Abstract Background Metagenomic next-generation sequencing (mNGS) of microbial cell-free DNA (mcfDNA) allows for non-invasive broad-range pathogen detection from plasma. The Karius® test that emerged in 2016 made mNGS widely available. However, there is little data describing the optimal role for this assay in clinical decision making. Methods We performed a single-center retrospective cohort study of adult patients for whom a Karius test was sent between May 2019 and February 2021 to assess clinical utility. We predefined criteria for clinical impact categories (Table 1) and stratified data by patient comorbidities, infectious syndromes, duration of antimicrobial therapy prior to Karius testing, reasons for sending the test, and final clinical diagnoses. Clinical impact was arbitrated by all authors after review and discussion of each case. Results A total of 80 patients were included. 45 patients (56%) were immunocompromised, and 14 (18%) had prosthetic hardware or grafts. The most common clinical syndrome was pneumonia/respiratory failure (31%), followed by sepsis/septic shock (15%) and endocarditis (13%). 72 patients (90%) received antibiotics prior to sending the Karius assay. The most common reason for sending the assay was unknown microbiologic diagnosis (78%), followed by avoiding invasive procedures (14%). The test was consistent with the final diagnosis in 65% of cases and had a positive impact in 34 (43%), a negative impact in 2 (3%), and uncertain or no impact in 44 (55%) (Table 2). A positive impact was observed in solid organ transplant recipients (SOTR, 71.4%, p=0.003), sepsis (71.4%, p=0.003), and those receiving antimicrobial agents for less than 7 days prior to Karius testing (i.e., 61.8%, p=0.004) (Table 3). Conclusion In our cohort, clinical utility of Karius testing was highest in SOTR and in patients with sepsis. Prolonged antimicrobial use ( > 7 days) prior to Karius testing limited the utility of the assay. Prospective studies evaluating the utility of mNGS mcfDNA assays should be performed to further clarify its role in clinical management. Disclosures All Authors: No reported disclosures