From Chart Biopsy to Liquid Biopsy: Evaluating the Diagnostic Yield and Clinical Impact of Plasma Microbial Cell-Free DNA Next-Generation Sequencing in the Management of Fever of Unknown Origin

Abstract

Background: The underlying cause of fever of unknown origin (FUO) remains unidentified in up to 51% of cases despite systematic evaluation. Microbial cell-free DNA next-generation sequencing (mcfDNA-NGS) offers an agnostic, noninvasive approach to pathogen identification, but the utility and clinical impact of this assay in FUO remain unknown. Methods: This retrospective cohort study evaluated adult patients referred for FUO evaluation at a tertiary medical center between November 2019 and November 2023. Patients underwent both standard microbiologic testing (ST) and mcfDNA-NGS. Diagnostic impact was assessed in 4 domains: new diagnoses, earlier time to diagnosis, avoidance of invasive procedures, and non-hypothesis-driven diagnoses. Logistic regression was used to identify predictors of positive mcfDNA-NGS testing. Results: Among 176 patients, mcfDNA-NGS was positive in 44.3%, with 49% of these cases considered clinically significant. Infectious cause of FUO was identified in 39% of patients, noninfectious in 35%, and unknown in 26%. mcfDNA-NGS contributed to a positive diagnostic impact in 30% of cases, mainly by earlier diagnosis (16%) and potential for avoidance of invasive procedures (10%). Positive mcfDNA-NGS was significantly associated with higher Charlson comorbidity index score (odds ratio [OR], 1.22; P