Collaborative Study of the NCCLS and Flow Cytometry Methods for Antifungal Susceptibility Testing of Candida albicans

Abstract

One hundred clinical isolates of Candida albicans were tested for amphotericin B and fluconazole susceptibilities by the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution test at center 1 (C1). The same isolates were tested blinded at center 2 (C2) by NCCLS and flow cytometry (FC) methods. The agreement between NCCLS and FC methods ranged from 96 to 99%.In 2002, the National Committee for Clinical Laboratory Standards (NCCLS) published a standardized method for antifungal susceptibility testing of Candida spp. and Cryptococcus neoformans (M27-A2 [7]). This is a reliable method for susceptibility testing of yeasts for interlaboratory correlation; standardization of variables like medium, inoculum size, MIC endpoints, and temperature and duration of incubation; and correlation of the MICs (fluconazole and itraconazole) with clinical outcome in candidiasis (14). However, this method is somewhat labor-intensive, unreliable for detection of amphotericin B resistance, and prone to the trailing-growth phenomenon with azole antifungals (9,14). Alternative methods such as spectrophotometry (1, 3), colorimetry (10, 12), agar-based assay (2, 11), and flow cytometry (FC) (4,5,6,8,13,17) are being evaluated. The objectives of the present study were to evaluate the FC method by using isolates previously tested in another laboratory and to determine interlaboratory agreements between center 1 (C1; University of Iowa College of Medicine, Iowa City) and center 2 (C2; Mycology Laboratory, Wadsworth Center, Albany, N.Y.).One hundred Candida albicans isolates were received blinded at C2 from C1. These isolates were routinely tested by the NCCLS M27-A2 protocol at C1. The cultures were maintained in sterile water at 4°C. Before the assays, the cultures were passaged twice on Sabouraud dextrose agar at 35°C. Amphotericin B was purchased from Sigma (St. Louis, Mo.), and fluconazole was a gift from Pfizer Pharmaceuticals (New York, N.Y.). Stock solutions of amphotericin B and fluconazole were prepared in dimethyl sulfoxide (Amresco, Solon, Ohio) and water, respectively, and were stored at Ϫ70°C. The broth microdilution test for amphotericin B (0.03 to 16.0 g/ml) and fluconazole (0.06 to 64.0 g/ml) was done according to the NCCLS M27-A protocol (7). The endpoints were a 100% optically clear well for amphotericin B and 50% growth inhibition compared to drug-free wells for fluconazole (7).The FC assay was performed as described earlier (13). Briefly, serial twofold dilutions of two drugs were prepared in RPMI 1640 containing L-glutamine without bicarbonate, buffered to pH 7.0 with MOPS (morpholinepropanesulfonic acid). Yeast suspensions were prepared in 0.85% sterile saline and adjusted spectrophotometrically to match an 0.5 McFarland density. One-half milliliter of the yeast suspension was added to 0.5 ml of serial drug dilutions and incubated at 35°C for 2 h for amphotericin B and 4 h for fluconazole according to the rationale provided in an earlier publication from our laboratory (13). The growth control ...

Comparison of Two Probes for Testing Susceptibilities of Pathogenic Yeasts to Voriconazole, Itraconazole, and Caspofungin by Flow Cytometry

“…This effect only occurred in the resistant strains when challenged with the highest drug concentrations (4 to 16 g/ml); the effect was similar to that observed previously with fluconazole (14) and flucytosine (19,14). We were able to identify itraconazole categorical endpoints after 1 h of incubation with this agent; the overall time in other studies for this incubation period is 4 to 6 h for yeasts (20,2) and 2 to 3 h for A. fumigatus (21).…”

“…An optimized cytometric assay to study the susceptibility of Candida spp. with incubation for 2 h with amphotericin B and 4 h with fluconazole has been described recently and shows a good correlation with the CLSI method (2).…”

Novel Method for Evaluating In Vitro Activity of Anidulafungin in Combination with Amphotericin B or Azoles

“…Flow cytometry (FC) is a valuable tool for studying antifungal susceptibility, since it can be used to detect different physiological cell stages by using the appropriate fluorescent markers (5,8). FC susceptibility testing to azoles, amphotericin B (AMB), and echinocandins has already been described (23-25, 27, 30).…”

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

“…However, this technique introduces a subjective decision that causes variable results from person to person and between laboratories [18]. To overcome this limitation, several modifications have been adopted as broth-based alternative approaches that may better serve practical clinical laboratory needs; such as spectrophotometric method [14,15], colorimetric method [11][12][13], flow cytometry [6,7], and E test [8][9][10]. Among these, the spectrophotometric reading of broth microdilution tests provides a more accurate and objective result and eliminates the subjective judgments that can confuse visual assessment of MIC endpoints [15].…”

“…Recently, several alternative methods such as flow cytometry [6,7], E test [8][9][10], colorimetric assay [11][12][13], and spectrophotometric reading of broth microdilution [14,15] have been used in an attempt to obtain an objective and convenient interpretation of antifungal susceptibility results. Among these options, a modified broth microdilution format using spectrophotometry complemented by visual reading was accepted in CLSI M27-A2 for determining the MICs of such organisms, which is the first well that shows a 50% decrease in optical density (OD) relative to the drug-free growth control well [4].…”