JOURNAL ARTICLE
Plasma Microbial Cell-free DNA Metagenomic Sequencing for Diagnosis of Invasive Fungal Diseases Among High-risk Outpatient and Inpatient Immunocompromised Hosts
Beatrice Z Sim, Jordan K Mah, Madeleine R Heldman, et al
Clinical Infectious Diseases, Volume 81, Issue 5, 15 November 2025, Pages 1008–1014, https://doi.org/10.1093/cid/ciaf170
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%).
Table 1. Demographic and Clinical Characteristics
| Characteristic | IFDsa (N = 65) | Controls (N = 65) |
|---|---|---|
| Age in years, median (IQR) | 63 (18) | 60.0 (15.5) |
| Female | 24.6% (16) | 36.9% (24) |
| Race, White | 84.6% (55) | 92.3 (60) |
| Underlying condition | ||
| Solid organ transplant | 47.7% (31) | 52.3% (34) |
| Lung | 35.4% (23) | 52.3% (34) |
| Heart | 10.8% (7) | 0.0% (0) |
| Kidney | 1.5% (1) | 0.0% (0) |
| Hematopoietic stem cell transplant | 20.0% (13) | 24.6% (16) |
| Allogeneic | 13.8% (9) | 23.1% (15) |
| Autologous | 6.2% (4) | 0.0% (0) |
| Syngeneic | 0.0% (0) | 1.5% (1) |
| HIV/AIDS | 1.5% (1) | 0.0% |
| Hematologic malignancy | 24.6% (16) | 23.1% (15) |
| Pulmonary disease | 6.2% (4) | 0.0% (0) |
| Neutropenic at the time of specimen collectionb | 15.4% (10) | 21.9% (14) |
| Prior IFDc | 16.9% (11) | 4.6% (3) |
| On systemic antifungal therapy at time of specimen collection | 89.2%% (58) | 49.2%% (32) |
| On active antifungal therapy at time of specimen collection | 76.9% (50) | NA |
| Specimen collected inpatient | 81.5% (53) | 64.6% (42) |
aDefined per 2008 MSG/EORTC definitions.
bNeutropenia defined as absolute neutrophil count <500 per microliter.
cPrior IFD was defined as an IFD that occurred ≥8 weeks before the index IFD or <8 weeks before the index IFD and with appropriate treatment and resolution of symptoms and documented mycologic clearance before index IFD diagnosis.
Table 2. Invasive Fungal Diseases (N = 65)
| MSG/EORTC Disease Category | ||
|---|---|---|
| Proven (n = 30) | Probable (n = 35) | |
| Causative pathogen by usual care diagnostics | ||
| Mold | ||
| Aspergillusa,b | 36.7% (11) | 77.1% (27) |
| Fusarium | 0.0% (0) | 2.9% (1) |
| Scedosporium | 3.3% (1) | 0.0% (0) |
| Mucoralesc | 16.7% (5) | 2.9% (1) |
| Otherd | 13.3% (4) | 2.9% (1) |
| Multiple fungal pathogense | 16.7% (5) | 14.3% (5) |
| Yeast | ||
| Candida | 6.7% (2) | 0.0% (0) |
| Cryptococcus | 3.3% (1) | 0.0% (0) |
| Trichosporon | 3.3% (1) | 0.0% (0) |
| Infection site | ||
| Lung | 83.3% (25) | 97.1% (34) |
| Sinus | 3.3% (1) | 2.9% (1) |
| Blood | 0.0% (0) | 0.0% (0) |
| Disseminatedf | 13.3% (4) | 0.0% (0) |
| Response to therapy 8 wk after MSG/EORTC diagnosis date | ||
| Complete/partial | 60.0% (18) | 60.0% (21) |
| Stable | 10.0% (3) | 11.4% (4) |
| Progression/death | 30.0% (9) | 28.6% (10) |
| Overall mortalityg | 20.0% (6) | 28.6% (8) |
aAspergillus species included: A. fumigatus, A. terreus, A. flavus, A. niger, A. ustus.
bThree invasive Aspergillosis episodes included 2 or more Aspergillus species (A. fumigatus + A. terreus; A. fumigatus + A. flavus + A. niger; A. fumigatus + A. terreus).
cMucorales included: Rhizopus species, Mucor species, Rhizomucor species.
dOther included: Mold, not otherwise specified; hyaline hyphomycete mold.
eMultiple fungal pathogens was defined as pathogens recovered from same specimen or on separate specimens collected for the same or worsening clinical syndrome within 8 weeks of index IFD MSG diagnosis date and included: Aspergillus niger + yeast (probable Candida); Aspergillus fumigatus + Aspergillus terreus + Scopulariopsis species; Aspergillus terreus + Penicillium NOS; NOS Aspergillus flavus + Aspergillus terreus + Aspergillus niger + Aspergillus ochraceous group + Curvularia NOS + Penicillium NOS; Aspergillus niger + Penicillium NOS + Chrysosporium NOS; Aspergillus flavus + Cryptococcus neoformans; Aspergillus flavus + Geotrichum species; Aspergillus fumigatus + Paecilomyces lilacinus; Penicillium citrinum + Paecilomyces variotti; Cunninghamella + hyaline hyphomycete mold NOS.
fTwo or more sites of infection: sites of dissemination included lungs, skin, peritoneum, sinus, brain, mediastinum, kidney.
gAt 8 weeks after MSG/EORTC diagnosis date; discharge to hospice contributed to overall mortality.
Table 3. Performance of Plasma mcfDNA Sequencing for Diagnosis of IFD Based on Specimen Collection Time Relative to MSG/EORTC Diagnosis Date
| N | Sensitivity | Specificity | |
|---|---|---|---|
| Timing of specimen collection (median [IQR] days) | |||
| All specimens (median 6 [9] days) | 65 | 47.7% | 100% |
| Within 14 (median 5 [5]) days of MSG/EORTC diagnosis | 56 | 51.8% | 100% |
| Within 7 (median 3 [3]) days of MSG/EORTC diagnosis | 40 | 52.5% | 100% |
| Proven versus probable infection (all specimens, n = 65) | |||
| Proven IFD | 30 | 60.0% | 100% |
| Probable IFD | 35 | 37.1% | 100% |
| Causative pathogen (all specimens, n = 65) | |||
| Aspergillus speciesa | 46 | 45.7% | 100% |
| Mucoralesa | 7 | 100% | 100% |
aIncluded samples with multiple fungal pathogens.
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.
