Isavuconazole, micafungin, and 8 comparator antifungal agents' susceptibility profiles for common and uncommon opportunistic fungi collected in 2013: temporal analysis of antifungal drug resistance using CLSI species-specific clinical breakpoints and proposed epidemiological cutoff values.

The in vitro activities of isavuconazole, micafungin, and 8 comparator antifungal agents were determined for 1613 clinical isolates of fungi (1320 isolates of Candida spp., 155 of Aspergillus spp., 103 of non-Candida yeasts, and 35 non-Aspergillus molds) collected during a global survey conducted in 2013. The vast majority of the isolates of the 21 different species of Candida, with the exception of Candida glabrata (MIC90, 2 µg/mL), Candida krusei (MIC90, 1 µg/mL), and Candida guilliermondii (MIC90, 8 µg/mL), were inhibited by ≤0.25 µg/mL of isavuconazole. C. glabrata and C. krusei were largely inhibited by ≤1 µg/mL of isavuconazole. Resistance to fluconazole was seen in 0.5% of Candida albicans isolates, 11.1% of C. glabrata isolates, 2.5% of Candida parapsilosis isolates, 4.5% of Candida tropicalis isolates, and 20.0% of C. guilliermondii isolates. Resistance to the echinocandins was restricted to C. glabrata (1.3-2.1%) and C. tropicalis (0.9-1.8%). All agents except for the echinocandins were active against 69 Cryptococcus neoformans isolates, and the triazoles, including isavuconazole, were active against the other yeasts. Both the mold active triazoles as well as the echinocandins were active against 155 Aspergillus spp. isolates belonging to 10 species/species complex. In general, there was low resistance levels to the available systemically active antifungal agents in a large, contemporary (2013), global collection of molecularly characterized yeasts and molds. Resistance to azoles and echinocandins was most prominent among isolates of C. glabrata, C. tropicalis, and C. guilliermondii.

In vitro activity of a Hos2 deacetylase inhibitor, MGCD290, in combination with echinocandins against echinocandin-resistant Candida species.

MGCD290, a Hos2 fungal histone deacetylase inhibitor, showed modest activity when tested alone (MIC range, 0.12-4 µg/mL; MIC50/90, 0.5/4 µg/mL) against Candida glabrata (n=15; 14 fks mutants; 5 also fluconazole resistant), Candida albicans (8 fks mutants; 2 also fluconazole resistant), Candida tropicalis (4 fks mutants), and Candida krusei (3 fks mutants). However, MGCD290 showed synergy or partial synergy for 33.3%, 30.1%, 36.7%, and 80.0% of the isolates when tested with anidulafungin, caspofungin, micafungin, and fluconazole, respectively. Favorable interactions were achieved with low concentrations of MGCD290 (0.015-0.25 µg/mL), and categorical shifts were observed in 2 of 8 (25.0%) isolates of C. albicans and 2 of 3 (66.7%) isolates of C. krusei and in 4 of the 5 (80.0%) fluconazole-resistant isolates of C. glabrata. MGCD290 exerts a distinctly favorable influence on the MICs of fluconazole and the echinocandins, resulting in conversion from resistance to susceptibility regardless of fks mutations.

Comparison of the Sensititre YeastOne colorimetric antifungal panel with CLSI microdilution for antifungal susceptibility testing of the echinocandins against Candida spp., using new clinical breakpoints and epidemiological cutoff values.

