Development and evaluation of a Pan-Mucorales Real-time PCR and a multiplex Real-time PCR for detection and identification of <i>Rhizopus arrhizus, Rhizopus microsporus</i> , and <i>Mucor</i> spp. in clinical specimens

Mucorales fungi, identified collectively as a high-priority pathogen on the World Health Organization fungal priority pathogens list, are the causative agents of mucormycosis. Mortality is high (up to 80%), and early, accurate diagnosis is critical to enable timely initiation of targeted antifungal therapy and surgical debridement for source control to optimize patient outcomes. In our laboratory, as in many others, the current standard for the diagnosis of mucormycosis is histopathology and culture-based methods supplemented by panfungal PCR assay/DNA sequencing; however, this process may take 7 days, with considerable labor and cost implications. Here, we present two Mucorales-specific real-time PCR assays, which when used sequentially, reduce diagnostic turnaround time and costs to detect three common agents of mucormycosis-<i>Rhizopus microsporus</i>, <i>Rhizopus arrhizus</i>, and <i>Mucor</i> species. This approach not only improves diagnostic efficiency and integration into workflow but can facilitate surveillance through accurate genus- and species-level identification.