Airborne Mucorales in the Netherlands largely mirror Dutch clinical isolates causing mucormycosis

We determined the optimal method to culture Mucorales species from air samples. Subsequently, we investigated the diversity of Mucorales species in Dutch air and compared these species with those causing mucormycosis in patients. We optimized the Mucorales culturing protocol by testing different growth conditions with samples from a newly developed air sampling approach. We used 120 air samples taken throughout the Netherlands in the project called Schimmelradar (September-October 2023). These samples were supplemented with additional air samples taken in the Netherlands (February 2024). The Mucorales species cultured from these air samples were compared to 17 clinical isolates (2016-2023) from a Dutch university medical center. Mucormycosis infections were classified using the European Organisation for Research and Treatment of Cancer (EORTC) criteria. All Mucorales were identified at genus level by culture morphology, and a subset was analyzed at species level using matrix-assisted laser desorption ionization-time of flight mass spectrometry, microscopy, and Internal Transcribed Spacer Polymerase Chain Reaction (ITS PCR). A combination of Sabouraud dextrose agar, voriconazole and incubation at 30°C yielded the broadest variety of Mucorales species from air samples. Species found in Dutch air included Rhizomucor pusillus, Rhizopus microsporus, and Mucor circinelloides. Clinical data showed that Rhizop. microsporus and M. circinelloides were most frequently identified in mucormycosis infections. We validated a selective method for the culture of Mucorales species from air samples using a delta trap air sampling method. Three of the 10 species cultured from air samples were also detected in clinical isolates. Although inhalation is assumed as primary route of infection, this is the first study demonstrating the similarity of Mucorales species between air and clinical samples.