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Journal of Clinical Microbiology, October 2004, p. 4577-4580, Vol. 42, No. 10
0095-1137/04/$08.00+0 DOI: 10.1128/JCM.42.10.4577-4580.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Clinical Evaluation of the Sensititre YeastOne Colorimetric Antifungal Plate for Antifungal Susceptibility Testing of the New Triazoles Voriconazole, Posaconazole, and Ravuconazole
M. A. Pfaller,1* A. Espinel-Ingroff,2 and R. N. Jones3,4
University of Iowa College of Medicine and College of Public Health, Iowa City, Iowa,1
Medical College of Virginia of Virginia Commonwealth University, Richmond, Virginia,2
JMI Laboratories, North Liberty, Iowa,3
Tufts University School of Medicine, Boston, Massachusetts4
Received 19 April 2004/
Returned for modification 27 May 2004/
Accepted 13 June 2004

ABSTRACT
A commercially prepared dried colorimetric microdilution panel
(Sensititre YeastOne, TREK Diagnostic Systems, Cleveland, Ohio)
was compared in three different laboratories with the National
Committee for Clinical Laboratory Standards (NCCLS) reference
microdilution method by testing two quality control strains
and 300 clinical isolates of
Candida spp. against fluconazole,
voriconazole, posaconazole, and ravuconazole. Reference MIC
endpoints were established after 48 h of incubation and YeastOne
colorimetric endpoints were established after 24 h of incubation.
YeastOne endpoints were determined to be the lowest concentration
at which the color in the well changed from red (indicating
growth) to purple (indicating growth inhibition) or blue (indicating
no growth). Excellent agreement (within two dilutions) between
the reference and colorimetric MICs was observed. Overall agreement
was 95.4%. Agreement ranged from 92.3% with posaconazole to
98.0% with fluconazole. The YeastOne colorimetric method appears
to be comparable to the NCCLS reference method for testing the
susceptibility of
Candida spp to the new triazoles voriconazole,
posaconazole, and ravuconazole.

INTRODUCTION
The National Committee for Clinical Laboratory Standards (NCCLS)
reference broth microdilution antifungal susceptibility testing
method has been established since 1997 (NCCLS M27-A) and is
now used in laboratories worldwide (
1,
3,
9,
17,
18). The second
edition of NCCLS document M27-A2 was published in 2002 and includes
quality control ranges for microdilution testing of both established
(e.g., fluconazole and itraconazole) and newly introduced (voriconazole)
or investigational (posaconazole and ravuconazole) agents (
8).
Commercial development of microdilution antifungal panels conforming
to the NCCLS M27 guidelines has been gradual (
7,
13); however,
one product, the Sensititre YeastOne colorimetric plate (Trek
Diagnostic Systems, Cleveland, Ohio), has been cleared by the
U.S. Food and Drug Administration for patient care testing of
fluconazole, itraconazole, and flucytosine. Subsequent to U.S.
Food and Drug Administration clearance, the YeastOne system
has been used widely in the United States and elsewhere with
good results in terms of accuracy and reproducibility (4, 7;
R. N. Jones, minutes of the NCCLS Antifungal Testing Subcommittee
meeting, 10 January 2004, Phoenix, Ariz.).
The YeastOne system is available in a dry-form 96-well panel with the colorimetric growth indicator Alamar Blue and has a shelf life of approximately 24 months at ambient temperature. Based on previous multicenter evaluations (4, 10-12), the MICs of fluconazole and other agents may be read following 24 h of incubation with the YeastOne system. Recently, the new extended-spectrum triazoles (voriconazole, posaconazole, and ravuconazole) have been added to a panel containing fluconazole in anticipation of these agents' gaining agency approval for the treatment of candidiasis. A study designed to establish the interlaboratory reproducibility of the YeastOne system for testing the three triazoles has shown excellent agreement (96 to 99%) when a set of 100 isolates were tested in three different laboratories and MIC results were read after 24 h of incubation (5).
The purpose of the present study was to validate the performance of the YeastOne panel with voriconazole, posaconazole, and ravuconazole against a broad range of clinical isolates of Candida spp. in three independent laboratories and to compare the results from these panels with those from a frozen reference microdilution method performed according to NCCLS guidelines.

