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ABSTRACTME1111 is a novel small molecule antifungal agent under development for the topical treatment of onychomycosis. Standardization of the susceptibility testing method for this candidate antifungal is needed. Toward this end, 8 independent laboratories determined the interlaboratory reproducibility of ME1111 susceptibility testing. In addition, we subsequently identified 2 strains as quality control (QC) isolates for the method. In the reproducibility study, 5 blinded clinical strains each of Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermophyton floccosumwere tested, while the QC study tested 6 blinded T. rubrumor T. mentagrophytesATCC strains. Testing was performed in frozen microtiter panels according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 methodology. In the reproducibility study, 9 of 15 clinical strains showed interlaboratory agreement of >90% at the 80% inhibition endpoint, with a range of agreement of 76.2% to 100%. In the QC study, 4 of the 6 ATCC strains showed interlaboratory agreement of >90%. ME1111 demonstrated excellent interlaboratory agreement when tested against dermatophytes. Based on this data, the CLSI Subcommittee on Antifungal Susceptibility Tests approved the susceptibility testing of ME1111 against dermatophytes according to M38-A2 methodology, which stipulates RPMI 1640 as the test medium, an inoculum size of 1 to 3 × 103CFU/ml, and an incubation time and temperature of 96 h at 35°C. The MIC endpoint should be 80% inhibition compared with the growth control. T. rubrumATCC MYA-4438 and T. mentagrophytesATCC 28185 were selected as QC isolates, with an acceptable range of 0.12 to 1 µg/ml for the two strains.

ABSTRACTA fully automated antifungal susceptibility test system recently updated to reflect the new species-specific clinical breakpoints (CBPs) of fluconazole for Candida(Vitek 2 AF03 yeast susceptibility test; bioMérieux, Inc., Durham, NC) was compared in three different laboratories with the Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution (BMD) method by testing 2 quality control strains, 10 reproducibility strains (4 Candidaspecies and 6 Cryptococcus neoformansstrains), and 746 isolates of Candidaspecies (702 isolates, 13 species) and 44 isolates of C. neoformansagainst fluconazole. Excellent essential agreement (EA) (within 2 dilutions) between the reference and Vitek 2 MICs was observed for fluconazole and Candidaspecies (94.0%). The EA was lower for fluconazole and C. neoformansat 86.4%. The mean times to a result with the Vitek 2 test were 9.1 h for Candidaspecies and 12.1 h for C. neoformans. Categorical agreement (CA) between the two methods was assessed by using the new species-specific CBPs. For less common species without fluconazole CBPs, the epidemiological cutoff values (ECVs) were used to differentiate wild-type (WT; MIC, =ECV) from non-WT (MIC, >ECV) strains. The CAs between the two methods were 92.0% for Candidaspecies (0.3% very major errors [VME] and 2.6% major errors [ME]) and 84.1% for C. neoformans(4.5% VME and 11.4% ME). The updated Vitek 2 AF03 IUO yeast susceptibility system is comparable to the CLSI BMD reference method for testing the susceptibility of clinically important yeasts to fluconazole when using the new (lower) CBPs and ECVs.

ABSTRACTSince epidemiological cutoff values (ECVs) using CLSI MICs from multiple laboratories are not available for Candidaspp. and the echinocandins, we established ECVs for anidulafungin and micafungin on the basis of wild-type (WT) MIC distributions (for organisms in a species-drug combination with no detectable acquired resistance mechanisms) for 8,210 Candida albicans, 3,102 C. glabrata, 3,976 C. parapsilosis, 2,042 C. tropicalis, 617 C. krusei, 258 C. lusitaniae, 234 C. guilliermondii, and 131 C. dubliniensisisolates. CLSI broth microdilution MIC data gathered from 15 different laboratories in Canada, Europe, Mexico, Peru, and the United States were aggregated to statistically define ECVs. ECVs encompassing 97.5% of the statistically modeled population for anidulafungin and micafungin were, respectively, 0.12 and 0.03 μg/ml for C. albicans, 0.12 and 0.03 μg/ml for C. glabrata, 8 and 4 μg/ml for C. parapsilosis, 0.12 and 0.06 μg/ml for C. tropicalis, 0.25 and 0.25 μg/ml for C. krusei, 1 and 0.5 μg/ml for C. lusitaniae, 8 and 2 μg/ml for C. guilliermondii, and 0.12 and 0.12 μg/ml for C. dubliniensis. Previously reported single and multicenter ECVs defined in the present study were quite similar or within 1 2-fold dilution of each other. For a collection of 230 WT isolates (no fksmutations) and 51 isolates with fksmutations, the species-specific ECVs for anidulafungin and micafungin correctly classified 47 (92.2%) and 51 (100%) of the fksmutants, respectively, as non-WT strains. These ECVs may aid in detecting non-WT isolates with reduced susceptibility to anidulafungin and micafungin due to fksmutations.