A commercially prepared dried colorimetric microdilution panel (Sensititre Yeast One, TREK Diagnostic Systems, Cleveland, OH, USA) was compared in 3 different laboratories with the Clinical and Laboratory Standards Institute (CLSI) reference microdilution method by testing 2 quality control strains, 25 reproducibility strains, and 404 isolates of Candida spp. against anidulafungin, caspofungin, and micafungin. Reference CLSI BMD MIC end points and YeastOne colorimetric end points were read after 24 h of incubation. Excellent (100%) essential agreement (within 2 dilutions) between the reference and colorimetric MICs was observed. Categorical agreement (CA) between the 2 methods was assessed using the new species-specific clinical breakpoints (CBPs): susceptible (S), ≤0.25 µg/mL; intermediate (I), 0.5 µg/mL; and resistant (R), ≥1 µg/mL, for C. albicans, C. tropicalis, and C. krusei, and ≤2 µg/mL (S), 4 µg/mL (I), and ≥8 µg/mL (R) for C. parapsilosis and all 3 echinocandins. The new CBPs for anidulafungin and caspofungin and C. glabrata are ≤0.12 µg/mL (S), 0.25 µg/mL (I), and ≥0.5 µg/mL (R), whereas those for micafungin are ≤0.06 µg/mL (S), 0.12 µg/mL (I), and ≥0.25 µg/mL (R). Due to the lack of CBPs for any of the echinocandins and C. lusitaniae, the epidemiological cutoff values (ECVs) were used for this species to categorize the isolates as wild-type (WT; MIC ≤ECV) and non-WT (MIC >ECV), respectively, for anidulafungin (≤2 µg/mL/>2 µg/mL), caspofungin (≤1 µg/mL/>1 µg/mL), and micafungin (≤0.5 µg/mL/>0.5 µg/mL). CA ranged from 93.6% (caspofungin) to 99.6% (micafungin) with less than 1% very major or major errors. The YeastOne colorimetric method remains comparable to the CLSI BMD reference method for testing the susceptibility of Candida spp. to the echinocandins when using the new (lower) CBPs and ECVs. Further study using defined fks mutant strains of Candida is warranted.

Frequency of decreased susceptibility and resistance to echinocandins among fluconazole-resistant bloodstream isolates of Candida glabrata.

The echinocandin class of antifungal agents is considered to be the first-line treatment of bloodstream infections (BSI) due to Candida glabrata. Recent reports of BSI due to strains of C. glabrata resistant to both fluconazole and the echinocandins are of concern and prompted us to review the experience of two large surveillance programs, the SENTRY Antimicrobial Surveillance Program for the years 2006 through 2010 and the Centers for Disease Control and Prevention population-based surveillance conducted in 2008 to 2010. The in vitro susceptibilities of 1,669 BSI isolates of C. glabrata to fluconazole, voriconazole, anidulafungin, caspofungin, and micafungin were determined by CLSI broth microdilution methods. Fluconazole MICs of ≥64 µg/ml were considered resistant. Strains for which anidulafungin and caspofungin MICs were ≥0.5 µg/ml and for which micafungin MICs were ≥0.25 µg/ml were considered resistant. A total of 162 isolates (9.7%) were resistant to fluconazole, of which 98.8% were nonsusceptible to voriconazole (MIC > 0.5 µg/ml) and 9.3%, 9.3%, and 8.0% were resistant to anidulafungin, caspofungin, and micafungin, respectively. There were 18 fluconazole-resistant isolates that were resistant to one or more of the echinocandins (11.1% of all fluconazole-resistant isolates), all of which contained an acquired mutation in fks1 or fks2. By comparison, there were no echinocandin-resistant strains detected among 110 fluconazole-resistant isolates of C. glabrata tested in 2001 to 2004. These data document the broad emergence of coresistance over time to both azoles and echinocandins in clinical isolates of C. glabrata.

Validation of 24-hour posaconazole and voriconazole MIC readings versus the CLSI 48-hour broth microdilution reference method: application of epidemiological cutoff values to results from a global Candida antifungal surveillance program.

We performed 24- and 48-h MIC determinations of posaconazole and voriconazole against more than 16,000 clinical isolates of Candida species. By using the 24- and 48-h epidemiological cutoff values (ECVs), the categorical agreement between the 24-h and reference 48-h broth microdilution results ranged from 97.1% (C. parapsilosis and voriconazole) to 99.8% (C. krusei and voriconazole), with 0.0 to 2.9% very major discrepancies (VMD). The essential agreement (within 2 log(2) dilutions) between the 24- and 48-h results was 99.6% for both posaconazole and voriconazole. The MIC results obtained for both posaconazole and voriconazole after only 24 h of incubation may be used to determine the susceptibilities of Candida spp. to these important antifungal agents. The applications of ECVs to this large collection of Candida isolates suggests the potential to develop 24-h species-specific clinical breakpoints for both posaconazole and voriconazole.

Comparison of the broth microdilution (BMD) method of the European Committee on Antimicrobial Susceptibility Testing with the 24-hour CLSI BMD method for testing susceptibility of Candida species to fluconazole, posaconazole, and voriconazole by use of epidemiological cutoff values.

