An evaluation of seven methods of testing in vitro susceptibility of clinical yeast isolates to fluconazole

Schmalreck, A.; Kottmann, I.; Reiser, Anita; Ruffer, U.; Scharr, E.; Vanca, E.

doi:10.1111/j.1439-0507.1995.tb00065.x

Cited by 18 publications

(8 citation statements)

References 11 publications

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“…On the other hand, based on the results of a multicentre study of the working group ‘Clinical Mycology’ of the German Speaking Mycological Society concerning the susceptibility testing of fluconazole a proposal for a standardized microdilution method using the semisynthetic HR (high resolution) medium (Oxoid, CM 845) has been developed and published [5, 11]. Contrary to the above mentioned NCCLS method, HR medium supplemented with glucose and asparagine but without sodium hydrogen carbonate is used.…”

Section: Discussionmentioning

confidence: 99%

In vitro susceptibility of yeasts for fluconazole and itraconazole. Evaluation of a microdilution test

Nenoff

Oswald

Haustein

1999

Mycoses

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In vitro susceptibilities were determined for a total of 159 clinical isolates and 12 reference strains of yeasts belonging to different Candida species including 94 Candida albicans strains, and further genera such as Cryptococcus, Trichosporon, Geotrichum and Saccharomyces. Minimum inhibitory concentration (MIC) values for fluconazole and itraconazole were assessed using a microdilution technique with the semisynthetic high resolution (HR) medium supplemented with glucose and asparagine but without sodium hydrogen carbonate (pH 7.0), according to a proposal of the working group 'Clinical Mycology' of the German Speaking Mycological Society. Fluconazole MIC values for C. albicans were between 0.125 and > or = 128 micrograms ml-1. Thus, the median of 1 microgram ml-1 showed that the overall fluconazole susceptibility was good. As expected, Candida krusei (seven strains) exhibited diminished in vitro susceptibility with MIC values for fluconazole of 8 to 128 micrograms ml-1 with a median of 64 micrograms ml-1. Some Candida kefyr strains seemed to be less susceptible against fluconazole which was indicated by a MIC90 of 64 micrograms ml-1. Surprisingly, no Candida glabrata isolate exhibited a MIC value greater than 16 micrograms ml-1. Other Candida species, Trichosporon cutaneum, Geotrichum candidum and Saccharomyces cerevisiae showed low MICs to fluconazole. In vitro susceptibility testing of itraconazole revealed that all Candida species except C. albicans, but also Trichosporon cutaneum, Geotrichum candidum, and Saccharomyces cerevisiae exhibited acceptable low MIC values against itraconazole (0.03-2 micrograms ml-1). Their MIC90 values for itraconazole were in the close range between 0.125 and 2 micrograms ml-1. MIC values between 0.125 and 2 micrograms ml-1 were obtained, even for C. krusei strains. On the other hand, the range of C. albicans MICs was between 0.0125 and > or = 16 micrograms ml-1 with MIC50 and MIC90 values of 0.125 and > or = 16 micrograms ml-1, respectively, indicating that a considerable number of yeast strains have high MICs. The comparative evaluation of different experimental conditions revealed that there exists a marked influence both of inoculum size and incubation time on the results of susceptibility testing. Therefore, for routine usage 10(2) CFU ml-1 and 18-24 h incubation time for this microdilution method with HR medium are recommended.

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Section: Discussionmentioning

confidence: 99%

In vitro susceptibility of yeasts for fluconazole and itraconazole. Evaluation of a microdilution test

Nenoff

Oswald

Haustein

1999

Mycoses

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“…To this end, in vitro conditions, including medium (14) and use of an 80% inhibition (as opposed to complete inhibition) endpoint (5), have been manipulated in attempts to define conditions under which the FCZ antifungal effect can be better expressed and which might better correlate with in vivo results. Our data would suggest testing in serum, which more closely approximates physiologic conditions, would represent a direction for such attempts.…”

Section: Discussionmentioning

confidence: 99%

Different components in human serum inhibit multiplication of Cryptococcus neoformans and enhance fluconazole activity

