Abstract

Objectives: We have investigated the use of a viability dye, chloromethylfluorescein di-acetate (CMFDA), for antifungal susceptibility testing in a fluorescence microplate (FM) assay format.

Methods: For this FM assay, conidia were incubated in increasing concentrations of antifungal drug for 16 h and stained with CMFDA. Fluorescence, measured as mean fluorescence units (MFU) in a fluorescence microplate reader, was graphed relative to that of a drug-free control, and the MIC was defined as the lowest concentration of the drug that resulted in complete reduction (100%) in MFU for amphotericin B, or 90% reduction in MFU for itraconazole and voriconazole. Susceptibilities of 10 clinical isolates of Aspergillus fumigatus, Aspergillus terreus and Aspergillus niger to amphotericin B, itraconazole and voriconazole were tested in a blinded fashion using the FM and the NCCLS methods.

Results and conclusions: Reproducibility of the FM assay was excellent, and results correlated with those of the NCCLS microdilution method. The FM assay appears to be a rapid, objective method for testing fungal susceptibilities to itraconazole, voriconazole and amphotericin B.

Introduction

Verification of Aspergillus susceptibility to antifungals may be important to guide therapy, yet methods of measurement, such as the one proposed by the NCCLS are subjective and rely on assessment of culture turbidity.1 It is not clear whether culture turbidity provides an accurate measure of filamentous colony growth, as moulds, unlike yeasts and bacteria, do not multiply by binary division, and clumping of filamentous forms might yield spurious measures of overall growth.

We have previously described a flow cytometer-based assay that measures conidial viability after exposure to amphotericin B by quantifying fluorescence of the viability dye FUN-1.2 Although this method is rapid and reproducible for evaluating susceptibility to amphotericin B, susceptibilities to less fungicidal drugs, such as itraconazole and voriconazole, were more difficult to determine. Also, a flow cytometry-based assay that requires expensive equipment and trained personnel would be challenging to implement in a clinical microbiology laboratory. Herein, we describe a new assay that measures susceptibilities by chloromethylfluorescein di-acetate (CMFDA) fluorescence using a fluorescence microplate reader (FM assay).

Materials and methods

Media and antifungal drugs

RPMI-1640 medium (with glutamine and phenol red, without bicarbonate) buffered with 0.165 M MOPS (RPMI; Sigma Chemical Co., St Louis, MO, USA), Sabouraud's dextrose agar (Becton Dickinson, Sparks, MD, USA) and sodium-HEPES buffer (NHG buffer; 0.6 g sodium chloride, 10 mM HEPES, 20 g/L glucose, pH 7.2) were used in this study. CMFDA was purchased from Molecular Probes (Eugene, OR, USA). Amphotericin B (Sigma), itraconazole (Ortho-Biotech, Bridgewater, NJ, USA) and voriconazole (Pfizer, New York, NY, USA) were dissolved in dimethyl sulphoxide to yield stock solutions of 1600 mg/L. Further drug dilutions were prepared in RPMI, as outlined in the NCCLS M38-A document.1

Antifungal drug susceptibility testing

MICs for Aspergillus isolates of itraconazole, voriconazole and amphotericin B were obtained by the NCCLS microdilution method, as described elsewhere.2

CMFDA is a colourless, non-fluorescent probe that passes freely through cell membranes and is cleaved by cytosolic esterases into a brightly fluorescent product only in a living and growing cell. For optimization of assay conditions, experiments were first performed using variables such as dye concentrations, inoculum sizes and incubation times; Aspergillus fumigatus and Aspergillus terreus conidia and hyphae in different concentrations (104, 105, 106 and 107) were incubated for 15, 30 and 45 min and 1 h with various dye concentrations (2.5, 5, 10 and 15 μM). To identify the optimal incubation time, conidial suspensions (106 conidia/mL in RPMI) of one representative strain of A. fumigatus, one Aspergillus niger and one A. terreus (all clinical specimens) were inoculated in 96-well plates and incubated at 35°C for 0, 1, 4, 6, 8 and 16 h. After the respective incubation periods, the plates were centrifuged at 1200 g for 10 min, and the supernatant was discarded. The wells were filled with 200 μL of NHG buffer containing CMFDA (final concentration 5 μM). The plates were incubated for an additional 45 min and fluorescence was read as mean fluorescence units (MFU) at an excitation of 485 nm and an emission of 520 nm in a fluorescence microplate reader (Bio-Tek—Synergy HT Microplate reader, Bio-Tek Instruments, Winooski, VT, USA).