MATERIALS AND METHODS
Study design.
The study was designed to compare MIC results for fluconazole,
voriconazole, posaconazole, and ravuconazole obtained by the
YeastOne system to those obtained by the M27-A2 broth microdilution
method (
8) in the three participating laboratories. Each laboratory
tested 100 clinical isolates of
Candida spp. by the YeastOne
system and the NCCLS frozen reference broth microdilution panel
(a total of 300 isolates). The MIC results obtained with the
YeastOne system following 24 h of incubation were compared with
those obtained with the reference broth microdilution panel
read after 48 h of incubation.
Test organisms.
The test organisms include two American Type Culture Collection (ATCC) strains that have been established as quality control strains (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) by the NCCLS (2, 8). These isolates have well-defined microdilution MIC reference ranges for fluconazole, voriconazole, posaconazole, and ravuconazole (2). An additional 300 clinical isolates of Candida spp. were also tested. The collection included 75 isolates of Candida albicans, 59 of Candida parapsilosis, 62 of Candida krusei, 61 of Candida lusitaniae, 15 of Candida tropicalis, 16 of Candida glabrata, 6 of Candida guilliermondii, 3 of Candida rugosa, and 1 each of Candida kefyr, Candida pelliculosa, and Candida lipolytica. These were all recent clinical isolates and were selected to represent the clinically prevalent species, including azole-resistant strains, to provide a broad range of MICs, and to maximize the on-scale MIC endpoints for the various antifungal agents tested. Isolates were identified by standard methods (6). Prior to testing, each isolate was passaged at least twice on potato dextrose agar (Remel, Lenexa, Kans.) to ensure purity and viability.
Antifungal agents and microdilution panels.
The YeastOne panels and the frozen reference broth microdilution trays containing serial twofold dilutions of fluconazole (0.12 to 256 µg/ml), voriconazole (0.008 to 16 µg/ml), posaconazole (0.004 to 8 µg/ml), and ravuconazole (0.008 to 16 µg/ml) were provided by Trek Diagnostic Systems. The YeastOne panels were shipped in sealed packages and stored at ambient temperature until testing was performed. The broth microdilution trays, which were prepared by following the M27-A2 additive procedure (8), were shipped frozen to each participant laboratory and stored at 70°C until the day of the test.
Inoculum preparation.
Stock inoculum suspensions of the Candida spp. were obtained from 24-h cultures on Sabouraud dextrose agar at 35°C. The turbidity of each yeast suspension was adjusted by the spectrophotometric method by following the M27-A2 guidelines (8).
NCCLS broth microdilution method.
Reference broth microdilution testing was performed exactly as outlined in NCCLS document M27-A2 (8) with a final inoculum concentration of 1.5 x 103 ± 1.0 x 103 cells/ml and RPMI 1640 medium buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer. The panels were incubated in air at 35°C and observed for the presence or absence of growth at 48 h. The MICs of all four agents were read as the lowest concentration that produced a prominent decrease in turbidity (approximately 50% reduction in growth) relative to the growth control (8).
Sensititre YeastOne colorimetric MIC procedure.
On the day of the test, a working yeast suspension of approximately 1.5 x 103 cells/ml was prepared in YeastOne broth (Trek). The dried YeastOne panels were rehydrated with the working yeast suspension with a multichannel pipetting device by dispensing 100 µl into each well. The YeastOne panels were covered with seal strips and incubated at 35°C for 24 h in a non-CO2 incubator. The colorimetric MIC endpoints were read with the aid of a reading mirror. Yeast growth was evident as a color change from blue (no growth) to red (growth). Colorimetric MIC results for all of the test agents were defined as the lowest concentration of antifungal agent that prevented the development of a red color (the first blue or purple well) (4, 5, 12).
Quality control.
Quality control was ensured by testing the NCCLS-recommended quality control strains C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 (2, 8). These isolates were tested between 6 and 22 times in each of the three laboratories, and all (100%) MICs were within the respective reference ranges.
Analysis of results.
The MIC results obtained with the YeastOne panels read at 24 h were compared with those of the reference panels read at 48 h. Both on-scale and off-scale results were included in the analysis. As with previous studies (4, 5, 15), high off-scale MIC results were converted to the next highest concentration, and low off-scale MIC results were left unchanged. Overall, 85.0% of MICs were on-scale. Discrepancies among MIC endpoints of no more than two dilutions (two wells) were used to calculate the percent agreement. The analysis of percent agreement included both total results (on- and off-scale) and on-scale results only.

