Precision and Accuracy of Fluconazole Susceptibility Testing by Broth Microdilution, Etest, and Disk Diffusion Methods

Abstract

Interpretive agreements among the results of fluconazole broth microdilution tests, Etests, and disk diffusion tests were documented by evaluating 495 Candida spp. Microdilution reference test results were in agreement with 96% of the Etest results; most discrepancies were minor differences. Fluconazole resistance of Candida krusei strains often required a full 48 h of incubation in order to be observed by the standard method. For the disk diffusion tests that were performed on Mueller-Hinton agar with glucose and methylene blue, 97% of results were in agreement with those of the reference test, especially when zones of inhibition were measured after the first 24 h of incubation. Some Candida glabrata isolates failed to grow satisfactorily until a full 48 h of incubation was completed. Precision was determined by testing 50 selected isolates in triplicate in each of three laboratories. The reproducibility of results of disk diffusion tests was comparable to that of the reference method. With all procedures, determination of test results was particularly challenging with some strains, and new methods are needed in order to improve endpoint definition.Compromised patients, such as those infected with human immunodeficiency virus, are often colonized by and/or infected with fungi, especially Candida spp. Consequently, they frequently receive antifungal agents such as fluconazole for relatively long periods of time during hospitalization. Prolonged exposure to an azole such as fluconazole can select strains with diminished susceptibilities (6, 7), and consequently, increasing doses may be necessary. For this reason, the susceptibilities of isolates recovered from compromised patients receiving prolonged fluconazole treatment should be monitored in order to determine when strains with decreased susceptibilities have been selected (3). In order to do this in a cost-effective way, simple, inexpensive testing procedures are needed, but such tests must be accurate and precise. The purpose of this report is to document the relative levels of accuracy and precision of three procedures for testing fluconazole: broth microdilution, Etest, and disk diffusion. The accuracy of each procedure was assessed by comparing the results to those obtained by the 48-h microdilution reference method of the National Committee for Clinical Laboratory Standards (NCCLS) (8). Precision was measured by performing replicate tests in three separate laboratories. MATERIALS AND METHODSMicroorganisms. For this study, M. A. Pfaller selected 495 clinical isolates of Candida spp. for which there was a broad range of fluconazole MICs. The five species that were represented are shown in Table 1. For testing reproducibility, 50 of the 495 isolates were selected in order to maximize the number of strains for which MICs were near the interpretive breakpoints of Յ8.0 g/ml (susceptible) and Ն64 g/ml (resistant). This subset of strains included 26 strains of Candida albicans and 6 strains each of Candida glabrata, Candida krusei, Candida parapsilosis, and Cand...

Susceptibilities of Candida albicans Mouth Isolates to Antifungal Agents, Essentials Oils and Mouth Rinses

“…Disk diffusion (DD) testing of amphotericin B, nystatin, ketoconazole, econazole, fluconazole and miconazole was performed as described by CLSI guidelines (M44-A protocol [21]) and [22] except the use of methylene blue at 0.5 lg/ml. Amphotericin B (10 lg), nystatin (50 lg), ketoconazole (15 lg), econazole (10 lg), fluconazole (25 lg) and miconazole (10 lg) were from Neo-sensitabs TM ROSCO Ò .…”

“…Quality control (QC) for DD and ET was performed by using C. albicans ATCC 90028 and Candida parapsilosis ATCC 22019 [22]. These species were included in all runs, and the results were within published limits.…”

Evaluation of the Etest and Disk Diffusion Methods for Determining Susceptibilities of 235 Bloodstream Isolates of Candida glabrata to Fluconazole and Voriconazole

“…Barry et al (3) demonstrated that both the disk diffusion and the Etest methods were accurate and precise when they were used to determine the fluconazole susceptibilities of 495 isolates of Candida spp. Although published data for the disk diffusion and Etest methods with voriconazole are limited (4, 9, 12), the results of both methods show good agreement with those of the reference broth microdilution (BMD) method.The studies to date that have documented the efficacies of agar-based methods for the testing of susceptibilities to fluconazole and voriconazole have generally included adequate numbers of Candida albicans isolates but relatively few C. glabrata isolates (1,3,8,17). Among the four most common species of Candida causing bloodstream infections (BSIs; C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis) (10), C. glabrata alone tends to be less susceptible to fluconazole, with a significant percentage of isolates classified as susceptible-dose dependent (S-DD; MIC, 16 to 32 g/ml) or resistant (R; MIC, Ն64 g/ml) (11).…”

Synthesis of 3-Substituted-clavams: Antifungal Properties, DD-Peptidase and β-Lactamase Inhibition

“…The mixture was then evaporated and the residue was purified on silica gel column using hexane - (3R,5S) and (3R,5R) 3-(Furyl-2Ј)-4-dethia-4-oxa-penam (32 and 33) A 6 : 1 mixture of 27 and 28 was subjected to intramolecular alkylation following procedure described for 29. The mixture of 32 and 33 was separated into pure (3R,5S,6R) 3-Benzyloxycarbonyl-6-methyl-4-dethia-4-oxapenam (34) To the solution of 29 (0.10 g, 0.5 mmol) in the mixture of CCl 4 (0.7 ml), acetonitrile (0.7 ml) and water (1.1 ml) sodium metaperiodate (1.5 g, 7.0 mmol) and RuCl 2 (0.02 g) were added. The mixture was stirred at room temperature for 40 minutes.…”

“…(Table 2) [34,35] All studies were conducted with Candida albicans ATTC 90028. The strain was grown 48 hours onto solid Sabourand's medium (Difco) at 35°C.…”