Effects of Treated versus Untreated Polystyrene on Caspofungin In Vitro Activity against Candida Species

Abstract

Significant interlaboratory variability is observed in testing the caspofungin susceptibility of Candida species by both the CLSI and EUCAST broth microdilution methodologies. We evaluated the influence of treated versus untreated polystyrene microtiter trays on caspofungin MICs using 209 isolates of four Candida species, including 16 C. albicans and 11 C. glabrata isolates with defined FKS mutations. Caspofungin MICs were also determined using the commercially available YeastOne and Etest assays and 102 isolates. All C. glabrata isolates had caspofungin MICs of >0.5 g/ml, the clinical breakpoint for caspofungin resistance in this species, measured using trays made of treated polystyrene, regardless of the FKS status. In contrast, susceptible isolates could readily be distinguished from resistant/non-wild-type isolates when caspofungin MICs were measured using untreated polystyrene trays and both the YeastOne and Etest assays. Similar results were also observed for C. krusei isolates, as all isolates had caspofungin MICs above the threshold for resistance measured using treated polystyrene trays. In contrast, C. albicans isolates could be correctly identified as susceptible or resistant when caspofungin MICs were measured with treated or untreated trays and with the YeastOne and Etest assays. MICs falsely elevated above the resistance breakpoint were also not observed for C. tropicalis isolates. These results demonstrated that the use of treated polystyrene may be one factor that leads to falsely elevated caspofungin in vitro susceptibility results and that this may also be a greater issue for some Candida species than for others. Significant interlaboratory variability has been reported for caspofungin susceptibility testing, with some laboratories reporting MIC values elevated above the revised CLSI clinical breakpoints (1). The interlaboratory variability is not without consequences, as it may result in major errors for caspofungin use (i.e., falsely interpreting an isolate as resistant to this echinocandin). In a retrospective review of our antifungal susceptibility database using data from 2008 to 2012, we found that 95.6% of C. glabrata and 74.3% of C. krusei isolates would be classified as resistant to caspofungin on the basis of the revised CLSI echinocandin clinical breakpoints (2). Other laboratories have reported similar results (3). As the factors that lead to interlaboratory variability are unknown, we sought to determine if the choice of plastic used in the microtiter trays, either treated or untreated polystyrene, could affect caspofungin MIC values. These trays are designed for cell cultures, and treatment of the plastic, either by coating or corona discharge to impart an electrical charge to the plastic, allows the cells to adhere. Our laboratory has historically used 96-well microtiter trays made of treated polystyrene, while other laboratories have used those made of untreated plastic. The CLSI M27-A3 standard for yeast antifungal susceptibility testing has not addressed plastic type or recomm...

EUCAST Reference Testing of Rezafungin Susceptibility and Impact of Choice of Plastic Plates

“…The wavelength range for plate reading used by the laboratories generating high MICs overlapped the wavelength range used by the laboratories generating low MICs. Finally, all four centers used treated (tissue or cell culture-treated) polystyrene plates (TC plates) to avoid the variation that has previously been associated with echinocandin MIC testing across TC plates and untreated polystyrene plates (UT plates) (6). One consideration was that the pure substance was shipped without temperature control.…”

Effects of Culture Conditions (Microplate and Solvent) on <i>in vitro</i> Antifungal Activity of Caspofungin Against <i>Candida</i> Species

“…In addition, these issues may affect revisions of the future CBPs based on accurate and stable susceptibility surveillance data. No clear single factor-such as CPFG lot, stock solution solvent, CPFG powder storage method, medium, or evaluation method-was found to influence CPFG MICs 9) , while there were some reports of the effects of the surface treatment of microplates and the solvent for preparation of drug substance 11,12) . The present study focused on the surface treatment of the microplates (treated or untreated) and the solvent used to dissolve the drug (water or DMSO) as factors that may affect the susceptibility of three Candida species to CPFG.…”

“…6) recommends that susceptibility testing to other echinocandins or genetic screening in resistant mutations should be conducted if a strain is found to be resistant or have intermediate resistance to CPFG because resistance to echinocandins is mainly induced by mutation of FKS genes, and cross-resistance has been reported in the echinocandin antifungal agent class. Recently, Fothergill et al showed that surface-treated microplates were associated with higher CPFG MICs than non-surface-treated microplates in susceptibility against Candida species 11) . Different range and variability of CFPG MICs were also reported between the solvents for stock solution 12) .…”

“…3870-096; plate was treated using discharge apparatus to increase hydrophobicity) and surface-untreated (untreated; Code No. 3875-096) U-shaped bottom microplates manufactured by IWAKI (AGC TECHNO GLASS Co., Ltd.) were compared 11) . Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258 were assayed as quality control strains.…”

Hot Topics in Antifungal Susceptibility Testing: a New Drug, a Bad Bug, Sweeping Caspofungin Testing under the Rug, and Solving the Epidemiological Cutoff Value Shrug

“…This is not seen when testing the same isolates with micafungin or anidulafungin. Although some studies have attempted to determine the cause of this discrepancy such as testing different lots of caspofungin and using different types of microtiter plates, the overall cause has remained elusive (42). This phenomenon is an in vitro artifact and has no bearing on the clinical treatment of patients: there is no evidence that an isolate can be resistant to one echinocandin but susceptible to another.…”