For the development of the susceptibility assay, one isolate of A. fumigatus (B5233) and one of A. terreus (F11 I6), which were recovered from clinical specimens at the National Institutes of Health (B5233) and the Fred Hutchinson Cancer Research Center (FHCRC, F11 I6) were used. A. fumigatus (B5233) had low MICs of amphotericin B, itraconazole and voriconazole (amphotericin B=0.25 mg/L; itraconazole=0.125 mg/L; voriconazole=0.125 mg/L) and the A. terreus isolate F11 I6 had relatively high MICs of amphotericin B, itraconazole and voriconazole (amphotericin B=2 mg/L; itraconazole=1 mg/L; voriconazole=2 mg/L) as measured using the NCCLS methods of susceptibility testing.2 Conidial suspensions of these Aspergillus isolates were plated in 96-well plates containing increasing concentrations of the various drugs (amphotericin B, itraconazole and voriconazole) as per the NCCLS M38-A recommendations. For inoculum preparation, the conidia were counted on a haemocytometer and adjusted to a final concentration of 0.5 × 106 conidia/mL instead of the NCCLS recommended turbidity adjustment. One well containing conidial suspensions with no drug (growth control) and one well containing only RPMI (background control) were included in every plate. The plates were incubated at 35°C for 16 h, centrifuged at 1200 g for 10 min, the supernatant discarded and the wells filled with 200 μL of NHG buffer containing CMFDA (5 μM). After incubation for 45 min in the dark, the cells were visualized with a Deltavision wide field fluorescence microscope (Applied Precision Inc., Issaquah, WA, USA) fitted with an FITC filter. Fluorescence of the drug-treated and drug-free fungi were also read as MFU in a fluorescence plate reader.

For determining the MICs for the fungi by the FM assay, the ratio of the MFU of drug-treated Aspergillus isolates relative to MFU of the drug-free controls was calculated, after subtraction of background fluorescence. The MIC of amphotericin B was defined as the lowest drug concentration at which there was 100% reduction in relative MFU, and the MICs of voriconazole and itraconazole were defined as the concentrations that yielded 90% reduction in MFU relative to drug-free control.

Six isolates of A. fumigatus, two A. terreus and two A. niger were randomly selected from a frozen culture bank of clinical samples obtained from patients at the Fred Hutchinson Cancer Research Center. Susceptibilities of these fungi to amphotericin B, itraconazole and voriconazole were measured using the NCCLS and the FM assay, as described above. Experiments were repeated at least three times on three different days. Each time the experiment was repeated for both the methods, the conidial inoculum was prepared fresh from a recently subcultured Aspergillus colony. Mean MICs were calculated from three different experiments to determine reproducibility of the FM assay. Percentage agreement between the MICs obtained by the NCCLS and the FM assay was calculated as the percentage of MICs that were within one dilution of the NCCLS MICs of the various drugs tested. Differences between the assays were tested for significance using the Student's t-test with P < 0.05 considered significant.