ABSTRACTAntifungal susceptibility testing of Candidahas been standardized and refined and now may play a useful role in managing Candidainfections. Important new developments include validation of 24-h reading times for all antifungal agents and the establishment of species-specific epidemiological cutoff values (ECVs) for the systemically active antifungal agents and both common and uncommon species of Candida. The clinical breakpoints (CBPs) for fluconazole, voriconazole, and the echinocandins have been revised to provide species-specific interpretive criteria for the six most common species. The revised CBPs not only are predictive of clinical outcome but also provide a more sensitive means of identifying those strains with acquired or mutational resistance mechanisms. This brief review serves as an update on the new developments in the antifungal susceptibility testing of Candidaspp. using Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) methods.

ABSTRACTClinical breakpoints (CBPs) and epidemiological cutoff values (ECVs) have been established for several Candidaspp. and the newer triazoles and echinocandins but are not yet available for older antifungal agents, such as amphotericin B, flucytosine, or itraconazole. We determined species-specific ECVs for amphotericin B (AMB), flucytosine (FC) and itraconazole (ITR) for eight Candidaspp. (30,221 strains) using isolates from 16 different laboratories in Brazil, Canada, Europe, and the United States, all tested by the CLSI reference microdilution method. The calculated 24- and 48-h ECVs expressed in µg/ml (and the percentages of isolates that had MICs less than or equal to the ECV) for AMB, FC, and ITR, respectively, were 2 (99.8)/2 (99.2), 0.5 (94.2)/1 (91.4), and 0.12 (95.0)/0.12 (92.9) for C. albicans; 2 (99.6)/2 (98.7), 0.5 (98.0)/0.5 (97.5), and 2 (95.2)/4 (93.5) for C. glabrata; 2 (99.7)/2 (97.3), 0.5 (98.7)/0.5 (97.8), and 05. (99.7)/0.5 (98.5) for C. parapsilosis; 2 (99.8)/2 (99.2), 0.5 (93.0)/1 (90.5), and 0.5 (97.8)/0.5 (93.9) for C. tropicalis; 2 (99.3)/4 (100.0), 32 (99.4)/32 (99.3), and 1 (99.0)/2 (100.0) for C. krusei; 2 (100.0)/4 (100.0), 0.5 (95.3)/1 (92.9), and 0.5 (95.8)/0.5 (98.1) for C. lusitaniae; -/2 (100.0), 0.5 (98.8)/0.5 (97.7), and 0.25 (97.6)/0.25 (96.9) for C. dubliniensis; and 2 (100.0)/2 (100.0), 1 (92.7)/-, and 1 (100.0)/2 (100.0) for C. guilliermondii. In the absence of species-specific CBP values, these wild-type (WT) MIC distributions and ECVs will be useful for monitoring the emergence of reduced susceptibility to these well-established antifungal agents.

ABSTRACTWe performed 24- and 48-h MIC determinations of posaconazole and voriconazole against more than 16,000 clinical isolates of Candidaspecies. 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. parapsilosisand voriconazole) to 99.8% (C. kruseiand voriconazole), with 0.0 to 2.9% very major discrepancies (VMD). The essential agreement (within 2 log2dilutions) 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 Candidaspp. to these important antifungal agents. The applications of ECVs to this large collection of Candidaisolates suggests the potential to develop 24-h species-specific clinical breakpoints for both posaconazole and voriconazole.

ABSTRACTThe 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 Candidawas 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 Candidawhen 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.

ABSTRACTWe tested 16,191 strains of Candidaagainst 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. kefyrwere 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 Candidaspp. 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 Candidaspecies encountered clinically. The CBPs for voriconazole should be reassessed, with consideration for development of species-specific CBPs.

