Interlaboratory Evaluation of Etest Method for Testing Antifungal ...

JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1996, p. 848–852 0095-1137/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 34, No. 4

Interlaboratory Evaluation of Etest Method for Testing Antifungal Susceptibilities of Pathogenic Yeasts to Five Antifungal Agents by Using Casitone Agar and Solidified RPMI 1640 Medium with 2% Glucose A. ESPINEL-INGROFF,1* M. PFALLER,2 M. E. ERWIN,2

AND

R. N. JONES2

Medical College of Virginia/Virginia Commonwealth University, Richmond, Virginia,1 and University of Iowa, Iowa City, Iowa2 Received 28 August 1995/Returned for modification 9 December 1995/Accepted 11 January 1996

An interlaboratory evaluation (two centers) of the Etest method was conducted for testing the antifungal susceptibilities of yeasts. The MICs of amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole were determined for 83 isolates of Candida spp., Cryptococcus neoformans, and Torulopsis glabrata. Two buffered (phosphate buffer) culture media were evaluated: solidified RPMI 1640 medium with 2% glucose and Casitone agar. MIC endpoints were determined after both 24 and 48 h of incubation at 35&C. Analysis of 3,420 MICs demonstrated higher interlaboratory agreement (percentage of MIC pairs within a 2-dilution range) with Casitone medium than with RPMI 1640 medium when testing amphotericin B (84 to 90% versus 1 to 4%), itraconazole (87% versus 63 to 74%), and ketoconazole (94 to 96% versus 88 to 90%). In contrast, better interlaboratory reproducibility was determined between fluconazole MIC pairs when RPMI 1640 medium rather than Casitone medium was used (96 to 98% versus 77 to 90%). Comparison of the flucytosine MICs obtained with RPMI 1640 medium revealed greater than 80% reproducibility. The study suggests the potential value of the Etest as a convenient alternative method for testing the susceptibilities of yeasts. It also indicates the need for further optimization of medium formulations and MIC endpoint criteria to improve interlaboratory agreement.

the AIDS pandemic also yielded more immunosuppressed patients, who are highly predisposed to life-threatening fungal infections (8). The increased frequency and severity of mycotic diseases prompted the development and higher level of use of antifungal agents, which led to the recognition of antifungal resistance since the early years of antifungal drug development (5, 27). Furthermore, with the wider use of azole antifungal therapy, fungal pathogens for which MICs were higher began to emerge (2, 28, 34). This combination of factors warrants testing of the antifungal susceptibilities of yeasts and emerging filamentous fungal pathogens. Although the M27-P reference method improved the interlaboratory agreement of MIC results, a macrodilution procedure is not a practical testing tool in the clinical laboratory. More convenient, efficient, and cost-effective alternative approaches that have compared favorably with the M27-P method have been evaluated during the past 5 years (3, 7, 9, 11, 13, 14, 18, 24, 25, 30, 31). Among the commercial alternatives, the Etest for yeasts is a novel agar diffusion procedure which is based on the diffusion of a continuous concentration gradient of the antifungal agent tested from a plastic strip into an agar medium. Previous comparisons by independent laboratories of the Etest with the NCCLS M27-P method have shown excellent agreement rates (.90%) for fluconazole and flucytosine (9, 31) and good agreement rates for the azoles (7). The present study represents the first interlaboratory comparison (two centers) of MIC endpoints determined by using the newly adapted Etest procedure for yeasts. The MICs of amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole were determined for 83 pathogenic yeasts and four American Type Culture Collection (ATCC) isolates in each laboratory by the Etest with two media (buffered [phosphate] RPMI 1640 medium with 2% glucose and Casitone agar).

