1 Clinical Microbiology Laboratory, and 2 Infectious Disease Unit, Shaare Zedek Medical Center, Jerusalem, affiliated with the Faculty of Health Sciences, Ben Gurion University of the Negev, Be'er Sheva; 3 Infectious Disease Unit, Sheba Medical Center, Tel Aviv, Israel
Received 15 March 2004; returned 9 August 2004; revised 14 September 2004; accepted 4 October 2004
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Abstract |
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Methods: Reference and laboratory strains of Candida were tested for susceptibility to fluconazole and echinocandin by fluorescent flow cytometry using Acridine Orange as indicator of viability. Flow cytometry results were compared with MICs as determined by macrodilution and/or Etest.
Results: Seventy Candida strains were tested for susceptibility to fluconazole, and 74 strains for susceptibility to echinocandin. Minimal concentration of fluconazole causing 40% cell damage, as determined by flow cytometry, showed excellent association with MIC, as determined by other methods. The flow method, completed within 5 h, had excellent sensitivity and specificity to distinguish between sensitive, susceptible dose-dependent and resistant strains. The flow cytometry method for echinocandin was completed within 3 h, and minimal concentration causing 50% cell damage was associated with MIC as determined by macrodilution.
Conclusions: Antifungal susceptibility testing by FACS is a reliable, rapid method for determining susceptibility of Candida to fluconazole and echinocandin. The method allows same-day results, assisting in the selection of appropriate antifungal therapy.
Keywords: fungal , MIC , FACS , fluconazole , echinocandin
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Introduction |
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The standard method for fungal susceptibility testing for azoles is the broth dilution method proposed by the NCCLS.5 However, because the method is labour intensive and often presents problems with interpretation, especially the trailing phenomenon observed with azoles, other methods have been explored.6 The most intensively studied alternative method has been the Etest and good correlation with the NCCLS method has been reported. Despite the good correlation, the broth dilution and Etest methods require 2448 h for final results, and attempts have been made to develop reliable, more rapid tests. Several years ago, it was suggested that flow cytometry might be useful for susceptibility testing of microorganisms, as drug-induced cell damage could be assessed by use of various fluorescent dyes on a cell-by-cell basis.7 Some investigators have assessed cell damage by measuring the extent of penetration of vital dyes, by changes in membrane potential as measured by membrane potential-sensitive dyes, or by using dyes sensitive to intracellular metabolic changes.6 After using various flow cytometry protocols, which proved to be technically difficult, we tested whether the simple method of Kirk et al.,8 using Acridine Orange (AO), could yield rapid accurate results.
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Materials and methods |
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A total of 62 clinical isolates were tested. These included 31 isolates of C. albicans, 14 of Candida tropicalis, seven of Candida parapsilosis, eight of Candida glabrata, and two of Candida krusei.
Reference strains
Reference strains included C. albicans ATCC 26278, C. albicans ATCC 24433, C. albicans ATCC 90028, C. albicans ATCC 90029, C. tropicalis ATCC 750, C. parapsilosis ATCC 22019, C. krusei ATCC 6528 and a fluconazole-resistant mutant C. albicans, kindly provided by Merck Research Laboratories (USA). In addition, for our echinocandin experiments, four strains of C. albicans were kindly provided by Merck Research Laboratories (USA), and included a laboratory-generated echinocandin-resistant mutant.
Antifungal agents
Fluconazole was provided as stock solution by the manufacturer (Pfizer, Orsay, France). Echinocandin (L774967, MSD) was kindly provided as a pure powder by the manufacturer (Merck Research Laboratories, USA). The powder was dissolved in sterile distilled water and frozen as a stock solution at 20°C.
Standard susceptibility tests
Yeast antifungal susceptibility was tested by either broth macrodilution or by Etest, and some strains by both methods. Broth macrodilution was performed according to NCCLS instructions.5
Briefly, tubes containing two-fold dilutions of fluconazole, with concentrations in the range 0.25256 mg/L, and tubes containing two-fold dilutions of echinocandin, with concentrations in the range 0.068.0 mg/L, were prepared in RPMI broth. Yeast suspension was added to each of the tubes, which were incubated at 35°C for 48 h. MIC was defined as the lowest concentration demonstrating complete growth inhibition for echinocandin and growth of 20% of the control tube for fluconazole.
The Etest diffusion method was performed according to the manufacturer's instructions (AB Biodisk, Solna, Sweden). Briefly, a yeast suspension adjusted to a turbidity equivalent to that of a 0.5 McFarland standard was inoculated by swab onto casitone agar plates, which were incubated at 35°C. Plates were read at 24 and 48 h and the MIC of fluconazole determined as the point of intersection of zone edge and strip at 80% growth inhibition.
