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

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2011, p. 2245–2255 0066-4804/11/$12.00 doi:10.1128/AAC.01350-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved. Vol. 55, No. 5 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䌤† Guillermo Garcia-Effron,‡ Steven Park, and David S. Perlin* Public Health Research Institute, UMDNJ, Newark, New Jersey Received 4 October 2010/Returned for modification 1 December 2010/Accepted 23 February 2011 15, 16), which led us to propose new interpretative MIC breakpoints for the echinocandins against Candida species (for echinocandins against C. albicans, C. tropicalis, and C. krusei, ⱕ0.25 ␮g/ml for susceptibility, 0.5 ␮g/ml for intermediacy, and ⱖ1.00 ␮g/ml for resistance; for anidulafungin [ANF] and caspofungin [CSF] against C. glabrata, ⱕ0.25 ␮g/ml for susceptibility, 0.50 ␮g/ml for intermediacy, and ⱖ1.00 ␮g/ml for resistance; for micafungin [MCF] against C. glabrata, ⱕ0.06 ␮g/ml for susceptibility, 0.12 ␮g/ml for intermediacy, and ⱖ0.25 ␮g/ml for resistance; and for echinocandins against C. parapsilosis and C. guilliermondii, ⱕ2.00 ␮g/ml for susceptibility, 4.00 ␮g/ml for intermediacy, and ⱖ8.00 ␮g/ml for resistance) (27). In order to improve the detection of fks hot spot mutants with the CLSI susceptibility breakpoint, different options have been proposed: (i) reduce the ANF and MCF susceptibility breakpoints for C. albicans and C. glabrata (15, 16), (ii) use CSF as a surrogate marker for echinocandin cross-resistance and as the agent for the detection of fks hot spot mutations (5, 15, 16), (iii) identify fks hot spot mutants by testing for susceptibility to ANF with a susceptibility breakpoint defined as two 2-fold dilutions higher than the MIC50 (MIC at which 50% of isolates are inhibited) for the wild-type population of each Candida species (2), or (iv) add 50% human serum to the MIC assay medium in order to improve the identification of echinocandin-resistant Candida spp. harboring FKS mutations (15, 16). The last proposal more closely simulates the physiochemical properties of the active drug following intravenous administration, but it is difficult to apply in a clinical assay due to safety and standardization difficulties associated with human serum. Echinocandin drugs inhibit the 1,3-␤-D-glucan synthase complex (EC 2.4.1.34) (GS), which catalyzes the biosynthesis of 1,3-␤-D-glucan, the major glucan component of fungal cell walls. GS is an enzyme complex with at least two subunits, Fksp and Rho1p. Fksp, encoded by three related genes, FKS1, FKS2, and FKS3, is the catalytic subunit, and it is the target of the echinocandin drugs. Echinocandin resistance resulting in clinical failure has been linked to dominant mutations in the Fksp subunit of GS (27). These amino acid substitutions have been mapped onto two conserved regions of Fks1p (Candida spp.) and Fks2p (in Candida glabrata only) (13–16, 19, 27) named hot spot regions (e.g., C. albicans Fks1p hot spot 1 [Phe-641 to Pro-649] and hot spot 2 [Asp-1357 to Leu-1364]) (26). Recently, the CLSI Antifungal Subcommittee established a MIC of ⱕ2 ␮g/ml as an interpretative MIC breakpoint for the susceptibility of Candida spp. to the three echinocandin drugs (7, 33). However, it has been demonstrated that the CLSI breakpoints are not able to distinguish all the echinocandin-resistant fks mutants from wild-type (WT) isolates (2, * Corresponding author. Mailing address: Public Health Research Institute, New Jersey Medical School—UMDNJ, Newark, NJ 071033535. Phone: (973) 854-3200. Fax: (973) 854-3101. E-mail: perlinds @umdnj.edu. † Supplemental material for this article may be found at http://aac .asm.org/. ‡ Present address: Laboratorio de Micología y Diagnóstico Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral—CONICET, Santa Fe, Argentina. 䌤 Published ahead of print on 7 March 2011. 2245 Downloaded from http://aac.asm.org/ on November 25, 2019 by guest 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 (ⴚ2, ⴚ1, and ⴚ3 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. 2246 GARCIA-EFFRON ET AL. The aim of this study was to overcome this issue by evaluating commercial fatty-acid-free bovine serum albumin (BSA) as a safe and standardized alternative to human serum for better discrimination between susceptible strains and resistant clinical isolates harboring fks mutations. MATERIALS AND METHODS 1,3-␤-D-Glucan synthase complex inhibition curves. Inhibition curves and 50% inhibitory concentrations (IC50s) were determined using a sigmoidal response (variable-slope) curve and a two-site competition fitting algorithm with GraphPad Prism software, version 4.0 (13). The IC50s were obtained with and without 50% serum or 50 mg/ml BSA. Definitions. The isolates were classified into three groups depending on the FKS hot spot sequence. The Candida strains included in the wild-type (WT) group harbor the same FKS hot spot sequence as that described in the GenBank accession number for each of the Candida species. The speci (...truncated)