ABSTRACTIn the absence of clinical breakpoints, epidemiological cutoff values (ECVs) have been established to distinguish wild-type (WT) isolates of Aspergillusspp. from those that may harbor resistance mutations. Recently, the CLSI has developed ECVs for triazoles (itraconazole, posaconazole, and voriconazole) and common Aspergillusspecies. We applied the triazole ECVs to 1,789 Aspergillusisolates collected from 63 centers worldwide from 2001 to 2009 to determine the frequency of non-WT strains of each species. Temporal trends were evaluated for Aspergillus fumigatusand Aspergillus flavusover the 9-year period for each drug. The collection included 1,312 isolates of A. fumigatus, 235 of A. flavus, 162 of Aspergillus niger, 64 of Aspergillus terreus, and 15 of Aspergillus versicolor. Using the ECVs, the percentages of non-WT isolates for itraconazole, posaconazole, and voriconazole, respectively, were as follows: A. fumigatus(2.0%, 3.5%, and 1.4%), A. flavus(0.8%, 5.1%, and 1.7%), A. niger(17.3%, 3.7%, and 0.6%), A. terreus(0.0%, 1.6%, and 3.2%), and A. versicolor(6.3%, 0.0%, and 0.0%). Among 49 Aspergillusisolates for which itraconazole MICs were >2 µg/ml, the posaconazole and voriconazole MICs were greater than the ECVs for 14 and 12 isolates, respectively. The percentages of isolates for which MICs were greater than the ECVs ranged from 1.1 to 5.7% for posaconazole, 0.0 to 1.6% for voriconazole, and 0.7 to 4.0% for itraconazole. There was no consistent trend toward decreased susceptibility for any triazole and A. fumigatusor A. flavusover time. Decreased susceptibility among Aspergillusspp. was observed for each of the extended-spectrum triazoles and varied by species over the 9-year study period.

ABSTRACTThe CLSI clinical breakpoint (CBP) for echinocandin susceptibility (S; MICs of =2 µg/ml) may classify isolates with acquired resistance (R) mutations as susceptible. Epidemiological cutoff values (ECVs) have been established to distinguish wild-type (WT) Candidastrains from those that may exhibit R mutations. The CLSI-developed ECVs for anidulafungin, caspofungin, and micafungin were applied to 15,269 isolates of Candidaspp. collected from over 100 centers worldwide between 2001 and 2009 to determine the frequency of non-WT strains of each species. The collection included 8,378 isolates of Candida albicans, 2,352 isolates of C. glabrata, 2,195 isolates of C. parapsilosis, 1,841 isolates of C. tropicalis, and 503 isolates of C. krusei. The mean percentages of non-WT isolates per year for anidulafungin, caspofungin, and micafungin, respectively, were as follows: for C. albicans, 0.3, 0.1, and 2.1; for C. glabrata, 0.8, 1.3, and 1.6; for C. parapsilosis, 0.0, 1.5, and 0.5; for C. tropicalis, 0.9, 0.7, and 0.9; and for C. krusei, 0.5, 6.4, and 3.5. We noted increases in the percentage of non-WT isolates, from 0.5% (2001) to 3.1% (2009) for caspofungin and C. parapsilosis, from 0.4% (2004) to 1.8% (2009) for anidulafungin and C. glabrata, from 2.4% (2004) to 5.7% (2009) for micafungin and C. krusei, and from 0.0% (2004) to 3.1% (2009) for micafungin and C. parapsilosis. No trends were noted for any species and drug when we used the CBP. Echinocandin CBPs are insensitive for detecting emerging R. Although uncommon, decreased S among Candidaisolates was observed for each of the echinocandins and varied by species. Using ECVs is important in determining R trends among echinocandins and Candida.

