Echinocandin and Triazole Antifungal Susceptibility Profiles for Clinical Opportunistic Yeast and Mold Isolates Collected from 2010 to 2011: Application of New CLSI Clinical Breakpoints and Epidemiological Cutoff Values for Characterization of Geographic and Temporal Trends of Antifungal Resistance

Abstract

The SENTRY Antimicrobial Surveillance Program monitors global susceptibility and resistance rates of newer and established antifungal agents. We report the echinocandin and triazole antifungal susceptibility patterns for 3,418 contemporary clinical isolates of yeasts and molds. The isolates were obtained from 98 laboratories in 34 countries during 2010 and 2011. Yeasts not presumptively identified by CHROMagar, the trehalose test, or growth at 42°C and all molds were sequence identified using internal transcribed spacer (ITS) and 28S (yeasts) or ITS, translation elongation factor (TEF), and 28S (molds) genes. Susceptibility testing was performed against 7 antifungals (anidulafungin, caspofungin, micafungin, fluconazole, itraconazole, posaconazole, and voriconazole) using CLSI methods. Rates of resistance to all agents were determined using the new CLSI clinical breakpoints and epidemiological cutoff value criteria, as appropriate. Sequencing of fks hot spots was performed for echinocandin non-wild-type (WT) strains. Isolates included 3,107 from 21 Candida spp., 146 from 9 Aspergillus spp., 84 from Cryptococcus neoformans, 40 from 23 other mold species, and 41 from 9 other yeast species. Among Candida spp., resistance to the echinocandins was low (0.0 to 1.7%). Candida albicans and Candida glabrata that were resistant to anidulafungin, caspofungin, or micafungin were shown to have fks mutations. Resistance to fluconazole was low among the isolates of C. albicans (0.4%), Candida tropicalis (1.3%), and Candida parapsilosis (2.1%); however, 8.8% of C. glabrata isolates were resistant to fluconazole. Among echinocandin-resistant C. glabrata isolates from 2011, 38% were fluconazole resistant. Voriconazole was active against all Candida spp. except C. glabrata (10.5% non-WT), whereas posaconazole showed decreased activity against C. albicans (4.4%) and Candida krusei (15.2% non-WT). All agents except for the echinocandins were active against C. neoformans, and the triazoles were active against other yeasts (MIC₉₀, 2 μg/ml). The echinocandins and triazoles were active against Aspergillus spp. (MIC₉₀/minimum effective concentration [MEC₉₀] range, 0.015 to 2 μg/ml), but the echinocandins were not active against other molds (MEC₉₀ range, 4 to >16 μg/ml). Overall, echinocandin and triazole resistance rates were low; however, the fluconazole and echinocandin coresistance among C. glabrata strains warrants continued close surveillance.

INTRODUCTION

The spectrum of opportunistic yeasts and molds causing invasive fungal infections (IFIs) is clearly increasing in diversity throughout the world (1–3). Accurate identification (ID) and antifungal resistance testing of these organisms are important for guiding therapy and determining prognosis in these IFIs, as well as in epidemiologic surveys (2, 4–12). One of the most important aspects of global and regional surveillance programs is to use state-of-the-art methods for ID and antifungal resistance testing of the organisms that are implicated in IFIs (2, 6–8, 11, 13). Accurate ID of opportunistic fungi is important in identifying geographic trends in the resistance profiles of common species and in assessing the epidemiology, pathogenicity, and antifungal susceptibility profiles of less common or “cryptic” species (1, 6–8, 13, 14).

Antifungal testing has progressed from humble beginnings with limited available drugs to the current arsenal of agents exhibiting broad-spectrum activities against a wide variety of fungi (11, 15, 16). The introduction of these newer agents (triazoles and echinocandins) and the development of standardized testing methods by the Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antifungal Susceptibility Testing (17–19) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (20) have proven to be invaluable to the clinician (21–28), particularly with the introduction of clinical breakpoints (CBPs) (11, 29–31). The advent of global surveillance programs, backed by molecular ID and resistance detection, has resulted in a vast accumulation of data that has presented a clearer view of changing patterns in fungal species distributions and the emergence of less-susceptible and less-common species (2, 6–8, 12, 14, 32, 33). It also became clear that the previously established breakpoints for available antifungal agents needed refinement to more specifically address reduced reporting times and broad differences in susceptibilities among species to the different agents, as well as to specifically address those non-wild-type (non-WT) strains within a given species exhibiting reduced susceptibilities to the respective agents (11). Antifungal susceptibility testing has departed from a “one breakpoint fits all” approach (15, 34, 35) to using more-refined species-specific CBPs at the shorter 24-h reporting times (11, 12, 29). The recent surveillance focus has been on how best to detect the emergence of resistance within a population of a given fungal species (2, 7, 8, 12–14, 36). The development of epidemiological cutoff values (ECVs) has aided in the detection of non-WT strains in given populations, allowing a more focused approach to the molecular definition of strains presenting less-susceptible profiles (6, 8, 11, 13, 33, 37).

The SENTRY Antimicrobial Surveillance Program has been active since 1997 and has reported the frequency of pathogen occurrence and the susceptibilities to various antifungal agents on a global scale (12, 14, 38). In the present study, we summarize the results of the global SENTRY Program for 2010 and 2011, comparing the activities of 3 echinocandins and 4 triazoles tested against a collection of 3,418 clinical isolates of Candida (3,107 isolates, 21 species), non-Candida yeasts (125 isolates, 10 species), Aspergillus (146 isolates, 9 species), and non-Aspergillus molds (40 isolates, 23 species). This study is unique in that molecular methods were used to confirm the IDs of the less-common species of Candida, as well as those of the non-Candida yeasts and all of the filamentous fungi (8). Furthermore, we applied the newly revised CBPs for the echinocandins, fluconazole, and voriconazole to determine the resistance profiles of various Candida species (11, 29–31) and the ECVs for these agents, as well as itraconazole and posaconazole, to detect emerging resistance among less-common species of Candida (11) and among isolates of Aspergillus fumigatus (37) and Cryptococcus neoformans (39). Furthermore, we provide a molecular characterization of the mechanisms of echinocandin resistance among isolates of Candida spp. showing elevated echinocandin MIC results.

MATERIALS AND METHODS

Organisms.

