A Plasma Cell-Free DNA PCR Panel Test for Invasive Mold Disease
March 1, 2025
By Jake Scott, MD
Synopsis: Lieu and colleagues performed a retrospective study of patients with suspected invasive mold disease that showed a high degree of concordance between noninvasive plasma cell-free deoxyribonucleic acid (DNA) polymerase chain reaction (PCR) testing and invasive specimen fungal test results.
Source: Lieu A, Zimmet AN, Pozdol J, et al. Concordance of non-invasive plasma cell-free DNA with invasive diagnostics for diagnosis of invasive fungal disease. Clin Infect Dis. 2025; Jan 17:ciaf021. doi:10.1093/cid/ciaf02. [Online ahead of print].
Lieu and colleagues performed a retrospective single-center cohort study of patients hospitalized at Stanford Health Care facilities between August 2020 and August 2023 with suspected invasive mold disease (IMD) to determine the diagnostic value of noninvasive mold plasma cell-free deoxyribonucleic acid (cfDNA) polymerase chain reaction (PCR) panel testing added to invasive specimen testing with conventional fungal diagnostics.1 The study included patients who underwent mold plasma cfDNA PCR testing up to seven days before or two days after an invasive specimen collection for testing with conventional fungal diagnostics. Invasive specimens included deep tissue biopsies and bronchoalveolar lavage fluid (BAL). The mold PCR panel included testing for Aspergillus species, Mucorales agents, Fusarium species, and Scedosporium species. Specimens collected invasively were tested with culture, galactomannan (GM), histopathology, cytology, mold PCR, and/or targeted fungal PCR amplicon sequencing.
The primary endpoint of the study was the concordance between the results of noninvasive mold plasma cfDNA PCR and those of invasive specimen fungal test. The secondary endpoint was the identification of clinical factors associated with the concordance of the two testing methods. Patient information was collected from electronic medical records and included data on demographics, underlying comorbidities and immunosuppression, anti-mold therapy, test results, and clinical outcomes. Cases were categorized as proven, probable, possible, or no IMD based on the European Organization for Research and Treatment of Cancer and Mycoses Study Group Education and Research Consortium (EORTC/MSGERC) definitions.2 Two consecutive positive plasma cfDNA PCR results were considered mycological evidence for probable IMD. The mold cfDNA PCR panel was developed by the Stanford Health Care clinical microbiology laboratory and was performed using previously optimized preanalytical parameters.3,4
The study included data from a total of 438 unique patients with 506 noninvasive mold plasma cfDNA PCR tests and concurrent conventional fungal tests performed on deep tissue (80), BAL (383), or both (43). Results were available sooner for mold plasma cfDNA PCR than for invasive specimen tests, with a median turnaround time difference of 44.9 hours (interquartile range [IQR], 12.0-82.8), with 84.4% (427) of plasma samples collected before the invasive specimens.
The majority (61.1%) of the patients were males. The median age was 55 years (IQR, 30-67 years); 14.8% of patients were pediatric. Most patients (86.4%) were immunosuppressed due to conditions, including hematopoietic stem cell transplantation (23.5%), solid organ transplantation (30.2%), hematologic malignancy (44.1%), solid organ malignancy (7.5%), rheumatologic disorders (4.7%), and other causes (5.7%). Slightly less than half of patients (47.8%) were on mold prophylaxis at the time of mold plasma cfDNA PCR testing.
The most common source of infection was pulmonary (87.6%); 3.6% had sinus infections, 4.4% had non-pulmonary or non-sinus localized infections, and 4.2% had disseminated infections. Based on the EORTC/MSGERC criteria, 7.4% had proven IMD, 11.7% had probable IMD, 23.0% had possible IMD, and 57.8% had no IMD. Antifungal therapy was modified within seven days of mold plasma cfDNA PCR testing in 45.1% of cases. Thirty-day and six-month all-cause mortality rates were 17.0% and 37.2%, respectively.
After adjudicating discordant results based on the EORTC/MSGERC definitions for IMD, mold plasma cfDNA PCR was 88.5% (448/506) concordant and 11.5% (58/506) discordant with invasive fungal test results. Of those that were concordant, 7.9% (40/506) were negative by both methods and 74.3% (376/506) were positive. Of those that were discordant, 6.3% (32/506) had positive mold plasma cfDNA PCR results and negative invasive fungal tests (26 BAL, five tissue, and one BAL and tissue). Analysis of these discordant results determined that 18.8% (6/32) had proven, 15.6% (5/32) had probable, 37.5% (12) had possible, and 28.1% (9/32) had no IMD. A total of 11.3% (57/506) had negative mold plasma cfDNA PCR results and positive invasive fungal tests (41 BAL, 12 tissue, and four BAL and tissue). Of these, analysis found that 29.8% (17/57) had proven, 38.6% (22/57) had probable, 15.8% (9/57) had possible, and 15.8 (99/57) had no IMD. Among the 17 with proven IMD with negative mold plasma cfDNA PCR results and positive tissue results, the most common sources of infection were sino-orbital (47.1%, 8/17), pulmonary (29.4%, 5/17), limb (17.6%, 3/17), and liver (5.9%, 1/17).