Nassar

Brummer

Stevens

1995

Antimicrob Agents Chemother

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The inhibitory effect of human serum on the multiplication of Cryptococcus neoformans and the interaction with fluconazole were studied. Compared with cryptococcal multiplication in RPMI 1640 medium alone, 5% human serum in medium inhibited multiplication by 76% ؎ 6% (n ‫؍‬ 8). The inhibitory effect of human serum was donor independent heat stable (56؇C, 30 min), and not due to albumin or globulin. Bovine and murine sera were not inhibitory at that concentration. A fungistatic concentration of fluconazole (5.0 g/ml) in medium plus 5% human serum resulted in 40% ؎ 5% (n ‫؍‬ 8) killing (reduction of inoculum CFU) in a 24-h assay. Bovine or murine sera did not have the enhancing effect, and this human serum activity was heat stable and donor independent. At 2.5 g of fluconazole per ml, fungistasis by fluconazole plus human serum was significantly greater than with either alone. Higher concentrations in serum potentiated fluconazole more. At higher fluconazole concentrations (e.g., 20 g/ml) fluconazole alone could kill, but serum potentiated this. A fluconazole-resistant isolate (MIC, 100 g/ml) was not killed by fluconazole (5.0 g/ml) in 5% human serum, but human serum potentiated the partial fluconazole inhibition. When human serum was dialyzed (molecular weight cutoff, 6,000 to 8,000) against phosphate-buffered saline, it lost the ability to synergize with fluconazole for killing Cryptococcus organisms but not the capacity to inhibit multiplication. Filtration of serum suggested the filtrate with a molecular weight of <10,000 could interact synergistically with fluconazole for killing but could not inhibit cryptococcal multiplication. These findings indicate that human serum has two components, one (macromolecular) with a unique ability to inhibit C. neoformans and a low-molecular-weight component that enhances fluconazole anticryptococcal activity.In earlier studies we showed that fungistatic concentrations of fluconazole (FCZ) acted synergistically with fungistatic murine macrophages for killing Cryptococcus neoformans (2, 3). In subsequent studies with human macrophages (12), we used human serum in the culture system and noted that FCZ concentrations previously found to be fungistatic were now fungicidal. In the present study, we systematically investigated this phenomenon, in light of prior observations (1, 9, 11) that serum alone is inhibitory to C. neoformans. MATERIALS AND METHODSC. neoformans. C. neoformans (CDC 9759) was transferred from storage at 4ЊC or room temperature and subcultured twice on blood agar plates (BAP) at 35ЊC before use in experiments. Yeast cells grown on BAP for 48 h at 35ЊC were collected, washed, counted, and suspended in test media. RPMI 1640 containing penicillin (100 U/ml) and streptomycin (100 g/ml) was the basic test medium (referred to as RPMI). Fresh frozen human serum, human serum heated (56ЊC, 30 min) to inactivate complement (referred to as heat-inactivated serum), commercial pooled blood group AB serum (Gibco, Gaithersburg, Md.), fetal bovine serum (FBS), or mouse serum w...

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“…These kits provide reagents for testing a limited number of drug concentrations selected as critical breakpoint values. Direct comparisons have found these methods to possess limited correlation with the M27-A reference method (42,165,196,208,226,229). ATB Fungus (API-bioMerieux, Marcy l'Etoile, France), Mycostandard (Institut Pasteur, Paris, France), and Mycototal (Behring Diagnostic, Rueil-Malmaison, France) are similarly designed systems for which only limited data are available on correlation with the M27-A methodology (51,84,174,196).…”

Section: Methods For Susceptibility Testing Nccls Broth-based Methodomentioning

confidence: 99%

“…A commercial disk system (Neo-Sensitabs; Rosco Diagnostica, Taastrup, Denmark) is available, but the results were not found to correlate well with the reference method (25,202,208,226). Reports on the Diff Test (Diagnostics Pasteur, Paris, France) disk diffusion system and a spiral gradient method did not include comparisons with the reference method (51,196).…”

Section: Other Agar-based Testing Methodsmentioning

confidence: 99%

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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An evaluation of seven methods of testing in vitro susceptibility of clinical yeast isolates to fluconazole

Cited by 18 publications

References 11 publications

In vitro susceptibility of yeasts for fluconazole and itraconazole. Evaluation of a microdilution test

In vitro susceptibility of yeasts for fluconazole and itraconazole. Evaluation of a microdilution test

Different components in human serum inhibit multiplication of Cryptococcus neoformans and enhance fluconazole activity

Antifungal Susceptibility Testing: Practical Aspects and Current Challenges

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