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. 2025 Dec 24;81(5):1008-1014.
doi: 10.1093/cid/ciaf170.

Plasma Microbial Cell-free DNA Metagenomic Sequencing for Diagnosis of Invasive Fungal Diseases Among High-risk Outpatient and Inpatient Immunocompromised Hosts

Beatrice Z Sim  1 , Jordan K Mah  1 , Madeleine R Heldman  1 , Kelly L Stanly  1 , Kimberly E Hanson  2   3 , Angela M Caliendo  4 , David Andes  5 , Luis Ostrosky-Zeichner  6 , John R Wingard  7 , Barbara D Alexander  1
Affiliations

Plasma Microbial Cell-free DNA Metagenomic Sequencing for Diagnosis of Invasive Fungal Diseases Among High-risk Outpatient and Inpatient Immunocompromised Hosts

Beatrice Z Sim et al. Clin Infect Dis. .

Abstract

Background: New and minimally invasive tools to aid the diagnosis of invasive fungal diseases (IFD) are urgently needed as the immunocompromised population at highest risk increases. Advancements in molecular technology have rendered new diagnostics more readily available for clinical use.

Methods: This case-control study used prospectively collected archived plasma specimens and data from the Aspergillus Technology Consortium Repository to investigate the diagnostic performance of microbial cell-free DNA (mcfDNA) sequencing as a minimally invasive diagnostic for IFDs in a population of high-risk immunocompromised hosts including hematologic malignancy, stem cell, and solid organ transplants patients. The 2008 Mycoses Study Group/European Organization for the Research and Treatment of Cancer diagnostic criteria served as the gold standard for test performance.

Results: Sixty-five adult subjects with proven or probable IFD and 65 controls without IFD were included. Among IFD episodes Aspergillus was the most common pathogen (70.8%, 46/65), followed by Mucorales (10.8%, 7/65). Overall, sensitivity was 47.7% and specificity was 100%. Sensitivity varied based on disease certainty and pathogen; sensitivity was higher in proven versus probable IFD (60.0% vs 37.1%, respectively) and higher for subjects with invasive mucormycosis (100%) compared with aspergillosis (45.7%).

Conclusions: A positive result by mcfDNA sequencing may reduce the need for invasive sampling in patients with suspected IFD. In this exploratory analysis, its high sensitivity and specificity for invasive mucormycosis suggests it could be useful for early treatment and intervention of this IFD. Future studies should focus on understanding how specific factors impact the sensitivity of mcfDNA sequencing for invasive aspergillosis.

Keywords: fungal diagnostics; fungal infections; immunocompromised host; microbial cell-free DNA metagenomic sequencing; minimally invasive diagnostics.

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Conflict of interest statement

Potential conflicts of interest. K. E. H. has served as a consultant for Karius and HealthtrackRx Laboratories. A. C. M. has served as an advisor to Danaher/Cepheid/Backman Coulter, Visb, and VedaBio. D. A. has served as a consultant for Astellas, Basilea, Elion, and Melinta. L. O. Z. has received consulting honoraria from Eurofins Viracor and research funding from Eurofins Viracor, IMMY, and T2 Biosystems. J. W. R. has served as a consultant to Ansun, BMS, Celgene, Elion, F2G, Karius, Mundipharma, Orca, Pearl Diagnostics, Pulmotect, and Takeda. B. D. A. has received research funding to her institution from Karius and serves as an advisor to HealthTrackRx Laboratories. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

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