Amphotericin B susceptibility testing of Candida lusitaniae isolates by flow cytofluorometry: comparison with the Etest and the NCCLS broth macrodilution method

Favel, Anne; Peyron, F; Méo, Michel De; Michel-Nguyen, A.; Carrière, Jessica; Chastin, Christiane; Régli, P.

doi:10.1093/jac/43.2.227

Cited by 20 publications

(13 citation statements)

References 18 publications

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“…(2,69,79,146,(161)(162)(163)(164). Staining (or lack of staining) with suitable dyes permits the rapid detection of damaged fungi.…”

Section: Flow Cytometry and Use Of Viability Dyesmentioning

confidence: 99%

“…Recent work has further explored this possibility (80,88,97,151,175,176,223) and shown the potential for correlation of flow cytometry-based techniques with the reference method. One study suggested that flow cytometry might be especially useful for detection of amphotericin B resistance (69). In conceptually related studies, fluorescent viability dyes have been used to examine the nature of drug-induced damage to yeasts and moulds (50, 111) and to estimate MICs and minimal fungicidal concentrations (MFCs) for molds (105).…”

Section: Flow Cytometry and Use Of Viability Dyesmentioning

confidence: 99%

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Antifungal Susceptibility Testing: Practical Aspects and Current Challenges

Rex¹,

et al. 2001

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Development of standardized antifungal susceptibility testing methods has been the focus of intensive research for the last 15 years. Reference methods for yeasts (NCCLS M27-A) and molds (M38-P) are now available. The development of these methods provides researchers not only with standardized methods for testing but also with an understanding of the variables that affect interlaboratory reproducibility. With this knowledge, we have now moved into the phase of (i) demonstrating the clinical value (or lack thereof) of standardized methods, (ii) developing modifications to these reference methods that address specific problems, and (iii) developing reliable commercial test kits. Clinically relevant testing is now available for selected fungi and drugs: Candida spp. against fluconazole, itraconazole, flucytosine, and (perhaps) amphotericin B; Cryptococcus neoformans against (perhaps) fluconazole and amphotericin B; and Aspergillus spp. against (perhaps) itraconazole. Expanding the range of useful testing procedures is the current focus of research in this area

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“…(2,69,79,146,(161)(162)(163)(164). Staining (or lack of staining) with suitable dyes permits the rapid detection of damaged fungi.…”

Section: Flow Cytometry and Use Of Viability Dyesmentioning

confidence: 99%

Section: Flow Cytometry and Use Of Viability Dyesmentioning

confidence: 99%

Antifungal Susceptibility Testing: Practical Aspects and Current Challenges

Rex¹,

et al. 2001

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“…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.).…”

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confidence: 99%

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

2004

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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 ...

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“…The laboratory detection of amphotericin B-resistant strains remains problematic, notably with the widely used National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 procedure (12,13). Alternatives, such as antibiotic medium 3 (AM3) with the M27-A2 method, Etest with a predefined gradient of drug concentration, Iso-Sensitest broth, and flow cytometry (FC) have been used to discriminate between amphotericin B-susceptible and -resistant strains (2,4,5,7,9,11,14). Etest with AM3 agar did not yield good results with susceptible and resistant strains, but Etest with RPMI provided reliable discrimination (9).…”

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confidence: 99%