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

## TL;DR

Researchers developed a safer and more efficient method for screening antifungal drugs against Coccidioides posadasii, using multi-well plates and a less dangerous strain of the fungus.

## Contribution

The novel contribution is the development of BSL-2-compatible 96- and 384-well colorimetric assays for high-throughput antifungal screening against Coccidioides.

## Key findings

- A BSL-2 attenuated C. posadasii strain was used to enable safer antifungal screening.
- 96-well and 384-well plate methods were optimized for high-throughput screening.
- The approach supports large-scale discovery of novel antifungal therapeutics.

## Abstract

Coccidioides immitis and Coccidioides posadasii, 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 Coccidioides spp. has been hampered by the requirement for Biosafety Level 3 (BSL-3) containment. To overcome these barriers, we leveraged an attenuated C. posadasii 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.