Collaborative investigation of broth microdilution and semisolid agar dilution for in vitro susceptibility testing of Candida albicans

Abstract

A study was performed in two laboratories to evaluate the effect of growth medium and test methodology on interand intralaboratory variations in the MICs of amphotericin B (AMB), flucytosine (5FC), fluconazole (FLU), itraconazole (ITRA), and the triazole Sch 39304 (SCH) against 14 isolates of Candida albicans. Testing was performed by broth microdilution and semisolid agar dilution with the following media, buffered to pH 7.0 with morpholinepropanesulfonic acid (MOPS): buffered yeast nitrogen base (BYNB), Eagle's minimal essential medium (EMEM), RPMI 1640 medium (RPMI), and synthetic amino acid medium for fungi (SAAMF). Inocula were standardized spectrophotometrically, and endpoints were defined by the complete absence of growth for AMB and by no more than 25% of the growth in the drug-free control for all other agents. Comparative analyses of median MICs, as determined by each test method, were made for all drug-medium combinations. Both methods yielded similar (±1 twofold dilution) median MICs for AMB in EMEM and RPMI, 5FC in all media, and FLU in EMEM, RPMI, and SAAMF. In contrast, substantial between-method variations in median MICs were seen for AMB in BYNB and SAAMF, FLU in BYNB, and ITRA and SCH in all media. Interlaboratory concordance of median MICs was good for AMB, 5FC, and FLU but poor for fTRA and SCH in all media. Endpoint determinations were analyzed by use of kappa statistical analyses for evaluating the strength of observer agreement. Moderate to almost perfect interlaboratory agreement occurred with AMB and 5FC in all media and with FLU in EMEM, RPMI, and SAAMF, irrespective of the test method. Slight to almost perfect interlaboratory agreement occurred with ITRA and SCH in EMEM, RPMI, and SAAMF when tested by semisolid agar dilution but not broth microdilution. Kappa values assessing intralaboratory agreement between methods were high for 5FC in all media, for AMB in BYNB, EMEM, and RPMI, and for FLU in EMEM, RPMI, and SAAMF. One laboratory, but not the other, reported substantial to almost perfect agreement between methods for ITRA and SCH in EMEM, RPMI, and SAAMF. Both laboratories reported poor agreement between methods for the azoles in BYNB. Discrepancies noted in azole-BYNB combinations were largely due to the greater inhibitory effect of these agents in BYNB than in other media. These results indicate that the semisolid agar dilution and broth microdilution methods with EMEM or RPMI yield equivalent and reproducible MICs for AMB, 5FC, and FLU but not ITRA and SCH.

Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance

“…The MICs of fluconazole, itraconazole, ketoconazole, and amphotericin B for FR2 and other azole-resistant strains of C. albicans were determined ( Table 2). The strains were designated as susceptible or resistant to each antifungal agent according to the reported ranges of the MICs of the antifungal agents for susceptible C. albicans strains (14,33,39,47). Resistant organisms were defined as those for which the MICs of on July 16, 2020 by guest http://aac.asm.org/ the following antifungal agents were as indicated: fluconazole, Ͼ10 g/ml; itraconazole, Ͼ2 g/ml; ketoconazole, Ͼ0.5 g/ml; and amphotericin B, Ͼ1 g/ml.…”

Emergence of fluconazole-resistant strains of Candida albicans in patients with recurrent oropharyngeal candidosis and human immunodeficiency virus infection

“…Antifungal susceptibility tests with FCZ and other azoles were performed in large collaborative studies (8,29,36,41) which showed good inter-and intralaboratory reproducibilities for certain testing methods (e.g., the macrodilution technique with HR medium (27). The major sources of antifungal susceptibility test variation for FCZ and the other azoles arise from differences in the manner in which the test is conducted (e.g., the pH of the medium, medium composition, inoculum size, incubation time, and incubation temperature) (35).…”

“…Some earlier reports have already described azole resistance in Candida species (17,30,38,44), but recently, concerns have been raised regarding the development of resistance to FCZ in C. albicans strains isolated from AIDS patients. However, in the majority of those studies, no correlations were established between in vitro susceptibility data and in vivo treatment response (7,10,18,25,30,36,46). Some studies have presented data on C. albicans isolates and compared the in vitro susceptibility findings with the clinical findings from patients with HIV infection and so-called fluconazole-resistant OPC (1,2,31).…”

Comparison of broth macrodilution, broth microdilution, and E test antifungal susceptibility tests for fluconazole

“…A broth microdilution method performed according to NCCLS guidelines demonstrated good agreement with the reference broth macrodilution method (10,14). A recent study comparing colorimetric broth microdilution testing of fluconazole with reference broth macrodilution and broth microdilution methods demonstrated excellent agreement among the three methods (10).…”