The publication in 1992 by the National Committee for Clinical Standards (NCCLS) of the proposed broth macrodilution reference method for yeasts (NCCLS M27-P document [20], which was moved to the tentative level, or M27-T, in 1995 [21]) is an important landmark in the field of testing antifungal susceptibilities. This standard method was developed through a series of collaborative studies (12, 15, 16, 26) and provides guidelines for testing the antifungal susceptibilities of Candida spp., Cryptococcus neoformans, and Torulopsis glabrata. Prior to this event, the few reference laboratories that performed antifungal susceptibility testing followed a variety of methods and testing parameters. As a consequence, interlaboratory consensus of MIC endpoints was unacceptably low (17). This situation posed a problem, because more clinical laboratories were requested to perform antifungal susceptibility testing. Several factors account for the greater role assumed by the clinical laboratory in the selection and monitoring of antifungal chemotherapy. The higher incidence of severe fungal infections that was noticed in the late 1940s with the advent of antibacterial agents first and steroid therapy later (19, 32, 35) steadily continued to increase throughout the ensuing decades. Unfortunately, the more aggressive and frequent use of broad-spectrum antibiotics and antineoplastic and immunosuppressive chemotherapies for larger numbers of oncology and transplantation patients also augmented the volume of systemic mycoses after 1980, especially nosocomial yeast infections (4, 22, 33). By the 1980s,

* Corresponding author. Mailing address: Medical Mycology Laboratory, Division of Infectious Diseases, Medical College of Virginia, Box 980049, MCV, Richmond, VA 23298-0049. Phone: (804) 8289711. Fax: (804) 828-3097. 848

