Received 12 December 2010/Returned for modification 5 January 2011/Accepted 16 March 2011

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1 JOURNAL OF CLINICAL MICROBIOLOGY, May 2011, p Vol. 49, No /11/$12.00 doi: /jcm Copyright 2011, American Society for Microbiology. All Rights Reserved. Multicenter Comparison of the Vitek 2 Antifungal Susceptibility Test with the CLSI Broth Microdilution Reference Method for Testing Caspofungin, Micafungin, and Posaconazole against Candida spp. Jess F. Peterson, 1 Michael A. Pfaller, 3 Daniel J. Diekema, 3 Michael G. Rinaldi, 4 Katherine M. Riebe, 2 and Nathan A. Ledeboer 1,2 * Department of Pathology, Medical College of Wisconsin, Milwaukee, Wisconsin 1 ; Dynacare Laboratories, Milwaukee, Wisconsin 2 ; University of Iowa, Carver College of Medicine, Iowa City, Iowa 3 ; and University of Texas Health Science Center, San Antonio, Texas 4 Received 12 December 2010/Returned for modification 5 January 2011/Accepted 16 March 2011 The performance of the automated Vitek 2 (biomérieux, Inc., Marcy l Etoile, France) antifungal susceptibility system was compared to that of broth microdilution (BMD) for the determination of MICs of various antifungal drugs. A total of 112 challenge strains and 755 clinical isolates of Candida spp. were tested against caspofungin and micafungin. An additional 452 clinical isolates of Candida albicans were tested against posaconazole. Reference BMD MIC endpoints were established after 24 h of incubation for caspofungin and micafungin and after 48 h of incubation for posaconazole. Essential agreements (EAs) between the Vitek 2 and BMD methods for caspofungin and micafungin were 99.5% and 98.6%, respectively. EA between the Vitek 2 and BMD methods was 95.6% for posaconazole. The overall categorical agreements (CAs) between the Vitek 2 system and BMD were 99.8% for caspofungin, 98.2% for micafungin, and 98.1% for posaconazole. The Vitek 2 system reliably determined caspofungin and micafungin MICs among Candida spp. and posaconazole MICs among C. albicans isolates and demonstrated excellent quantitative and qualitative agreement with the reference BMD method. Human disease attributed to Candida spp. has been an increasing concern over the last few decades. Disease can range from non-life-threatening mucocutaneous to life-threatening invasive disease (11, 16). To date, Candida spp. are the most common cause of invasive fungal infections in hospitalized patients and the third most common cause of nosocomial bloodstream infections in the United States (8). Modern health care advances in immunosuppressive therapy, particularly related to bone marrow transplantation, and increasing utilization of implantable devices have contributed to the rise of invasive Candida infections in recent decades (6, 11, 16). The increased incidence of candidiasis has resulted in a significant increase in morbidity and mortality rates among at-risk patients and has contributed to increased health care costs (11, 12). Due to the increase in incidence and severity of disease, the need for rapid and accurate methods for testing antifungal susceptibilities has never been greater. Several antifungal agents have recently been approved by the FDA for treatment or prophylaxis of candidiasis. Caspofungin and micafungin, both echinocandins that inhibit -1,3- glucan synthesis, are effective in the treatment of invasive candidiasis (caspofungin) and prophylaxis in subjects with neutropenia (micafungin) (9, 24). Posaconazole, a triazole, decreases ergosterol synthesis by inhibiting 14- -demethylase and has been shown to be effective in preventing invasive * Corresponding author. Mailing address: Department of Pathology, Medical College of Wisconsin, 9200 West Wisconsin Ave., Milwaukee, WI Phone: (414) Fax: (414) nledeboe@mcw.edu. Published ahead of print on 23 March fungal infections (particularly aspergillosis) in immunosuppressed patients while improving overall survival (4, 23). Currently, the Clinical and Laboratory Standards Institute (CLSI) Antifungal Subcommittee has established breakpoint MICs based on the broth microdilution (BMD) method for caspofungin and micafungin (susceptible, 2 g/ml; nonsusceptible, 4 g/ml) (3). Breakpoints for posaconazole