A total of 3,418 clinical isolates from patients with IFI were collected during 2010 and 2011 from 98 laboratories (34 countries) in North America (1,349 isolates), Europe (1,191 isolates), Latin America (492 isolates), and the Asia-Pacific region (384 isolates) as part of the SENTRY Program. In each case, collection was approved by the appropriate institutional review board. Each participating center recovered consecutive nonduplicated isolates from patients with bloodstream infections (2,227 isolates), normally sterile body fluid, abscess, and tissue samples (425 isolates), respiratory tract infections (352 isolates), and other or unknown sites (414 isolates). Isolates were identified at the participating institutions using methods routinely employed at the submitting laboratory, including Vitek, MicroScan, API, and AuxaColor, supplemented by classical methods for yeast and mold ID (40, 41). Isolates were submitted to JMI Laboratories (North Liberty, IA), where the ID was confirmed by morphological, biochemical, and molecular methods (6, 8, 42). Yeast isolates were subcultured and screened using CHROMagar Candida (Becton, Dickinson, Sparks, MD) to ensure purity and to differentiate Candida albicans/Candida dubliniensis, Candida tropicalis, and Candida krusei. Biochemical tests, including Vitek 2 (bioMérieux, Hazelwood, MO), trehalose assimilation (for Candida glabrata), or growth at 45°C (for C. albicans/C. dubliniensis), were additionally used to identify common Candida species. Molecular methods were performed on common species of Candida that could not be definitively identified using phenotypic methods or that presented unusual phenotypic or biochemical profiles, as well as for all uncommon species of Candida, non-Candida yeasts, and all molds. Candida spp. and other yeasts were identified using sequence-based methods for the internal transcribed spacer (ITS) region, 28S ribosomal subunit (D1/D2), and the intergenic spacer (IGS) (Debaryomyces spp.) and IGS1 (Trichosporon spp.) (6, 8). All mold isolates were subcultured and analyzed by ITS sequencing, followed by specific molecular species ID within genera: β-tubulin for Aspergillus spp., translation elongation factor (TEF) for Fusarium spp., and 28S for all other genera of molds (8). Nucleotide sequences were examined using Lasergene software (DNAStar, Madison, WI) and then compared to database sequences using BLAST (http://www.nbeti.nlmnih.gov.blast). Fusarium spp. isolates were analyzed for TEF sequence using the Fusarium-ID database (http://www.isolate.fusariumdb.org) and the Fusarium multilocus sequence typing (MLST) database (http://chs.knaw.nl/fusarium/) (8). Results were considered acceptable if homology was >99.5% with other entries in the databases used for comparison. Available sequences that were considerably different from the majority of entries for one species were considered outliers and were discarded in the analysis. Additionally, if no match was found in the database, the ID was based on species complex (SC), genus, family, or order, according to the most current classification systems.

Among the 3,107 isolates of Candida, there were 1,405 isolates of C. albicans, 571 of C. glabrata, 565 of Candida parapsilosis, 318 of C. tropicalis, 79 of C. krusei, and 169 of miscellaneous Candida spp. (55 Candida lusitaniae, 50 Candida dubliniensis, 19 Candida guilliermondii, 16 Candida kefyr, 8 Candida fermentati, 4 Candida lipolytica, 3 Candida intermedia/Candida pseudointermedia, 2 Candida catenulata, 2 Candida haemulonii, 2 Candida inconspicua, 2 Candida nivariensis, 2 Candida pararugosa, and 1 each of Candida bracarensis, Candida fabianii, Candida norvegensis, and Candida pelliculosa). The collection also included C. neoformans (84 isolates), Trichosporon asahii (9 isolates), Saccharomyces cerevisiae (6 isolates), Rhodotorula mucilaginosa (5 isolates), Trichosporon spp. (5 isolates), Dipodascus capitatus (3 isolates), Lodderomyces elongisporus (2 isolates), and 1 isolate each from Aureobasidium pullulans, Debaryomyces fabryi, and Trichosporon mycotoxinivorans. Molds included 97 isolates from A. fumigatus sensu stricto, 49 from Aspergillus spp. (19 Aspergillus flavus species complex [SC], 15 Aspergillus niger SC, 7 Aspergillus terreus SC, 2 Aspergillus alabamensis, 2 Aspergillus nidulans, 2 Aspergillus sydowii, and 1 each of Aspergillus foetidus and Aspergillus ustus SC), and 40 from other molds (6 Gibberella fujikuroi SC, 4 Scedosporium apiospermum, 3 Fusarium solani SC, 3 Penicillium subgenus Aspergilloides, 2 Penicillium subgenus Terverticillata, 2 Alternaria spp., 2 Paecilomyces variotii, 2 Sarocladium [Acremonium] kiliense, and 1 each of Absidia [Lichtheimia] corymbifera, Acremonium strictum genogroup III, Coprinellus radians, Penicillium roqueforti group, Penicillium subgenus Fureatum, Penicillium variabile, Phialemonium curvatum, Rhizomucor pusillus, Rhizopus microsporus group, Rhizopus oryzae, a Curvularia sp., a Fusarium sp., a Penicillium sp., a Trichoderma sp., and Exophiala dermatitidis).

In addition to these isolates, we included the echinocandin MIC distributions from an earlier (2003 to 2007) collection of 8,023 Candida spp. (36) in order to assess any temporal change in echinocandin resistance between 2003 to 2007 and 2010 to 2011. P values were calculated by the χ² test for trend.

Antifungal susceptibility testing.

All yeast isolates were tested for in vitro susceptibility to the echinocandins (anidulafungin, caspofungin, and micafungin) and the triazoles (fluconazole, itraconazole, posaconazole, and voriconazole) using CLSI (17) broth microdilution (BMD) methods. The MIC results for all agents were read following 24 h of incubation when the agents were tested against Candida spp., whereas MIC endpoints for the triazoles were read after 48 h when the drugs were tested against non-Candida yeasts. In all instances, the MIC values were determined visually as the lowest concentration of drug that caused significant growth diminution levels (17, 18).

In vitro susceptibility testing of Aspergillus spp. and other molds against the echinocandins and triazoles (itraconazole, posaconazole, and voriconazole) was performed by BMD as described in CLSI document M38-A2 (19). The triazole MICs and echinocandin minimum effective concentrations (MEC) were determined as described in the CLSI reference method (19).

We used the recently revised CLSI CBP values to identify strains of the 5 most common species of Candida (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei) that were resistant to the echinocandins, as well as those that were resistant to fluconazole and voriconazole (11): anidulafungin, caspofungin, and micafungin MIC values of >0.5 μg/ml were considered resistant for C. albicans, C. tropicalis, and C. krusei, and MIC results of >4 μg/ml were categorized as resistant for C. parapsilosis; anidulafungin and caspofungin MIC values of >0.25 μg/ml and micafungin MIC values of >0.12 μg/ml were considered resistant for C. glabrata; fluconazole MIC results of >4 μg/ml were defined as resistant for C. albicans, C. parapsilosis, and C. tropicalis, and MICs of >32 μg/ml were considered resistant for C. glabrata. All isolates of C. krusei were defined as resistant to fluconazole. The CLSI resistance breakpoint for voriconazole was >0.5 μg/ml for C. albicans, C. tropicalis, and C. parapsilosis, and MIC results of >1 μg/ml were categorized as resistant for C. krusei; CLSI has not assigned CBPs for voriconazole and C. glabrata and recommends the ECV of 0.5 μg/ml to be used to differentiate wild-type (WT) from non-WT strains of this species (11, 30).