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MATERIALS AND METHODS Yeast isolates. A total of 83 well-characterized yeast isolates from the culture collections of the Medical College of Virginia, Richmond, and the University of Iowa, Iowa City, were evaluated. This set of isolates included Candida albicans (n 5 33), Candida guilliermondii (n 5 2), Candida krusei (n 5 4), Candida lusitaniae (n 5 8), Candida parapsilosis (n 5 7), Candida rugosa (n 5 2), Candida tropicalis (n 5 13), Cryptococcus neoformans (n 5 7), and Torulopsis glabrata (n 5 7). The following ATCC strains also were tested: C. albicans ATCC 90028, C. krusei ATCC 6258, and C. parapsilosis ATCC 90018 and ATCC 22019. The reference MIC ranges of amphotericin B, flucytosine, and fluconazole for these four ATCC strains are well defined; C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 are the two strains selected by NCCLS (M27-T method [21]) as quality control (QC) isolates for testing the antifungal susceptibilities of yeasts (23). All isolates were primarily recovered from the bloodstreams or body fluids of 83 individual patients. Each isolate was stored as a suspension in water at ambient temperature until testing was performed. Antifungal agents. The Etest antifungal gradient strips were provided by the manufacturer (AB BIODISK, Solna, Sweden). The concentration gradient for each drug ranged from 0.004 to 32 mg/ml. The strips were stored at 2208C until the day that the MICs were determined. Etest strips for antifungal susceptibility testing are now commercially available, but only for investigational purposes. The antifungal agents evaluated were amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole. Media. The culture media used for the testing of each isolate in each laboratory were solidified (1.5% agar) RPMI 1640 medium with 2% glucose and Casitone agar. The latter medium was not used for the determination of flucytosine MICs because of the presence of interfering metabolites. Both media were buffered with phosphate buffer to pH 7.0 (0.7% potassium monobasic and 0.1% sodium hydroxide), which has been shown to provide sharper endpoints (AB Biodisk in-house evaluations). The media were prepared by the manufacturer of the Etest strips (AB Biodisk). Inoculum suspensions. The stock yeast inoculum suspensions were prepared as described previously (20) and were adjusted to 1 3 106 to 5 3 106 CFU/ml (except for C. neoformans suspensions) by the spectrophotometric method. Briefly, each yeast was subcultured twice onto Sabouraud dextrose agar (Difco Laboratories, Detroit, Mich.), and inoculum suspensions were prepared for each experiment from 24-h-old (Candida spp. and T. glabrata) and 48-h-old (C. neoformans) cultures grown at 358C. The turbidities of the resulting yeast suspensions were measured with a spectrophotometer at 530 nm and were adjusted to the percent transmission that matched a 0.5 McFarland standard when testing Candida spp. and T. glabrata isolates and a 1 McFarland standard when testing C. neoformans isolates. The Etest procedure. The 83 clinical isolates and the four ATCC QC and reference strains were tested at each center by the Etest by using the two media and the same standardized inoculum described above. The Etest was performed by following the manufacturer’s instructions (1). Each solidified medium was inoculated by dipping a nontoxic sterile swab into the respective undiluted inoculum suspension and evenly streaking it in three directions over the entire surface of a 150-mm petri plate containing 60 ml of each medium. The agar surface was allowed to dry for 15 min, and the strips were placed onto the inoculated agar. The plates were incubated at 358C, and MICs were determined following incubation times of 24 and 48 h (Candida spp. and T. glabrata) or 48 and 72 h (C. neoformans). Determination of MIC endpoints. The MIC by the Etest was the lowest drug concentration at which the border of the elliptical inhibition zone intercepted the scale on the antifungal strip. C. albicans and certain isolates of other Candida spp. (e.g., C. tropicalis) gave diffuse endpoints with flucytosine and especially with the azoles. That is, instead of the colony-free and sharp inhibition ellipses observed with amphotericin B, either large to minute colonies or a decreasing intensity of growth (double halo) was observed at the endpoint or within the whole inhibition ellipse. Determination of MIC endpoints was difficult because of these diffuse inhibition endpoints. Analysis of the data. A total of 3,428 MICs for the ATCC isolates and the 83 clinical yeast isolates were obtained and analyzed. Each isolate was tested with the two media at each center with four of the five antifungal agents; flucytosine MICs were obtained only with the RPMI 1640 medium since Casitone agar contains compounds antagonistic to this antifungal agent. In addition, there were two MIC readings (24 and 48 h) per medium with each drug for each isolate. Because the Etest strips contain a continuous gradient of each antifungal agent tested instead of the established twofold drug dilution schema, the MIC endpoint evaluated by the Etest was elevated to the next twofold dilution concentration which matched the drug dilution schema of the NCCLS M27-P method to facilitate comparisons and the presentation of results. Both on-scale and off-scale MICs were included in the analysis; the high off-scale MICs of .32 mg/ml (4%) were converted to 64 mg/ml, and the low-off scale MICs of ,0.004 mg/ml (0.4%) were left alone. Discrepancies between MIC endpoints of no more than 2 dilutions from the two centers were used to obtain percent agreements. The percentage of the MICs that were within 2 dilutions was evaluated for each combination of isolate, drug, medium, and incubation time. In addition, the MIC range was determined for each of the above combinations of

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testing variables for the 83 clinical isolates. MIC ranges for the four ATCC strains also were obtained.