have not been established per CLSI document M27-A3 (3). In lieu of clinical correlative data and CLSI-established breakpoints, an internally defined breakpoint (susceptible, 1 g/ml; nonsusceptible, 1 g/ml) for posaconazole was selected based on published MIC distributions and the pharmacokinetic properties of the agent (20). Several commercial tests are available to determine MICs for Candida spp. These systems include Sensititre YeastOne (Trek, Cleveland, OH), Etest (biomérieux, Inc., Marcy l Etoile, France), and disk diffusion methods (1, 5, 15, 21, 22). These methods require manual setup and reading, which can delay turnaround and introduce variability into the system. The development of a fully automated identification and susceptibility system such as the Vitek 2 system is a major advancement, which significantly reduces technologist hands-on time, turnaround time, and variability by utilizing a standardized format (18, 19). The Vitek 2 system has previously demonstrated the ability to effectively identify Candida spp. and establish fluconazole and voriconazole MICs (18, 19). The purpose of this study was to evaluate the performance of the Vitek 2 system in determining MICs of caspofungin and micafungin against a broad range of Candida isolates and MICs of posaconazole against C. albicans isolates from three geographically distinct sites. The 1765

2 1766 PETERSON ET AL. J. CLIN. MICROBIOL. results obtained from 3 clinical test sites using the Vitek 2 system were compared with those obtained using a frozen BMD panel according to CLSI guidelines (3). MATERIALS AND METHODS Study design. We compared the MICs obtained using the Vitek 2 system to those obtained using the CLSI M27-A3 (3) BMD method for caspofungin, micafungin, and posaconazole at three geographically distinct trial sites. Each site tested a minimum of 250 clinical isolates of Candida spp. (at least 150 Candida albicans strains and 100 strains of Candida spp. other than C. albicans) with the Vitek 2 system and BMD method (755 clinical isolates for caspofungin and micafungin and 452 C. albicans clinical isolates for posaconazole). In addition, a challenge set of well-characterized stock isolates (112 Candida species isolates for caspofungin and micafungin and 28 C. albicans isolates for posaconazole) were tested by both methods at all of the participating sites to provide MICs and to represent clinically important species. The intra- and interlaboratory reproducibilities were determined at each participating site using a panel of 10 Candida species isolates tested in triplicate on three separate days. The MICs obtained using the Vitek 2 system were compared to those obtained using the BMD method after 24 h for caspofungin and micafungin and 48 h for posaconazole. This study was approved by the institutional review board of each participating institution. Test organisms. The test organisms included two American Type Culture Collection (ATCC) strains established as quality control strains (C. krusei ATCC 6258 and C. parapsilosis 22019) by the CLSI (3). The challenge set consisted of 112 Candida isolates including 28 isolates of C. albicans, 6 of C. dubliniensis, 50 of C. glabrata, 2 of C. guilliermondii, 6 of C. krusei, 5 of C. lusitaniae, 1 of C. norvegensis, 7ofC. parapsilosis, 2ofC. pelliculosa, and5ofc. tropicalis. Posaconazole was tested against 28 C. albicans challenge isolates. Clinical isolates of Candida spp. tested at the three clinical sites (755 total Candida isolates for caspofungin and micafungin and 452 total C. albicans isolates for posaconazole) included 452 isolates of C. albicans, 3 of C. dubliniensis, 128 of C. glabrata, 7 of C. guilliermondii, 1ofC. haemulonii, 8ofC. kefyr, 24ofC. krusei, 14ofC. lusitaniae, 75 of C. parapsilosis, 1 of C. pelliculosa, 1 of C. rugosa, and 41 of C. tropicalis. Reproducibility within and among clinical trial sites was assessed using a panel of the following organisms: 1 isolate of C. albicans, 1 of C. glabrata, 3 of C. guilliermondii, 2ofC. haemulonii, 3ofC. krusei, 1ofC. lusitaniae, 3ofC. norvegensis, and 7 of C. parapsilosis. All isolates were identified by the Vitek 2 yeast identification card or the API 20C AUX (biomérieux) system according to the manufacturer s