A novel and highly accurate diagnostic assay platform was established for rapid identification of FKS mutations associated with echinocandin resistance in Candida glabrata. The assay platform uses allele-specific molecular beacon and DNA melt analysis following asymmetric PCR. A dual assay for FKS1 and FKS2 was developed to identify within 3 h the most common and clinically relevant resistance-associated mutations, including 8 FKS1 HS1 (wild type [WT], S629P, F625S, D632Y, D632E [T1896G], D632E [T1896A], I634V, and F625F) and 7 FKS2 HS1 (WT, F659del, F659S, F659V, F659L, S663P, and S663F) genotypes. A blinded panel of 188 C. glabrata clinical isolates was tested by both assays. The molecular diagnostic results from the dual assay were 100% concordant with data obtained from DNA sequencing. This platform has the potential to overcome the deficiencies of existing in vitro susceptibility-based assays to identify echinocandin-resistant C. glabrata and holds promise as a surrogate diagnostic method to better direct echinocandin therapy.C andida glabrata is the second leading cause of candidemia in the United States, as well as in northern and eastern areas of Europe (1-3). With rapidly expanded usage of echinocandins, the first-line therapy for invasive candidiasis, an alarming trend of rising echinocandin resistance in C. glabrata has posed a serious clinical challenge (4-6). Detection of echinocandin resistance can be assessed phenotypically, using either microdilution or disc diffusion MIC assays performed in accordance with guidance of the Clinical and Laboratory Standards Institute (CLSI) M27-A3 standard (7) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) Definitive Document Edef 7.2 (8). Current echinocandin clinical breakpoints for C. glabrata were established by the CLSI for all echinocandins and by EUCAST for micafungin and anidulafungin. Breakpoints were specifically developed to identify C. glabrata harboring FKS mutations by considering multiple factors, such as -1,3-D-glucan synthase enzyme kinetics and echinocandin pharmacokinetic and pharmacodynamic data (6, 9). However, despite standardized methodologies, important limitations with the in vitro susceptibility testing cannot be ignored: first, the assay has a time-consuming setup and intrinsically slow turnaround time, requiring 24 to 48 h after isolate recovery; second, interlaboratory variability of caspofungin MICs has limited direct testing of this drug (10); and third, susceptible and resistant populations overlap (11).Clinical echinocandin resistance in C. glabrata is associated with amino acid substitutions caused by mutations in specific hotspot (HS) regions of FKS1 and FKS2, which encode the drug target -1,3-D-glucan synthase. A recent study performed among patients with invasive candidiasis due to C. glabrata has shown that the FKS genotype is a better indicator of echinocandin therapeutic failure than MIC (12).To date, DNA sequencing is the only means to identify mutations within FKS1 and FKS2 genes. Sequence data a...