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Resistance of clinical isolates of Candida albicans to the echinocandin drug caspofungin is slowly emerging and is linked to mutations in short conserved regions in the FKS1 gene. The most prominent changes occurred at the serine 645 position in Fks1p with substitutions of proline, tyrosine, and phenylalanine. An allele-specific real-time PCR molecular-beacon assay was developed for rapid identification of drug resistance by targeting FKS1 mutations. Mutations altering serine 645 were reliably identified in both heterozygous and homozygous states. The molecular-beacon assay was used to evaluate two large collections of spontaneous mutants from separate strains of C. albicans with resistance (MICs, &gt;16 microg/ml) to caspofungin with the goal of understanding the relationship between FKS1 mutations and echinocandin resistance. Of 85 resistant isolates recovered, all were identified with mutations in FKS1; 93% showed changes at Ser645, with 62% displaying a characteristic S645P substitution expressed as either a homozygous or a heterozygous mutation in FKS1. Two other prominent amino acid substitutions, S645Y and S645F, were found at frequencies of 22% and 8%, respectively. Three new mutations were also identified: T1922C, G1932T, and C1934G, encoding F641S, L644F, and S645C substitutions, respectively. One strain had the double amino acid substitution L644F and S645C. Allele-specific probes were combined in a multiplex assay for reliable screening of known FKS1 mutations. These data support the importance of FKS1p substitutions in echinocandin resistance and demonstrate the feasibility of applying molecular screening for routine resistance assessment.

Assessing Resistance to the Echinocandin Antifungal Drug Caspofungin in Candida albicans by Profiling Mutations in FKS1