CBPs have not been established for any antifungal agent and the less-common species of Candida, non-Candida yeasts, Aspergillus spp., or the non-Aspergillus molds; however, ECVs have been established for echinocandins and triazoles and 6 species of Candida that are encountered less frequently (C. lusitaniae, C. guilliermondii, C. dubliniensis, C. kefyr, C. orthopsilosis, and C. pelliculosa) (11). ECVs have been derived for C. neoformans and fluconazole (8 to 16 μg/ml), itraconazole (1 μg/ml), posaconazole (0.5 μg/ml), and voriconazole (0.25 μg/ml) (39). ECVs have also been developed for A. fumigatus, A flavus, A. terreus, and A. niger and itraconazole, posaconazole, and voriconazole (37): itraconazole and voriconazole MIC values of >1 μg/ml were considered non-WT for A. fumigatus, A. flavus, and A. terreus; itraconazole MIC values of >1 μg/ml and voriconazole MIC values of >2 μg/ml were considered non-WT for A. nidulans, whereas itraconazole and voriconazole MIC values of >2 μg/ml were non-WT for A. niger. Posaconazole MIC values of >0.5 μg/ml were considered non-WT for A. fumigatus, A. terreus, and A. niger, and MIC results of >0.25 μg/ml were non-WT for A. flavus; posaconazole MIC values of >1 μg/ml were non-WT for A. nidulans. Isolates of these Aspergillus spp. for which triazole MIC results exceed the ECV are considered to be non-WT and may harbor acquired mutations in the cyp51A gene (43, 44).

Quality control was performed as recommended in CLSI documents M27-A3 (17) and M38-A2 (19) using the strains C. krusei ATCC 6258 and C. parapsilosis ATCC 22019.

All isolates of Candida spp. that were resistant to ≥1 of the echinocandins were further characterized regarding the presence or absence of a mutation in the hot spot (HS) regions of fks1 and fks2 (C. glabrata only), as described previously (13, 33).

RESULTS AND DISCUSSION

Table 1 shows species distribution by geographic region of the fungi implicated in IFIs during 2010 and 2011. As expected, Candida spp. accounted for the vast majority of IFIs in all four regions (91.0%; range, 88.0% [Latin America] to 95.3% [Asia-Pacific]). C. albicans was most common in Europe (50.3%) and least common in North America (41.5%), whereas C. glabrata was most common in North America (25.3%) and least common in Latin America (8.8%). C. parapsilosis and C. tropicalis were most common in Latin America (24.0% and 17.6%, respectively) and the Asia-Pacific region (25.4% and 12.0%, respectively), and C. krusei was most common in North America (3.3%), as were other miscellaneous species of Candida (6.6%).

Table 1.

Geographic distribution of organisms collected during 2010 to 2011 in medical centers participating in the SENTRY Antimicrobial Surveillance Program

Organism/organism group	No. of isolates in each indicated geographic region:				Total
	North America	Europe	Latin America	Asia-Pacific
Total, yeasts and molds	1,349	1,191	492	384	3,418
Yeast
All Candida spp.	1,211	1,097	433	366	3,107
C. albicans	503	552	186	164	1,405
C. glabrata	306	175	38	52	571
C. parapsilosis	173	195	104	93	565
C. tropicalis	109	89	76	44	318
C. krusei	40	28	8	3	79
Other Candida spp.a	80	58	21	10	169
Cryptococcus neoformans	36	9	29	10	84
Other yeastsb	8	21	10	2	41
Mold
All Aspergillus spp.	74	51	15	6	146
Aspergillus fumigatus	45	34	13	4	97
Other Aspergillus spp.c	29	16	2	2	49
Other moldsd	20	15	5		40

^aOther Candida spp. include C. bracarensis (1 strain), C. catenulata (2 strains), C. dubliniensis (50 strains), C. fabianii (1 strain), C. fermentati (8 strains), C. guilliermondii (19 strains), C. haemulonii (2 strains), C. inconspicua (2 strains), C. kefyr (16 strains), C. lipolytica (4 strains), C. lusitaniae (55 strains), C. nivariensis (2 strains), C. norvegensis (1 strains), C. pararugosa (2 strains), C. pelliculosa (1 strain), and C. intermedia/C. pseudointermedia (3 strains).

^bOther yeasts include Aureobasidium pullulans (1 strain), Debaryomyces fabryi (1 strain), Dipodascus capitatus (3 strains), Lodderomyces elongisporus (2 strains), Rhodotorula mucilaginosa (5 strains), Saccharomyces cerevisiae (6 strains), Trichosporon asahii (9 strains), Trichosporon mycotoxinivorans (1 strain), Trichosporon spp. (5 strains), and unidentified yeasts (6 strains).

^cOther Aspergillus spp. include A. alabamensis (2 strains), A. flavus species complex (SC) (19 strains), A. foetidus (1 strain), A. nidulans (2 strains), A. niger SC (15 strains), A. sydowii (2 strains), A. terreus SC (7 strains), and A. ustus SC (1 strain).

^dOther molds include Absidia (Lichtheimia) corymbifera (1 strain), Acremonium strictum genogroup III (1 strain), Coprinellus radians (1 strain), Fusarium solani SC (3 strains), Gibberella fujikuroi SC (6 strains), Paecilomyces variotii (2 strains), Penicillium roqueforti group (1 strain), Penicillium subgenus Aspergilloides (3 strains), Penicillium subgenus Furcatum (1 strain), Penicillium subgenus Terverticillata (2 strains), Penicillium variabile (1 strain), Phialemonium curvatum (1 strain), Rhizomucor pusillus (1 strain), Rhizopus microsporus group (1 strain), Rhizopus oryzae (1 strain), Sarocladium (Acremonium) kiliense (2 strains), Scedosporium apiospermum (4 strains), Alternaria spp. (2 strains), Curvularia spp. (1 strain), Fusarium spp. (1 strain), Penicillium spp. (1 strain), Trichoderma spp. (1 strain), and Exophiala dermatitidis (1 strain).

Non-Candida yeasts accounted for 3.7% of all IFI isolates, dominated by C. neoformans (2.5% of all IFI isolates [range, 0.8% to 5.9%] and 67.2% of all non-Candida yeasts).

