Determination of Echinocandin MICs for Candida Species in Less than 8 Hours: Comparison of the Rapid Susceptibility Assay with the Clinical and Laboratory Standards Institute's Broth Microdilution Assay

The echinocandins prevent fungal cell wall synthesis by inhibiting β-1,3-glucan synthesis, a significant glucose-consuming process. Previous studies suggested that echinocandin inhibitory activity is evident within 1 h of exposure. We hypothesized that a susceptibility assay based on glucose consumption may provide clinically useful MICs rapidly. The rapid susceptibility assay (RSA), which provides MICs in less than 8 h, was compared with the standard broth microdilution susceptibility assay (Clinical and Laboratory Standards Institute, document M27-A3, 2008) for 56 Candida species strains. Variables which are known to influence MICs determined by the M27-A3 method were also assessed for their effects on the RSA results. Excellent agreement (>90%) between the results of the RSA and M27-A3 methods was achieved for all three FDA-approved echinocandins (micafungin, caspofungin, and anidulafungin). Candida lusitaniae strains were responsible for most of the discordant results. Assay variables such as the test medium, the age of the inoculum culture, and the presence of human serum affected MIC results from the RSA and the M27-A3 method similarly. The RSA is equivalent to the standard M27-A3 method for determining echinocandin MICs for Candida species. The RSA provides MIC results in less than 8 h and can be applied to old and young yeast colonies. The assay could potentially provide clinically useful MICs on the same day that yeast growth from a specimen is first detected on solid medium.Echinocandins are the newest class of FDA-approved drugs for the treatment of systemic fungal infections and the first approved class of antifungals that target the cell wall, specifically the synthesis of β-1,3-glucan. The spectrum of fungi which are susceptible to echinocandins is narrower than the spectrum of fungi susceptible to the polyenes, despite the presence of β-1,3-glucan in the cell walls of many nonzygomycetous fungi. However, the echinocandins have proven to be excellent agents for the treatment of candidiasis. In 2004, the Infectious Diseases Society of America published a practice guideline in which echinocandins are listed as one of the first-line options for the treatment of candidemia (8).Similar to the actions of β-lactam antibacterial agents, the inhibition of cell wall polysaccharide synthesis by echinocandins potentially results in cell death. Echinocandins inhibit β-1,3-glucan synthase (GS) in the cell membrane by targeting the Fks1p and Fks2p components of GS complexes. As a consequence, β-1,3-glucan, which is the predominant polysaccharide in the cell walls of Candida species, is not produced while cell wall remodeling and cell expansion continue to occur, resulting in weakness in the wall and susceptibility to cell lysis due to overwhelming turgor pressure (6). A potential outcome of the events prior to cell death is diminution of glucose uptake or consumption.Clancy et al. (2) have shown that cells exposed to echinocandins for only 1 h at levels in excess of the known MIC for the strain exhibit growth inhibition, as demonstrated by decreased numbers of CFU compared to those of untreated controls. This observation suggested that it may be possible to determine MICs of echinocandins for different yeast strains on the same day of testing by using the rapid susceptibility assay (RSA), especially because the RSA is based on levels of glucose consumption by cells exposed to the drug relative to those by control cells (4, 11). The RSA is similar to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 broth microdilution assay except that the RSA inoculum concentration is higher, the incubation period is shorter, and the end point is based on glucose uptake measured spectrophotometrically (3, 4, 11).The RSA was originally used by Cutler et al. (4, 11) to provide MICs of fluconazole and amphotericin in less than 24 h. Their results showed that MICs of the rapidly acting antifungal agent amphotericin B can be obtained in 6 h but that the slower-acting triazole fluconazole requires at least 8 to 18 h. The RSA has since been applied to Aspergillus fumigatus to obtain MICs of voriconazole, itraconazole, and amphotericin B (12, 13).Here, we evaluate the ability of the RSA to provide echinocandin MICs equivalent to those obtained by the “gold standard” CLSI M27-A3 method, which requires an incubation period of 24 h, for Candida species on the same day of testing. Further, the RSA and M27-A3 method were compared to determine if the MICs provided are similarly affected by variations in inoculum growth conditions, the test medium, and the presence of human serum. The results demonstrate that the RSA method is comparable to the M27-A3 method but provides MICs in less than 8 h regardless of whether inoculum cells were prepared from 4- or 1-day-old cultures.