Received 18 December 2008/Returned for modification 9 February 2009/Accepted 9 April 2009

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1 JOURNAL OF CLINICAL MICROBIOLOGY, June 2009, p Vol. 47, No /09/$ doi: /jcm Copyright 2009, American Society for Microbiology. All Rights Reserved. Activity of Contemporary Antifungal Agents, Including the Novel Echinocandin Anidulafungin, Tested against Candida spp., Cryptococcus spp., and Aspergillus spp.: Report from the SENTRY Antimicrobial Surveillance Program (2006 to 2007) Shawn A. Messer, 1 * Gary J. Moet, 1 Jeffrey T. Kirby, 1 and Ronald N. Jones 1,2 JMI Laboratories, North Liberty, Iowa, 1 and Tufts University School of Medicine, Boston, Massachusetts 2 Received 18 December 2008/Returned for modification 9 February 2009/Accepted 9 April 2009 Results from the SENTRY international fungal surveillance program for 2006 to 2007 are presented. A total of 1,448 Candida sp., 49 Aspergillus fumigatus, and 33 Cryptococcus neoformans isolates were obtained from infected sterile-site sources in patients on five continents. Reference susceptibility was determined for anidulafungin, caspofungin, 5-flucytosine, fluconazole, itraconazole, posaconazole, voriconazole, and amphotericin B by CLSI methods. The implementation of standardized antifungal testing methods for Candida spp. and Cryptococcus spp.(1, 2) and the recent development of testing methods for the filamentous fungi (3, 4) have facilitated the generation of meaningful data to detect and assess resistance to the current clinical armamentarium of antifungal agents. The use of these methods in global longitudinal surveillance programs has been useful in detecting emerging, rarely encountered yeast or mold organisms displaying decreased susceptibility to contemporary antifungal compounds (12, 16) and for monitoring the distribution of yeast and mold species with innate resistance profiles. We summarize here the results of the global SENTRY Antimicrobial Surveillance Program (2006 to 2007) comparing the activities of eight currently marketed antifungal agents tested against clinical isolates from North America, Europe, Latin America, and the Asia-Pacific (APAC) region, with susceptibilities interpreted by using established CLSI breakpoint criteria, where available (1, 3). The collection of yeasts included Candida albicans (771 isolates), C. parapsilosis (238 isolates), C. glabrata (202 isolates), C. tropicalis (157 isolates), C. krusei (29 isolates), C. lusitaniae (14 isolates), C. guilliermondii (9 isolates), C. dubliniensis (7 isolates), C. famata (6 isolates), C. kefyr (5 isolates), and C. pelliculosa (4 isolates). The collection also included C. neoformans (33 isolates), and of the 61 molds, only Aspergillus fumigatus (49 isolates) is represented. All yeast and mold isolates were identified at the participating medical centers by the established methods in use at each institution. Isolates were requested to be from bloodstream infections, taken in consecutive order, and limited to one isolate per patient. The rates of occurrence in participating laboratories could then be detected * Corresponding author. Mailing address: Molecular Epidemiology and Fungus Testing Laboratory, Department of Pathology, University of Iowa, Iowa City, IA Phone: (319) Fax: (319) messers@healthcare.uiowa.edu. Published ahead of print on 22 April by a prevalence mode. Confirmation of species identification was performed at the central reference laboratory by using Vitek (biomérieux, St. Louis, MO) and by conventional reference methods (5, 17). Anidulafungin (4, 6, 7, 9, 10, 12, 14; Eraxis package insert [Pfizer, Inc., New York, NY, 2007]), voriconazole (Pfizer, Inc., New York, NY), amphotericin B, fluconazole, itraconazole, and 5-flucytosine (5-FC) (Sigma Chemical Co., St. Louis, MO) were obtained as standard powders. Caspofungin (Merck Research Laboratories, Rahway, NJ), and posaconazole (Schering-Plough Research Institute, Kenilworth, NJ) were prepared according to CLSI guidelines at TREK Diagnostics (Cleveland, OH) (1, 3). The final concentration ranges were as follows: anidulafungin, to 32 g/ml; caspofungin, to 16 g/ml; amphotericin B, 0.12 to 8 g/ml; 5-FC, 0.5 to 64 g/ml; fluconazole, 0.5 to 64 g/ml; itraconazole, to 2 g/ml; posaconazole, 0.06 to 8 g/ml; voriconazole, 0.06 to 8 g/ml. The dilution scheme was selected to maximize capture of the MIC 50 (MIC for 50% of the strains tested) and MIC 90 for wild-type and resistant mutant populations and varied by agent. Further, the ranges of newer or investigational agents were expanded in order to identify populations resistant to these newer agents. Micafungin powder was not yet available from the manufacturer at the commencement of testing. The absence of micafungin in comparison with the other available echinocandins is a limitation of this study. Broth microdilution testing for yeasts followed standardized procedures described in the CLSI M27-A3 reference method (1, 2). All of the filamentous fungi were tested under conditions described for the CLSI M38-A2 reference method (3, 4). Quality control isolates C. krusei ATCC 6258, C. parapsilosis ATCC 22019, and C. tropicalis ATCC 750 from the American Type Culture Collection were used as recommended (CLSI), and all results were observed within previously published ranges (1 3, 4, 7). All panels were incubated in enclosed, humid containers at 35 C and visualized with a reading mirror at 24 h (echinocandins) and 1942

2 VOL. 47, 2009 NOTES 1943 TABLE 1. In vitro antifungal agent susceptibilities of Candida and Cryptococcus isolates collected by the SENTRY Program in 2006 to 2007 Species (no. of isolates) and drug MIC 50 /MIC 90 ( g/ml) MIC range ( g/ml) % by category a S SDD R b All Candida spp. (1,448) c Anidulafungin 0.03/ Caspofungin 0.12/ Amphotericin B 0.5/ FC 0.5/ (2.2) 1.9 Fluconazole 0.5/ Itraconazole 0.06/ Posaconazole 0.06/ Voriconazole 0.06/ C. albicans (771) Anidulafungin 0.015/ Caspofungin 0.12/ Amphotericin B 0.5/ FC 0.5/ (0.1) 2.0 Fluconazole 0.5/ Itraconazole 0.03/ Posaconazole 0.06/ Voriconazole 0.06/ C. parapsilosis (238) Anidulafungin 2/ Caspofungin 0.5/ Amphotericin B 1/ FC 0.5/ (0.0) 1.3 Fluconazole 1/ Itraconazole 0.25/ Posaconazole 0.12/ Voriconazole 0.06/ C. glabrata (202) Anidulafungin 0.015/ Caspofungin 0.25/ Amphotericin B 1/ FC 0.5/ (0.0) 0.0 Fluconazole 8/ Itraconazole 1/ Posaconazole 1/ Voriconazole 0.25/ C. tropicalis (157) Anidulafungin 0.03/ Caspofungin 0.12/ Amphotericin B 1/ FC 0.5/ (0.6) 4.5 Fluconazole 0.5/ Itraconazole 0.12/ Posaconazole 0.12/ Voriconazole 0.06/ C. krusei (29) Anidulafungin 0.06/ Caspofungin 0.5/ d Amphotericin B 1/ FC 16/ (93.2) 3.4 Fluconazole 32/ Itraconazole 0.5/ Posaconazole 0.5/ Voriconazole 0.25/ C. lusitaniae (14) Anidulafungin 0.25/ Caspofungin 0.5/ Amphotericin B 0.5/ FC 0.5/ (0.0) 0.0 Fluconazole 0.5/ Continued on following page

3 1944 NOTES J. CLIN. MICROBIOL. TABLE 1 Continued Species (no. of isolates) and drug MIC 50 /MIC 90 ( g/ml) MIC range ( g/ml) % by category a S SDD R b Itraconazole 0.12/ M Posaconazole 0.06/ Voriconazole 0.06/ C. neoformans (33) Anidulafungin 32/ Caspofungin 16/ Amphotericin B 0.25/ FC 4/ Fluconazole 4/ Itraconazole 0.06/ Posaconazole 0.06/ Voriconazole 0.06/ a The breakpoint criteria used are those of the CLSI (2008). When testing amphotericin B, a susceptibility breakpoint of 1 g/ml was used. Each value in parentheses is the percent intermediate (CLSI, 2008), and a dash indicates that there is no established breakpoint. b There is no resistant category for echinocandins. The CLSI interpretive guidelines state that strains with echinocandin MICs of 2.0 g/ml are susceptible. MICs of 2.0 g/ml result in classification as not susceptible. c Data are not shown for C. dubliniensis (seven isolates), C. famata (six isolates), C. guilliermondii (nine isolates), C. kefyr (five isolates), C. pelliculosa (four isolates), and four Candida sp. isolates plus two unspecified isolates. d Represents a single isolate. 