A MALDI-TOF MS-based multiple detection panel of drug resistance-associated multiple single-nucleotide polymorphisms in Candida tropicalis

Candida tropicalis is one of the main causes of invasive candidiasis. Rapid identification of antifungal resistance is crucial for selection of an appropriate antifungal to improve patient outcomes. Mutations at specific loci are strongly correlated with resistance to antifungal agents. In this study, we developed a multi-single-nucleotide polymorphism (SNP) panel to accurately identify 36 mutation sites across seven genes of C. tropicalis that are associated with resistance to azoles and/or echinocandins. Ten isolates were selected to test repeatability, and another 20 isolates of C. tropicalis were selected to validate consistency. Intra-assay and inter-assay repeatability of the panel was 100%, with the loci accuracy being 99.44% (716 of 720). Furthermore, 109 isolates were examined for clinical research, and the most commonly detected mutations were G751A and A866T of UPC2, A491T of TAC1, and A395T and C461T of ERG11. The G751A and A866T mutations of UPC2 as well as the A395T and C461T mutations of ERG11 co-existed. The SNP panel enables identification of specific mutations at critical sites of drug-resistant strains to facilitate the rapid selection of appropriate antifungal agents and efficient monitoring of the regional epidemiological trends of resistance of C. tropicalis.IMPORTANCEC. tropicalis infections pose a growing global public health challenge, with mortality rates approaching 40%. C. tropicalis is one of the top four Candida spp. responsible for candidiasis, particularly in the Asia-Pacific region and Latin America, notably affecting patients with neutropenia and malignancies. The azole resistance rate of C. tropicalis ranges from 0% to 30%. Between 2009 and 2018, the China Hospital Invasive Fungal Surveillance Network reported an increase in fluconazole and voriconazole resistance from 5.7% to ~30%. Although resistance to echinocandins and amphotericin B remains low, multi-resistance to echinocandins and azoles has been observed. Current methods for detecting drug resistance are limited by the long turnaround time of antifungal susceptibility testing, low throughput of Sanger sequence to target resistance mutations, complex data analysis, and high costs of second-generation sequencing. We developed and validated a rapid, high-throughput, and cost-effective panel to detect and monitor drug-resistance mutations of C. tropicalis.