Results

Preliminary experiments identified an inoculum size of 106 cells incubated for 45 min with 5 μM CMFDA, as optional assay conditions to generate reproducible decreases in MFU with increasing antifungal drug concentrations (data not shown). Higher dye concentrations resulted in high background staining, requiring additional washing steps, and lower concentrations of dye yielded less-uniform staining of the fungal cells. Inoculum sizes <105 yielded very low MFU, resulting in MICs that did not approximate NCCLS results and a high amount of run-to-run variability (data not shown). Under these conditions, all the Aspergillus spp. could cleave CMFDA into a fluorescent product and the fluorescence intensity increased over time (Figure 1). The steepest increase in fluorescence intensity occurred during 6–16 h of growth, the period corresponding to conidial germination and early hyphal elongation (data not shown). Fungal conidia that were heat-killed (incubated at 85°C for 30 min) and stained with CMFDA could not cleave the probe into a fluorescent product (data not shown). Thus it appeared that CMFDA could be used as an indicator of viability and maturation of early fungal forms.

To investigate whether CMFDA fluorescence can be used for drug susceptibility testing, two isolates with known NCCLS MICs were assayed against amphotericin B, voriconazole and itraconazole. Mean fluorescence units of drug-treated and drug-free cells were measured after 16 h. This relatively long incubation time was chosen in order to achieve a reasonably high MFU to allow for measured differences; drug-treated Aspergillus species demonstrated ∼2 h lag in growth.

In the absence of drug, and in the presence of subinhibitory concentrations of amphotericin B, itraconazole and voriconazole, Aspergillus spp. appeared as a network of branching hyphae that stained uniformly and brightly with CMFDA. In the presence of inhibitory concentrations of voriconazole and itraconazole, most cells appeared as swollen conidia with or without short hyphae that stained dimly with CMFDA. In the presence of inhibitory concentrations of amphotericin B, very small, ungerminated, dimly stained conidia were observed. Thus, the mean fluorescence intensity of drug-exposed cells was lower than drug-free cells, with relative MFU decreasing with increased concentrations of amphotericin B, voriconazole and itraconazole (Figure 2). At the drug concentration comparable with the MIC values (derived by the NCCLS method) for these fungi, amphotericin B exposure yielded 100% reduction in MFU relative to growth control, and the azole antifungals typically yielded 90% reduction in MFU. Therefore, the MIC was defined as the lowest concentration of drug that caused 100% reduction in MFU relative to growth control for amphotericin B and 90% reduction in MFU relative to growth control for itraconazole and voriconazole. Amphotericin B, voriconazole and itraconazole MICs thus calculated by the FM assay correlated with NCCLS MICs for the representative isolates with low and high MICs (Figure 2).

Since the amphotericin B, itraconazole and voriconazole MICs obtained by the FM assay and the NCCLS assay correlated within one drug dilution when testing two isolates with known MICs, susceptibilities of a large number of Aspergillus isolates were tested using both methods (Table 1). Results of the FM assay were reproducible between experiments and correlated to within one dilution of the NCCLS microdilution MICs (Table 1). The percentage correlation approximated 90% for amphotericin B and 100% for itraconazole and voriconazole. There was no statistical difference between MICs obtained by NCCLS and FM methods (P values >0.05, not shown).

Discussion

Determining antifungal susceptibilities of filamentous fungi is fraught with difficulties associated with slow growth of filamentous forms, and subjectivity of interpreting visual turbidimetric endpoints. In the present study, we have developed a rapid, objective method to test susceptibility using the fluorochrome CMFDA, a viability dye that is cleaved into a green fluorescent product only in a living/growing cell thus ensuring that both the ‘cidal’ and ‘static’ effects of antifungals can be assessed.

In the presence of inhibitory concentrations of itraconazole, voriconazole and amphotericin B, Aspergillus spp. demonstrated germination and hyphal elongation defects that were accompanied by decreased fluorescence when stained with CMFDA. Drug-induced inhibition of germination and hyphal growth has been recently described as the basis for the development of a micro-broth kinetic assay to measure antifungal susceptibility of filamentous fungi.3 Although accurate measures of antifungal susceptibilities could be obtained with this kinetic assay, the assay was found to be too cumbersome for use as a standard susceptibility testing method. We have built on this principle of measuring viability and growth defects of early cellular forms by using the CMFDA fluorescence-based microplate assay. Comparative testing of multiple Aspergillus spp. using both the NCCLS and the FM assay showed that the results of the FM assay were highly comparable with the NCCLS MICs.