48 h (all other agents). The MICs of anidulafungin, caspofungin, 5-FC, fluconazole, itraconazole, and voriconazole were read as the lowest concentration at which a significant decrease in turbidity ( 50%) was discerned compared to that of the growth control. Amphotericin B MICs were determined as the lowest concentration at which no visible growth was detected (1). The interpretive breakpoints for susceptibility to anidulafungin and caspofungin (susceptible [S], 2 mg/liter; nonsusceptible [NS], 2 mg/liter), fluconazole (S, 8 mg/liter; susceptible dose dependent [SDD], 16 to 32 mg/liter; resistant [R], 64 mg/liter), 5-FC (S, 4 mg/liter; intermediate [I], 8 to 16 mg/liter; R, 32 mg/liter), itraconazole (S, mg/liter; SDD, 0.25 to 0.5 mg/liter; R, 1 mg/liter), and voriconazole S, 1 mg/liter; SDD, 2 mg/liter; R, 4 mg/liter) were those published by the CLSI (2). Interpretive criteria for amphotericin B and posaconazole have not been established, but for comparison, isolates inhibited by amphotericin B at 1 g/ml were considered susceptible. Mold testing panels were aerobically incubated at 35 C in an enclosed container and visualized with a reading mirror at 24 h (echinocandins) and 48 h (all other agents) under a biosafety hood. MICs of amphotericin B, itraconazole, posaconazole, and voriconazole were determined as the concentration at which no discernible growth was detected. MICs of 5-FC were determined as the lowest concentration at which a prominent decrease in growth ( 50%) was visualized compared to the growth control (3, 4). As described for anidulafungin (10) and caspofungin (11), minimal effective concentrations (MECs) were determined as the lowest concentration at which a pronounced morphological change from filamentous growth to nonfilamentous growth was observed. The rank order of the 1,448 Candida sp. isolates collected during the 2006 to 2007 SENTRY Program surveillance period (Table 1) from all geographic regions was C. albicans (53.2%) C. parapsilosis (16.4%), C. glabrata (13.9%), C. tropicalis (10.8%), C. krusei (2.0%), and other Candida spp. (3.5%). The species distribution was consistent with Candida spp. collected in prior SENTRY Program monitoring periods (1997 to 1999, 2003) (8, 9, 13). The antifungal activities (MIC 50 s and MIC 90 s, percent categorical interpretation) of the eight compounds tested against Candida spp. and C. neoformans are summarized in Table 1. The CLSI has not established breakpoints for agents in use against C. neoformans. However, posaconazole demonstrated the greatest activity (MIC 90, 0.12 g/ml) against this species. A. fumigatus was the most common (80.3%) of the Aspergillus spp. tested (Table 2). The agents most active against Aspergillus spp. included anidulafungin (MEC 90, g/ml), caspofungin (MEC 90, 0.12 g/ml), and posaconazole (MIC 90, 0.5 g/ml). An analysis of Candida species by continent of origin is summarized in Table 3 (APAC region data not shown). Among the four most common Candida species reported, those from North America and Europe had the same rank order (C. albicans C. glabrata C. parapsilosis C. tropicalis), which represents a shift in European species distribution from that in the previous SENTRY Program report (8, 9, 13). C. glabrata rose in prevalence rank from third to second in Europe, and TABLE 2. Activities of seven antifungal agents against 49 A. fumigatus strains collected by the SENTRY Program in 2006 to 2007 Agent MIC 50 /MIC 90 ( g/ml) MIC range ( g/ml) % of isolates inhibited by 1 g/ml a Anidulafungin 0.004/ Caspofungin 0.12/ Amphotericin B 2/ FC 64/ Itraconazole 1/ Posaconazole 0.5/ Voriconazole 0.5/ a Breakpoint criteria have not been established by the CLSI (2008). For comparison, the percent inhibited by 1 g/ml was used (3).