Aspergillus spp. accounted for 4.3% of all IFI isolates, with A. fumigatus sensu stricto being the most common species of the genus in all regions (range, 60.8% of all Aspergillus spp. isolates [North America] to 86.7% [Latin America]). The other molds were largely reported from North America and Europe (Table 1).

Geographic trends in resistance to the echinocandins and azoles for the five most common species of Candida are shown in Table 2. Resistance to the echinocandins was distinctly uncommon (overall range, 0.0 to 1.2%) among C. albicans (0.0 to 0.6%), C. tropicalis (0.0%), C. parapsilosis (0.0 to 1.2%), and C. krusei (0.0%) isolates from all four geographic regions, using the new CLSI CBP values. Resistance to anidulafungin (3.8%), caspofungin (1.9%), micafungin (1.9%), and the triazoles (5.8% to 13.5%) was most prominent among isolates of C. glabrata from the Asia-Pacific region, whereas none of the C. glabrata isolates from Latin America were resistant to the echinocandins (Table 2). Fluconazole resistance was uncommon among isolates of C. albicans and C. tropicalis from all four regions and among C. parapsilosis isolates from North America, Europe, and Latin America. Similar to C. glabrata, the highest rate of fluconazole resistance among C. parapsilosis isolates was seen in the Asia-Pacific region. Aside from C. glabrata, resistance to voriconazole was minimal in all four geographic regions. Decreased susceptibility (MIC > ECV) to posaconazole was most prominent (>5%) among C. albicans isolates (ECV, 0.06 μg/ml) from Europe (8.3%) and Latin America (6.5%), C. glabrata isolates (ECV, 2 μg/ml) from Latin America (5.3%) and the Asia-Pacific region (5.8%), C. parapsilosis isolates (ECV, 0.25 μg/ml) from the Asia-Pacific region (6.5%), and C. krusei isolates (ECV, 0.5 μg/ml) from all regions (15.2% overall; range, 7.1% [Europe] to 33.3% [Asia-Pacific]). Among the 12 isolates of C. krusei characterized as being non-WT to posaconazole (ECV, 0.5 μg/ml), the MIC was 1 μg/ml for 10 isolates (83%) and 2 μg/ml for 2 isolates. Thus, the posaconazole MIC was ≤1 μg/ml for 77 of 79 isolates (97.5%) of C. krusei, comparable to the activity seen with voriconazole against this species. Isolates of C. krusei for which posaconazole MICs are 1 μg/ml (non-WT) yet which are classified as WT for voriconazole are very unusual and not explained by known mechanisms of resistance (45), suggesting that the ECV for posaconazole and C. krusei may be set too low or that technical factors may have led to falsely elevated MIC values in the present study. It should also be noted that the posaconazole ECVs for C. krusei and the other common species of Candida were derived from MIC distributions obtained from a single laboratory and may differ from those derived from a multicenter study. Furthermore, these ECVs are not meant to serve as a measure of susceptibility or resistance in vivo (11).

Table 2.

Frequency of antifungal resistance among clinical isolates of Candida spp. by geographic region in the 2010-2011 SENTRY Surveillance Program

Candida species	Antifungal agent	No. of isolates (% resistanta to each antifungal agent) by region
		North America	Europe	Latin America	Asia-Pacific	Total
C. albicans	Anidulafungin	503 (0.0)	552 (0.0)	186 (0.0)	164 (0.0)	1,405 (0.0)
	Caspofungin	503 (0.0)	552 (0.4)	186 (0.0)	164 (0.6)	1,405 (0.2)
	Micafungin	503 (0.0)	552 (0.4)	186 (0.0)	164 (0.0)	1,405 (0.1)
	Fluconazole	503 (0.6)	552 (0.2)	186 (0.0)	164 (0.6)	1,405 (0.4)
	Posaconazolec	503 (0.6)	552 (8.3)	186 (6.5)	164 (0.6)	1,405 (4.4)
	Voriconazole	503 (0.6)	552 (0.2)	186 (0.0)	164 (0.6)	1,405 (0.4)
C. glabrata	Anidulafungin	306 (1.6)	175 (1.7)	38 (0.0)	52 (3.8)	571 (1.8)
	Caspofungin	306 (1.6)	175 (1.7)	38 (0.0)	52 (1.9)	571 (1.6)
	Micafungin	306 (1.3)	175 (1.1)	38 (0.0)	52 (1.9)	571 (1.2)
	Fluconazole	306 (9.8)	175 (6.3)	38 (5.3)	52 (13.5)	571 (8.8)
	Posaconazolec	306 (3.3)	175 (2.9)	38 (5.3)	52 (5.8)	571 (3.5)
	Voriconazoleb	306 (11.1)	175 (9.1)	38 (7.9)	52 (13.5)	571 (10.5)
C. parapsilosis	Anidulafungin	173 (1.2)	195 (0.0)	104 (1.0)	93 (0.0)	565 (0.5)
	Caspofungin	173 (0.0)	195 (0.0)	104 (0.0)	93 (0.0)	565 (0.0)
	Micafungin	173 (0.0)	195 (0.0)	104 (0.0)	93 (0.0)	565 (0.0)
	Fluconazole	173 (0.0)	195 (3.1)	104 (1.0)	93 (5.4)	565 (2.1)
	Posaconazolec	173 (1.2)	195 (0.5)	104 (3.9)	93 (6.5)	565 (2.3)
	Voriconazole	173 (0.0)	195 (0.0)	104 (0.0)	93 (1.1)	565 (0.2)
C. tropicalis	Anidulafungin	109 (0.0)	89 (0.0)	76 (0.0)	44 (0.0)	318 (0.0)
	Caspofungin	109 (0.0)	89 (0.0)	76 (0.0)	44 (0.0)	318 (0.0)
	Micafungin	109 (0.0)	89 (0.0)	76 (0.0)	44 (0.0)	318 (0.0)
	Fluconazole	109 (2.7)	89 (1.1)	76 (0.0)	44 (0.0)	318 (1.3)
	Posaconazolec	109 (7.3)	89 (7.9)	76 (2.6)	44 (0.0)	318 (5.3)
	Voriconazole	109 (0.9)	89 (0.0)	76 (0.0)	44 (0.0)	318 (0.3)
C. krusei	Anidulafungin	40 (0.0)	28 (0.0)	8 (0.0)	3 (0.0)	79 (0.0)
	Caspofungin	40 (0.0)	28 (0.0)	8 (0.0)	3 (0.0)	79 (0.0)
	Micafungin	40 (0.0)	28 (0.0)	8 (0.0)	3 (0.0)	79 (0.0)
	Posaconazolec	40 (17.5)	28 (7.1)	8 (25.0)	3 (33.3)	79 (15.2)
	Voriconazole	40 (2.5)	28 (0.0)	8 (0.0)	3 (0.0)	79 (1.3)

^aResistance is defined as an MIC of >0.5 μg/ml for anidulafungin, caspofungin, and micafungin against C. albicans, C. tropicalis, and C. krusei, an MIC of >4 μg/ml against C. parapsilosis, an MIC of >0.25 μg/ml for anidulafungin and caspofungin, and an MIC of >0.12 μg/ml for micafungin against C. glabrata; an MIC of >4 μg/ml for fluconazole against C. albicans, C. tropicalis, and C. parapsilosis, an MIC of >32 μg/ml against C. glabrata; and an MIC of >0.5 μg/ml for voriconazole against C. albicans, C. tropicalis, and C. parapsilosis.