Clinical factors statistically associated with negative mold plasma cfDNA PCR results in patients with proven or probable IMD were non-hematologic malignancy, non-neutropenic states, and lower 30-day all-cause mortality, when compared with proven or probable IMD in those with concordant positive cfDNA and invasive fungal tests. There was a non-significant trend toward a lower proportion of immunosuppressed patients and a higher proportion of sinus infection among those with negative cfDNA but positive invasive fungal tests.
Commentary
This study provides important information about the clinical value of plasma cfDNA PCR testing for IMD. The high degree of concordance between plasma cfDNA PCR and invasive fungal test results, particularly among patients without fungal sinusitis or limb infections, indicates that a diagnosis of IMD often can be made more rapidly and safely with plasma cfDNA. These findings are especially pertinent given that there has been an increasing trend in the prevalence of IMD and since early diagnosis and targeted treatment is associated with better outcomes.5-7 Adjunctive non-invasive fungal tests have mainly included serum Aspergillus GM and serum 1,3-β-D-Glucan (BDG), both of which have significant limitations. GM is a polysaccharide that makes up Aspergillus cell walls and often is used to aid with the diagnosis of invasive aspergillosis, but its diagnostic accuracy is suboptimal (sensitivity, 71.0%; specificity, 89.0%) and is affected by non-neutropenic states and receipt of antifungal therapy.8,9 1,3-β-D-Glucan is a cell wall component of many fungi, but the specificity of the BDG assay is poor.10,11 Testing with cfDNA PCR is a novel noninvasive diagnostic modality that has been demonstrated in previous studies to be highly sensitive and specific for IMD.3,12
Given that invasive diagnostic tests carry risks, such as hemorrhage and pneumothorax, as Lieu and colleagues discuss, the high performance of plasma cfDNA PCR testing for IMD indicates that invasive procedures potentially may be avoided, where cfDNA is available, thereby optimizing clinical outcomes in addition to healthcare resources.1 However, invasive procedures still may be required in certain cases, particularly those with fungal sinusitis and other scenarios where debridement is necessary. In conclusion, this study provides further evidence that plasma cfDNA PCR testing is an important advance in patient care and diagnostic stewardship.
Jake Scott, MD, is Clinical Associate Professor, Infectious Diseases and Geographic Medicine, Stanford University School of Medicine; Antimicrobial Stewardship Program Medical Director, Stanford Health Care Tri-Valley.
References
- Lieu A, Zimmet AN, Pozdol J, et al. Concordance of non-invasive plasma cell-free DNA with invasive diagnostics for diagnosis of invasive fungal disease. Clin Infect Dis. 2025; Jan 17:ciaf021. doi:10.1093/cid/ciaf02. [Online ahead of print].
- Donnelly JP, Chen SC, Kauffman CA, et al. Revision and update of the consensus definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium. Clin Infect Dis. 2020;71(6):1367-1376.
- Senchyna F, Hogan CA, Murugesan K, et al. Clinical accuracy and impact of plasma cell-free DNA fungal polymerase chain reaction panel for noninvasive diagnosis of fungal infection. Clin Infect Dis. 2021;73(9):1677-1684.
- Murugesan K, Hogan CA, Palmer Z, et al. Investigation of preanalytical variables impacting pathogen cell-free DNA in blood and urine. J Clin Microbiol. 2019;57(11):e00782-19.
- Webb BJ, Ferraro JP, Rea S, et al. Epidemiology and clinical features of invasive fungal infection in a US health care network. Open Forum Infect Dis. 2018;5(8):ofy187.
- Bitar D, Lortholary O, Le Strat Y, et al. Population-based analysis of invasive fungal infections, France, 2001-2010. Emerg Infect Dis. 2014;20(7):1149-1155.
- Chamilos G, Lewis RE, Kontoyiannis DP. Delaying amphotericin B-based frontline therapy significantly increases mortality among patients with hematologic malignancy who have zygomycosis. Clin Infect Dis. 2008;47(4):503-509.
- Terrero-Salcedo D, Powers-Fletcher MV. Updates in laboratory diagnostics for invasive fungal infections. J Clin Microbiol. 2020;58(6):e01487-19.
- Leeflang MMG, Debets-Ossenkopp YJ, Wang J, et al. Galactomannan detection for invasive aspergillosis in immunocompromised patients. Cochrane Database Syst Rev. 2015;2015(12):CD007394.
- Sulahian A, Porcher R, Bergeron A, et al. Use and limits of (1-3)-β-d-glucan assay (Fungitell), compared to galactomannan determination (Platelia Aspergillus), for diagnosis of invasive aspergillosis. J Clin Microbiol. 2014;52(7):2328-2333.
- White SK, Schmidt RL, Walker BS, Hanson KE. (13)-β-D-glucan testing for the detection of invasive fungal infections in immunocompromised or critically ill people. Cochrane Database Syst Rev. 2020;7(7):CD009833.
- Mah J, Nicholas V, Tayyar R, et al. Superior accuracy of Aspergillus plasma cell-free DNA polymerase chain reaction over serum galactomannan for the diagnosis of invasive aspergillosis. Clin Infect Dis. 2023;77(9):1282-1290.
Lieu and colleagues performed a retrospective study of patients with suspected invasive mold disease that showed a high degree of concordance between noninvasive plasma cell-free deoxyribonucleic acid (DNA) polymerase chain reaction (PCR) testing and invasive specimen fungal test results.
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