instructions. Before the tests were performed, each isolate was subcultured twice onto Sabouraud dextrose agar (Remel, Lenexa, KS) to ensure purity and viability. Antifungal agents and microdilution panels. The Vitek 2 susceptibility card and BMD reference panels containing serial 2-fold dilutions of caspofungin, micafungin, and posaconazole were provided by biomérieux. The Vitek 2 cards were shipped in sealed packages and were stored at 2 to 8 C until testing was performed. The BMD panels were shipped frozen and stored at 70 C until testing. Inoculum preparation. Inoculum suspensions of the Candida isolates were obtained from 24-h cultures on Sabouraud dextrose agar at 35 C. Inoculum suspensions for the Vitek 2 system were prepared in 0.45% sterile saline to a density equal to a 2.0 McFarland standard (acceptable range of 1.8 to 2.2) using the DensiChek (biomérieux) instrument. Inoculum suspensions for the reference BMD panels were prepared by diluting the 2.0 McFarland standard used in the Vitek 2 system to a 0.5 McFarland standard per CLSI guidelines. Reference broth microdilution method. Reference BMD testing was performed according to CLSI document M27-A3 (3). Panels were incubated in ambient air at 35 C for 24 to 48 h. MICs were automatically interpreted with the Opsys MR microplate reader (Dynex Technologies, Chantilly, VA) at 24 h for caspofungin and micafungin and 48 h for posaconazole. MICs were also manually interpreted by reading the lowest concentration of antifungal that inhibited growth by 50% compared to the drug-free control well. Vitek 2 system. The Vitek 2 system was set up according to the manufacturer s instructions. Briefly, a 2.0 McFarland standard of each test organism was placed on the instrument with a susceptibility card. The instrument was set to automatic dilution mode, which dilutes the suspension to the appropriate density, inoculates the card, and incubates the card according to the preprogrammed algorithm. The time of incubation varied based on the rate of growth in the drug-free control well, and the results were expressed as MICs in g/ml. Statistical analysis. Discrepancies among MIC endpoints of more than 2 dilutions (two wells) were used to calculate the essential agreement (EA) between the MICs determined with the Vitek 2 system and by the reference BMD method. Inter- and intralaboratory agreements, assessed with the 10-isolate reproducibility panel, were defined when the MICs were within a 3-dilution range (mode 1 log 2 dilution). The CLSI interpretive breakpoints for caspofungin and for micafungin (susceptible, 2 g/ml; nonsusceptible, 4 g/ml) were used to obtain categorical agreement (CA) percentages between the MICs determined with the Vitek 2 system and by the reference BMD method (3). Interpretive breakpoints for posaconazole were internally defined (susceptible, 1 g/ml; nonsusceptible, 1 g/ml). Very major errors were defined as instances when the reference MIC indicated resistance and the Vitek 2 system MIC indicated susceptibility. Major errors were defined as instances when the isolate was classified resistant by the Vitek 2 system and susceptible by the reference method. RESULTS Three clinical sites compared MICs of the Vitek 2 antifungal susceptibility system to those of the BMD method for clinical and challenge Candida isolates (867 total) against caspofungin and micafungin at 24 h. Posaconazole was tested against clinical and challenge C. albicans isolates (480 total) at 48 h. Table 1 summarizes the combined in vitro susceptibilities of the 867 isolates of Candida spp. to caspofungin and micafungin and of the 480 C. albicans isolates to posaconazole. The essential agreements (EAs) between the Vitek 2 and BMD MICs for caspofungin and micafungin were 99.5% and 98.6%, respectively. The EA between the Vitek 2 and BMD MICs was 95.6% for posaconazole (Table 1). Of the discrepancies noted between Vitek 2 and BMD results, the MICs generated by BMD were higher than those obtained by the Vitek 2 system in 32 of 37 (86.5%) instances (3 of 4 with caspofungin, 8 of 12 with micafungin, and 21 of 21 with posaconazole). The remaining 5 of 37 (13.5%) discrepancies were attributed to lower MICs generated by BMD than by the Vitek 2 system (1 of 4 with