Precision and Accuracy of Fluconazole Susceptibility Testing by Broth Microdilution, Etest, and Disk Diffusion Methods

Interpretive agreements among the results of fluconazole brothmicrodilution tests, Etests, and disk diffusion tests were documentedby evaluating 495 Candida spp. Microdilution reference testresults were in agreement with 96% of the Etest results; mostdiscrepancies were minor differences. Fluconazole resistanceof Candida krusei strains often required a full 48 h of incubationin order to be observed by the standard method. For the diskdiffusion tests that were performed on Mueller-Hinton agar withglucose and methylene blue, 97% of results were in agreementwith those of the reference test, especially when zones of inhibitionwere measured after the first 24 h of incubation. Some Candidaglabrata isolates failed to grow satisfactorily until a full48 h of incubation was completed. Precision was determined bytesting 50 selected isolates in triplicate in each of threelaboratories. The reproducibility of results of disk diffusiontests was comparable to that of the reference method. With allprocedures, determination of test results was particularly challengingwith some strains, and new methods are needed in order to improveendpoint definition.

INTRODUCTION

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Introduction

Compromised patients, such as those infected with human immunodeficiencyvirus, are often colonized by and/or infected with fungi, especiallyCandida spp. Consequently, they frequently receive antifungalagents such as fluconazole for relatively long periods of timeduring hospitalization. Prolonged exposure to an azole suchas fluconazole can select strains with diminished susceptibilities(6, 7), and consequently, increasing doses may be necessary.For this reason, the susceptibilities of isolates recoveredfrom compromised patients receiving prolonged fluconazole treatmentshould be monitored in order to determine when strains withdecreased susceptibilities have been selected (3). In orderto do this in a cost-effective way, simple, inexpensive testingprocedures are needed, but such tests must be accurate and precise.The purpose of this report is to document the relative levelsof accuracy and precision of three procedures for testing fluconazole:broth microdilution, Etest, and disk diffusion. The accuracyof each procedure was assessed by comparing the results to thoseobtained by the 48-h microdilution reference method of the NationalCommittee for Clinical Laboratory Standards (NCCLS) (8). Precisionwas measured by performing replicate tests in three separatelaboratories.

MATERIALS AND METHODS

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Materials and Methods

Microorganisms. For this study, M. A. Pfaller selected 495 clinical isolatesof Candida spp. for which there was a broad range of fluconazoleMICs. The five species that were represented are shown in Table1. For testing reproducibility, 50 of the 495 isolates wereselected in order to maximize the number of strains for whichMICs were near the interpretive breakpoints of $<=$ 8.0 µg/ml(susceptible) and $>=$ 64 µg/ml (resistant). This subset ofstrains included 26 strains of Candida albicans and 6 strainseach of Candida glabrata, Candida krusei, Candida parapsilosis,and Candida tropicalis. Quality control strains of C. parapsilosis(ATCC 22019) and C. krusei (ATCC 6258) were included in everytest run.

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TABLE 1. Susceptibility of 495 Candida spp. isolates to fluconazole as determined by two different methods and two incubation times

Microdilution susceptibility tests. The NCCLS procedure (8) was followed as carefully as possible.Serial dilutions of fluconazole (from 256 to 0.03 µg/ml)were prepared in RPMI 1640 broth (HyClone lot no. AHC 7888A)and then dispensed into microdilution test panels. The trayswere then stored at -70°C until needed. The inoculum wasadjusted to provide 0.5 x 10³ to 2.5 x 10³ CFU/ml (4, 8, 9)as confirmed by periodic colony counts throughout the study.The trays were incubated at 35°C in ambient air. After 24h and again after 48 h, MICs were recorded as the lowest concentrationsthat were visually determined to result in substantial decreasesin the amount of growth (about 50 to 80% inhibition comparedto that in a growth control well). Quality control strains ofCandida spp. were evaluated throughout the study, and the fluconazoleMICs fell within the expected ranges (2).