^bIn lieu of clinical breakpoints for voriconazole against C. glabrata, the epidemiological cutoff value (ECV) of >0.5 μg/ml was used to identify non-wild-type (non-WT) isolates.

^cPosaconazole ECVs were used to identify non-WT isolates of C. albicans (ECV > 0.06 μg/ml), C. glabrata (ECV > 2 μg/ml), C. parapsilosis (ECV > 0.25 μg/ml), C. tropicalis (ECV > 0.12 μg/ml), and C. krusei (ECV > 0.5 μg/ml).

Table 3 provides a comparison of the MIC distributions for each of the echinocandins according to the species of Candida that were collected in the years 2003 to 2007 (36) and the present collection representing 2010 to 2011. Results from the two time periods were obtained with CLSI reference BMD methods in two different laboratories (2003 to 2007 at the University of Iowa and 2010 to 2011 at JMI Laboratories); however, testing was performed by common personnel in both time periods, and quality control procedures were rigorously performed during both studies, suggesting that testing conditions did not influence the differences that we noted.

Table 3.

Comparison of in vitro susceptibilities of three echinocandins versus isolates of Candida collected during two different time periods^a

Candida species	Antifungal agentb	Yearsc	No. of isolates tested	% of isolates at an MIC (μg/ml) of:
				≤0.008	0.015	0.03	0.06	0.12	0.25	0.5	1	2	4	≥8
C. albicans	ANF	2003-2007	4,283	7.9	29.8	36.0	20.9	5.0	0.3		<0.1
		2010-2011	1,405	9.3	22.6	35.4	24.7	7.0	0.5	0.4
	CSF	2003-2007	4,283	2.1	27.6	47.6	21.0	1.6	0.1
		2010-2011	1,405	0.7	16.2	45.7	25.9	10.0	0.9	0.4	0.1	0.1
	MCF	2003-2007	4,283	14.2	68.9	14.6	2.1	0.1
		2010-2011	1,405	4.2	40.2	47.6	6.3	1.1	0.2	0.3	0.1
C. glabrata	ANF	2003-2007	1,236		0.6	13.0	57.8	25.9	2.1	0.2	0.2	0.2	0.1
		2010-2011	571		0.7	15.2	52.0	25.7	4.6	0.4	1.1	0.2	0.2
	CSF	2003-2007	1,236		10.7	59.1	26.6	2.1	0.6	0.6	0.1			0.2
		2010-2011	571		1.2	58.5	23.3	13.0	2.5	0.5	0.7		0.2	0.2
	MCF	2003-2007	1,236	16.8	75.6	5.7	1.0	0.3	0.2	0.1	0.2	0.1
		2010-2011	571	9.8	68.5	17.5	2.1	0.9	0.2	0.7	0.2	0.2
C. parapsilosis	ANF	2003-2007	1,238	0.1	0.2	0.1	0.1	1.1	4.0	25.8	61.8	6.9		0.1
		2010-2011	565		0.2		0.5	4.2	10.1	32.0	39.3	13.1	0.5
	CSF	2003-2007	1,238	0.2	0.4	2.5	10.2	44.0	32.2	9.1	1.3	0.1		0.2
		2010-2011	565		0.4	1.4	10.4	52.4	26.2	8.8	0.4
	MCF	2003-2007	1,238	0.2	0.2	0.1	0.8	5.3	21.1	54.6	17.8			0.2
		2010-2011	565				0.2	0.9	2.5	13.5	52.9	29.6	0.5
C. tropicalis	ANF	2003-2007	996	4.1	25.5	49.5	17.4	2.4	0.7	0.1		0.3
		2010-2011	318	11.9	37.1	38.7	10.7	1.3	0.3
	CSF	2003-2007	996	1.7	31.9	48.4	16.2	1.2	0.4		0.1			0.1
		2010-2011	318	0.6	18.9	57.9	17.0	4.7	0.9
	MCF	2003-2007	996	4.6	40.2	37.7	15.0	1.7	0.6	0.1	0.2
		2010-2011	318	2.2	27.7	50.3	17.3	1.9	0.6
C. krusei	ANF	2003-2007	270		1.5	58.9	33.7	5.2	0.4	0.4
		2010-2011	79	1.3	7.6	31.6	45.6	12.7	1.3
	CSF	2003-2007	270		0.4		51.9	29.3	14.8	3.0	0.7	0.4
		2010-2011	79	1.3		2.5	30.4	21.5	39.2	12.9
	MCF	2003-2007	270		1.5	10.4	78.1	7.8	2.2
		2010-2011	79	1.3	1.3		27.8	68.4	1.3

^aAll isolates were tested using CLSI broth microdilution methods (17).

^bANF, anidulafungin; MCF, micafungin; CSF, caspofungin.

^cData from the 2003-2007 period were compiled from the work of Pfaller et al. (36).

Among the 4,283 isolates of C. albicans from 2003 to 2007, 1 (0.02%) was resistant to anidulafungin and intermediate to both caspofungin and micafungin, whereas 3 isolates (0.2%) (P < 0.001) from 2010 to 2011 were resistant to caspofungin and 2 (0.1%) (P < 0.001) were resistant to micafungin (Table 3). The 3 caspofungin-resistant isolates in 2010 to 2011 were from Europe (2 isolates) and China (1 isolate), 2 of which were intermediate to anidulafungin (MIC, 0.5 μg/ml) and resistant to micafungin (MIC, 1 μg/ml), and 1 of which was susceptible to anidulafungin (MIC, 0.25 μg/ml) and intermediate to micafungin (MIC, 0.5 μg/ml). All 3 isolates were found to have a mutation in HS1 of fks1 (Table 4).

Table 4.