The antifungal broth microdilution (BMD) method of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) was compared with CLSI BMD method M27-A3 for fluconazole, posaconazole, and voriconazole susceptibility testing of 1,056 isolates of Candida. The isolates were obtained in 2009 from more than 60 centers worldwide and included 560 isolates of C. albicans, 175 of C. glabrata, 162 of C. parapsilosis, 124 of C. tropicalis, and 35 of C. krusei. The overall essential agreement (EA) between EUCAST and CLSI results ranged from 96.9% (voriconazole) to 98.6% (fluconazole). The categorical agreement (CA) between methods and species of Candida was assessed using previously determined epidemiological cutoff values (ECVs). The ECVs (expressed as µg/ml) for fluconazole, posaconazole, and voriconazole, respectively, were as follows: 0.12, 0.06, and 0.03 for C. albicans; 32, 2, and 0.5 for C. glabrata; 2, 0.25, and 0.12 for C. parapsilosis; 2, 0.12, and 0.06 for C. tropicalis; 64, 0.5, and 0.5 for C. krusei. Excellent CA was observed for all comparisons between the EUCAST and CLSI results for fluconazole, posaconazole, and voriconazole, respectively, for each species: 98.9%, 93.6%, and 98.6% for C. albicans; 96.0%, 98.9%, and 93.7% for C. glabrata; 90.8%, 98.1%, and 98.1% for C. parapsilosis; 99.2%, 99.2%, and 96.8% for C. tropicalis; 97.1%, 97.1%, and 97.1% for C. krusei. We demonstrate high levels of EA and CA between the CLSI and EUCAST BMD methods for testing of triazoles against Candida when the MICs were determined after 24 h and ECVs were used to differentiate wild-type (WT) from non-WT strains. These results provide additional data in favor of the harmonization of these two methods.

Wild-type MIC distributions and epidemiological cutoff values for posaconazole and voriconazole and Candida spp. as determined by 24-hour CLSI broth microdilution.

We tested 16,191 strains of Candida against posaconazole and voriconazole, using the CLSI M27-A3 broth microdilution (BMD) method (24-h incubation), in order to define wild-type (WT) populations and epidemiological cutoff values (ECVs). From 2001 to 2009, 8,619 isolates of Candida albicans, 2,415 isolates of C. glabrata, 2,278 isolates of C. parapsilosis, 1,895 isolates of C. tropicalis, 508 isolates of C. krusei, 205 isolates of C. lusitaniae, 177 isolates of C. guilliermondii, and 93 isolates of C. kefyr were obtained from over 100 centers worldwide. The modal MICs (µg/ml) for posaconazole and voriconazole, respectively, were as follows: for C. albicans, 0.016 and 0.007; for C. glabrata, 0.5 and 0.06; for C. parapsilosis, 0.06 and 0.007; for C. tropicalis, 0.03 and 0.015; for C. krusei, 0.25 and 0.12; for C. lusitaniae, 0.03 and 0.007; for C. guilliermondii, 0.12 and 0.03; and for C. kefyr, 0.06 and 0.007. The ECVs (µg/ml [% of isolates that had MICs equal to or less than the ECV]) for posaconazole and voriconazole, respectively, were as follows: 0.06 (98.5) and 0.03 (98.9) for C. albicans, 2 (96.2) and 0.5 (90.4%) for C. glabrata, 0.25 (99.3) and 0.12 (97.9) for C. parapsilosis, 0.12 (97.6) and 0.06 (97.2) for C. tropicalis, 0.5 (99.8) and 0.5 (99.4) for C. krusei, 0.12 (95.6) and 0.03 (96.6) for C. lusitaniae, 0.5 (98.9) and 0.25 (98.3) for C. guilliermondii, and 0.25 (100.0) and 0.015 (100.0) for C. kefyr. In the absence of clinical breakpoints (CBPs) for posaconazole, these WT distributions and ECVs will be useful in surveillance for emergence of reduced susceptibility to posaconazole among Candida spp. Whereas a CBP for susceptibility of ≤ 1 µg/ml has been established for voriconazole and all species of Candida, it is notable that ECVs for this agent range from 10- to >100-fold lower than the CBP, depending on the species of Candida. The CBP is inadequate in detecting the emergence of voriconazole resistance among most Candida species encountered clinically. The CBPs for voriconazole should be reassessed, with consideration for development of species-specific CBPs.

