41614P-10551-01-US) and receives consulting fees from Eurofins Viracor. C.A.R. has received institutional support from ModernaTX, Inc., Pfizer Inc., BioFire Inc., GSK plc, MedImmune, Micron Technology Inc., Janssen Pharmaceuticals, Merck & Co., Inc., Novavax, PaxVax, Regeneron, and Sanofi Pasteur. She is co-inventor of patented RSV vaccine technology which has been licensed to Meissa Vaccines, Inc. C.Y.C. receives grant funding for research unrelated to this work from the Bay Area Lyme Disease Foundation, the Chan-Zuckerberg Biohub, and Abbott Laboratories, Inc. C.y.C. is on the scientific advisory board for Mammoth Biosciences, Poppy Health, BiomeSense, BioMeme, FlightPath Biosciences, and Delve Bio, and is a co-founder of Delve Bio.

This study was funded by the National Institutes of Health (NIH) / National Institute of Child Health and Human Development (NICHD) grants R61HD105618, R33HD105618, and R33HD105593. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Institutional Review Board (IRB) (#21-33403) of University of California San Francisco (UCSF) gave ethical approval for this work. The Institutional Review Board (IRB) of Emory University (STUDY00000723) gave ethical approval for this work. The Institutional Review Board (IRB) of Children's National Medical Center (Pro00010632) gave ethical approval for this work The Institutional Review Board (IRB) or the University of California San Diego (IRB #140220) gave ethical approval for this work. The Institutional Review Board (IRB) of Cornell University IRB for Human Participants (2012010003) gave ethical approval for this work.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

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De-identified RNA-seq count matrices have been uploaded to the NCBI (National Center for Biotechnology Information) GEO (Gene Expression Omnibus) database and will be publicly available upon publication (GSE255555). All code has been deposited on GitHub and will be available upon publication.