Next Generation Sequencing of free pathogenic DNA in blood samples of a critically-ill pediatric population: A single-center experience Short title: NGS of pathogenic cfDNA in critically ill children

Background: Rapid and accurate pathogen detection is critical for optimizing outcomes in pediatric sepsis. Next-generation sequencing (NGS) of cell-free DNA (cfDNA) from blood enables culture-independent identification of microbial DNA from bacteria, viruses, fungi, and parasites. We evaluated the diagnostic yield and clinical impact of cfDNA-based NGS in critically ill and predominantly immunocompromised pediatric patients (≤ 18 years) with suspected infection. This retrospective single-center study included pediatric patients who underwent plasma cfDNA-NGS at a tertiary care hospital in Germany. Following computational removal of human DNA, remaining sequences were aligned to curated microbial reference databases. Diagnostic performance was compared with blood cultures and viral PCR, and clinical relevance was assessed by pediatric infectious disease specialists. Results: 111 tests in 78 pediatric patients, mostly with systemic inflammatory response syndrome of unknown etiology, were performed. Overall, 61 tests (54.5%) were positive for pathogenic cfDNA. Compared with conventional microbiological diagnostics, NGS demonstrated a sensitivity of 64.7% and specificity of 88.2% when blood cultures and viral PCR served as the reference standard. NGS identified additional pathogens in a substantial proportion (41.1%) of cases that remained negative by standard testing. Of those pathogens only found by NGS, over 60% were deemed clinically relevant. In 14.8% of positive NGS results, a pathogen-specific therapy was started, while 40.2% of tests led to a discontinuation of therapy (51.0% of negative tests). Out of all positive NGS, 38 (62.3%) were classified as clinically relevant. NGS testing also detected rare infections with fungi and parasites in four cases each. Conclusion: Detection of pathogenic cfDNA through NGS from blood shows promising results as an additional diagnostic tool in critically ill pediatric patients with suspected infections. Clinical utility is currently still limited by its high cost, undetermined diagnostic validity and limitations in testing for resistances and restricted availability of raw sequencing data due to data-protection constraints.