4 VOL. 47, 2009 NOTES 1945 TABLE 3. In vitro antifungal agent susceptibilities of Candida isolates collected by the SENTRY Program in 2006 to 2007 Species (no. tested) No. (%) of isolates or MIC 50/90 in g/ml (% susceptible) a North America Europe Latin America All Candida spp. 726 (50.1) 429 (29.6) 274 (18.9) Anidulafungin 0.03/2 (98.9) 0.03/1 (99.3) 0.03/1 (98.5) Caspofungin 0.12/0.5 (99.7) 0.12/0.5 (100.0) 0.12/0.5 (99.6) Amphotericin B 0.5/1 (99.9) 0.5/1 (99.5) 1/1 (98.9) 5-FC 0.5/ 0.5 (95.7) 0.5/ 0.5 (95.3) 0.5/ 0.5 (97.1) Fluconazole 0.5/8 (91.3) 0.5/8 (94.4) 0.5/2 (97.1) Itraconazole 0.06/1 (64.9) 0.06/1 (72.3) 0.06/0.5 (68.6) Posaconazole 0.06/1 0.06/1 0.06/0.25 Voriconazole 0.06/0.25 (97.9) 0.06/0.25 (98.4) 0.06/ 0.06 (99.3) C. albicans 377 (48.9) 260 (33.7) 126 (16.3) Anidulafungin 0.015/0.06 (100.0) 0.015/0.06 (100.0) 0.015/0.06 (100.0) Caspofungin 0.12/0.25 (100.0) 0.12/0.25 (100.0) 0.12/0.25 (100.0) Amphotericin B 0.5/1 (100.0) 0.5/1 (100.0) 0.5/1 (100.0) 5-FC 0.5/ 0.5 (96.8) 0.5/ 0.5 (98.8) 0.5/ 0.5 (99.2) Fluconazole 0.5/ 0.5 (99.5) 0.5/ 0.5 (100.0) 0.5/ 0.5(100.0) Itraconazole 0.03/0.12 (97.3) 0.03/0.06 (98.1) 0.03/0.12 (97.6) Posaconazole 0.06/ / /0.12 Voriconazole 0.06/ 0.06 (100.0) 0.06/ 0.06 (100.0) 0.06/ 0.06 (100.0) C. parapsilosis 122 (51.3) 53 (22.3) 60 (25.2) Anidulafungin 2/2 (93.4) 2/2 (94.3) 1/2 (98.3) Caspofungin 0.5/1 (99.2) 0.5/1 (100.0) 0.5/1 (100.0) Amphotericin B 1/1 (100.0) 1/1 (100.0) 1/1 (100.0) 5-FC 0.5/ 0.5 (99.2) 0.5/ 0.5 (100.0) 0.5/ 0.5 (96.7) Fluconazole 1/4 (95.9) 1/2 (100.0) 1/4 (95.0) Itraconazole 0.25/0.5 (43.4) 0.25/0.25 (43.4) 0.25/0.5 (30.0) Posaconazole 0.12/ / /0.25 Voriconazole 0.06/0.12 (100.0) 0.06/ 0.06(100.0) 0.06/0.12 (98.3) C. glabrata 133 (65.8) 57 (28.2) 11 (0.05) Anidulafungin 0.06/0.12 (100.0) 0.06/0.12 (100.0) 0.06/0.12 (100.0) Caspofungin 0.12/0.25 (100.0) 0.25/0.25 (100.0) 0.25/0.5 (100.0) Amphotericin B 1/1 (100.0) 1/1 (100.0) 1/1 (100.0) 5-FC 0.5/ 0.5 (100.0) 0.5/ 0.5 (100.0) 0.5/ 0.5 (100.0) Fluconazole 8/ 64 (71.4) 8/16 (78.9) 4/16 (81.8) Itraconazole 1/ 2 (2.3) 1/ 2 (5.3) 1/2 (9.1) Posaconazole 1/4 1/4 0.5/1 Voriconazole 0.25/2 (88.7) 0.25/1 (93.0) 0.12/0.5 (90.9) C. tropicalis 53 (33.7) 36 (22.9) 62 (39.5) Anidulafungin 0.03/0.06 (100.0) 0.03/0.6 (100.0) 0.03/0.06 (100.0) Caspofungin 0.12/0.5 (100.0) 0.12/0.25 (100.0) 0.12/0.5 (100.0) Amphotericin B 1/1 (100.0) 1/1 (100.0) 1/1 (95.2) 5-FC 0.5/ 0.5 (96.2) 0.5/ 0.5 (91.7) 0.5/ 0.5 (95.2) Fluconazole 0.5/1 (100.0) 0.5/1 (97.2) 0.5/ 0.5 (100.0) Itraconazole 0.12/0.5 (62.3) 0.12/0.5 (72.2) 0.12/0.5 (66.1) Posaconazole 0.12/ / /0.25 Voriconazole 0.06/ 0.06 (100.0) 0.06/ 0.06 (100.0) 0.06/ 0.06 (100.0) a The breakpoint criteria used are those of the CLSI (2008). When testing amphotericin B, a susceptibility breakpoint of 1 g/ml was used. No results are shown for categories with 10 isolates. the C. parapsilosis rank order dropped to third. The impact of the more frequent occurrence of C. glabrata in Europe