ABSTRACTClinical breakpoints have not been established for mold testing. Wild-type (WT) MIC distributions (organisms in a species/drug combination with no detectable acquired resistance mechanisms) were defined in order to establish epidemiologic cutoff values (ECVs) for five Aspergillusspp. and itraconazole, posaconazole, and voriconazole. Also, we have expanded prior ECV data for Aspergillus fumigatus.The number of available isolates varied according to the species/triazole combination as follows: 1,684 to 2,815 for A. fumigatus, 323 to 592 for A. flavus, 131 to 143 for A. nidulans, 366 to 520 for A. niger, 330 to 462 for A. terreus, and 45 to 84 for A. versicolor. CLSI broth microdilution MIC data gathered in five independent laboratories in Europe and the United States were aggregated for the analyses. ECVs expressed in µg/ml were as follows (percentages of isolates for which MICs were equal to or less than the ECV are in parentheses): A. fumigatus, itraconazole, 1 (98.8%); posaconazole, 0.5 (99.2%); voriconazole, 1 (97.7%); A. flavus, itraconazole, 1 (99.6%); posaconazole, 0.25 (95%); voriconazole, 1 (98.1%); A. nidulans, itraconazole, 1 (95%); posaconazole, 1 (97.7%); voriconazole, 2 (99.3%); A. niger, itraconazole, 2 (100%); posaconazole, 0.5 (96.9%); voriconazole, 2 (99.4%); A. terreus, itraconazole, 1 (100%); posaconazole, 0.5 (99.7%); voriconazole, 1 (99.1%); A. versicolor, itraconazole, 2 (100%); posaconazole, 1 (not applicable); voriconazole, 2 (97.5%). Although ECVs do not predict therapy outcome as clinical breakpoints do, they may aid in detection of azole resistance (non-WT MIC) due to cyp51Amutations, a resistance mechanism in some Aspergillusspp. These ECVs should be considered for inclusion in the future CLSI M38-A2 document revision.

Previous pharmacodynamic studies using in vivo candidiasis models have demonstrated that the 24-h area under the concentration-time curve (AUC)/MIC is a good descriptor of the echinocandin exposure-response relationship. Further studies investigating the 24-h AUC/MIC target for a stasis endpoint identified free-drug 24-h AUC/MIC against Candida albicans and were similar for two echinocandins, anidulafungin and micafungin. The current studies expand investigation of a third echinocandin (caspofungin) and compare the pharmacodynamic target among C. albicans, Candida glabrata, and Candida parapsilosis. Treatment studies were conducted with six C. albicans, nine C. glabrata, and 15 C. parapsilosis strains with various MICs (anidulafungin, 0.015 to 4.0 µg/ml; caspofungin, 0.03 to 4.0 µg/ml; and micafungin, 0.008 to 1.0 µg/ml). Efficacy was closely tied to MIC and the 24-h AUC/MIC. Therapy against C. parapsilosis required more of each echinocandin on a mg/kg basis. Caspofungin required less drug on a mg/kg basis for efficacy against all of the organisms than did the other two drugs. However, the 24-h AUC/MIC targets were similar among the echinocandins when free drug concentrations were considered, suggesting the relevance of protein binding. The targets for C. parapsilosis (mean, 7) and C. glabrata (mean, 7) were significantly lower than those for C. albicans (mean, 20) for each echinocandin. The results suggest that current susceptibility breakpoints and the consideration of organism species in these determinations should be reexplored.

ABSTRACTPrevious pharmacodynamic studies using in vivocandidiasis models have demonstrated that the 24-h area under the concentration-time curve (AUC)/MIC is a good descriptor of the echinocandin exposure-response relationship. Further studies investigating the 24-h AUC/MIC target for a stasis endpoint identified free-drug 24-h AUC/MIC against Candida albicansand were similar for two echinocandins, anidulafungin and micafungin. The current studies expand investigation of a third echinocandin (caspofungin) and compare the pharmacodynamic target among C. albicans, Candida glabrata, and Candida parapsilosis. Treatment studies were conducted with six C. albicans, nine C. glabrata, and 15 C. parapsilosisstrains with various MICs (anidulafungin, 0.015 to 4.0 μg/ml; caspofungin, 0.03 to 4.0 μg/ml; and micafungin, 0.008 to 1.0 μg/ml). Efficacy was closely tied to MIC and the 24-h AUC/MIC. Therapy against C. parapsilosisrequired more of each echinocandin on a mg/kg basis. Caspofungin required less drug on a mg/kg basis for efficacy against all of the organisms than did the other two drugs. However, the 24-h AUC/MIC targets were similar among the echinocandins when free drug concentrations were considered, suggesting the relevance of protein binding. The targets for C. parapsilosis(mean, 7) and C. glabrata(mean, 7) were significantly lower than those for C. albicans(mean, 20) for each echinocandin. The results suggest that current susceptibility breakpoints and the consideration of organism species in these determinations should be reexplored.

ABSTRACTThe 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 Candidaspecies. The isolates were characterized for the presence or absence of fks1and/or fks2gene 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. glabrataand caspofungin (85.3%) and C. kruseiand caspofungin (54.5%). The CA between the Etest and CLSI results was also excellent for all comparisons, with the exception of C. kruseiand caspofungin (81.8%). All three methods were able to differentiate wild-type (WT) strains from those with fksmutations. 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 fksmutant strains of Candidaspecies from WT strains using each of the echinocandins.