Comparison of European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Etest methods with the CLSI broth microdilution method for echinocandin susceptibility testing of Candida species.

The antifungal broth microdilution (BMD) method of the European Committee on Antibiotic Susceptibility Testing (EUCAST) and the Etest agar diffusion method were compared with the Clinical and Laboratory Standards Institute (CLSI) BMD method M27-A3 for anidulafungin, caspofungin, and micafungin susceptibility testing of 133 clinical isolates of Candida species. The isolates were characterized for the presence or absence of fks1 and/or fks2 gene mutations and included 34 isolates of C. glabrata (4 mutant strains), 32 of C. albicans (1 mutant strain), 25 of C. parapsilosis, 19 of C. guilliermondii, 12 of C. tropicalis (2 mutant strains), and 11 of C. krusei. Excellent essential agreement (EA; within 2 dilutions) between the CLSI and EUCAST and CLSI and Etest MIC results was observed. The overall EA between the EUCAST and CLSI results ranged from 89.5% (caspofungin) to 99.2% (micafungin), whereas the EA between the Etest and CLSI results ranged from 90.2% (caspofungin) to 93.2% (anidulafungin). The categorical agreement (CA) between methods for each antifungal agent was assessed using previously determined epidemiological cutoff values (ECVs). Excellent CA (>90%) was observed for all comparisons between the EUCAST and CLSI results with the exceptions of C. glabrata and caspofungin (85.3%) and C. krusei and caspofungin (54.5%). The CA between the Etest and CLSI results was also excellent for all comparisons, with the exception of C. krusei and caspofungin (81.8%). All three methods were able to differentiate wild-type (WT) strains from those with fks mutations. With anidulafungin as the test reagent, the CLSI method identified 5 of 7 mutant strains, whereas the EUCAST method and the Etest identified 6 of 7 mutant strains. With either caspofungin or micafungin as the test reagent, the CLSI method identified all 7 mutant strains and the EUCAST method identified 6 of 7 mutant strains. The Etest identified all 7 mutant strains using caspofungin as the reagent. All three test methods showed a high level of agreement and of ability to distinguish fks mutant strains of Candida species from WT strains using each of the echinocandins.

Wild-type MIC distributions and epidemiological cutoff values for the echinocandins and Candida spp.

We tested a global collection of Candida sp. strains against anidulafungin, caspofungin, and micafungin, using CLSI M27-A3 broth microdilution (BMD) methods, in order to define wild-type (WT) populations and epidemiological cutoff values (ECVs). From 2003 to 2007, 8,271 isolates of Candida spp. (4,283 C. albicans, 1,236 C. glabrata, 1,238 C. parapsilosis, 996 C. tropicalis, 270 C. krusei, 99 C. lusitaniae, 88 C. guilliermondii, and 61 C. kefyr isolates) were obtained from over 100 centers worldwide. The modal MICs (in microg/ml) for anidulafungin, caspofungin, and micafungin, respectively, for each species were as follows: C. albicans, 0.03, 0.03, 0.015; C. glabrata, 0.06, 0.03, 0.015; C. tropicalis, 0.03, 0.03, 0.015; C. kefyr, 0.06, 0.015, 0.06; C. krusei, 0.03, 0.06, 0.06; C. lusitaniae, 0.05, 0.25, 0.12; C. parapsilosis, 2, 0.25, 1; and C. guilliermondii, 2, 0.5. 05. The ECVs, expressed in microg/ml (percentage of isolates that had MICs that were less than or equal to the ECV is shown in parentheses) for anidulafungin, caspofungin, and micafungin, respectively, were as follows: 0.12 (99.7%), 0.12 (99.8%), and 0.03 (97.7%) for C. albicans; 0.25 (99.4%), 0.12 (98.5%), and 0.03 (98.2%) for C. glabrata; 0.12 (98.9%), 0.12 (99.4%), and 0.12 (99.1%) for C. tropicalis; 0.25(100%), 0.03 (100%), and 0.12 (100%) for C. kefyr; 0.12 (99.3%), 0.25 (96.3%), and 0.12 (97.8%) for C. krusei; 2 (100%), 0.5 (98.0%), and 0.5 (99.0%) for C. lusitaniae; 4 (100%), 1 (98.6%), and 4 (100%) for C. parapsilosis; 16 (100%), 4 (95.5%), and 4 (98.9%) for C. guilliermondii. These WT MIC distributions and ECVs will be useful in surveillance for emerging reduced echinocandin susceptibility among Candida spp. and for determining the importance of various FKS1 or other mutations.

