Discussion

In this single-center inpatient series of paired molecular testing, plasma mcfDNAseq produced substantially higher diagnostic yield than an amplicon-based blood NGS assay: 8/10 plasma tests were positive (7 clinically meaningful) whereas all amplicon tests were negative. Rapid organism identification is central to management of endovascular infection, and culture-negative presentations remain common, often after antibiotic exposure, or with fastidious organisms. ^{Reference Delgado, Ajmone Marsan and de Waha1–Reference Lin, Yeh, Chuang, Wang, Fu and Liu3}

Detected pathogens were clinically plausible for culture-negative or diagnostically complex endovascular syndromes. Notably, C. burnetii—a classic cause of culture-negative endocarditis typically diagnosed by serology—is emphasized in diagnostic frameworks. ^{Reference DeSimone, Esquer Garrigos and Marx2,Reference Lin, Yeh, Chuang, Wang, Fu and Liu3} Culture-negative endocarditis has been associated with worse outcomes than culture-positive disease, reinforcing the importance of establishing an etiology when possible. ^{Reference Kong, Salsano and Giacobbe4}

These findings are consistent with prior analytic validation and clinical experience showing that plasma mcfDNA sequencing can detect a broad range of pathogens, including organisms that may be missed by conventional culture. ^{Reference Blauwkamp, Thair and Rosen5} Multicenter real-world studies suggest that plasma mcfDNA testing can be clinically impactful when used in appropriately selected patients. ^{Reference Hogan, Yang and Garner6}

The lack of yield from amplicon-based blood NGS in our cohort may reflect differences in analyte (cell-free vs cell-associated nucleic acids), assay target scope, and low microbial burden in intermittently bacteremic syndromes. Although confirmatory studies are needed, these results suggest that concurrent ordering of amplicon-based blood NGS alongside plasma mcfDNAseq may add limited value in similar inpatient evaluations.

From an antimicrobial stewardship perspective, plasma mcfDNAseq may support earlier pathogen-directed therapy in culture-negative or complex endovascular infection, but results require clinical correlation. The single adjudicated falsepositive (S. epidermidis) highlights the need to interpret potential contaminants and incidental detections cautiously. ^{Reference Blauwkamp, Thair and Rosen5,Reference Hogan, Yang and Garner6} Viral co-detections should similarly be interpreted in the context of syndrome and host factors, as detection does not necessarily indicate causation or need for therapy. ^{Reference Hogan, Yang and Garner6}

This study is limited by a small sample size and a short accrual window, a retrospective single-center design, and selection bias because inclusion required paired testing. Clinical adjudication is an imperfect reference standard, and we did not assess turnaround time, treatment changes, costs, or outcomes.

In conclusion, plasma mcfDNAseq provided greater clinically actionable yield than an amplicon-based blood NGS assay and appeared highly sensitive for adjudicated infections; when a broad blood-based molecular test is indicated, plasma mcfDNAseq may be preferred, with stewardship-guided interpretation to mitigate falsepositive and incidental results.