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

Abstract

Rezafungin is a new long-acting echinocandin currently in phase 3 development. Epidemiological cutoff values are necessary for breakpoint setting but have not been established due to unexplained interlaboratory MIC variations observed in a prior multicenter study. Here we investigated if the choice of microtiter plates affected the variability when anidulafungin was included as a comparator. Testing by the EUCAST E.Def 7.3.1 reference method using tissue and cell culture-treated polystyrene plates (TC plates) and untreated polystyrene plates (UT plates) from four manufacturers was performed. Six control strains (Candida albicans, n = 3; C. krusei, n = 2; C. parapsilosis, n = 1) were tested (520 MICs). Subsequently, 5 or 6 wild-type isolates and 4 or 5 fks mutants of C. albicans, C. glabrata, C. krusei, C. parapsilosis (wild type only), and C. tropicalis were tested (930 MICs). For each strain-plate combination, ≥98% of the repetitive MICs were within 3 dilutions. The rezafungin modal MICs for the collated C. albicans control strain distributions were 0.016 mg/liter across TC plates but 0.03 mg/liter across UT plates, whereas they were 0.004 mg/liter and 0.016 mg/liter, respectively, for anidulafungin. The difference was most pronounced with Falcon plates and was not observed for C. krusei and C. parapsilosis. Eleven rezafungin MICs for mutants overlapped with the MICs for wild-type isolates (TC plates, n = 4; UT plates, n = 7). For anidulafungin, five overlaps (all UT plates) were observed. Most overlaps (rezafungin, n = 5; anidulafungin, n = 3) were caused by fks mutants of C. tropicalis (Fks1, F650F/L) and C. glabrata (Fks2. D666Y; rezafungin, n = 2; anidulafungin, n = 1). Interlaboratory variation was low. The use of TC plates resulted in lower MICs, particularly for C. albicans and Falcon plates, ad this was more often the case for anidulafungin than for rezafungin. Adoption of TC plates for EUCAST antifungal susceptibility testing would improve interlaboratory reproducibility and the separation of non-wild-type and wild-type strains.

Rezafungin In Vitro Activity against Contemporary Nordic Clinical Candida Isolates and Candida auris Determined by the EUCAST Reference Method

“…The echinocandins are highly potent compounds with a tendency to adhere to plastic. Microdilution testing of echinocandins has previously been associated with variation, as previously reported for caspofungin, anidulafungin, and rezafungin, and the data found here suggest that may also be the case occasionally for micafungin, a challenge that needs attention (6)(7)(8).…”

Activity of a Long-Acting Echinocandin, Rezafungin, and Comparator Antifungal Agents Tested against Contemporary Invasive Fungal Isolates (SENTRY Program, 2016 to 2018)

“…Although an essential agreement rate (Ϯ2 dilution steps) of 92.0% for C. albicans and 100.0% for C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei between CLSI and EUCAST methods for rezafungin was observed previously (31), alignment between CLSI and EUCAST susceptibility profiles and breakpoints is yet to be determined, as significant interlaboratory EUCAST MIC variability (likely attributed to nonspecific binding of the drug to plastics) has been identified for rezafungin against a more susceptible collection of Candida spp. (28,44).…”

Antimicrobial Susceptibility Testing of Antimicrobial Peptides to Better Predict Efficacy

“…The choice of laboratory plasticware is similarly problematic as there are a number of publications that indicate that cationic AMP bind to polypropylene, polystyrene and borosilicate glass, the most commonly used materials used in the manufacture of labware (Chico et al, 2003 ; Kristensen et al, 2015 ), although the extent of binding may be similar regardless of the type of material used (Sanchez-Gomez et al, 2008 ; Kristensen et al, 2015 ). This situation also applies to some antibiotics (see above) and even antifungals such as the echinocandins (Fothergill et al, 2016 ; Arendrup et al, 2019 ). In the study of Kristensen and co-workers, binding to the surface of the tubes (irrespective of material) was saturable and relatively more peptide bound at low concentrations, so a simple solution may be to keep peptide concentrations as high as possible to minimise the percentage lost to protein binding.…”