Activity of MGCD290, a Hos2 histone deacetylase inhibitor, in combination with azole antifungals against opportunistic fungal pathogens.

We report on the in vitro activity of the Hos2 fungal histone deacetylase (HDAC) inhibitor MGCD290 (MethylGene, Inc.) in combination with azoles against azole-resistant yeasts and molds. Susceptibility testing was performed by the CLSI M27-A3 and M38-A2 broth microdilution methods. Testing of the combinations (MGCD290 in combination with fluconazole, posaconazole, or voriconazole) was performed by the checkerboard method. The fractional inhibitory concentrations were determined and were defined as <0.5 for synergy, >or=0.5 but <4 for indifference, and >or=4 for antagonism. Ninety-one isolates were tested, as follows: 30 Candida isolates, 10 Aspergillus isolates, 15 isolates of the Zygomycetes order, 10 Cryptococcus neoformans isolates, 8 Rhodotorula isolates, 8 Fusarium isolates, 5 Trichosporon isolates, and 5 Scedosporium isolates. MGCD290 showed modest activity when it was used alone (MICs, 1 to 8 microg/ml) and was mostly active against azole-resistant yeasts, but the MICs against molds were high (16 to >32 microg/ml). MGCD290 was synergistic with fluconazole against 55 (60%) of the 91 isolates, with posaconazole against 46 (51%) of the 91 isolates, and with voriconazole against 48 (53%) of the 91 isolates. Synergy between fluconazole and MGCD290 was observed against 26/30 (87%) Candida isolates. All 23 of the 91 Candida isolates that were not fluconazole susceptible demonstrated a reduced fluconazole MIC that crossed an interpretive breakpoint (e.g., resistant [MIC, >or=64 microg/ml] to susceptible [MIC,

In vitro susceptibility of clinical isolates of Aspergillus spp. to anidulafungin, caspofungin, and micafungin: a head-to-head comparison using the CLSI M38-A2 broth microdilution method.

We determined the in vitro activities of anidulafungin, caspofungin, and micafungin against 526 isolates of Aspergillus spp. (64 A. flavus, 391 A. fumigatus, 46 A. niger, and 25 A. terreus isolates) collected from over 60 centers worldwide from 2001 through 2007. Susceptibility testing was performed according to the CLSI M38-A2 method. All three echinocandins--anidulafungin (50% minimum effective concentration [MEC50], 0.007 microg/ml; MEC90, 0.015 microg/ml), caspofungin (MEC50, 0.015 microg/ml; MEC90, 0.03 microg/ml), and micafungin (MEC50, 0.007 microg/ml; MEC90, 0.015 microg/ml)-were very active against Aspergillus spp. More than 99% of all isolates were inhibited by < or = 0.06 microg/ml of all three agents.

In vitro activity of seven systemically active antifungal agents against a large global collection of rare Candida species as determined by CLSI broth microdilution methods.