caspofungin and 4 of 12 with micafungin). Discrepant EAs between the Vitek 2 and BMD methods for drug-organism combinations are as follows: caspofungin, 2 isolates of C. glabrata, 1 of C. albicans, and 1 of C. parapsilosis; micafungin, 5 isolates of C. parapsilosis, 3 of C. albicans, 2 of C. glabrata, 1ofC. kefyr, and1ofc. rugosa; and posaconazole, 21 isolates of C. albicans. Essential agreement was also calculated for individual Candida spp. between the Vitek 2 system and BMD. All drugorganism combinations for the Vitek 2 system and BMD (caspofungin and micafungin) showed EAs of 93.0%, with the exceptions of micafungin and C. kefyr (8 isolates, 87.5% EA) and micafungin and C. rugosa (1 isolate, 0.0% EA). However, due to the low sample size of C. kefyr and C. rugosa isolates tested against micafungin, further evaluation with a larger study is necessary to determine the statistical significance. Overall, the drug-organism combination results were highly reproducible between the Vitek 2 and reference BMD methods. For caspofungin, 17 of 270 (6.3%) autodilution reproducibility results (all for C. parapsilosis; 7 from site A, 6 from site B, and 4 from site C) and 12 of 270 (4.4%) manual dilution reproducibility results (all for C. parapsilosis; 2 from site A, 7 from site B, and 3 from site C) were reported greater than the 3-dilution range (for acceptable reproducibility). For micafungin, 100% of the auto- and manual dilution reproducibility results were within the 3-dilution range. Posaconazole resulted

3 VOL. 49, 2011 VITEK 2 ANTIFUNGAL SUSCEPTIBILITY TEST TRIALS 1767 TABLE 1. In vitro susceptibilities of 867 isolates of Candida spp. against caspofungin and micafungin and 480 C. albicans isolates against posaconazole, as determined with the Vitek 2 system and reference BMD method a Species (no. of isolates tested) Antifungal agent Test method b MIC ( g/ml) Range 50% 90% EA (%) c C. albicans (480) Caspofungin Vitek BMD Micafungin Vitek BMD Posaconazole Vitek BMD C. glabrata (178) Caspofungin Vitek BMD Micafungin Vitek BMD C. parapsilosis (82) Caspofungin Vitek BMD Micafungin Vitek BMD C. tropicalis (46) Caspofungin Vitek BMD Micafungin Vitek BMD C. krusei (30) Caspofungin Vitek BMD Micafungin Vitek BMD C. lusitaniae (19) Caspofungin Vitek BMD Micafungin Vitek BMD C. dubliniensis (9) Caspofungin Vitek BMD Micafungin Vitek BMD C. guilliermondii (9) Caspofungin Vitek BMD Micafungin Vitek BMD C. kefyr (8) Caspofungin Vitek BMD Micafungin Vitek BMD C. pelliculosa (3) Caspofungin Vitek BMD Micafungin Vitek BMD C. haemulonii (1) Caspofungin Vitek BMD Micafungin Vitek BMD C. norvegensis (1) Caspofungin Vitek BMD Micafungin Vitek BMD C. rugosa (1) Caspofungin Vitek BMD Micafungin Vitek BMD All Candida spp. (867) Caspofungin Vitek BMD Micafungin Vitek BMD a Isolates include both clinical (n 755) and challenge (n 112) sets. b BMD-24 and BMD-48, BMD incubation for 24 and 48 h, respectively. c EA ( 2 log 2 dilutions) between Vitek 2 and BMD MICs.

4 1768 PETERSON ET AL. J. CLIN. MICROBIOL. TABLE 2. Categorical agreements of the Vitek 2 system and BMD MICs for 867 isolates of Candida spp. against caspofungin and micafungin and 480 C. albicans isolates against posaconazole, from three geographically distinct clinical trial sites a Species (no. of isolates tested) Antifungal agent Test method % of MICs by category CA (%) % errors S NS VME ME C. albicans (480) Caspofungin Vitek BMD Posaconazole Vitek BMD C. glabrata (178) Caspofungin Vitek BMD BMD C. parapsilosis (82) Caspofungin Vitek BMD C. tropicalis (46) Caspofungin Vitek C. krusei (30) Caspofungin Vitek C. lusitaniae (19) Caspofungin Vitek C. dubliniensis (9) Caspofungin Vitek C. guilliermondii (9) Caspofungin Vitek C. kefyr (8) Caspofungin Vitek Micafungin Vitek BMD C. pelliculosa (3) Caspofungin Vitek C. haemulonii (1) Caspofungin Vitek C. norvegensis (1) Caspofungin Vitek C. rugosa (1) Caspofungin Vitek All Candida spp. (867) Caspofungin Vitek BMD Micafungin Vitek BMD a Abbreviations: S, susceptible; NS, nonsusceptible; CA, categorical agreement; VME, very major error; ME, major error; BMD-24 and BMD-48, BMD method results read at 24 h and 48 h of incubation, respectively. Isolates include both clinical (n 755) and challenge (n 112) sets.