has yet to be clarified, and further longitudinal surveillance to detect sustained trends in species distribution and antifungal susceptibility appears warranted. Shifts in species distribution were also observed in Latin America and the APAC region, where the 2006 to 2007 rank order of Candida spp. was C. albicans C. tropicalis C. parapsilosis C. glabrata. Among the 19 Candida isolates from the APAC region, no more than 8 were of one species (C. albicans). These yeasts had susceptibility rates ranging from 73.7% (itraconazole) to 100% (anidulafungin, caspofungin, amphotericin B, 5-FC). This report extends the longitudinal global surveillance results for yeast and mold species collected from bloodstream and sterile body site infections and closely monitors developments in species occurrences with antifungal susceptibility processed by established CLSI reference methods (1 3, 4, 15). As the use of routine antifungal testing by commercial or reference procedures remains a practice performed by a distinct minority of clinical laboratories (15), the use of national or

5 1946 NOTES J. CLIN. MICROBIOL. global antifungal surveillance networks clearly is a necessity to guide formulary choices (8, 9, 12, 13, 16). We thank K. Mellen, T. R. Fritsche, H. S. Sader, and A. Fuhrmeister for significant contributions to study analysis, technical support, and manuscript preparation. This work was funded by a grant from Pfizer, Inc. REFERENCES 1. Clinical and Laboratory Standards Institute M27-A3 reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard, third edition. Clinical and Laboratory Standards Institute, Wayne, PA. 2. Clinical and Laboratory Standards Institute M27-S3 reference method for broth dilution antifungal susceptibility testing of yeasts; third informational supplement. Clinical and Laboratory Standards Institute, Wayne, PA. 3. Clinical and Laboratory Standards Institute M38-A2 reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard, second edition. Clinical and Laboratory Standards Institute, Wayne, PA. 4. Espinel-Ingroff, A., A. Fothergill, M. A. Ghannoum, E. Manavathu, L. Ostrosky-Zeichner, M. A. Pfaller, M. G. Rinaldi, W. A. Schell, and T. J. Walsh Quality control and reference guidelines for CLSI broth microdilution method (M38-A document) for susceptibility testing of anidulafungin against molds. J. Clin. Microbiol. 45: Hazen, K. C., and S. A. Howell Candida, Cryptococcus, and other yeasts of medical importance, p In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. L. Landry, and M. A. Pfaller (ed.), Manual of clinical microbiology, 9th ed. American Society for Microbiology, Washington, DC. 6. Jones, R. N., J. T. Kirby, S. A. Messer, and D. J. Sheehan Development of anidulafungin for disk diffusion susceptibility testing against Candida spp. Diagn. Microbiol. Infect. Dis. 58: Krisher, K., S. Brown, and M. Traczewski Quality control parameters for broth microdilution tests of anidulafungin. J. Clin. Microbiol. 