Summary of fks alterations detected in echinocandin-resistant Candida sp. strains

Organism	State or country	Year	MIC (μg/ml) for each indicated antifungal:			1,3-β-d-Glucan synthase alterations ina:
			Anidulafungin	Caspofungin	Micafungin	fks1 HS1	fks1 HS2	fks2 HS1b	fks2 HS2b
Candida glabrata	Indiana	2010	1	4	0.06	WT	WT	F641V	WT
C. glabrata	Belgium	2010	1	1	0.06	WT	WT	WT	WT
C. glabrata	Michigan	2010	0.25	0.5	0.03	WT	WT	D648E	WT
C. glabrata	Texas	2010	0.5	0.25	0.03	WT	WT	F641Y	WT
C. glabrata	Germany	2010	1	0.5	0.5	WT	WT	L644W	WT
C. glabrata	New York	2010	0.25	0.25	0.5	WT	WT	WT	WT
Candida parapsilosis	New York	2010	8	2	1	WT	WT	NT	NT
C. parapsilosis	New York	2010	8	1	1	WT	WT	NT	NT
C. parapsilosis	Argentina	2010	8	1	1	WT	WT	NT	NT
Candida albicans	Sweden	2011	0.5	1	1	S654P	WT	NT	NT
C. albicans	Scotland	2011	0.5	2	1	S629P	WT	NT	NT
C. albicans	China	2011	0.25	1	0.5	S645P	WT	NT	NT
C. glabrata	Australia	2011	0.5	0.25	0.12	F625S	WT	WT	WT
C. glabrata	Canada	2011	1	1	0.25	WT	WT	S659Y	WT
C. glabrata	Indiana	2011	1	0.5	0.5	WT	WT	S663Y	WT
C. glabrata	Australia	2011	1	1	0.5	WT	WT	S663P	WT
C. glabrata	Greece	2011	2	1	1	WT	WT	S663P	WT
C. glabrata	Louisiana	2011	4	16	2	S629P	WT	WT	WT

^aWT, wild-type.

^bNT, not tested.

Among the C. glabrata isolates from 2003 to 2007, 7 (0.6%) were resistant to anidulafungin (MIC, ≥0.5 μg/ml), 10 (0.8%) were resistant to caspofungin (MIC, ≥0.5 μg/ml), and 6 (0.5%) were resistant to micafungin (MIC, ≥0.25 μg/ml) (Table 3). In contrast, 10 isolates (1.8%) (P < 0.005) from 2010 to 2011 were resistant to anidulafungin, 9 isolates (1.6%) (P = 0.0013) were resistant to caspofungin, and 7 isolates (1.5%) (P = 0.056) were resistant to micafungin (Table 3). Taken together, the data indicate that a total of 12 isolates (2.1%) of C. glabrata from 2010 to 2011 were resistant to one or more of the echinocandins, 7 of which were from North America, 3 were from Europe, and 2 were from the Asia-Pacific region (Australia) (Table 4). Among these 12 isolates, 8 were found to contain mutations in HS1 of fks2, 2 were found to contain mutations in HS1 of fks1, and 2 did not have mutations in either fks1 or fks2 (Table 4). Of the latter 2 isolates, 1 was resistant to anidulafungin and caspofungin and susceptible to micafungin, and 1 was intermediate to anidulafungin and caspofungin and resistant to micafungin (Table 4). Although the clinical importance of such differential susceptibilities to the echinocandins in the isolates of C. glabrata is not clear, a recent study by Arendrup and colleagues (46) found such differences to be meaningful in an in vivo model of invasive candidiasis, irrespective of the presence or absence of fks mutations. Taken together, these results suggest that resistance to echinocandins may be increasing in C. glabrata isolates and that in vitro susceptibility test results are predictive of fks resistance mutations using the CLSI CBPs. It should also be noted that 38% of the echinocandin-resistant isolates of C. glabrata were also resistant to fluconazole.

Similar to that seen with C. albicans, echinocandin resistance was very uncommon among isolates of C. parapsilosis, C. tropicalis, and C. krusei from both time periods (Table 3). Three isolates of C. parapsilosis from 2010 to 2011 were resistant to anidulafungin (MIC, >4 μg/ml) and were susceptible (MIC, ≤2 μg/ml) to both caspofungin and micafungin, none of which contained an fks resistance mutation (Table 4).

Table 5 shows the frequency of decreased susceptibility of A. fumigatus to itraconazole, posaconazole, and voriconazole across the four geographic regions. Using the ECV of 1 μg/ml for both itraconazole and voriconazole, the only non-WT isolates in the present collection were from North America, for an overall frequency of non-WT strains of 1.0 to 2.0%. In contrast, 0.0% to 15.6% of isolates (9.4% overall) were classified as non-WT to posaconazole using the ECV of 0.5 μg/ml established by Espinel-Ingroff et al. (37). Among the 9 isolates characterized as non-WT to posaconazole, the MIC was 1 μg/ml for 8 isolates (89%) and 4 μg/ml for 1 isolate. The latter isolate was also non-WT to both itraconazole and voriconazole, suggesting a rare cross-resistance among the three triazoles. Isolates for which posaconazole MICs are 1 μg/ml (non-WT) yet which are classified as WT for both itraconazole and voriconazole do not conform to known mechanisms of resistance (44), suggesting that the ECV for posaconazole and A. fumigatus may be set too low, or that technical factors in testing, such as solubility of the drug, may have led to falsely elevated MIC values in the present study.

Table 5.

Frequency of decreased susceptibility of Aspergillus fumigatus to azole antifungal agents by geographic region using CLSI epidemiological cutoff values

Antifungal agent	ECVb (μg/ml)	No. of isolatesa (% non-wild-type to each antifungal agent) by region
		North America	Europe	Latin America	Asia-Pacific	Total
Itraconazole	1	45 (4.4)	34 (0.0)	13 (0.0)	4 (0.0)	96 (2.0)
Posaconazole	0.5	45 (15.6)	34 (2.9)	13 (7.7)	4 (0.0)	96 (9.4)
Voriconazole	1	45 (2.2)	34 (0.0)	13 (0.0)	4 (0.0)	96 (1.0)

^aAll isolates tested using CLSI broth microdilution methods (19).

^bECVs as published by Espinel-Ingroff et al. (37).

It should be noted that whereas azole resistance in A. fumigatus is generally considered to be quite uncommon (47), reports from The Netherlands (48) and the United Kingdom (Manchester) (49) suggest that azole resistance may have increased in recent years. Recent estimates of the frequency of azole (itraconazole) resistance among European isolates of A. fumigatus range from 0.8% in France (50) to 5% in The Netherlands (48) and from 14 to 20% in a specialty referral laboratory in Manchester, United Kingdom (49). The lack of azole-resistant (non-WT) isolates in the present European collection may be explained in part by the fact that only 34 isolates were obtained from European study sites and that there were no isolates submitted from The Netherlands or from Manchester. The finding of 4% non-WT isolates of A. fumigatus in North America is similar to the findings of Baddley et al. (51), who reported 4% resistance to azoles in isolates of Aspergillus in the North American-based Transplant-Associated Infection Surveillance Network (TRANSNET).

