Development and Optimization of Multi-Well Colorimetric Assays for Growth of Coccidioides posadasii Spherules and Their Application in Large-Scale Screening

<i>Coccidioides immitis</i> and <i>Coccidioides posadasii</i>, the causative agents of coccidioidomycosis, represent a major public health concern in endemic regions of North and South America. The disease spectrum ranges from mild respiratory illness to severe disseminated infections, with thousands of cases reported annually in the United States and an increasing recognition of its global impact. Despite existing antifungal therapies, treatment remains challenging due to toxicity, drug resistance, and limited therapeutic options. High-throughput screening platforms have revolutionized drug discovery for infectious diseases; however, progress in antifungal screening for <i>Coccidioides</i> spp. has been hampered by the requirement for Biosafety Level 3 (BSL-3) containment. To overcome these barriers, we leveraged an attenuated <i>C. posadasii</i> strain that can be safely handled under BSL-2 conditions. Here, we describe the development and optimization of 96-well and 384-well plate screening methodologies, providing a safer and more efficient platform for antifungal discovery. This approach enhances the feasibility of large-scale screening efforts and may facilitate the identification of novel therapeutics for coccidioidomycosis.