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Life sciences · Journal article
Journal of Clinical Microbiology · September 25, 2026
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ABSTRACT Timely diagnosis of invasive aspergillosis (IA) is hindered by moderate sensitivity of conventional diagnostic methods. Increasing plasma input volumes has been shown to improve sensitivity of plasma polymerase chain reaction (PCR), and using plasma-derived fungal exosomes (nanosized extracellular vesicles) may do the same. This study aimed to determine whether using large plasma input volumes and exosome-derived DNA enhances the diagnostic performance of Aspergillus PCR. We included hematology patients from whom plasma was collected within 48 h of bronchoalveolar lavage in the prospective multicenter ARPO-study (NCT06069505); plasma from healthy donors served as controls. Sensitivity, specificity, positive predictive values, and negative predictive values of the PCR on DNA extracted from plasma-derived exosomes, large plasma volumes of around 4.0 mL, and small plasma volumes of 0.2 mL were assessed using the European Organization for Research and Treatment of Cancer/Mycoses Study Group Education and Research Consortium diagnostic criteria. The IA prevalence was 34.2% (38/111), and the median plasma volume used in the large-volume group was 4.0 mL (IQR 3.1 mL–4.0 mL). Aspergillus fumigatus large-volume plasma PCR yielded 22 positive results with a sensitivity of 52.6%, significantly higher than with small plasma volume (29.7%, P = 0.039) without loss of specificity (97.2%), improving further in patients not receiving versus receiving mold-active antifungals (66.7% versus 40.0%, P = 0.013). Sensitivity of the PCR on plasma-derived exosomes was low (29.4%). A. flavus DNA was detected in five patients and three healthy controls; all were considered false positives. DNA extraction from larger plasma volumes improved the detection of A. fumigatus. The sensitivity of plasma-derived exosomes was limited; their use is not recommended. IMPORTANCE Invasive fungal infection caused by Aspergillus fumigatus remains a life-threatening complication in patients with hematological malignancies, while early diagnosis is often challenging with the current tools. This study demonstrates that increasing the volume of blood plasma used for fungal DNA detection significantly improves diagnostic sensitivity without compromising specificity. As blood sampling is less invasive than bronchoalveolar lavage, a more sensitive plasma-based approach may facilitate earlier detection and initiation of targeted therapy when utilizing a diagnostic-driven approach. Our findings provide practical guidance for optimizing molecular diagnostic protocols in clinical laboratories.