ABSTRACTFluconazole in vitrosusceptibility test results for 256,882 isolates of Candidaspp. were collected from 142 sites in 41 countries from June 1997 to December 2007. Data were collected for 197,619 isolates tested with voriconazole from 2001 to 2007. A total of 31 different species of Candidawere isolated. Increased rates of isolation of the common non-albicansspecies C. glabrata(10.2% to 11.7%), C. tropicalis(5.4% to 8.0%), and C. parapsilosis(4.8% to 5.6%) were noted when the time periods 1997 to 2000 and 2005 to 2007 were compared. Investigators tested clinical isolates of Candidaspp. by the CLSI M44-A disk diffusion method. Overall, 90.2% of Candidaisolates tested were susceptible (S) to fluconazole; however, 13 of 31 species identified exhibited decreased susceptibility (<75% S), similar to that seen with the resistant (R) species C. glabrataand C. krusei. Among 197,619 isolates of Candidaspp. tested against voriconazole, 95.0% were S and 3% were R. About 30% of fluconazole-R isolates of C. albicans, C. glabrata, C. tropicalis, C. rugosa, C. lipolytica, C. pelliculosa, C. apicola, C. haemulonii, C. humicola, C. lambica, and C. ciferriiremained S to voriconazole. An increase in fluconazole resistance over time was seen with C. parapsilosis, C. guilliermondii, C. lusitaniae, C. sake, and C. pelliculosa. Among the emerging fluconazole-R species were C. guilliermondii(11.4% R), C. inconspicua(53.2% R), C. rugosa(41.8% R), and C. norvegensis(40.7% R). The rates of isolation of C. rugosa, C. inconspicua, and C. norvegensisincreased by 5- to 10-fold over the 10.5-year study period. C. guilliermondiiand C. rugosawere most prominent in Latin America, whereas C. inconspicuaand C. norvegensiswere most common in Eastern European countries. This survey identifies several less-common species of Candidawith decreased susceptibility to azoles. These organisms may pose a future threat to optimal antifungal therapy and underscore the importance of prompt and accurate species identification and antifungal susceptibility testing.

ABSTRACTWe tested a global collection of Candidasp. 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 Candidaspp. (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. kefyrisolates) were obtained from over 100 centers worldwide. The modal MICs (in µg/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 µg/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 Candidaspp. and for determining the importance of various FKS1or other mutations.

ABSTRACTWe 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, =0.5 but <4 for indifference, and =4 for antagonism. Ninety-one isolates were tested, as follows: 30 Candidaisolates, 10 Aspergillusisolates, 15 isolates of the Zygomycetes order, 10 Cryptococcus neoformansisolates, 8 Rhodotorulaisolates, 8 Fusariumisolates, 5 Trichosporonisolates, and 5 Scedosporiumisolates. MGCD290 showed modest activity when it was used alone (MICs, 1 to 8 µg/ml) and was mostly active against azole-resistant yeasts, but the MICs against molds were high (16 to >32 µg/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%) Candidaisolates. All 23 of the 91 Candidaisolates that were not fluconazole susceptible demonstrated a reduced fluconazole MIC that crossed an interpretive breakpoint (e.g., resistant [MIC, =64 µg/ml] to susceptible [MIC, =8 µg/ml]) when fluconazole was combined with MGCD290 at 0.12 to 4 µg/ml. The activity of fluconazole plus MGCD290 was also synergistic against 6/10 Aspergillusisolates. Posaconazole plus MGCD290 demonstrated synergy against 14/15 Zygomycetes (9 Rhizopusisolates and 5 Mucorisolates). Voriconazole plus MGCD290 demonstrated synergy against six of eight Fusariumisolates. Thus, MGCD290 demonstrated in vitro synergy with azoles against the majority of clinical isolates tested, including many azole-resistant isolates and genera inherently resistant to azoles (e.g., Mucorand Fusarium). Further evaluation of fungal HDAC inhibitor-azole combinations is indicated.

