Activities of Fluconazole and Voriconazole against 1,586 Recent Clinical Isolates of Candida Species Determined by Broth Microdilution, Disk Diffusion, and Etest Methods: Report from The ARTEMIS Global Antifungal Susceptibility Program, 2001

Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Activities of Fluconazole and Voriconazole against 1,586 Recent

Clinical Isolates of

Candida

Species Determined by Broth

Microdilution, Disk Diffusion, and Etest Methods: Report

from The ARTEMIS Global Antifungal Susceptibility

Program, 2001

M. A. Pfaller,

_{* D. J. Diekema,}

_{S. A. Messer,}

_{L. Boyken,}

_{and R. J. Hollis}

and College of Public Health, University of Iowa, Iowa City, Iowa 52242

Received 11 October 2002/Returned for modification 5 December 2002/Accepted 22 December 2002

The ARTEMIS Global Antifungal Susceptibility Program (ARTEMIS Program) was initiated in 2001 to provide focused surveillance of the activities of fluconazole and voriconazole againstCandidaspp. isolated from blood and other normally sterile sites. A total of 1,586 episodes of infection were detected at 61 international study sites. Overall, 57.7% of the infections were due toCandida albicans, followed byC. glabrata(14.8%),C. parapsilosis(12.5%),C. tropicalis(9.4%),C. krusei (2.7%), andC. lusitaniae (1.5%). Isolates ofC. albicans,C. parapsilosis, andC. tropicaliswere all highly susceptible to fluconazole (for 99% of the isolates the MICs were

<_{8␮g/ml). Likewise, 99 to 100% of these species were inhibited by}<_{1␮g of voriconazole per ml. Voriconazole}

was also active againstC. glabrata(93% of the isolates were susceptible [MICs<_{1␮g/ml]) andC. krusei(100%}

of the isolates were susceptible). The agar-based Etest and disk diffusion methods performed well for the testing of both fluconazole and voriconazole compared to the broth microdilution MIC reference method. These observations establish the continued importance ofC. albicansas a pathogen and the sustained activity of fluconazole and the broad spectrum of activity of voriconazole and will serve as the first-year benchmark for the ARTEMIS Program. Continued surveillance and refinement of broth- and agar-based test methods will help to identify susceptibility trends and improve the laboratory capability for antifungal susceptibility testing.

Antimicrobial resistance surveillance serves many purposes. The most common is detection and tracking of resistance trends and emerging new resistance threats (10, 15, 16, 18). Clearly, this is very important and serves as the basis for both empirical treatment recommendations and as a means to as-sess interventional efforts (8, 10, 14, 16). In addition to these potentially far-reaching objectives, surveillance programs also serve as a means to monitor the prevalent pathogens causing serious infections (e.g., bloodstream infections [BSIs]). The isolates collected in those programs that use a central labora-tory can be used to assess the activities of new antimicrobial agents and to conduct postmarketing surveillance of the activ-ities of established agents and aid in the development and validation of new susceptibility testing methods (8–10, 16). In order to address effectively any of these secondary objectives, the availability of a geographically diverse collection of isolates from clinically important sites of infection (e.g., blood and normally sterile body fluids [NSBFs]) is essential (16, 18, 31). Although there are numerous ongoing antibacterial resis-tance surveillance programs that address with various degrees of success the objectives noted above (10, 15, 16, 31), very few programs provide similar information for fungal infections and antifungal resistance (5, 21, 31). The ARTEMIS Global

Anti-fungal Susceptibility Program (ARTEMIS Program) was initi-ated in 2001 in order to provide focused surveillance of the activities of fluconazole and voriconazole againstCandidaspp. causing invasive infections and to provide continuous develop-ment and validation of various broth- and agar-based antifun-gal susceptibility test systems. The ARTEMIS Program uses a central reference laboratory and an international network of 105 participating centers as sources of clinical isolates.