RESULTS Interlaboratory agreement for the QC and reference isolates. The MICs for each ATCC isolate were determined four times with the five drugs at both incubation times and with both media. A total of 400 MIC endpoints for the ATCC strains were analyzed; a summary of the 48-h MIC ranges stratified by ATCC strain, drug, and medium formulation is presented in Table 1. The recommended reference MIC ranges (M27-T document [21]) of amphotericin B, fluconazole, and flucytosine are also provided in Table 1. Interlaboratory agreement was excellent, because most MICs obtained at both centers were within a 3-dilution range for each isolate. Etest MICs for the two yeasts which were recommended by NCCLS as QC isolates (C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 [21]) were within the established reference values on Casitone agar for amphotericin B and fluconazole and on RPMI 1640 medium for fluconazole and flucytosine (C. parapsilosis only). Lower MIC ranges of amphotericin B were obtained for the reference isolates C. parapsilosis and C. albicans (on both media), and higher MIC ranges of fluconazole were obtained for C. albicans (on Casitone agar). Interlaboratory agreement for the clinical isolates. Table 2 presents a summary of the interlaboratory agreement between the two laboratories of MIC endpoints for the 83 pathogenic yeasts determined by the Etest with solidified RPMI 1640 medium with 2% glucose and Casitone agar. The results are stratified by species or genera, antifungal agents, and incubation times. Flucytosine MICs were not determined on Casitone agar, because this agar formulation contains compounds antagonistic to this drug. The values in Table 2 represent the percentage of isolates of each species and drug combination for which MIC endpoints were within 2 doubling dilutions between the two laboratories when each medium was used. The amphotericin B MICs determined with RPMI 1640 medium showed the lowest level of reproducibility between the two centers at both incubation times; the agreement ranged from 0 to 6% at 24 and 48 h (overall agreement, 1 to 4%). On the other hand, when Casitone agar was used, the agreement for amphotericin B MICs ranged from 76 to 100%, depending on the species or genus (overall agreement, 84 to 90%). In contrast, when testing on RPMI 1640 medium was performed, fluconazole MICs demonstrated excellent agreement (94 to 100% at 24 and 48 h), and interlaboratory agreement was independent of incubation time and the species or genus tested. The agreement was lower with Casitone agar (52 to 100% at 24 and 48 h) and was more dependent on incubation time and the species or genus tested. The highest discrepancies among fluconazole MICs on Casitone agar were found when testing C. albicans at 48 h (52 versus 100% at 24 h) and T. glabrata at 24 h (71 versus 100% at 48 h). The agreement for the other azoles was lower on RPMI 1640 medium (57 to 91% for itraconazole and 69 to 100% for ketoconazole) than on Casitone agar (57 to 100% for itraconazole and 86 to 100% for ketoconazole). The interlaboratory reproducibilities of the ketoconazole and itraconazole MICs were also dependent on the length of incubation and, to a certain extent, on the species or genus tested. The lowest level of reproducibility was mostly found among the MICs determined after the shorter incubation times. On Casitone agar, the exceptions were itraconazole and ketoconazole MICs for C. albicans and itraconazole MICs for T. glabrata. The comparison of flucytosine MIC pairs from the two laboratories

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TABLE 1. MIC ranges of five antifungal agents for four ATCC strains (two QC) determined by Etest at two laboratories Fungusa

Antifungal agent

Reference MIC range (mg/ml) by reference M27-T method

MIC ranges (mg/ml) by Etest at both centers onb: RPMI 1640 medium–2% glucose

Casitone agar

C. krusei ATCC 6258 (QC)

Amphotericin B Fluconazole Flucytosine Itraconazole Ketoconazole

0.5–2 16–64 4–16 NAd NA

C. parapsilosis ATCC 22019 (QC)


0.25–1.0 2–8 0.12–0.5 NA NA

0.12 2 0.12 0.12 0.008

Candida albicans ATCC 90028


0.5–2 0.25–1.0 0.5–2 NA NA

0.03–1 0.25–1.0 0.5–2 0.06–0.25 0.008–0.03

0.25–0.5 0.5–4 ND 0.03–0.12 0.004–0.12

C. parapsilosis ATCC 90018


0.5–2 0.25–1.0 #0.12–0.25 NA NA

0.12–2 0.5–1.0 0.016–0.25 0.03–0.12 0.008–0.016

0.25–1.0 0.5–1.0 ND 0.03–0.06 0.008–0.016

0.03–4 .32 .32 0.5 0.12–0.5

0.5–1 .32 NDc 0.12–1.0 0.25–0.5 0.5 2 ND 0.25 0.03

a

ATCC 6258 and ATCC 22019 were recommended as QC isolates and ATCC 90028 and ATCC 90018 were recommended as reference strains by NCCLS (21). Each isolate was tested four times. ND, not determined because of interfering metabolites in Casitone agar. d NA, Reference MIC ranges have not been established for itraconazole and ketoconazole. b c