Methods to generate MICs in very short (6–8 h) incubation times have been described using fluorescent markers in yeasts.4 In our study, the shortest incubation time that would provide reproducible results was 16 h, largely because drug-treated fungi demonstrated a lag time prior to production of enough MFU to allow for drug-induced changes in growth.

The FM assay was more rapid and objective with regards to endpoint determination compared with the conventional method. The automated platform and simplicity of the FM assay might make it more amenable for use as a susceptibility test. However, the complexities of the FM assay include the incorporation of a centrifugation step and require a fluorescence plate reader. Larger studies will need to be performed to optimize the FM assay and to test a wide range of filamentous fungi to examine inter-laboratory reproducibility.

Figure 1.

Increase in fluorescence for all Aspergillus species tested over time. Conidia of A. niger (diamonds), A. terreus (squares) and A. fumigatus (triangles) were incubated at 37°C and stained with CMFDA, and MFU were read. All the Aspergillus species tested increased in MFU, with the steepest increase occurring ∼8 h after incubation in the absence of drug.

Figure 1.

Increase in fluorescence for all Aspergillus species tested over time. Conidia of A. niger (diamonds), A. terreus (squares) and A. fumigatus (triangles) were incubated at 37°C and stained with CMFDA, and MFU were read. All the Aspergillus species tested increased in MFU, with the steepest increase occurring ∼8 h after incubation in the absence of drug.

Figure 2.

Mean fluorescence of drug-treated fungi relative to growth controls. MIC curves were generated by measurement of mean fluorescence units (MFU). MIC curves for representative Aspergillus isolates with low MICs (left-hand panels) and high MICs (right-hand panels) are shown for three different experiments. Curves generated from individual experiments are represented by different symbols (triangles, squares or diamonds). MFU relative to controls (Relative MFU) are graphed for (a) amphotericin B (AMB), (b) itraconazole (ITZ) and (c) voriconazole (VRZ). The respective NCCLS MICs are indicated with arrows.

Figure 2.

Mean fluorescence of drug-treated fungi relative to growth controls. MIC curves were generated by measurement of mean fluorescence units (MFU). MIC curves for representative Aspergillus isolates with low MICs (left-hand panels) and high MICs (right-hand panels) are shown for three different experiments. Curves generated from individual experiments are represented by different symbols (triangles, squares or diamonds). MFU relative to controls (Relative MFU) are graphed for (a) amphotericin B (AMB), (b) itraconazole (ITZ) and (c) voriconazole (VRZ). The respective NCCLS MICs are indicated with arrows.

Table 1.

MICs for Aspergillus isolates of multiple antifungals as determined by the NCCLS and the FM methods

