Correlation between E-Test, Disk Diffusion, and Microdilution Methods for Antifungal Susceptibility Testing of Fluconazole and Voriconazole

Abstract

The activities of fluconazole and voriconazole against isolates of Candida spp. (n ‫؍‬ 400) were tested by the E-test, disk diffusion, and the National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 broth microdilution-based reference methods. More than 96% of isolates found to be susceptible to fluconazole by the reference method were identified as susceptible by the agar-based methods. Lesser degrees of correlation with the reference method were seen for isolates identified as resistant by the agar-based methods. Interpretive categories are not available for voriconazole, but results qualitatively similar to those for fluconazole were seen. The agar-based E-test and disk diffusion methods are reliable alternatives to the NCCLS M27-A2 reference microdilution method for isolates that test susceptible to fluconazole.The development of standardized antifungal susceptibility testing methods has been the subject of numerous studies during the last decade. Reference methods for yeasts (the National Committee for Clinical Laboratory Standards [NCCLS] M27-A2 method) and molds (the NCCLS M38-A method) are now available (16). Agar-based susceptibility testing methods have been a focus of interest for many researchers and include the classical disk diffusion (DD) methods and the E-test (ET) method (3, 6-10, 13, 14, 16-18). Those tests are very attractive due to their simplicity, reproducibility, and lack of requirements for specialized equipment (11,16). Recent studies have documented comparable results between those methods and the results of standard reference broth microdilution (MD) susceptibility testing (7,11,13).In this study, we compared the NCCLS M27-A2 MD method with the ET and DD methods for determination of the susceptibilities of 400 Candida species isolates to fluconazole and voriconazole. The ET and DD methods are well studied for fluconazole (3,(6)(7)(8)(9)(10)(11)14), and this work extends their usage to include voriconazole. MATERIALS AND METHODSIsolates. Four hundred bloodstream isolates of Candida species were randomly selected for testing. These included 205 isolates of Candida albicans, 56 isolates of C. tropicalis, 39 isolates of C. glabrata, 66 isolates of C. parapsilosis, 24 isolates of C. krusei, and 10 isolates of other species. The isolates were identified with the API 20C AUX system (Biomerieux Vitek, Hazelwood, Mo.) and were subsequently stored in sterile distilled water at room temperature until susceptibility tests were performed. Each isolate was subcultured at least twice on Sabouraud dextrose agar and incubated at 35°C prior to testing to ensure purity and optimal growth.Inoculum suspensions. Yeast inoculum suspensions were prepared as described for the NCCLS M27-A2 method (12). The turbidity was measured with a spectrophotometer at 530 nm and was adjusted to match a 0.5 McFarland density standard, resulting in a concentration of 1 ϫ 10 6 to 5 ϫ 10 6 yeast cells/ml. This inoculum was used directly for inoculation of agar plates (see below) or was diluted as needed for the MD ...

Evaluation of glucose-methylene-blue-mueller-hinton agar for E-test minimum inhibitory concentration determination in <i>Candida </i>spp.

“…Requirement of additional equipment (spectrophotometer) is also eliminated. 4 The results obtained by using both media were in acceptable concordance (>93%) with those obtained by MBD method. Similar Þ ndings were corroborated by previous studies.…”

“…5 It was observed in the current and prior studies that GMB-MHA has enhanced growth, simpliÞ ed reading and minimal microcolonies around zone of inhibition. 1,4 Thus GMB-MHA appears to be a useful medium for E-test, as it compared well with BMD and RPMI-G (except a single C. tropicalis outlier despite repeat testing). The stock-solution of GMB can be refrigerated and added to the molten and cooled MHA which is made routinely.…”

“…Similar Þ ndings were corroborated by previous studies. 4 Disparate readings were attributable to 'trailing growth phenomenon' in an isolate of C. tropicalis. This discrepancy was also observed in prior studies.…”

Resistência a antifúngicos de Candida tropicalis isoladas no Estado do Ceará

“…A resistência a azólicos entre cepas de Candida tropicalis isoladas no estado do Ceará ainda é baixa e o teste de disco de difusão é uma alternativa ao método de referência sendo mais prático e menos oneroso 8 . …”

The Optimal Wavelength of Spectrophotometric Broth Microdilution Antifungal Susceptibility Test for Flucytosine and Three Azoles

“…However, in previous articles, the wavelength was diverse: 405 nm [15,19], 450 nm [20], 492 nm [14], 530 nm [21,22], and 570 nm [23]. There was no reason explained for the selection of a specific wavelength.…”