Fluconazole is a very widely used systemic antifungal agent with a broad therapeutic range and little toxicity (34). Con-cerns regarding the inappropriate usage of fluconazole (e.g., poor indications and inadequate dosing practices) abound (3, 20), coupled with concerns of increased resistance due to ei-ther the emergence of resistance in previously susceptible spe-cies or the emergence of spespe-cies with primary resistance to fluconazole (3, 7, 28, 30, 36). Likewise, there is a need for broad and ongoing assessment of the potency and spectrum of activity of voriconazole both prior to and after its introduction into clinical practice. Assessment of susceptibility and resis-tance to these agents amongCandidaspp. is dependent upon the use of standardized antifungal susceptibility testing meth-ods. For surveillance purposes the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution (BMD) method (19) is ideal, as it is standardized, provides quantitative MICs, and is well suited for use in a high-volume reference laboratory. The NCCLS method may not be optimal for use in many clinical laboratories, as there are few validated commercial MIC systems and many laboratories test only a few

* Corresponding author. Mailing address: Molecular Epidemiology & Fungus Testing Laboratory, Departments of Pathology and Epide-miology, C606 GH, University of Iowa College of Medicine and Col-lege of Public Health, Iowa City, IA 52242. Phone: (319) 384-9566. Fax: (319) 356-4916. E-mail: michael-pfaller@uiowa.edu.

1440

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isolates per year. Thus, development of agar-based tests such as disk diffusion and Etest methods has been deemed desirable (27, 30). Both methods have been evaluated, and Etest appears to perform well in providing quantitative MICs of both flucon-azole and voriconflucon-azole (22, 25). The disk diffusion method has only recently been shown to be useful for the testing of flu-conazole (2, 11, 17, 19a), and the published data for voricon-azole disk testing is limited (13). In the present study we report the first data from the ARTEMIS Program, including data on both the surveillance of antifungal resistance amongCandida

spp. and the continued development and validation of the disk diffusion and Etest methods for fluconazole and voriconazole.

MATERIALS AND METHODS

Study design.The ARTEMIS Program was established in 2001 to monitor the species and antifungal susceptibility patterns ofCandidaspp. isolated from blood and other NSBFs via a broad network of 105 sentinel hospital sites in North America, Latin America, Europe, Africa, and Asia.Candidaspp. causing inva-sive disease (BSIs and infections of NSBFs) were reported from 61 of 105 medical centers monitored in 2001 (January through December).

Each participant hospital contributed results (organism identification, date of isolation, and hospital location) for consecutive blood and NSBF culture isolates (one isolate per patient) ofCandidaspp. judged to be clinically significant by local criteria and detected in each calendar month during the study period. All isolates were saved on agar slants and were sent to the University of Iowa College of Medicine (Iowa City) for storage and further characterization by reference identification methods and susceptibility testing (19, 37). Isolates were also subjected to testing by the disk diffusion and Etest methods (2, 19a, 22, 25).

Organism identification.AllCandidasp. isolates were identified at the par-ticipating institutions by the routine method used in each laboratory. Upon receipt at the University of Iowa, the isolates were subcultured onto potato dextrose agar (Remel, Lenexa, Kans.) and CHROMagar Candida medium (Har-dy Laboratories, Santa Maria, Calif.) to ensure viability and purity. Confirmation of species identification was performed with Vitek and API products (bi-oMerieux, St. Louis, Mo.), as recommended by the manufacturer or by conven-tional methods, as required (37). Isolates were stored as suspensions in water or on agar slants at ambient temperature until needed.

Susceptibility testing.Reference antifungal susceptibility testing ofCandida

spp. was performed by the BMD method described by the NCCLS (19). Refer-ence powders of fluconazole and voriconazole were obtained from Pfizer Phar-maceuticals (Groton, Conn.).

Etest strips for fluconazole and voriconazole were provided by AB BIODISK (Solna, Sweden). MICs were determined by Etest as described previously (22, 25) with RPMI agar with 2% glucose (Remel), an inoculum suspension adjusted to the turbidity of a 0.5 McFarland standard (⬃106_{cells/ml), and incubation at} 35°C for 48 h. The MICs of both fluconazole and voriconazole were read as the lowest concentration at which the border of the elliptical inhibition zone inter-cepted the scale on the strip. Any growth, such as microcolonies, throughout a discernible inhibition ellipse was ignored.

