Development and Validation of a Direct Colony Real-Time PCR for Rapid and Specific Candida auris Detection

Candida auris, an emerging multidrug-resistant fungal pathogen, poses a significant diagnostic and infection control challenge with limited identification methodologies. To overcome this, we developed and validated a rapid, specific, and direct colony real-time PCR assay for C. auris identification without DNA extraction. Species-specific primers were designed using comparative genomics and subjected to rigorous experimental validations. The optimized assay using a selected primer pair (Candida-auris-5F/5R) successfully identified C. auris directly from the cultured colonies within two hours. It demonstrated high specificity against eight clinically relevant non-auris Candida species, and sufficient sensitivity (as low as 100 cells/reaction) for routine laboratory testing. This method significantly reduced the turnaround time, labor, and costs compared to conventional molecular techniques that require DNA extraction. Acknowledging the recent emergence of Clade I strains in Japan, the selected primer pairs and positive controls were distributed nationally to all 59 regional public health institutes by January 2025, establishing a standardized surveillance system. This assay is expected to play a key role in timely detection of C. auris, thereby improving patient care and outbreak management.