Five Candida species (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei) account for over 95% of invasive candidiasis cases. Some less common Candida species have emerged as causes of nosocomial candidiasis, but there is little information about their in vitro susceptibilities to antifungals. We determined the in vitro activities of fluconazole, voriconazole, posaconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against invasive, unique patient isolates of Candida collected from 100 centers worldwide between January 2001 and December 2007. Antifungal susceptibility testing was performed by the CLSI M27-A3 method. CLSI breakpoints for susceptibility were used for fluconazole, voriconazole, anidulafungin, caspofungin, and micafungin, while a provisional susceptibility breakpoint of < or = 1 microg/ml was used for amphotericin and posaconazole. Of 14,007 Candida isolates tested, 658 (4.7%) were among the less common species. Against all 658 isolates combined, the activity of each agent, expressed as the MIC50/MIC90 ratio (and the percentage of susceptible isolates) was as follows: fluconazole, 1/4 (94.8%); voriconazole, 0.03/0.12 (98.6%); posaconazole, 0.12/0.5 (95.9%); amphotericin, 0.5/2 (88.3%); anidulafungin, 0.5/2 (97.4%); caspofungin, 0.12/0.5 (98.0%); and micafungin, 0.25/1 (99.2%). Among the isolates not susceptible to one or more of the echinocandins, most (68%) were C. guilliermondii. All isolates of the less common species within the C. parapsilosis complex (C. orthopsilosis and C. metapsilosis) were susceptible to voriconazole, posaconazole, anidulafungin, caspofungin, and micafungin. Over 95% of clinical isolates of the rare Candida species were susceptible to the available antifungals. However, activity did vary by drug-species combination, with some species (e.g., C. rugosa and C. guilliermondii) demonstrating reduced susceptibilities to commonly used agents such as fluconazole and echinocandins.

Variation in susceptibility of bloodstream isolates of Candida glabrata to fluconazole according to patient age and geographic location in the United States in 2001 to 2007.

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (> or = 80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).

Validation of 24-hour fluconazole MIC readings versus the CLSI 48-hour broth microdilution reference method: results from a global Candida antifungal surveillance program.

We performed 24- and 48-h MIC determinations and disk diffusion testing of fluconazole against more than 11,000 clinical isolates of Candida species. By using the reference MIC breakpoints, the categorical agreement between the 24-h and reference 48-h broth microdilution results ranged from 93.8% (all Candida species) to 94.9% (all Candida species minus Candida krusei), with only 0.1% very major errors (VME). The essential agreement (within 2 log(2) dilutions) between the 24-h and 48-h results was 99.6%. The categorical agreement between the 24-h disk diffusion results and the 24-h MIC results, using the previously established breakpoints, was 94.4%, with 0.1% VME. Both the MIC and the disk diffusion results obtained for fluconazole after only 24 h of incubation may be used to determine the susceptibilities of Candida spp. to this widely used antifungal agent.

In vitro survey of triazole cross-resistance among more than 700 clinical isolates of Aspergillus species.

Few data exist to describe in vitro patterns of cross-resistance among large collections of clinical Aspergillus isolates, including those of species other than Aspergillus fumigatus. We examined 771 Aspergillus spp. clinical isolates collected from 2000 to 2006 as part of a global antifungal surveillance program (553 A. fumigatus, 76 A. flavus, 59 A. niger, 35 A. terreus, and 24 A. versicolor isolates and 24 isolates of other Aspergillus species). Antifungal susceptibility testing was performed by the Clinical and Laboratory Standards Institute (CLSI) M38-A broth dilution method with itraconazole (ITR), posaconazole (POS), ravuconazole (RAV), and voriconazole (VOR). We examined the potential for cross-resistance by using measures of correlation overall and by species. For most Aspergillus isolates (from 88% of isolates for ITR to 98% of isolates for VOR and POS), MICs of each triazole were < or = 1 microg/ml. When all 771 isolates were examined, there were statistically significant correlations for all six triazole-triazole pairs. For A. fumigatus, the strongest correlations seen were those between VOR and RAV MICs (r = 0.7) and ITR and POS MICs (r = 0.4). Similarly, for A. flavus, only VOR and RAV MICs and ITR and POS MICs demonstrated statistically significant positive correlations. We have demonstrated correlations among triazole MICs for Aspergillus, which for the most common species (A. fumigatus and A. flavus) were strongest between VOR and RAV MICs and ITR and POS MICs. However, Aspergillus species for which MICs of VOR or POS were >2 microg/ml remain extremely rare (<1% of isolates).

Clinical evaluation of the Sensititre YeastOne colorimetric antifungal panel for antifungal susceptibility testing of the echinocandins anidulafungin, caspofungin, and micafungin.