The MIC distributions for the echinocandins and triazoles for the uncommon species of Candida and Aspergillus, the non-Candida yeasts, and non-Aspergillus molds for which molecular ID was confirmed are shown in Table 6 and in the supplemental material (species for which there were <5 isolates). Given that these species are uncommon in most regions of the world and that data concerning their in vitro susceptibility to most antifungals are lacking, we have elected to display the results as the number of isolates at each MIC value so that these results may eventually be combined with similarly derived data to form a more robust understanding of the MIC profiles of these unusual species.

Table 6.

MIC distributions for azole and echinocandin antifungal agents tested against isolates of uncommon species of Candida and Aspergillus, as well as non-Candida yeasts and non-Aspergillus molds identified by molecular methods (≥5 isolates per species) in SENTRY, 2010 to 2011

Species (no. tested)	Antifungal agenta	No. at an MIC/MEC (μg/ml) of:
		0.007	0.015	0.03	0.06	0.12	0.25	0.5	1	2	4	8	16	32	64	128
Candida species
C. dubliniensis (50)	ANF	1	3	17	18	8	3
	CSF	2	2	14	23	8	1
	MCF	4	6	23	12	4	1
	FLC				16	16	17
	ITR	1	10	19	14	4		2
	PSC	2	7	28	5	7		1
	VRC	41	6	2		1
C. fermentati (8)	ANF								4	4
	CSF					4	2	2
	MCF						1	6	1
	FLC						1	1	1	3	1					1
	ITR						2	4	1			1
	PSC				3	3	1					1
	VRC			2	2	3						1
C. guilliermondii (19)	ANF						1		5	8	4	1
	CSF				3	3	3	4	4	2
	MCF				1	1	5	4	5	2		1
	FLC								1	7	4	3	2		1	1
	ITR						4	7	2	5		1
	PSC					5	5	4	5
	VRC			1	6	7	3		1	1
C. kefyr (16)	ANF			8	4	2	2
	CSF	1	13	1		1
	MCF			6	7	2		1
	FLC				1	5	8	2
	ITR		1	3	7	3	2
	PSC		1	3	7	3	2
	VRC	13	3
C. lusitaniae (55)	ANF			1	3	2	18	19	11	1
	CSF		1		4	12	20	13	5
	MCF		2	2	3	7	25	10	5	1
	FLC				12	13	11	8	4	4	1			1	1
	ITR	1		1	13	16	8	11	4	1
	PSC	1	5	10	19	7	11	1	1
	VRC	34	12	6	1		1	1
Non-Candida yeasts
Cryptococcus neoformans (84)	ANF												84
	CSF											11	73
	MCF												84
	FLC							3	14	24	26	15	2
	ITR			8	18	24	22	11	1
	PSC		1	4	19	30	16	12	2
	VRC	4	17	29	29	5
Rhodotorula mucilaginosa (5)	ANF												5
	CSF											2	3
	MCF												5
	FLC															5
	ITR								2	2		1
	PSC								2	2	1
	VRC							2			3
Saccharomyces cerevisiae (6)	ANF				1	1	2	2
	CSF					2	3	1
	MCF					5	1
	FLC								3	2	1
	ITR						2	2	2
	PSC						2	2	2
	VRC			3	2	1
Trichosporon asahii (9)	ANF												9
	CSF										1	3	5
	MCF												9
	FLC							2		5	1					1
	ITR					1	1	5	2
	PSC					2	3	3	1
	VRC		1	2	3	1	1	1
Trichosporon spp. (5)	ANF												5
	CSF											2	3
	MCF												5
	FLC								2	2	1
	ITR					1	1	2	1
	PSC				2	1	1	1
	VRC				3	2
Aspergillus species
A. flavus SC (19)	ANF	16	1	2
	CSF	4	9	6
	MCF	15	2	2
	ITR							12	7
	PSC						3	15	1
	VRC							11	7	1
A. niger SC (15)	ANF	10	3	2
	CSF		5	8	1	1
	MCF	12	1	2
	ITR								8	6
	PSC							9	5
	VRC							8	4	1
A. terreus SC (7)	ANF	3	2	1		1
	CSF		3	3			1
	MCF	5	1			1
	ITR						1	3	3
	PSC						4	3
	VRC						1	6
Non-Aspergillus molds
Gibberella fujikuroi SC (6)	ANF										1		5
	CSF												6
	MCF												6
	ITR							1				5
	PSC						1	2	1		1	1
	VRC		1						1	2	1	1

^aANF, anidulafungin; MCF, micafungin; CSF, caspofungin; ITR, itraconazole; PSC, posaconazole; VRC, voriconazole.

Among the 169 isolates of the less-common species of Candida encountered during the 2010 to 2011 time period, we identified 15 different species (Table 6; see also the supplemental material), including 5 species (C. dubliniensis, C. guilliermondii, C. kefyr, C. lusitaniae, and C. pelliculosa) for which ECVs have been derived for the echinocandins and triazoles (11). In general, the MIC values obtained for both classes and these species conform to the WT MIC distributions described previously. Notable observations include decreased echinocandin susceptibility (MIC > ECV) in C. guilliermondii (1 isolate) to anidulafungin and micafungin (MIC >4 μg/ml), in C. kefyr (1 isolate) to caspofungin (MIC, >0.03 μg/ml) and micafungin (MIC, >0.12 μg/ml), and in C. lusitaniae (6 isolates) to micafungin (MIC, >0.5 μg/ml). The MIC values for the echinocandins against the very rare species of Candida were generally low (<0.5 μg/ml), with the exceptions of C. fermentati (Table 6), C. haemulonii, C. lipolytica, and C. nivariensis (see the supplemental material).

Cross-resistance (MIC > ECV) among the triazoles was detected in 4 isolates of C. guilliermondii (fluconazole MIC, >8 μg/ml; itraconazole MIC, >1 μg/ml; posaconazole MIC, >0.5 μg/ml; voriconazole MIC, >0.25 μg/ml), 1 isolate of C. dubliniensis (fluconazole MIC, >0.5 μg/ml; itraconazole MIC, >0.25 μg/ml; posaconazole MIC, >0.12 μg/ml; voriconazole MIC, >0.06 μg/ml), and 3 isolates of C. lusitaniae (fluconazole MIC, >2 μg/ml; itraconazole MIC, >0.5 μg/ml; posaconazole MIC, >0.12 μg/ml; voriconazole MIC, >0.03 μg/ml). Additional species in which fluconazole MICs appeared to be elevated (MIC, ≥8 μg/ml) included C. catenulata (MIC, 8 μg/ml), C. fermentati (MIC, 128 μg/ml), C. inconspicua (MIC, 32 μg/ml), and C. lipolytica (MIC, 16 μg/ml).