provided agreements ranging from 71 to 88%, depending on the genus or species tested (Table 2). Etest antifungal susceptibilities of pathogenic yeasts. Table 3 summarizes the combined MIC ranges of the five antifungal agents for each species tested at the two laboratories. Since interlaboratory agreement was medium dependent, only amphotericin B, itraconazole, and ketoconazole MIC ranges determined with Casitone agar and fluconazole and flucytosine ranges derived from MICs obtained with RPMI 1640 medium are included in Table 3. In general, MIC ranges were similar to previously reported Etest data (7, 9, 31) and broth dilution MIC data (11–13, 16). Amphotericin B MICs range for C. lusitaniae were 0.016 to 1.0 mg/ml at 24 h (data not shown in Table 3) and 0.06 to 8 mg/ml at 48 h on Casitone agar. Both centers defined the amphotericin B MIC for an isolate of C. lusitaniae as 8 mg/ml at 48 h, while light growth at 24 h either precluded an MIC determination at one center or yielded an MIC of 1.0 mg/ml at the other center. DISCUSSION Although the NCCLS broth macrodilution methods (M27-T document [21]) improved the level of interlaboratory agreement of antifungal MIC endpoints, these procedures are not convenient and efficient testing tools for the clinical laboratory. Several tests that are alternatives to the NCCLS methodology have been developed and evaluated. Among them, the Etest is the second commercial method that has been investigated for the testing of pathogenic yeasts (7, 9, 31). The Etest is a simple, agar-based, quantitative diffusion method which provides MIC endpoints instead of inhibition zone diameters, which the disk diffusion methods provide. Earlier comparisons of antifungal MIC data obtained by the Etest and the NCCLS broth dilution methods have determined that the former test is a potential

candidate as an alternative choice for testing the antifungal susceptibilities of pathogenic yeasts (7, 9, 31). Because of this, we focused on the comparison of the MICs determined by the Etest by two independent laboratories, and this represents the first interlaboratory evaluation of the Etest procedure for testing antifungal susceptibilities. The present study included a smaller set of isolates, but a broader spectrum of genera. In addition, five of the six available antifungal drugs were simultaneously evaluated in the present study, whereas prior studies involved the evaluation of one (31) to three (7, 9) antifungal agents. Previous evaluations of the effect of medium formulation on MICs for yeasts obtained by the Etest (9) did not include Casitone agar or solidified RPMI 1640 medium with 2% glucose. However, in-house evaluations by the manufacturer of the Etest strips have indicated that these two media improved the reproducibility of the MICs determined by the Etest, while they retained a correlation of these values with the MICs obtained by both NCCLS methods. Elevating the concentration of glucose from 0.2 to 2% optimized the growth of certain species of yeasts, as described previously (30), and facilitated our examination of MIC endpoints at both 24 and 48 h. The same effect was achieved with Casitone agar. Our analysis of the data indicated that interlaboratory reproducibility was dependent on medium formulation. A higher level of interlaboratory agreement was achieved with amphotericin B, itraconazole, and ketoconazole MICs obtained on Casitone agar (84 to 96% overall agreement at 48 h) than on solidified RPMI 1640 medium (1 to 90% overall agreement at 48 h). In contrast, agreement was optimized when fluconazole testing was performed on RPMI 1640 medium (Table 2). The percent agreement with amphotericin B, itraconazole (Casitone agar), and fluconazole (RPMI 1640 medium) among the laboratories was similar to that observed in prior interlabora-

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TABLE 2. Percent agreement between two laboratories by Etest with RPMI 1640 medium–2% glucose and Casitone agara Fungus (no. of isolates tested)

Candida albicans (33)

Antifungal agent


Candida spp. other Amphotericin B than C. albicans (36) Fluconazole Flucytosine Itraconazole Ketoconazole Cryptococcus neoformans (7)


Torulopsis glabrata (7)


Overall % agreement


% Agreement withb: Incubation RPMI 1640 Casitone medium– time (h) agar 2% glucose