A commercially prepared, dried colorimetric microdilution panel (Sensititre YeastOne Trek Diagnostic Systems, Cleveland, OH) was compared in three different laboratories with the Clinical and Laboratory Standards Institute (CLSI) reference microdilution method by testing 2 quality control strains, 25 reproducibility strains, and 404 isolates of Candida spp. against anidulafungin, caspofungin, and micafungin. Reference MIC endpoints and YeastOne colorimetric endpoints were read after 24 h of incubation. YeastOne endpoints were determined to be the lowest concentration at which the color in the well changed from red (positive, indicating growth) to blue (negative, indicating no growth). Excellent essential agreement (within 2 dilutions) between the reference and colorimetric MICs was observed. Overall agreement was 100% for all three agents. Categorical agreement ranged from 99.3% (anidulafungin) to 100% (caspofungin, micafungin) and interlaboratory reproducibility was 99%. The YeastOne colorimetric method appears to be comparable to the CLSI reference method for testing the susceptibility of Candida spp. to the echinocandins anidulafungin, caspofungin, and micafungin.

Selection of a surrogate agent (fluconazole or voriconazole) for initial susceptibility testing of posaconazole against Candida spp.: results from a global antifungal surveillance program.

There are currently no FDA-approved broth microdilution antifungal susceptibility testing products or interpretive breakpoints for susceptibility testing of the new triazole posaconazole. Fluconazole and voriconazole are in the same triazole class as posaconazole, have CLSI-approved interpretive MIC breakpoints, and are available on some commercially available MIC panels. We investigated whether one or both of these agents may be useful as a surrogate marker for posaconazole susceptibility. Fluconazole, voriconazole, and posaconazole MIC results for 10,807 isolates of Candida spp. were analyzed to validate a potential surrogate marker for posaconazole activity against indicated species. For illustrative purposes, we applied the voriconazole MIC breakpoints to posaconazole (susceptible, < or =1 microg/ml; susceptible dose dependent, 2 microg/ml; resistant, > or =4 microg/ml) and compared these MIC results and categorical interpretations with those of fluconazole and voriconazole by using regression statistics and categorical agreement. For all 10,807 isolates, the absolute categorical agreement was 91.1% (0.1% very major errors [VME], 1.2% major errors [ME], and 7.6% minor errors [M]) using fluconazole as the surrogate marker and 97.7% (0.3% VME 0.1% ME, and 1.9% M) using voriconazole as the surrogate. The results with fluconazole improved to a categorical agreement of 93.7% (0.1% VME, 0.2% ME, and 6.0% M) when results for Candida krusei (not indicated for fluconazole testing) were omitted. Either fluconazole or voriconazole MIC results may serve as surrogate markers to predict the susceptibility of Candida spp. to posaconazole.

In vitro susceptibility of invasive isolates of Candida spp. to anidulafungin, caspofungin, and micafungin: six years of global surveillance.

The echinocandins are being used increasingly as therapy for invasive candidiasis. Prospective sentinel surveillance for the emergence of in vitro resistance to the echinocandins among invasive Candida sp. isolates is indicated. We determined the in vitro activities of anidulafungin, caspofungin, and micafungin against 5,346 invasive (bloodstream or sterile-site) isolates of Candida spp. collected from over 90 medical centers worldwide from 1 January 2001 to 31 December 2006. We performed susceptibility testing according to the CLSI M27-A2 method and used RPMI 1640 broth, 24-h incubation, and a prominent inhibition endpoint for determination of the MICs. Of 5,346 invasive Candida sp. isolates, species distribution was 54% C. albicans, 14% C. parapsilosis, 14% C. glabrata, 12% C. tropicalis, 3% C. krusei, 1% C. guilliermondii, and 2% other Candida spp. Overall, all three echinocandins were very active against Candida: anidulafungin (MIC50, 0.06 microg/ml; MIC90, 2 microg/ml), caspofungin (MIC50, 0.03 microg/ml; MIC90, 0.25 microg/ml), micafungin (MIC50, 0.015 microg/ml; MIC90, 1 microg/ml). More than 99% of isolates were inhibited by < or = 2 microg/ml of all three agents. Results by species (expressed as the percentages of isolates inhibited by < or = 2 microg/ml of anidulafungin, caspofungin, and micafungin, respectively) were as follows: for C. albicans, 99.6%, 100%, and 100%; for C. parapsilosis, 92.5%, 99.9%, and 100%; for C. glabrata, 99.9%, 99.9%, and 100%; for C. tropicalis, 100%, 99.8%, and 100%; for C. krusei, 100%, 100%, and 100%; and for C. guilliermondii, 90.2%, 95.1%, and 100%. There was no significant change in the activities of the three echinocandins over the 6-year study period and no difference in activity by geographic region. All three echinocandins have excellent in vitro activities against invasive strains of Candida isolated from centers worldwide. Our prospective sentinel surveillance reveals no evidence of emerging echinocandin resistance among invasive clinical isolates of Candida spp.

