Development and evaluation of a Pan-Mucorales Real-time PCR and a multiplex Real-time PCR for detection and identification of

Mucormycosis is a life-threatening infection associated with high morbidity and mortality. Rapid and accurate diagnosis is essential for improving patient outcomes. Conventional diagnostic methods, such as histopathology and culture, are limited by low sensitivity and prolonged turnaround times, while commercial polymerase chain reaction (PCR) assays are costly and may lack specific genus or species targets. Here, we present a novel molecular diagnostic workflow to facilitate the rapid detection of Mucorales directly from clinical specimens. This workflow integrates two in-house IMPORTANCE: 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-