24 48 24 48 24 48 24 48 24 48

6 0 100 97 88 85 67 91 94 97

76 82 100 52 NDc ND 97 88 100 97

24 48 24 48 24 48 24 48 24 48

6 0 94 97 83 83 58 69 69 78

86 92 86 92 ND ND 78 89 89 94

24 48 24 48 24 48 24 48 24 48

0 0 100 100 71 71 86 86 71 100

100 100 86 100 ND ND 100 100 80 100

24 48 24 48 24 48 24 48 24 48

0 0 100 100 86 86 57 71 100 86

100 100 71 100 ND ND 71 57 100 100

24 48 24 48 24 48 24 48 24 48

4 1 96 98 84 83 63 74 90 88

84 90 90 77 ND ND 87 87 94 96

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TABLE 3. MIC ranges of five antifungal agents for 83 clinical yeast isolates determined by Etest at two laboratories Fungus (no. of isolates tested)

Antifungal agent

Test mediuma

MIC range (mg/ml)b

Candida albicans (33)

Amphotericin B Itraconazole Ketoconazole Fluconazole Flucytosine

Casitone Casitone Casitone RPMI RPMI

0.06–0.5 0.016–2 ,0.008–0.03 0.12–16 0.03–32

Candida guilliermondii (2)



0.25–0.5 0.06–0.25 0.008–0.03 0.25–2 0.03–0.12

Candida krusei (4)



0.12–1.0 0.12–2 0.008–1.0 0.25–32 0.06–.32

Candida lusitaniae (8)



0.06–8 0.016–.32 #0.008–1.0 0.25–16 0.008–.32

Candida parapsilosis (7)



0.12–1.0 0.03–1.0 #0.008–0.12 0.12–8 0.03–4

Candida rugosa (2)



0.008–0.5 0.016–0.5 0.008–0.03 2–8 0.03–0.5

Candida tropicalis (13)



0.03–0.5 0.016–1.0 #0.008–0.06 0.12–.32 0.016–0.5

Cryptococcus neoformans (7)



0.12–0.5 0.12–2 0.03–0.25 .32 1–.32

Torulopsis glabrata



0.25–0.5 0.016–.32 #0.008–4 0.25–.32 0.008–1.0

a Etest MICs were determined by using Casitone agar (Casitone) or solidified (1.5% agar) RPMI 1640 medium with 2% glucose (RPMI). b Only the 48-h (Candida spp. and T. glabrata) and 72-h (C. neoformans) MIC ranges are listed.

a

A total of 83 clinical yeast isolates were tested in both laboratories. Results for MIC pairs from both laboratories were considered in agreement when the differences between MICs were within 2 doubling dilutions (61 dilution). c ND, not determined. b

tory evaluations of NCCLS broth dilution MICs for yeasts and mold-fungi (10, 12, 16). As reported previously in reports of studies that used other methods, the higher degree of variability observed with the azoles and flucytosine was associated with difficulty in examining the partial inhibition (growth trailing)

exhibited by these drugs. For the Etest, this growth trailing corresponds to the presence of microcolonies around or inside the whole inhibition ellipsis. This problem was partially overcome with fluconazole and ketoconazole (higher percent agreement) by testing on the two media that were evaluated. Although the MIC data for the more stable ATCC strains are closely in agreement with established reference ranges (Table 1), further research with other yeast isolates is needed to im-