Use of fluconazole as a surrogate marker to predict susceptibility and resistance to voriconazole among 13,338 clinical isolates of Candida spp. Tested by clinical and laboratory standards institute-recommended broth microdilution methods.

Clinical laboratories frequently face the problem of delayed availability of commercially prepared approved reagents for performing susceptibility testing of new antimicrobials. Although this problem is encountered more often with antibacterial agents, it is also an issue with antifungal agents. A current example is voriconazole, a new triazole antifungal with an expanded spectrum and potency against Candida spp., Aspergillus spp., and other opportunistic fungal pathogens. The present study addresses the use of fluconazole as a surrogate marker to predict the susceptibility of Candida spp. to voriconazole. Reference broth microdilution MIC results for 13,338 strains of Candida spp. isolated from more than 200 medical centers worldwide were used. Voriconazole MICs and interpretive categories (susceptible, < or =1 microg/ml; susceptible dose dependent, 2 microg/ml; resistant, > or =4 microg/ml) were compared with those of fluconazole by regression statistics and error rate bounding analyses. For all 13,338 isolates, the absolute categorical agreement was 91.6% (false susceptible or very major error [VME], 0.0%). Since voriconazole is 16- to 32-fold more potent than fluconazole, the performance of fluconazole as a surrogate marker for voriconazole susceptibility was improved by designating those isolates with fluconazole MICs of < or =32 microg/ml as being susceptible to voriconazole, resulting in a categorical agreement of 97% with 0.1% VME. Clinical laboratories performing antifungal susceptibility testing of fluconazole against Candida spp. can reliably use these results as surrogate markers until commercial FDA-approved voriconazole susceptibility tests become available.

Global surveillance of in vitro activity of micafungin against Candida: a comparison with caspofungin by CLSI-recommended methods.

Micafungin is an echinocandin antifungal agent that has recently been approved for the prevention of invasive fungal infection and the treatment of esophageal candidiasis. Prospective sentinel surveillance for the emergence of in vitro resistance to micafungin among invasive Candida sp. isolates is indicated. We determined the in vitro activity of micafungin against 2,656 invasive (bloodstream or sterile site) unique patient isolates of Candida spp. collected from 60 medical centers worldwide in 2004 and 2005. We performed antifungal susceptibility testing according to the Clinical and Laboratory Standards Institute (CLSI) M27-A2 method and used a 24-hour prominent inhibition endpoint for determination of the MIC. Caspofungin was tested in parallel against all isolates. Of 2,656 invasive Candida sp. isolates, species distribution was 55.6% Candida albicans, 14.4% Candida parapsilosis, 13.4% Candida glabrata, 10.1% Candida tropicalis, 2.4% Candida krusei, 1.7% Candida guilliermondii, 0.9% Candida lusitaniae, 0.6% Candida kefyr, and 0.9% other Candida species. Overall, micafungin was very active against Candida (MIC50/MIC at which 90% of the isolates tested are inhibited [MIC90], 0.015/1 microg/ml; 96% inhibited at a MIC of < or =1 microg/ml, 100% inhibited at a MIC of < or =2 microg/ml) and comparable to caspofungin (MIC50/MIC90, 0.03/0.25 mug/ml; 99% inhibited at a MIC of < or =2 microg/ml). Results by species, expressed as MIC50/MIC90 (micrograms per milliliter), were as follows: C. albicans, 0.015/0.03; C. glabrata, 0.015/0.015; C. tropicalis, 0.03/0.06; C. krusei, 0.06/0.12; C. kefyr, 0.06/0.06; C. parapsilosis, 1/2; C. guilliermondii, 0.5/1; C. lusitaniae, 0.12/0.25; other Candida spp., 0.25/1. Although the species distribution varied considerably among the different geographic regions, there was no difference in micafungin activity across the regions. Micafungin has excellent in vitro activity against invasive clinical isolates of Candida from centers worldwide.