Disk diffusion testing of fluconazole and voriconazole was performed as de-scribed by Barry et al. (2) and in NCCLS document M44-P (19a). Fluconazole (25␮g) and voriconazole (1␮g) disks were obtained from Becton Dickinson (Sparks, Md.). For disk diffusion testing, 150-mm-diameter plates containing Mueller-Hinton agar (Difco Laboratories) supplemented with 2% glucose and methylene blue (0.5␮g/ml) at a depth of 4.0 mm were used. The agar surface was inoculated by using a swab dipped in a cell suspension adjusted to the turbidity of a 0.5 McFarland standard. The plates were incubated in air at 35°C and read at 24 h. Zone diameter endpoints were read at 80% growth inhibition by using the BIOMIC image analysis plate reader system (version 5.9; Giles Scientific, Santa Barbara, Calif.) (17).

MIC interpretive criteria for fluconazole were those published by Rex et al. (29) and the NCCLS (19) and were as follows: susceptible, MICⱕ8␮g/ml; susceptible-dose dependent, MIC⫽16 to 32␮g/ml; resistant, MICⱖ64␮g/ml. The interpretive criteria for the fluconazole disk test were those published by Barry et al. (2) and the NCCLS (19a): susceptible, zone diameter ofⱖ19 mm; susceptible-dose dependent, zone diameter of 15 to 18 mm; resistant, zone diameter ofⱕ14 mm. Interpretive breakpoints have not yet been established for voriconazole.

QC.Quality control (QC) was performed for BMD and Etest in accordance with NCCLS document M27-A (19) by usingCandida kruseiATTC 6258 andC.

parapsilosisATCC 22019 (1, 19). QC determinations made on each day of testing

were within the control limits for fluconazole and voriconazole described by Barry et al. (1). QC for disk diffusion testing was performed by usingC. albicans

ATCC 90028 andC. parapsilosisATCC 22019 (2, 17, 19a).

Analysis of results.The Etest MICs of fluconazole and voriconazole read at 48 h were compared to the reference BMD MICs read at 48 h. The Etest MICs were rounded up to the next even log2concentration in order to simplify analysis (2, 22, 25). Discrepancies of no more than 2 dilutions were used to calculate the percent agreement.

The diameters of the zones of inhibition (in millimeters) surrounding the fluconazole and voriconazole disks at 24 h of incubation were plotted against their respective BMD MICs read at 48 h (2). The method of least squares was used to calculate a regression line for each comparison. The interpretive break-points described by Barry et al. (2) and the NCCLS (19a) were used to determine the categorical agreement between the disk diffusion and BMD results for flu-conazole. Major errors were identified as a classification of resistance by the disk diffusion test and susceptibility by BMD, very major errors were identified as a classification of susceptibility by the disk diffusion method and resistance by BMD, and minor errors occurred when the result of one of the tests was susceptibility or resistance and that of the other test was susceptible-dose de-pendent.

RESULTS AND DISCUSSION

During the 2001 study period, a total of 1,586 isolates of

Candida spp. from blood and other NSBFs were submitted from 61 participating centers in North America (20 centers), Latin America (8 centers), Europe (19 centers), Africa (5 cen-ters), and Asia (9 centers). The species identification provided from the submitting laboratory was confirmed for 92% of the isolates (range, 25% forC. guilliermondiito 97% forC. albi-cans). The frequencies of infections due to the various species ofCandida identified by the central reference laboratory are presented in Table 1. As observed in other surveys (17, 26), 97% of serious candidal infections were due to C. albicans

(57.7%),C. glabrata(14.8%),C. parapsilosis(12.5%),C. tropi-calis(9.4%), andC. krusei(2.7%). Likewise, the rank order of these species is essentially identical to that reported by the SENTRY Program (26) and by Meis and colleagues for the Global Antifungal Surveillance Group (17). As noted previ-ously (23, 24), these data further demonstrate the sustained importance of C. albicans as an etiologic agent of BSIs and infections of other normally sterile sites. Although the fre-quency of C. albicans as a cause of BSIs may vary among different institutions (26), these data indicate that the role ofC. albicans as a major fungal pathogen had not diminished in

