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

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 in vitro diagnostic PCR assays: a real-time, qualitative Pan-Mucorales PCR, followed by a real-time multiplex genus/species-specific PCR targeting Rhizopus arrhizus, Rhizopus microsporus, and Mucor spp. Specificity of the assays was validated using cultured isolates of Mucorales, as well as non-Mucorales fungi and bacteria. The diagnostic performance was assessed across 166 clinical specimens (70 Mucorales-positive and 96 negative), confirmed by an in-house panfungal PCR and DNA sequencing protocol. Specimens studied included fresh and formalin-fixed paraffin-embedded tissues, fluid, bronchoalveolar lavage/washing fluid, fine needle aspirate, cerebrospinal fluid, and bone. The Pan-Mucorales PCR demonstrated 98.6% sensitivity and 100% specificity, while the multiplex genus/species-specific PCR assay yielded sensitivities of 93.8% for R. arrhizus, 70.8% for R. microsporus, and 75% for Mucor spp., each with 100% specificity. Concordance with the panfungal PCR (>99% for Pan-Mucorales PCR and >89% for multiplex PCR) was high, supporting the robustness of the workflow. This diagnostic approach has the potential to significantly reduce turnaround times, labor and costs, while streamlining the diagnostic process through timely, precise diagnostics.

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-Rhizopus microsporus, Rhizopus arrhizus, and Mucor species. This approach not only improves diagnostic efficiency and integration into workflow but can facilitate surveillance through accurate genus- and species-level identification.