Agar-based susceptibility tests. Etest strips (AB Biodisk, Solna, Sweden) were tested on RPMI1640 broth with 2% glucose and 1.5% Bacto Agar (Difco Laboratories).For disk diffusion tests, 25-µg fluconazole disks (BBLlot no. 801553) were also applied to the same test plate. Inaddition, disk tests were also performed on Mueller-Hinton agarwith 2% glucose and methylene blue (0.5 µg/ml). The latterreagent was added to help clarify the zone of inhibition. Becausethis medium gave encouraging results with disk tests in theinitial phase of this study, it was added to the later evaluationsof reproducibility. For either test, agar plates were swab inoculatedwith a suspension of yeast cells in a manner that is currentlybeing used for testing antibacterial agents. When adjusted tothe turbidity of a 0.5 McFarland standard, a suspension of yeastscontains about 10⁶ CFU/ml (9). The diameter of each zone ofinhibition was taken as the area that showed a sharp declinein the density of growth. Etest MICs were determined accordingto the manufacturer's instructions and were rounded up to thenearest even log₂ concentration in order to simplify analysis.

Precision of test procedures. A challenge set of 50 strains was selected, and each strainwas subcultured to nine slants. These slants were then labeledwith random code numbers, and three slants of each strain weredistributed to the authors' laboratories. Broth microdilutiontrays, agar plates, 25-µg fluconazole disks, and Eteststrips were provided by a common source. Each participant wasasked to perform fluconazole susceptibility tests with subculturesof each of the 150 coded slants. In this way, triplicate testswere performed in a truly blind fashion that generated ninevalues for each strain. To evaluate the overall precision ofeach method, the median MIC or zone diameter was calculatedand the reported results were evaluated in terms of the numberof twofold dilution intervals away from the median MIC or thenumber of millimeters away from the median zone diameter forthe strain.

RESULTS AND DISCUSSION

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Results and Discussion

MICs. Table 1 shows the MICs for each species as determined by microdilutionand Etest procedures and incubation for 24 or 48 h. The twoprocedures gave MICs that were in general agreement (±one or two doubling concentrations). For C. albicans, C. tropicalis,and C. krusei, most Etest MICs were the same or 1 dilution higherthan those provided by microdilution tests. The reverse wastrue when we tested C. glabrata. With both methods, fluconazoleresistance among C. krusei isolates was more apparent when theisolates were incubated a full 48 h. For the purposes of thisstudy, the 48-h microdilution susceptibility test results (8)were used as the standard reference for evaluating other procedures.

Accuracy of tests. All strains tested for susceptibility were interpreted as belongingto one of three categories (1, 8, 10): susceptible (strainsfor which MICs were $<=$ 8 µg/ml or zone diameters were $>=$ 19mm), susceptible-dose dependent (strains for which MICs were16 to 32 µg/ml or zone diameters were 15 to 18 mm), orresistant (strains for which MICs were $>=$ 64 µg/ml or zonediameters were $<=$ 14 mm). Percentages of results that were interpretedas being in agreement or discrepant were then calculated. Table2 summarizes these estimates of accuracy for the different methods.When microdilution panels were observed after only 24 h of incubation,98% of the strains had grown sufficiently to permit estimationsof MICs and 94% of the MICs agreed with the results of the 48-hreference test. Very major discrepancies involved tests of C.krusei strains which were susceptible at 24 h but resistantat 48 h. This species also provided 20 of the 22 minor discrepancies.

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TABLE 2. Overall interpretive agreement between results of fluconazole susceptibility tests and of standard 48-h broth microdilution reference tests

On RPMI 1640-glucose-agar, 94% of the strains grew sufficientlyto permit the recording of test results after 24 h. The majorityof early-growth failures involved tests of the C. glabrata strainsthat required a full 48 h for testing, and even then, the lawnsof growth were rather light. Etest MICs with this medium showed96% agreement with the reference microdilution test results.The majority of discrepancies were minor differences, but resultsfor two C. krusei isolates showed very major discrepancies at24 h. Results of disk diffusion tests on the same agar platesdisplayed 93.5% agreement; all very major discrepancies involvedtests of C. krusei, and results for two C. albicans isolatesprovided major discrepancies but only when they were read at48 h.

