Droplet digital PCR for diagnosing invasive aspergillosis in hematological malignancies: a prospective cohort study

ABSTRACT Our objective was to evaluate the diagnostic accuracy of Aspergillus droplet digital PCR (ddPCR) in exhaled breath condensate (EBC) and bronchoalveolar lavage fluid (BALF) for diagnosing invasive aspergillosis (IA), and to compare its performance with quantitative real-time PCR (qPCR) in patients with hematologic malignancies. We conducted a prospective single-center study, including adults admitted to leukemia and allogeneic hematopoietic stem cell transplant units who underwent bronchoscopy for pulmonary infiltrates between October 2021 and December 2024. EBC and BALF were collected concurrently. ddPCR was performed on EBC and BALF (supernatant and cell pellet); qPCR was performed on BALF samples. IA was classified according to EORTC/MSGERC criteria without incorporating PCR into the reference standard. Diagnostic performance characteristics, including area under the receiver operating characteristic curve (AUC-ROC), were calculated. Among 203 participants, 26 (12.8%) were diagnosed with probable or proven IA. EBC ddPCR showed poor performance (AUC-ROC 0.55, 95% CI 0.43–0.67). BALF supernatant ddPCR demonstrated the best performance (AUC-ROC 0.88, 95% CI 0.80–0.96), with sensitivity of 0.65 (95% CI 0.43–0.84) and specificity of 0.96 (95% CI 0.92–0.99). BALF supernatant outperformed BALF cell pellet testing for both ddPCR and qPCR. Compared with qPCR, ddPCR showed higher sensitivity and similarly high specificity. Among participants without prior mold-active antifungal therapy, BALF supernatant ddPCR showed improved performance (AUC-ROC 0.95, 95% CI 0.89–1.00). In this large prospective study, EBC ddPCR demonstrated limited utility for the diagnosis of IA. BALF supernatant was the optimal specimen type, and ddPCR outperformed qPCR. Diagnostic performance improved substantially without prior mold-active antifungal exposure. IMPORTANCE Invasive aspergillosis is difficult to diagnose in patients with hematologic malignancies, and non-invasive diagnostic approaches are needed. In this prospective study of 203 patients, Aspergillus droplet digital PCR performed poorly in exhaled breath condensate (area under the receiver operating characteristic curve 0.55; 95% CI 0.43–0.67) but showed good diagnostic performance in bronchoalveolar lavage supernatant fluid (area under the receiver operating characteristic curve 0.88; 95% CI 0.80–0.96). Droplet digital PCR demonstrated higher sensitivity than quantitative PCR with similarly high specificity, and bronchoalveolar lavage supernatant fluid consistently outperformed cell pellet testing. Among patients without prior mold-active antifungal therapy, the area under the receiver operating characteristic curve was 0.95 (95% CI 0.89–1.00). These findings support bronchoalveolar lavage supernatant fluid as the preferred specimen for Aspergillus droplet digital PCR and warrant further evaluation of this approach for diagnosing invasive aspergillosis.