Antifungal susceptibility testing by flow cytometry
The conditions for the assay were those of Kirk et al.8
with slight modifications. Briefly, yeast strains that had been subcultured on Sabouraud agar were diluted in RPMI 1640 medium (Sigma, St. Louis, Mo, USA), with added L-glutamine, to a concentration of 5 x 105 cells per mL for the fluconazole assays. The strains were diluted to the same concentration in yeast peptone dextrose (YPD) (BD Biosciences, Sparks, MD, USA) medium for the echinocandin assays. Two-fold dilutions of fluconazole were prepared (0.125256 mg/L) in RPMI, and of echinocandin (0.068 mg/L) in YPD, and 50 µL of the antifungal drug solutions were inoculated with 50 µL of the yeast suspension. The test tubes were incubated at 35°C in a continuously shaking water bath at 200 rpm for 5 h for fluconazole and for 3 h for echinocandin. Following incubation, 400 µL of phosphate-buffered saline (pH 7.4) and 50 µL of AO (Sigma, St Louis, MO, USA), final concentration, 11 mg/L, were added. After vortexing, samples were incubated at room temperature for 5 min before analysis by flow cytometry (FACScan Flow Cytometer; Becton Dickinson Immunocytometry Systems, San Jose, CA, USA), using an argon laser emitting at 488 nm. Tubes with no antifungal drug served as the viable controls and similar tubes, heat-killed at 100°C for 10 min, at the end of the incubation, served as non-viable controls.
Acquisition of flow cytometric data
A gate that excluded debris and cell clusters was adjusted from a cytogram, derived from a forward-scatter versus side-scatter plot. A total of 5000 yeast cells were analysed. Data were analysed by Cell-Quest software (Becton Dickinson, California, USA) by quadrant statistical analysis of side-scatter versus FL3. Yeast moving into the red fluorescence region (R2) represented damaged cells. Five percent yeasts in this region were allowed as background in the viable control. The percentage of damaged yeastsas related to drug concentrationwas recorded, and correlated to MIC, as determined by standard methods. As the flow cytometry assay measures cell damage rather than inhibition, we prefer to relate to minimal damaging concentration (MDC) rather than MIC.
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Results |
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Discussion |
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Our attempt to use FUN-1 (Molecular Probes, Eugene, OR, USA)10 was also unsuccessful. This dye is converted into a red fluorescent probe in actively respiring cells, sequestering in vacuole structures. In non-respiring (dead) cells no accumulation of dye occurs in vacuoles, and the shift to red fluorescence should not be observed. Although we could easily distinguish live from dead cells by fluorescent microscopy, we could not distinguish them by flow cytometry, despite many attempted modifications. The small orangered vacuoles found in live cells did not produce a stronger red signal than the overflow into the region of red fluorescence by the green fluorescing dead cells.
We next attempted to use the method of Kirk et al.8 to test susceptibility to fluconazole and echinocandin, drugs that these authors had not tested. We also tested the feasibility of shortening the assay time to 5 h rather than the 824 h, as reported by Kirk et al.8 A 5 h incubation would realistically allow for results to be obtained on the same working day. In our hands, this method was very successful and we were able to complete the fluconazole assay in 5 h and the echinocandin assay in 3 h, allowing same-day results.
An additional aspect of the present study was the correlation between MIC results obtained by macrodilution and/or agar diffusion and flow cytometry results. All previous studies defined the parameter of MIC by flow cytometry, as the drug concentration producing 50% cell damage, measured by percent cells entering the dead cell region. We feel that the term MICminimal inhibitory concentrationis inappropriate, as inhibition is not being measured by flow cytometry. A more appropriate term would be MDC, minimal damaging concentration, and this is the parameter we chose to compare with conventional MIC. The present study demonstrated that 50% MDC might not be the best parameter for predicting the degree of fungal susceptibility to fluconazole under our test conditions. In fact, 30% and 40% MDC were more predictive of sensitive, susceptible dose-dependent and resistant levels of susceptibility of different Candida spp. The effect of echinocandin (L774967, MSD) was very dramatic and although we had only one resistant strain to test, repeated experiments indicated that 50% MDC could distinguish sensitive from resistant strains. The ability to obtain results for echinocandin susceptibility in such a short time, 24 h, is probably due to the mechanism of its actioninhibition of fungal cell wall synthesisresulting in rapid cellular damage.
In conclusion, we report a rapid flow cytometry assay for fluconazole and echinocandin fungal susceptibility testing, using AO. Results were obtained in 5 h or less, and compared very well with standard MIC determinations. The method is easy and reproducible and can be implemented in any laboratory with access to a flow cytometer.
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Acknowledgements |
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Footnotes |
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References |
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2
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