ABSTRACTFive Candidaspecies (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei) account for over 95% of invasive candidiasis cases. Some less common Candidaspecies 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 Candidacollected 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 =1 µg/ml was used for amphotericin and posaconazole. Of 14,007 Candidaisolates tested, 658 (4.7%) were among the less common species. Against all 658 isolates combined, the activity of each agent, expressed as the MIC50/MIC90ratio (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. parapsilosiscomplex (C. orthopsilosisand C. metapsilosis) were susceptible to voriconazole, posaconazole, anidulafungin, caspofungin, and micafungin. Over 95% of clinical isolates of the rare Candidaspecies were susceptible to the available antifungals. However, activity did vary by drug-species combination, with some species (e.g., C. rugosaand C. guilliermondii) demonstrating reduced susceptibilities to commonly used agents such as fluconazole and echinocandins.

ABSTRACTAntifungal susceptibility testing of Aspergillusspecies has been standardized by both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Recent studies suggest the emergence of strains of Aspergillus fumigatuswith acquired resistance to azoles. The mechanisms of resistance involve mutations in the cyp51A(sterol demethylase) gene, and patterns of azole cross-resistance have been linked to specific mutations. Studies using the EUCAST broth microdilution (BMD) method have defined wild-type (WT) MIC distributions, epidemiological cutoff values (ECVs), and cross-resistance among the azoles. We tested a collection of 637 clinical isolates of A. fumigatusfor which itraconazole MICs were =2 µg/ml against posaconazole and voriconazole using the CLSI BMD method. An ECV of =1 µg/ml encompassed the WT population of A. fumigatusfor itraconazole and voriconazole, whereas an ECV of =0.25 µg/ml was established for posaconazole. Our results demonstrate that the WT distribution and ECVs for A. fumigatusand the mold-active triazoles were the same when determined by the CLSI or the EUCAST BMD method. A collection of 43 isolates for which itraconazole MICs fell outside of the ECV were used to assess cross-resistance. Cross-resistance between itraconazole and posaconazole was seen for 53.5% of the isolates, whereas cross-resistance between itraconazole and voriconazole was apparent in only 7% of the isolates. The establishment of the WT MIC distribution and ECVs for the azoles and A. fumigatuswill be useful in resistance surveillance and is an important step toward the development of clinical breakpoints.

ABSTRACTWe examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrataand grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabratato 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. glabrataBSI 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. glabrataincreased with patient age, the rate of fluconazole resistance declined. The oldest age group (=80 years) had the highest proportion of BSI isolates that were C. glabrata(32%) and the lowest rate of fluconazole resistance (5%).

ABSTRACTFluconazole in vitro susceptibility test results determined by the CLSI M44-A disk diffusion method for 11,240 isolates of noncandidal yeasts were collected from 134 study sites in 40 countries from June 1997 through December 2007. Data were collected for 8,717 yeast isolates tested with voriconazole from 2001 through 2007. A total of 22 different species/organism groups were isolated, of which Cryptococcus neoformanswas the most common (31.2% of all isolates). Overall, Cryptococcus(32.9%), Saccharomyces(11.7%), Trichosporon(10.6%), and Rhodotorula(4.1%) were the most commonly identified genera. The overall percentages of isolates in each category (susceptible, susceptible dose dependent, and resistant) were 78.0%, 9.5%, and 12.5% and 92.7%, 2.3%, and 5.0% for fluconazole and voriconazole, respectively. Less than 30% of fluconazole-resistant isolates of Cryptococcusspp., Cryptococcus albidus, Cryptococcus laurentii, Trichosporon beigelii/Trichosporon cutaneum, Rhodotorulaspp., Rhodotorula rubra/Rhodotorula mucilaginosa, and Rhodotorula glutinisremained susceptible to voriconazole. Emerging resistance to fluconazole was documented among isolates of C. neoformansfrom the Asia-Pacific, Africa/Middle East, and Latin American regions but not among isolates from Europe or North America. This survey documents the continuing broad spectrum of activity of voriconazole against opportunistic yeast pathogens but identifies several of the less common species with decreased azole susceptibility. These organisms may pose a future threat to optimal antifungal therapy and emphasize the importance of prompt and accurate species identification.

ABSTRACTUsing blaZPCR as the “gold standard,” the sensitivities of CLSI penicillin zone edge and nitrocefin-based tests for ß-lactamase production in Staphylococcus aureuswere 64.5% and 35.5%, respectively, with specificity of 99.8% for both methods. In 2013, 13.5% of 3,083 S. aureusisolates from 31 U.S. centers were penicillin susceptible.