As expected, the echinocandins were inactive against many of the non-Candida yeasts (Table 6; see also the supplemental material). Echinocandin MIC results of ≤0.5 μg/ml were seen with A. pullulans, D. fabryi, L. elongisporus, and S. cerevisiae. In contrast, the triazoles, especially voriconazole, showed good activity against most of the non-Candida yeasts, with the exception of R. mucilaginosa. Among the 84 isolates of C. neoformans (Table 6), all (100.0%) were WT to fluconazole (ECV, 16 μg/ml) and voriconazole (ECV, 0.25 μg/ml); 97.6% were WT to posaconazole (ECV, 0.5 μg/ml), which is similar to our previous experience (12). The echinocandins were inactive against C. neoformans (Table 6).

The echinocandins were highly active against the non-fumigatus species of Aspergillus (Table 6; see also the supplemental material): only 1 isolate of A. niger SC and 1 of A. terreus SC exhibited an MEC of >0.06 μg/ml. Among the 4 species of Aspergillus for which triazole ECVs have been derived (A. flavus, A. terreus, A. niger, and A. nidulans), the MIC values obtained for both itraconazole and voriconazole conformed to the WT MIC distribution described by Espinel-Ingroff and colleagues (37). As seen with A. fumigatus (Table 5), the MIC distributions for posaconazole against A. flavus SC and A. niger SC were shifted 1 dilution higher than those described by Espinel-Ingroff et al. (37). Among the 4 remaining species of Aspergillus, only A. sydowii exhibited triazole MICs of >1 μg/ml (MIC, 2 μg/ml for both itraconazole and voriconazole).

Among the non-Aspergillus molds, only P. variotii, Penicillium spp., Sarocladium (Acremonium) kiliense, and Trichoderma spp. consistently exhibited echinocandin MECs of ≤0.06 μg/ml (Table 6; see also the supplemental material). Notably, MEC values of ≥8 μg/ml were observed for the vast majority of the Fusarium and Mucorales species in this molecularly characterized collection. Similarly, the triazoles showed poor activities against many of these rare molds.

Among the extended-spectrum triazoles, posaconazole stands apart from voriconazole in that it appears to be active against some clinical isolates of the mucoraceous molds both in vitro and in vivo (52, 53). Among the 4 molecularly identified isolates from the Mucormycetes genus in the present study, the voriconazole MICs were all 8 μg/ml, whereas the MICs for posaconazole ranged from 0.25 μg/ml (R. oryzae) to 1 μg/ml (R. pusillus and R. microsporus group) (see the supplemental material).

The typical antifungal susceptibility profiles of Fusarium spp. are of relative resistance to most antifungal agents (54). Among the small number of isolates in the present study, MICs were generally ≤4 μg/ml for both posaconazole and voriconazole against the 10 Fusarium isolates tested: G. fujikuroi (80% ≤4 μg/ml) (Table 6), F. solani (67%) (see the supplemental material), and Fusarium spp. (100%) (see the supplemental material). Although voriconazole and posaconazole exhibit only modest activity in vitro against isolates of Fusarium, both of these triazoles have been used successfully in some patients with amphotericin B-refractory fusariosis (55, 56).

S. apiospermum is generally considered to be resistant to amphotericin B, to which its clinical response is very poor (3), whereas both posaconazole and voriconazole have successfully been used for the treatment of central nervous system abscesses (57, 58). MICs for posaconazole ranged from 0.5 to 2 μg/ml and for voriconazole from 0.12 to 1 μg/ml against the 4 S. apiospermum isolates in the present study (see the supplemental material).

There are several important observations that can be made from this global survey. First, we have used molecular methods to document an amazing diversity of opportunistic fungal pathogens in four broad geographic regions of the world. Although the majority of IFIs were due to the five major species of Candida (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei), C. neoformans, and A. fumigatus, IFIs were associated with the isolation of an additional 15 species of Candida, 9 species of non-Candida yeasts, 8 species of Aspergillus, and 23 species of non-Aspergillus molds.

As reported previously (14) for Candida, the species distributions and antifungal susceptibility profiles varied across geographic regions. Whereas the common species of Candida remained largely susceptible to both the triazoles and echinocandins, resistance to both classes of agents continued to be detected among isolates of C. glabrata. In contrast to the results from the 2009 SENTRY Program, the highest frequency of resistance to both echinocandins and azoles was observed among isolates from the Asia-Pacific region. Conversely, we have yet to detect echinocandin resistance in C. glabrata isolates from Latin America. By comparison with earlier susceptibility data from 2003 to 2007, isolates of C. glabrata in the present collection showed an approximately 3-fold increase in resistance to the echinocandins, and the resistant phenotypes were associated with mutations in fks (P < 0.001).

We have included the antifungal susceptibility profiles of those species from the SENTRY Program that have undergone sequence-based ID in an effort to provide MIC data not only for the relatively common species of Candida and A. fumigatus but also for those that may be less frequently encountered but still pose problems for selecting optimal antifungal therapies. In doing so, we have identified decreased susceptibilities to both echinocandin and triazoles in several additional species of Candida, non-Candida yeasts, and non-Aspergillus molds. Whereas the emergence of azole resistance among A. fumigatus has been reported by others (43), we identified only 2% of A. fumigatus isolates (all from North America) that warrant further investigation regarding acquired resistance mechanisms (MIC > ECV).

In summary, we provide additional information demonstrating the excellent activity of the echinocandins (anidulafungin, caspofungin, and micafungin) and newer triazoles (posaconazole and voriconazole) against Candida and Aspergillus spp., and of the triazoles against C. neoformans and other non-Candida yeasts. We have applied the new (lower) species-specific CLSI CBPs and ECVs for the echinocandins and triazoles to this contemporary (2010 to 2011) collection of Candida spp. and documented measurable but low levels of resistance or decreased susceptibility (non-WT; MIC > ECV) for most species. We provide additional validation of the echinocandin CBPs by demonstrating that the vast majority of C. albicans and C. glabrata isolates that were phenotypically resistant to one or more echinocandin possessed an acquired resistance mutation in fks1 and/or fks2. Continued application of antifungal susceptibility testing combined with molecular characterization of species ID and resistance mechanisms for the available antifungal agents is critical in detecting the emergence of resistance among the ever-evolving spectrum of opportunistic fungal pathogens. Given the now-documented ability of C. glabrata to express resistance to both azoles and echinocandins (13, 59), this species must remain a focus of antifungal resistance surveillance in the coming years.