Development of a novel one-step thermostable heptaplex PCR assay for the detection of medically important Candida species

Invasive fungal infections, particularly candidemia, pose a significant public health challenge due to their high mortality rates. While Candida albicans remains the most common species in candidiasis, there has been a notable rise in non-albicans Candida (NAC) infections, each exhibiting different drug susceptibilities. This underscores the urgent need for rapid, user-friendly detection methods. Conventional molecular tests are limited by the need for expertise, cold-chain logistics, and specific storage conditions, restricting their widespread use. In this study, we developed a thermostabilized multiplex PCR assay to identify five medically significant Candida species: C. albicans, C. krusei, C. glabrata, C. tropicalis, and C. parapsilosis. This assay utilizes newly designed species-specific primers targeting the ITS1 and ITS2 regions. An Internal Amplification Control (IAC) using a specific primer for the SSP2 gene of P. falciparum (273 bp) was included to ensure accuracy. The assay demonstrated 100% sensitivity and specificity when tested with 63 genomic DNA templates from clinical isolates. In addition, the developed thermostabilized heptaplex PCR assay showed stability for at least 6 months as validated by Q10 method testing. In conclusion, this study demonstrated the development of a cold-chain-free PCR assay for the rapid, easy, and simultaneous detection of multiple Candida species. This advancement is intended to improve diagnostic efficiency, support targeted antifungal therapy, and enhance patient outcomes, particularly in resource-limited settings.