ABSTRACTWe performed 24- and 48-h MIC determinations and disk diffusion testing of fluconazole against more than 11,000 clinical isolates of Candidaspecies. 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 Candidaspecies) to 94.9% (all Candidaspecies minus Candida krusei), with only 0.1% very major errors (VME). The essential agreement (within 2 log2dilutions) 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 Candidaspp. to this widely used antifungal agent.

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 log2dilutions) 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.

ABSTRACTPrevious studies using in vivo candidiasis models have demonstrated that the concentration-associated pharmacodynamic indices, the maximum concentration of a drug in serum/MIC and 24-h area under the curve (AUC)/MIC, are associated with echinocandin treatment efficacy. The current investigations used a neutropenic murine model of disseminated Candida albicansand C. glabratainfection to identify the 24-h AUC/MIC index target associated with a stasis and killing endpoint for the echinocandin, micafungin. The kinetics after intraperitoneal micafungin dosing were determined in neutropenic infected mice. Peak levels and AUC values were linear over the 16-fold dose range studied. The serum drug elimination half-life ranged from 7.5 to 16 h. Treatment studies were conducted with 4 C. albicansand 10 C. glabrataisolates with micafungin MICs varying from 0.008 to 0.25 μg/ml to determine whether similar 24-h AUC/MIC ratios were associated with efficacy. The free drug AUC/MICs associated with stasis and killing (1-log) endpoints were near 10 and 20, respectively. The micafungin exposures associated with efficacy were similar for the two Candidaspecies. Furthermore, the free drug micafungin exposures required to produce stasis and killing endpoints were similar to those recently reported for another echinocandin, anidulafungin, against the identical Candidaisolates in this model.

ABSTRACTThe CLSI Antifungal Subcommittee followed the M23-A2 “blueprint” to develop interpretive MIC breakpoints for anidulafungin, caspofungin, and micafungin against Candidaspecies. MICs of =2 µg/ml for all three echinocandins encompass 98.8 to 100% of all clinical isolates of Candidaspp. without bisecting any species group and represent a concentration that is easily maintained throughout the dosing period. Data from phase III clinical trials demonstrate that the standard dosing regimens for each of these agents may be used to treat infections due to Candidaspp. for which MICs are as high as 2 µg/ml. An MIC predictive of resistance to these agents cannot be defined based on the data from clinical trials due to the paucity of isolates for which MICs exceed 2 µg/ml. The clinical data set included only three isolates from patients treated with an echinocandin (caspofungin) for which the MICs were >2 µg/ml (two C. parapsilosisisolates at 4 µg/ml and one C. rugosaisolate at 8 µg/ml). Based on these data, the CLSI subcommittee has decided to recommend a “susceptible only” breakpoint MIC of =2 µg/ml due to the lack of echinocandin resistance in the population of Candidaisolates thus far. Isolates for which MICs exceed 2 µg/ml should be designated “nonsusceptible” (NS). For strains yielding results suggestive of an NS category, the organism identification and antimicrobial-susceptibility test results should be confirmed. Subsequently, the isolates should be submitted to a reference laboratory that will confirm the results by using a CLSI reference dilution method.

ABSTRACTFew data exist to describe in vitro patterns of cross-resistance among large collections of clinical Aspergillusisolates, including those of species other than Aspergillus fumigatus. We examined 771 Aspergillusspp. 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. versicolorisolates and 24 isolates of other Aspergillusspecies). 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 Aspergillusisolates (from 88% of isolates for ITR to 98% of isolates for VOR and POS), MICs of each triazole were =1 µg/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. fumigatusand A. flavus) were strongest between VOR and RAV MICs and ITR and POS MICs. However, Aspergillusspecies for which MICs of VOR or POS were >2 µg/ml remain extremely rare (<1% of isolates).

ABSTRACTA 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 Candidaspp. 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 Candidaspp. to the echinocandins anidulafungin, caspofungin, and micafungin.

