A fully integrated sample-to-answer molecular diagnostic platform for rapid identification of four major Aspergillus species

Background Invasive pulmonary aspergillosis (IPA) is more common among immunocompromised patients. Timely diagnosis is crucial but is challenging because current diagnostic approaches are often limited by low sensitivity, prolonged turnaround times, and technical complexity. Here, we developed a fully automated, real-time PCR–based, sample-to-answer molecular diagnostic platform for the simultaneous identification of four major Aspergillus species ( A. fumigatus, A. flavus, A. niger , and A. terreus ). Materials and methods The sample-to-answer workflow was optimized using artificial bronchoalveolar lavage (BAL) samples spiked with Aspergillus species. The platform’s performance was evaluated through two primary validation steps: analytical consistency with MALDI-TOF using 21 patient-derived culture isolates, and clinical feasibility validation using 22 clinical BAL specimens from patients categorized as proven ( n = 11) or probable ( n = 11) IPA. Results The assay demonstrated 95.2% (20/21) overall agreement with MALDI-TOF for culture isolates, achieving 93.3% positive agreement and 100% negative agreement. In the feasibility validation using 22 patient BAL samples including 11 proven and 11 probable IPA cases, the platform detected 10 out of 11 proven IPA samples (90.9%) with 100% species-level consistency with MALDI-TOF results. Notably, this assay could add mycological evidence in 3 of 11 culture-negative probable IPA cases. Conclusion This fully automated, sample-to-answer platform enables rapid and accurate species-level identification of Aspergillus in suspicious IPA. We highlight that this assay offers a powerful molecular tool to overcome the limitations of conventional diagnostic methods in clinical settings.