TABLE 1. Species distributions ofCandidabloodstream and other normally sterile body fluid isolates in the ARTEMIS

Program, 2001

Species No. of isolates % of isolates

Candida albicans 916 57.7 Candida glabrata 235 14.8 Candida parapsilosis 198 12.5 Candida tropicalis 150 9.4 Candida krusei 43 2.7 Candida lusitaniae 24 1.5 Candidaspeciesa _{20 1.3}

Total 1,586 100

a_{IncludesC. kefyr(n⫽4),C. pelliculosa(n⫽8),C. rugosa(n⫽2),C. lipolytica} (n⫽2),C. guilliermondii(n⫽2),C. famata(n⫽1), andC. zeylanoides(n⫽1).

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2001 and in fact may be increasing in some locations, despite the widespread use of fluconazole (4, 20, 26, 32, 33, 35).

In vitro susceptibility testing by both the reference BMD method and Etest demonstrated that resistance to fluconazole (MIC ⱖ 64␮g/ml) remains rare among isolates of Candida

from blood and NSBFs (Table 2). By using the BMD results, 99% of C. albicans, C. parapsilosis, and C. tropicalis isolates were susceptible (MIC ⱕ 8 ␮g/ml) to fluconazole. Overall, 90.5% of isolates were susceptible and only 2.5% were resis-tant. As noted previously (26),C. glabrataand C. kruseiwere the least susceptible; however, only 7% ofC. glabrataisolates demonstrated high-level resistance. Although the Etest MIC results tended to be slightly higher than the BMD MIC results, especially forC. glabrata, the overall agreement between Etest and BMD was 96.4%, consistent with that reported previously (22).

Voriconazole demonstrated excellent potency and broad-spectrum activity against all species (Table 3). Overall, 99% of isolates were inhibited by voriconazole atⱕ1␮g/ml. As noted previously (26),C. albicanswas the most susceptible (MIC at which 90% of isolates are inhibited [MIC90], 0.015␮g/ml) and

C. glabratawas the least susceptible (MIC90, 1␮g/ml). All of

theC. kruseiisolates tested were inhibited by voriconazole at

ⱕ1␮g/ml. Similar to the results obtained with fluconazole, the agreement between BMD and Etest results was good (98.1%), with Etest MICs tending to be slightly higher than those de-termined by BMD. Again, these results are consistent with those reported previously (25).

Although the antifungal susceptibility data presented in Ta-bles 2 and 3 are very similar to those reported previously from other antifungal surveillance programs (17, 26), the manner of data presentation used in the ARTEMIS Program takes into

TABLE 2. In vitro susceptibilities ofCandidaspp. to fluconazole determined by BMD and Etest methods, ARTEMIS Program, 2001

Species (no. of isolates tested) Test Method Cumulative % susceptible at MIC (␮g/ml) of a_:

0.12 0.25 0.5 1 2 4 8 16 32 64

C. albicans(916) BMD 14 80 94 98 99 99 99 99 99 99

Etest 42 87 98 99 99 99 99 99 99 99

C. glabrata(235) BMD 0 0 0 1 6 25 57 89 93 95

Etest 0 0 0 1 6 15 32 66 86 91

C. parapsilosis(198) BMD 0 7 44 85 96 99 99 100

Etest 4 20 62 88 94 97 99 99 100

C. tropicalis(150) BMD 0 5 25 57 93 99 99 99 99 100

Etest 0 13 69 92 97 99 100

C. krusei(43) BMD 0 0 0 0 0 0 0 14 56 98

Etest 0 0 0 0 0 0 0 0 37 58

C. lusitaniae(24) BMD 4 21 67 88 92 96 96 96 96 100

Etest 8 29 54 83 92 96 96 96 96 96

a_{MICs were determined according to NCCLS guidelines (19).}

TABLE 3. In vitro susceptibilities ofCandidaspp. to voriconazole determined by BMD and Etest methods, ARTEMIS Program, 2001

