Improved Detection of Candida sp. fks Hot Spot Mutants by Using the Method of the CLSI M27-A3 Document with the Addition of Bovine Serum Albumin

Abstract

Echinocandins are highly bound to serum proteins, altering their antifungal properties. The addition of 50% human serum to the MIC assay improves the identification of echinocandin-resistant Candida spp. harboring fks hot spot mutations. However, this modification cannot readily be applied to the method of the CLSI M27-A3 document due to safety and standardization difficulties. The aim of this study was to evaluate commercial bovine serum albumin (BSA) as a safe and standardized alternative to human serum. A collection of 28 echinocandin-susceptible strains, 10 Candida parapsilosis sensu lato strains (with naturally reduced echinocandin susceptibility), and 40 FKS hot spot mutants was used in this work. When RPMI 1640 was used for susceptibility testing, wild-type strains and fks mutants showed MIC range overlaps (؊2, ؊1, and ؊3 2-fold-dilution steps separated these populations for anidulafungin, caspofungin, and micafungin, respectively). On the other hand, the addition of BSA to RPMI 1640 differentially increased echinocandin MIC values for these groups of strains, allowing better separation between populations, with no MIC range overlaps for any of the echinocandin drugs tested. Moreover, the use of RPMI-BSA reduced the number of fks hot spot mutant isolates for which MIC values were less than or equal to the upper limit for the wild type (very major errors) from 9, 2, and 7 with RPMI alone to 3, 0, and 3 for anidulafungin, caspofungin, and micafungin, respectively. When RPMI-BSA was used to study the susceptibility of C. parapsilosis sensu lato species to echinocandins, the strains behaved as anidulafungin-and micafungin-resistant isolates (MIC, >8 g/ml). These data support the need for a revision of the CLSI protocol for in vitro testing of echinocandin susceptibility in order to identify all or most of the fks hot spot mutants. Also, caspofungin could be used as a surrogate marker of reduced susceptibility to echinocandins.

Dose escalation studies with caspofungin against Candida glabrata

“…The decreased growth of our WT C. glabrata isolates in 50% serum is consistent with a previous report (Garcia-Effron et al, 2011), and the low in vivo growth rate suggests a relatively low virulence. Considering this, the extremely weak elimination capacity of the treatments was even more notable.…”

“…MICs of caspofungin (Sigma) in RPMI 1640 and in RPMI 1640 supplemented with 50% serum (hereafter referred to as 50% serum) (human serum from a male, type AB; Sigma) were determined simultaneously using the standard CLSI (2008) broth macrodilution method (Földi et al, 2012a, b;Garcia-Effron et al, 2011;Szilágyi et al, 2012). The caspofungin final concentration ranged between 0.03 and 32 mg l 21 .…”

“…Caspofungin activity was determined in RPMI 1640 and 50% human serum at 0.25, 1, 4, 8, 16 and 32 mg l 21 in a final volume of 10 ml (Földi et al, 2012a, b;Garcia-Effron et al, 2011;Kovács et al, 2014a, b;Nasar et al, 2013;Szilágyi et al, 2012). The starting inocula were 3.5|10 5 -5|10 5 c.f.u.…”

Comparison of Dimethyl Sulfoxide and Water as Solvents for Echinocandin Susceptibility Testing by the EUCAST Methodology

“…Two studies (4,12) have demonstrated that addition of bovine serum albumin to the medium increased the power of discrimination between wild-type and mutant isolates. It has also been suggested that the potency of these drugs may be affected by the solvent, given their relatively hydrophobic character.…”

Albumin Enhances Caspofungin Activity against Aspergillus Species by Facilitating Drug Delivery to Germinating Hyphae

“…In order to get mechanistic insight on the interaction of serum with caspofungin, we focused on albumin, the major serum protein component that binds highly to caspofungin (1,32,33). Indeed, we found that the positive interaction of serum with caspofungin was mediated almost exclusively by albumin.…”