5 VOL. 49, 2011 VITEK 2 ANTIFUNGAL SUSCEPTIBILITY TEST TRIALS 1769 TABLE 3. Categorical agreements of the Vitek 2 system and BMD method based on the proposed 2011 CLSI breakpoints for select Candida spp. against the echinocandins (including caspofungin and micafungin) a Species (no. of isolates tested) Antifungal agent Test method % of MICs by category % errors CA (%) S I NS VME ME Minor b C. albicans (480) Caspofungin Vitek BMD Micafungin Vitek BMD C. parapsilosis (82) Caspofungin Vitek BMD C. tropicalis (46) Caspofungin Vitek BMD C. krusei (30) Caspofungin Vitek BMD Micafungin Vitek BMD C. guilliermondii (9) Caspofungin Vitek a Candida spp. not included in the table lack sufficient data to modify previously established breakpoints. Abbreviations: S, susceptible; I, intermediate; NS, nonsusceptible; CA, categorical agreement; VME, very major error; ME, major error; Minor, minor error; BMD-24 and BMD-48, BMD method results read at 24 h and 48 h of incubation, respectively. b Minor errors were determined when the result of one of the test methods was either susceptible or resistant and that of the other was intermediate. in 100% auto- and manual dilution reproducibility within the 3-dilution range for C. albicans. The categorical agreement (CA) between the Vitek 2 system and BMD with caspofungin, micafungin, and posaconazole was assessed by combining the data obtained with the clinical and challenge organisms from the three clinical sites (Table 2). Overall, 99.9% of the 867 isolates were susceptible to caspofungin (MICs, 2 g/ml) as determined by the Vitek 2 system whereas 99.7% were susceptible by BMD. Of the 867 isolates tested against micafungin, 99.9% were susceptible ( 2 g/ml) using the Vitek 2 system, whereas 98.0% were susceptible by BMD. Of the 480 C. albicans isolates tested against posaconazole, 100.0% were susceptible (internally defined; 1 g/ml) by the Vitek 2 system, whereas 98.1% were susceptible by BMD. Categorical agreement for all drug-organism combinations was 98.0%, with the exception of micafungin and C. parapsilosis (82 isolates, 84.1% CA). Very major errors, defined as the reference MIC method indicating resistance and the Vitek 2 system MIC indicating susceptibility, occurred with the following drug-organism combinations: caspofungin, C. glabrata (2 of 178); micafungin, C. parapsilosis (13 of 82), C. glabrata (2 of 178), and C. albicans (1 of 480); and posaconazole, C. albicans (9 of 480). No major errors were reported with any drug-organism combinations. The mean times to results for the Vitek 2 system were 8.2 h with caspofungin (range, 5.6 to 19.2 h), 8.4 h with micafungin (range, 5.5 to 19.2 h), and 9.0 h with posaconazole (range, 8.2 to 13.6 h). Similar results were obtained at all three trial sites. DISCUSSION With the increasing prevalence of candidiasis, attributable mortality rates have risen considerably and have been reported as high as 49% (7). Of additional concern is the added cost of candidemia, estimated at an additional $40,000 per adult hospitalized patient (11), and an estimated cumulative hospital cost in the United States of $44 million to $320 million annually (10, 14). However, by providing clinicians with Candida species identification and antifungal susceptibility results in a timely fashion, patient outcomes and health care costs can be significantly improved through earlier treatment with appropriate antifungal agents (2, 13). Clinical laboratories currently have several options for antifungal susceptibility testing. The Sensititre YeastOne panel (Trek), a commercially prepared broth microdilution panel consisting of increasing concentrations of antifungal agents, relies on colorimetric change as the indicator of fungal growth and MIC determination (15). The Etest (biomérieux) is an agar-based assay consisting of a test strip impregnated with a predefined concentration gradient of a single antifungal agent. The MIC is evaluated by observation of growth inhibition in an elliptical zone corresponding to a specific concentration of antifungal agent (1). Another agar-based method, disk diffusion, is similar to the Etest in that a disk with a known concentration of a single antifungal agent is utilized to determine the zone of inhibition (22). Several drawbacks are inherent to all of the aforementioned susceptibility testing methods; extensive manual labor, combined with reader bias in MIC de-