42: Messer, S. A., R. N. Jones, and T. R. Fritsche International surveillance of Candida spp. and Aspergillus spp.: report from the SENTRY Antimicrobial Surveillance Program (2003). J. Clin. Microbiol. 44: Messer, S. A., J. T. Kirby, H. S. Sader, T. R. Fritsche, and R. N. Jones Initial results from a longitudinal international surveillance programme for anidulafungin (2003). J. Antimicrob. Chemother. 54: Odabasi, Z., V. L. Paetznick, J. R. Rodriguez, E. Chen, and L. Ostrosky- Zeichner In vitro activity of anidulafungin against selected clinically important mold isolates. Antimicrob. Agents Chemother. 48: Odds, F. C., M. Motyl, R. Andrade, J. Bille, E. Canton, M. Cuenca-Estrella, A. Davidson, C. Durussel, D. Ellis, E. Foraker, A. W. Fothergill, M. A. Ghannoum, R. A. Giacobbe, M. Gobernado, R. Handke, M. Laverdiere, W. Lee-Yang, W. G. Merz, L. Ostrosky-Zeichner, J. Peman, S. Perea, J. R. Perfect, M. A. Pfaller, L. Proia, J. H. Rex, M. G. Rinaldi, J. L. Rodriguez- Tudela, W. A. Schell, C. Shields, D. A. Sutton, P. E. Verweij, and D. W. Warnock Interlaboratory comparison of results of susceptibility testing with caspofungin against Candida and Aspergillus species. J. Clin. Microbiol. 42: Pfaller, M. A., L. Boyken, R. J. Hollis, S. A. Messer, S. Tendolkar, and D. J. Diekema In vitro activities of anidulafungin against more than 2,500 clinical isolates of Candida spp., including 315 isolates resistant to fluconazole. J. Clin. Microbiol. 43: Pfaller, M. A., D. J. Diekema, R. N. Jones, H. S. Sader, A. C. Fluit, R. J. Hollis, and S. A. Messer International surveillance of bloodstream infections due to Candida species: frequency of occurrence and in vitro susceptibilities to fluconazole, ravuconazole, and voriconazole of isolates collected from 1997 through 1999 in the SENTRY Antimicrobial Surveillance Program. J. Clin. Microbiol. 39: Pfaller, M. A., D. J. Diekema, L. Ostrosky-Zeichner, J. H. Rex, B. D. Alexander, D. Andes, S. D. Brown, V. Chaturvedi, M. A. Ghannoum, C. C. Knapp, D. J. Sheehan, and T. J. Walsh Correlation of MIC with outcome for Candida species tested against caspofungin, anidulafungin, and micafungin: analysis and proposal for interpretive MIC breakpoints. J. Clin. Microbiol. 46: Pfaller, M. A., and R. N. Jones Performance accuracy of antibacterial and antifungal susceptibility test methods: report from the College of American Pathologists Microbiology Surveys Program ( ). Arch. Pathol. Lab. Med. 130: Pfaller, M. A., S. A. Messer, L. Boyken, C. Rice, S. Tendolkar, R. J. Hollis, and D. J. Diekema In vitro survey of triazole cross-resistance among more than 700 clinical isolates of Aspergillus species. J. Clin. Microbiol. 46: Verweij, P. E., and M. E. Brandt Aspergillus, Fusarium, and other opportunistic moniliaceous fungi, p In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. L. Landry, and M. A. Pfaller (ed.), Manual of clinical microbiology, 9th ed. American Society for Microbiology, Washington, DC.