Disk tests were improved by using Mueller-Hinton agar supplementedwith glucose for better growth and a low concentration of methyleneblue to help clarify the zones of inhibition. At the first 24h, only five strains (three C. parapsilosis and two C. albicansstrains) grew too poorly to permit measurement of zone diametersbut they all grew sufficiently after 48 h. On this supplementedmedium, results of the 24-h disk tests showed the best (97%)agreement with results of the 48-h broth microdilution test:only one very major discrepancy was recorded (C. krusei). The14 minor discrepancies involved tests of eight C. krusei, threeC. parapsilosis, two C. glabrata, and one C. albicans isolate. When disks were held for 48 h before inhibitory zones weremeasured, five isolates (three C. glabrata, one C. albicans,and one C. parapsilosis isolate) were judged to be "false resistant"(major errors). The few strains that grew poorly on the firstday were accurately tested after an additional day of incubation.Figure 1 presents a scattergram plotted to compare the 24-hzones on this medium to the reference microdilution MICs foreach of the 490 strains that grew sufficiently well.

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FIG. 1. Zones of inhibition around 25-µg-fluconazole disks containing Mueller-Hinton agar supplemented with 2% glucose and methylene blue (0.5 µg/ml). Values are numbers of isolates from the 490 isolates of Candida spp. that grew on this medium after 24 h. The least-squares method was used to calculate a regression line (y = 55.4 - 2.66x, with r = 80).

Precision of MICs. Table 3 summarizes data concerning the reproducibility of fluconazoleMICs. By the reference microdilution test, 93 to 94% of allMICs varied over a broad range of ± two doubling concentrationson either side of the median. This range of variation is inagreement with observations made during the initial evaluationof the microdilution procedure (4). With the Etest, >90%of all MICs were no more than one dilution interval from theall-laboratory median. Compared to RPMI 1640-glucose-agar, Mueller-Hintonagar with glucose and methylene blue did not improve the precisionof Etests. The reference method used to evaluate the other proceduressuffers from inconsistencies due largely to difficulties indefining the endpoint when there is a trailing growth pattern.The agar-based Etest (5, 11) seems to have a slight advantageover broth dilution tests in this regard. By both methods, MICsrecorded after 24 h were more consistent than those determinedafter 48 h.

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TABLE 3. Precision of fluconazole MICs when 50 Candida isolates were tested in triplicate in each of three laboratories

Precision of disk tests. Table 4 describes the same type of data for disk diffusion tests.From the regression statistics calculated from the data in Fig.1, a twofold concentration change in MICs was accompanied bya 3- to 4-mm change in the diameters of the zones of inhibition.Consequently, the precision of disk tests can be compared tothat of MIC determinations. A range of MICs that represent themedian ± one doubling concentration is equal to a medianzone of ±3 or 4 mm, and a range of MICs that representthe median ± two doubling concentrations is equal toa median zone of ±6 to 8 mm. As can be seen in Table4, the precision of disk diffusion tests is similar to thatobserved for the standard reference procedure. When Mueller-Hintonagar with glucose and methylene blue was used, the zone diameterswere more reproducible than those on RPMI 1640-glucose-agar.Furthermore, zones of inhibition measured after 24 h of incubationwere more reproducible than those measured after 48 h.

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TABLE 4. Precision of fluconazole disk diffusion susceptibility tests on each of two agar media

We concluded that the results of fluconazole susceptibilitytests by the disk diffusion method were just as reproducibleas the results of the standard reference microdilution procedure.Furthermore, of the tests that we evaluated, the Etest producedthe most reproducible results. The precision of disk diffusiontests was maximized by using Mueller-Hinton agar with glucoseand methylene blue and by incubating the disks for only 24 h.The 1% of strains that failed to grow adequately at the 24-hexamination gave satisfactory results when they were incubatedfor another day. The results of disk diffusion tests done inthis way showed excellent (97%) agreement with the results ofthe reference broth microdilution method.

ACKNOWLEDGMENTS

This study was made possible by a grant from Pfizer Pharmaceuticals,New York, N.Y.

FOOTNOTES

* Corresponding author. Mailing address: The Clinical Microbiology Institute, 9725 SW Commerce Circle, Suite A-1, Wilsonville, OR 97070. Phone: (503) 682-3232. Fax: (503) 682-4548 or (503) 682-2065. E-mail: cmi{at}hevanet.com.

REFERENCES

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