Species (no. of isolates tested) Test method Cumulative % susceptible at MIC (␮g/ml) of a_:

0.007 0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8

C. albicans(916) BMD 79 94 99 99 99 99 99 99 99 100

Etest 73 98 99 99 99 99 99 99 99 100

C. glabrata(235) BMD 0 0 1 6 29 74 88 93 97 99 100

Etest 0 1 3 12 30 66 86 92 95 96 97

C. parapsilosis(198) BMD 18 63 83 96 99 99 100

Etest 25 64 85 97 99 99 100

C. tropicalis(150) BMD 4 11 41 87 98 100

Etest 3 22 71 92 99 100

C. krusei(43) BMD 0 0 0 0 16 65 93 100

Etest 0 0 0 0 7 72 95 100

C. lusitaniae(24) BMD 79 88 88 96 96 96 100

Etest 54 92 92 96 96 96 100

a_{MICs were determined according to NCCLS guidelines (19).}

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account the suggestions and critiques of several expert individ-uals regarding the usefulness of antimicrobial resistance sur-veillance programs (10, 15, 18).

The ARTEMIS Program susceptibility data are generated by a standardized quantitative reference method (NCCLS BMD method) and a validated reference quality quantitative method (Etest) and are presented in a continuous fashion as the cumulative percentages of organisms susceptible at each dilution throughout the full dilution series. The presentation of susceptibility data generated by standardized methods in a continuous fashion has several important advantages (10, 18): (i) the data may be compared over time, and subtle changes in susceptibility (trends) can be detected before changes in MIC50s, MIC90s, or interpretive categories are observed; (ii)

the data generated by the same methodology by different stud-ies may be compared; (iii) the data may be more generalizable, as continuous data are not restricted by interpretive criteria that may vary by nation or by international consensus groups; and (iv) continuous data provide antimicrobial susceptibility information for organisms or agents for which breakpoints are not available. Thus, the MIC data for fluconazole and

voricon-azole presented in Tables 2 and 3 represent the initial bench-mark results for the ARTEMIS Program. Testing ofCandida

spp. from defined sites of infection (blood and NSBFs) by a standardized reference method to generate full-scale MICs presented in a continuous fashion will ensure that the data from the ARTEMIS Program are truly international and not subject to the interpretive restrictions dictated by regulatory agencies, professional societies, or consensus standards orga-nizations (10, 18). The longitudinal nature of this program will also ensure that any trends in the susceptibility ofCandidaspp. to fluconazole or voriconazole are readily detected.

Comparison of susceptibility test methods by use of a refer-ence BMD method and disk diffusion testing of fluconazole and voriconazole is also an important aspect of the ARTEMIS Program. Figure 1 shows the correlation of the 25-␮g flucon-azole disk zone diameters read at 24 h compared to the BMD MIC results read at 48 h. The regression equation (y⫽49.6⫺

1.9x;R⫽0.7) is similar to that of Barry et al. (2) (y⫽55.4⫺

2.7x;R⫽0.8). The overall categorical agreement by use of the interpretive criteria of Barry et al. (2) and the NCCLS (19, 19a) was 93.1%, with 0.1% very major errors, 0.3% major

FIG. 1. Zones of inhibition around 25-␮g fluconazole disks tested on Mueller-Hinton agar supplemented with 2% glucose and methylene blue (0.5␮g/ml) plotted against the MICs at 48 h determined by the reference BMD method for 1,586 isolates ofCandidaspp. The method of least squares was used to calculate a regression line (y⫽49.6⫺1.9x;R⫽0.7).

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errors, and 6.5% minor errors. These data support the findings of Meis et al. (17) and Barry et al. (2) and provide further validation of the disk diffusion test as a useful method for determining the in vitro susceptibilities of Candida spp. to fluconazole.