6 1770 PETERSON ET AL. J. CLIN. MICROBIOL. TABLE 4. Performance summary of the Vitek 2 system and BMD method for 867 Candida species isolates against caspofungin and micafungin, and 480 C. albicans isolates against posaconazole, from three geographically distinct clinical trial sites a Parameter Caspofungin Micafungin Posaconazole Organism(s) evaluated Candida spp. Candida spp. Candida albicans No. of isolates b Breakpoints S, 2 g/ml; NS, 4 g/ml S, 2 g/ml; NS, 4 g/ml S, 1 g/ml; NS, 1 g/ml Overall EA (%) Overall CA (%) No. of VMEs (total no. of isolates) 2 (867) 16 (867) 9 (480) No. of MEs a Abbreviations: S, susceptible; NS, nonsusceptible; EA, essential agreement; CA, categorical agreement; VMEs, very major errors; MEs, major errors. b Isolates include both clinical and challenge sets. termination (due to trailing MICs and reader experience), leaves a significant potential for error. In addition, none of these methods include fungal identification capabilities. In contrast, the Vitek 2 system is a fully automated system that provides fungal identification, eliminates the inherent bias of manual MIC determination, and produces results in a timelier manner. Of significant importance is the upcoming modification of CLSI breakpoints (effective 2011). Modified echinocandin breakpoints are expected for several Candida spp., including C. albicans, C. krusei, and C. tropicalis. The modified breakpoints for C. albicans, C. krusei, and C. tropicalis will be as follows: susceptible, 0.25 g/ml; intermediate, 0.5 g/ml; nonsusceptible, 1.0 g/ml. Additionally, breakpoints for C. guilliermondii and C. parapsilosis will be as follows: susceptible, 2.0 g/ml; intermediate, 4.0 g/ml; nonsusceptible, 8.0 g/ml (17). Based on the updated breakpoints, categorical agreement for the following drug-organism combinations in our study would be modified from 100.0% (based on current CLSI guidelines) to the following: C. albicans (caspofungin), 72.9%; C. krusei (caspofungin and micafungin), 30.0% and 83.3%, respectively (Table 3). The majority of these discrepancies are attributable to the BMD method reporting intermediate resistance while the Vitek 2 system reports susceptibility. Candida spp. not included in the table lack sufficient data to modify previously established breakpoints set by the CLSI guidelines. In summary, compared to the reference BMD method, the Vitek 2 system yielded highly reproducible and accurate MIC results and excellent overall essential and categorical agreement for caspofungin (99.5% and 99.8%, respectively) and micafungin (98.6% and 98.2%, respectively) against Candida spp. (Table 4). Similarly, the essential and categorical agreement results were also impressive when comparing the Vitek 2 system to the BMD method for posaconazole (95.6% and 98.1%, respectively) against C. albicans. However, it is important to note that C. parapsilosis consistently produced discrepant MICs between the Vitek 2 and BMD methods for micafungin, in addition to low reproducibility from all trial sites. To address this issue, retesting of all resistant isolates producing discrepant results will be required. Overall, the Vitek 2 system provides a fully automated method for identifying organisms and determining the MICs for caspofungin and micafungin against Candida spp. and for posaconazole against C. albicans, based on spectrophotometry, thus eliminating the subjectivity that is inherent in systems relying on visual MIC determination. REFERENCES 1. Alexander, B. 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