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prove overall interlaboratory agreement of flucytosine and itraconazole MICs determined by the Etest. Overall, the distribution of the antifungal susceptibilities for the five drugs tested closely resemble prior MIC ranges obtained by the Etest (7, 31) and the NCCLS broth dilution methods (12, 16) after both incubation times (Table 3; data obtained at 24 h not shown). The MICs of ketoconazole for C. albicans determined by the Etest were lower than those determined by the M27-T methods (13), which have been reported before, when MICs were determined by other agar-based approaches and the Etest (6, 7). On the other hand, the fluconazole and flucytosine MICs for C. neoformans obtained by the Etest were higher than expected, which may be attributed to the increased growth rate induced by the two media used. It is noteworthy that testing of amphotericin B on Casitone agar clearly detected the isolate of C. lusitaniae that was resistant to this drug at 48 h (MICs, 8 mg/ml at both centers). This is an important breakthrough, because it is difficult to differentiate in vitro yeasts resistant to amphotericin B (MIC, .1.0 mg/ml) from yeasts susceptible to amphotericin B (MIC, ,1.0 mg/ml) when RPMI 1640 medium is used (29). This is one of the drawbacks of the NCCLS methodology. In conclusion, the results of the present interlaboratory evaluation of the Etest for testing the antifungal susceptibilities yeasts as well as previous comparisons of this method with the NCCLS procedures demonstrated that the Etest has potential as a reliable, practical, and easy method for use in the clinical laboratory. Further evaluations are deemed necessary to refine the testing conditions that will improve the level of interlaboratory agreement as well as to determine the reason for the unusually high flucytosine and fluconazole MICs for C. neoformans. ACKNOWLEDGMENTS We thank Anne Bolmstrom and Karen Mills at AB BIODISK for technical assistance during the study and also for the provision of the antifungal strips and media. We also thank Julie Rhodes for secretarial assistance in the preparation of the manuscript. This study was partially supported by a grant from Pfizer Inc. REFERENCES 1. AB Biodisk. 1993. Etest technical guide No. 4: antifungal susceptibility testing of yeasts. AB Biodisk, Solna, Sweden. 2. Anaissie, E. J., G. P. Bodey, and M. G. Rinaldi. 1989. Emerging fungal pathogens. Eur. J. Clin. Microbiol. Infect. Dis. 8:323–330. 3. Anaissie, E., V. Paetznick, and G. P. Bodey. 1991. Fluconazole susceptibility testing of Candida albicans: microtiter method that is independent of inoculum size, temperature, and time of reading. Antimicrob. Agents Chemother. 35:1641–1646. 4. Beck-Sague´, C. M., W. R. Jarvis, and the National Nosocomial Infections Surveillance System. 1993. Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980–1990. J. Infect. Dis. 167:1247–1251. 5. Block, E. R., A. E. Jennings, and J. E. Bennett. 1973. 5-Fluorocytosine resistance in Cryptococcus neoformans. Antimicrob. Agents Chemother. 3:647–656. 6. Brass, C., J. Z. Shainhouse, and D. A. Stevens. 1979. Variability of agar dilution-replicator method of yeast susceptibility testing. Antimicrob. Agents Chemother. 15:763–768. 7. Colombo, A. L., F. Barchiesi, D. A. McGough, and M. G. Rinaldi. 1995. Comparison of Etest and National Committee for Clinical Laboratory Standards broth macrodilution method for azole susceptibility testing. J. Clin. Microbiol. 33:535–540. 8. Dupont, B., J. R. Graybill, D. Armstrong, R. Laroche, J. E. Touze, and L. J. Wheat. 1992. Fungal infections in AIDS patients. J. Med. Vet. Mycol. 30(Suppl. 1):19–28. 9. Espinel-Ingroff, A. 1994. Etest for antifungal susceptibility testing of yeasts. Diagn. Microbiol. Infect. Dis. 19:217–220. 10. Espinel-Ingroff, A., K. Dawson, M. Pfaller, E. Anaissie, B. Breslin, D. Dixon, A. Fothergill, J. Peter, M. Rinaldi, and T. Walsh. 1995. Comparative and collaborative evaluation of standardization antifungal susceptibility testing for filamentous fungi. Antimicrob. Agents Chemother. 39:314–319. 11. Espinel-Ingroff, A., T. M. Kerkering, P. R. Goldson, and S. Shadomy. 1991. Comparison study of broth macrodilution and microdilution antifungal susceptibility tests. J. Clin. Microbiol. 29:1089–1094.

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