RESULTS AND DISCUSSION
Table
1 summarizes the in vitro susceptibilities of 300 clinical
isolates of
Candida spp. to fluconazole and the three extended-spectrum
triazoles as determined by the NCCLS reference broth microdilution
frozen panel. A broad range of on-scale MICs were observed with
each antifungal agent. In general, MIC results for each antifungal
agent were typical for each species of
Candida (
14-
16); however,
isolates of each species with decreased susceptibility to azoles
were included (55 fluconazole-resistant strains) in order to
expand the range of MIC results evaluated.
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TABLE 1. Antifungal susceptibilities of 300 clinical isolates of Candida spp. as determined by the NCCLS reference microdilution method
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Quality control determinations were performed on 6 to 22 different
occasions with each of the quality control strains recommended
by the NCCLS (
2,
8). These MICs were within control limits for
all four agents and both the reference and YeastOne panels.
The overall agreement between the results of the reference method and the YeastOne colorimetric method was 95.4% (94.6% for on-scale results) (Table 2). Agreement ranged from 92.3% for posaconazole to 98.0% for fluconazole. Similar high levels of agreement were observed for results from each of the three study sites; however, nine isolates tested in site 1 (eight C. lusitaniae and one C. krusei) did not grow sufficiently to be read after 24 h of incubation. The MIC results obtained after 48 h of incubation in both systems were compared for those isolates only. Study site 1 also recorded the lowest levels of agreement for voriconazole (93.0%) and posaconazole (85.0%).
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TABLE 2. Agreement between YeastOne colorimetric and reference microdilution MIC results of four agents tested against 300 isolates of Candida species in three different laboratories
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Regarding individual species of
Candida, the agreement in the
MIC endpoints for all drugs and all species was >90%, with
the exception of
C. albicans and posaconazole (89.3%),
C. lusitaniae and posaconazole (75.4%),
C. glabrata and voriconazole (87.5%),
and
Candida spp. and voriconazole (83.3%) (Table
3). Of the
27 discrepancies observed with these four organism-drug combinations,
15 (56%) were from a single study site. When present, discrepancies
were due to MIC tests determined with the YeastOne panels being
lower than the reference panel MIC results. A similar level
of agreement was observed regardless of whether all values or
only on-scale values were used (Table
3). Importantly, these
discrepancies resulted in only one false-susceptible (very major
error) and no false-resistant (major error) results in categorizing
isolates according to fluconazole susceptibility. Considering
only fluconazole-resistant strains, the agreement was 99% for
fluconazole, 96% for voriconazole and 96% for both ravuconazole
and posaconazole.
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TABLE 3. Agreement by species between YeastOne colorimetric and reference microdilution MIC results for four agents tested against 300 isolates of Candida spp.
|
The findings of the present study confirm and extend the results
of our previous interlaboratory reproducibility study (
5) and
demonstrate excellent performance of the YeastOne Colorimetric
system for antifungal MIC susceptibility testing of a wide variety
of clinical isolates against established (fluconazole), newly
introduced (voriconazole), and investigational (posaconazole
and ravuconazole) triazoles. As with previous studies, the colorimetric
MICs was read after 24 h of incubation (
4,
5,
12). Although
nine isolates did not grow sufficiently for the MIC results
to be read at 24 h in one laboratory, this problem was not observed
in the other study sites.
The overall level of agreement (95.4%) in this study is consistent with or superior to that reported from other studies of the YeastOne system (4, 5, 7). By testing several clinically relevant species with a broad range of susceptibilities to the azole antifungal agents, we have validated the YeastOne colorimetric MIC system for the testing of three additional antifungal agents that possess potent activity against Candida spp. Thus, the YeastOne system may be used for further investigation of the antifungal activity of voriconazole, posaconazole, and ravuconazole with results comparable to those obtained with the reference broth microdilution method (8). The establishment of interpretive breakpoints for these new agents is pending the results of clinical correlative studies.

ACKNOWLEDGMENTS
This study was supported in part by a grant from Trek Diagnostic
Systems.
We thank Linda Elliott for secretarial assistance in the preparation of the manuscript. We acknowledge the excellent assistance of the technical personnel in the Molecular Epidemiology and Fungus Testing Laboratory and JMI Laboratories. We appreciate the assistance with data analysis provided by Nikki Holliday.

FOOTNOTES
* Corresponding author. Mailing address: Medical Microbiology Division, C606 GH, Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242. Phone: (319) 384-9566. Fax: (319) 356-4916. E-mail:
michael-pfaller{at}uiowa.edu.


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Journal of Clinical Microbiology, October 2004, p. 4577-4580, Vol. 42, No. 10
0095-1137/04/$08.00+0 DOI: 10.1128/JCM.42.10.4577-4580.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
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