Isolate Species Method AMB MIC GM (range) mg/L ITZ MIC GM (range) mg/L VRZ MIC GM (range) mg/L 
10I7 A. fumigatus NCCLS 0.31 (0.25–0.5) 0.16 (0.125–0.2) 0.16 (0.125–0.25) 
  FM 0.20 (0.125–0.25) 0.16 (0.125–0.25) 0.125 (0.125) 
8H7 A. fumigatus NCCLS 0.12 (0.06–0.25) 0.31 (0.25–0.5) 0.31 (0.25–0.5) 
  FM 0.16 (0.125–0.25) 0.25 (0.25) 0.25 (0.25) 
8F8 A. fumigatus NCCLS 0.40 (0.25–0.5) 0.20 (0.125–0.25) 0.20 (0.125–0.25) 
  FM 0.31 (0.25–0.5) 0.20 (0.125–0.25) 0.10 (0.06–0.125) 
11A3 A. fumigatus NCCLS 0.25 (0.25) 0.25 (0.25) 0.20 (0.125–0.25) 
  FM 0.16 (0.125 –0.25) 0.32 (0.25–0.5) 0.125 (0.125) 
10I2 A. fumigatus NCCLS 0.40 (0.25–0.5) 0.31 (0.25–0.5) 0.125 (0.125) 
  FM 0.20 (0.125–0.25) 0.31 (0.25–0.5) 0.25 (0.25) 
B5233 A. fumigatus NCCLS 0.125 (0.125) 0.16 (0.125–0.25) 0.16 (0.125–0.25) 
  FM 0.25 (0.25) 0.20 (0.125–0.25) 0.25 (0.25) 
11I6 A. terreus NCCLS 1 (1) 1.26 (1–2) 1.6 (1–2) 
  FM 1 (1) 1 (1) 2 (2) 
12E4 A. terreus NCCLS 1.26 (1–2) 0.40 (0.25–0.5) 0.63 (0.5–10) 
  FM 1.26 (1–2) 0.40 (0.25–0.5) 1(1) 
7A3 A. niger NCCLS 0.25 (0.25) 0.25 (0.125–0.5) 0.25 (0.25) 
  FM 0.5 (0.5) 0.25 (0.125–0.5) 0.20 (0.125–0.25) 
Isolate Species Method AMB MIC GM (range) mg/L ITZ MIC GM (range) mg/L VRZ MIC GM (range) mg/L 
10I7 A. fumigatus NCCLS 0.31 (0.25–0.5) 0.16 (0.125–0.2) 0.16 (0.125–0.25) 
  FM 0.20 (0.125–0.25) 0.16 (0.125–0.25) 0.125 (0.125) 
8H7 A. fumigatus NCCLS 0.12 (0.06–0.25) 0.31 (0.25–0.5) 0.31 (0.25–0.5) 
  FM 0.16 (0.125–0.25) 0.25 (0.25) 0.25 (0.25) 
8F8 A. fumigatus NCCLS 0.40 (0.25–0.5) 0.20 (0.125–0.25) 0.20 (0.125–0.25) 
  FM 0.31 (0.25–0.5) 0.20 (0.125–0.25) 0.10 (0.06–0.125) 
11A3 A. fumigatus NCCLS 0.25 (0.25) 0.25 (0.25) 0.20 (0.125–0.25) 
  FM 0.16 (0.125 –0.25) 0.32 (0.25–0.5) 0.125 (0.125) 
10I2 A. fumigatus NCCLS 0.40 (0.25–0.5) 0.31 (0.25–0.5) 0.125 (0.125) 
  FM 0.20 (0.125–0.25) 0.31 (0.25–0.5) 0.25 (0.25) 
B5233 A. fumigatus NCCLS 0.125 (0.125) 0.16 (0.125–0.25) 0.16 (0.125–0.25) 
  FM 0.25 (0.25) 0.20 (0.125–0.25) 0.25 (0.25) 
11I6 A. terreus NCCLS 1 (1) 1.26 (1–2) 1.6 (1–2) 
  FM 1 (1) 1 (1) 2 (2) 
12E4 A. terreus NCCLS 1.26 (1–2) 0.40 (0.25–0.5) 0.63 (0.5–10) 
  FM 1.26 (1–2) 0.40 (0.25–0.5) 1(1) 
7A3 A. niger NCCLS 0.25 (0.25) 0.25 (0.125–0.5) 0.25 (0.25) 
  FM 0.5 (0.5) 0.25 (0.125–0.5) 0.20 (0.125–0.25) 

AMB, amphotericin B; ITZ, itraconazole; VRZ, voriconazole; GM, geometric mean.

This work was supported by National Institutes of Health grant R21 AI55928 to K.A.M.

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Author notes

1Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N. D3-100, Seattle, WA 98109; 2 Department of Medicine, University of Washington, Seattle, WA, USA