Although disk diffusion testing of voriconazole has not been fully developed (13), we used a 1-␮g voriconazole disk and the same method for the testing of fluconazole to generate zone diameters and MIC correlates for voriconazole. Figure 2 shows the correlation of the 1-␮g voriconazole disk zone diameters read at 24 h compared to the BMD MIC results read at 48 h. The regression statistics (y⫽43.8⫺2.4x;R⫽0.7) showed that there is a good level of agreement between the two methods. Although MIC breakpoints have not yet been established for voriconazole, the 17 isolates for which MICs wereⱖ2␮g/ml were either resistant (15 isolates) or susceptible-dose depen-dent (2 isolates) to fluconazole. Thirteen of these isolates were separated from the rest of the population by a zone diameter threshold ofⱕ13 mm. Although a single preliminary interpre-tive category of susceptible by use of an MIC threshold ofⱕ1

␮g/ml and a zone diameter of⬎13 mm may be supported by pharmacokinetic and pharmacodynamic profiles (6, 12, 34), the establishment of clinical correlates is imperative before break-points can be used clinically (30). Despite these concerns, the overall categorical agreement of the results obtained by using these threshold values was 99.4% for the voriconazole disk method and the BMD MIC test. On the basis of these findings, it appears that the disk diffusion test is a useful method for testing of the activity of voriconazole against Candida spp. Continued application of this test in the context of the ARTE-MIS Program will allow us to monitor the frequencies of clin-ical isolates surrounding these zone diameter and MIC thresh-olds. Depending on the frequency of occurrence and clinical response to therapy, more firmly established in vitro break-point criteria (susceptible, susceptible-dose dependent, and/or resistant) may be determined.

The data reported herein provide additional support to those from the SENTRY Program (24, 26) and the Global Antifungal Surveillance Group (17) regarding the sustained activity of fluconazole againstCandidaspp. causing BSIs and

FIG. 2. Zones of inhibition around 1-␮g voriconazole disks plotted against the MICs determined at 48 h by the reference BMD method for 1,586 isolates ofCandidaspp. The method of least squares was used to calculate a regression line (y⫽43.8⫺2.4x;R⫽0.7).

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other deeply invasive infections. The MIC data generated for both fluconazole and voriconazole in 2001 will serve as the benchmark for the ARTEMIS Program and the basis for fu-ture comparisons and the establishment of susceptibility trends. Continued monitoring of isolates from the interna-tional study sites by the BMD, disk diffusion, and Etest meth-ods will provide useful quantitative and qualitative data and allow continued refinement of all methods with a robust col-lection of geographically diverse strains ofCandidaspp. Thus far, the studies appear to validate the usefulness of both Etest and the disk diffusion test for determination of the in vitro susceptibilities of Candida spp. to fluconazole and voricon-azole.

ACKNOWLEDGMENTS

This study was supported in part by research and educational grants from Pfizer Pharmaceuticals and Giles Scientific, Inc.

We thank Linda Elliott for secretarial assistance in the preparation of the manuscript. We appreciate the contributions of all ARTEMIS site participants. The following participants contributed isolates to the study: Hershey Medical Center, Hershey, Pa. (P. Appelbaum); Stan-ford Hospitals and Clinics, StanStan-ford, Calif. (E. J. Baron); University of California Los Angeles Medical Center, Los Angeles (D. Brucker); New York State Department of Health, Albany (V. Chaturvedi); Wishard Health Services, Indianapolis, Ind. (T. Davis); Summa Health System, Akron, Ohio (J. DiPersio); Case Western Reserve University Hospital, Cleveland, Ohio (M. Ghannoum); Cleveland Clinic Founda-tion, Cleveland, Ohio (G. Hall); University of Rochester Medical Cen-ter, RochesCen-ter, N.Y. (D. Hardy); University of Virginia Health System, Charlottesville (K. Hazen); Veteran Affairs Medical Center, Ann Ar-bor, Mich. (C. Kauffman); and others, which are listed on the follow-ing website: http://www.medicine.uiowa.edu/pathology/path_folder /research/acknowledgements/artemis_participants.pdf.

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