ABSTRACTWe examined data from the ARTEMIS DISK Antifungal Surveillance Program to describe geographic and temporal trends in the isolation of Candida parapsilosisfrom clinical specimens and the in vitro susceptibilities of 9,371 isolates to fluconazole and voriconazole. We also report the in vitro susceptibility of bloodstream infection (BSI) isolates of C. parapsilosisto the echinocandins, anidulafungin, caspofungin, and micafungin. C. parapsilosisrepresented 6.6% of the 141,383 isolates of Candidacollected from 2001 to 2005 and was most common among isolates from North America (14.3%) and Latin America (9.9%). High levels of susceptibility to both fluconazole (90.8 to 95.8%) and voriconazole (95.3 to 98.1%) were observed in all geographic regions with the exception of the Africa and Middle East region (79.3 and 85.8% susceptible to fluconazole and voriconazole, respectively). C. parapsilosiswas most often isolated from blood and skin and/or soft tissue specimens and from patients hospitalized in the medical, surgical, intensive care unit (ICU) and dermatology services. Notably, isolates from the surgical ICU were the least susceptible to fluconazole (86.3%). There was no evidence of increasing azole resistance over time among C. parapsilosisisolates tested from 2001 to 2005. Of BSI isolates tested against the three echinocandins, 92, 99, and 100% were inhibited by concentrations of =2 µg/ml of anidulafungin (621 isolates tested), caspofungin (1,447 isolates tested), and micafungin (539 isolates tested), respectively. C. parapsilosisis a ubiquitous pathogen that remains susceptible to the azoles and echinocandins; however, both the frequency of isolation and the resistance of C. parapsilosisto fluconazole and voriconazole may vary by geographic region and clinical service.

ABSTRACTThere 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 Candidaspp. 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, =1 µg/ml; susceptible dose dependent, 2 µg/ml; resistant, =4 µg/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 Candidaspp. to posaconazole.

ABSTRACTCandida kruseiis well known as a fungal pathogen for patients with hematologic malignancies and for transplant recipients. Using the ARTEMIS Antifungal Surveillance Program database, we describe geographic and temporal trends in the isolation of C. kruseifrom clinical specimens and the in vitro susceptibilities of 3,448 isolates to voriconazole as determined by CLSI (formerly NCCLS) disk diffusion testing. In addition, we report the in vitro susceptibilities of bloodstream infection isolates of C. kruseito amphotericin B (304 isolates), flucytosine (254 isolates), anidulafungin (121 isolates), caspofungin (300 isolates), and micafungin (102 isolates) as determined by CLSI broth microdilution methods. Geographic differences in isolation were apparent; the highest frequency of isolation was seen for the Czech Republic (7.6%) and the lowest for Indonesia, South Korea, and Thailand (0 to 0.3%). Overall, 83% of isolates were susceptible to voriconazole, ranging from 74.8% in Latin America to 92.3% in North America. C. kruseiwas most commonly isolated from hematology-oncology services, where only 76.7% of isolates were susceptible to voriconazole. There was no evidence of increasing resistance of C. kruseito voriconazole from 2001 to 2005. Decreased susceptibilities to amphotericin B (MIC at which 90% of isolates were inhibited [MIC90], 4 µg/ml) and flucytosine (MIC90, 16 µg/ml) were noted, whereas 100% of isolates were inhibited by =2 µg/ml of anidulafungin (MIC90, 0.06 µg/ml), micafungin (MIC90, 0.12 µg/ml) or caspofungin (MIC90, 0.25 µg/ml). C. kruseiis an uncommon but multidrug-resistant fungal pathogen. Among the systemically active antifungal agents, the echinocandins appear to be the most active against this important pathogen.

ABSTRACTMultiple in vivo studies have characterized the pharmacodynamics of drugs from the triazole and polyene antifungal drug classes. Fewer studies have investigated these pharmacodynamic relationships for the echinocandin drug class. We used a neutropenic murine model of disseminated Candida albicans, Candida tropicalis, and Candida glabratainfection to characterize the time course of activity of the new echinocandin anidulafungin. The pharmacokinetic-pharmacodynamic (PK-PD) indices (the percentage of time that the drug concentration was above the MIC, the ratio of the area under the concentration-time curve from 0 to 24 h [AUC0-24] to the MIC, and the ratio of the maximum serum drug concentration [Cmax] to the MIC) were correlated with in vivo efficacy, as measured by organism numbers in kidney cultures after 96 h of therapy. The kinetics following intraperitoneal anidulafungin dosing in neutropenic infected mice were monitored. Peak levels and AUCs were linear over the 16-fold dose range studied. The drug elimination half-life in serum ranged from 14 to 24 h. Single-dose postantifungal-effect studies demonstrated prolonged suppression of organism regrowth after serum anidulafungin levels had fallen below the MIC. Of the four dosing intervals studied, treatment with the more widely spaced dosing regimens was most efficacious, suggesting the Cmax/MIC ratio as the PK-PD index most predictive of efficacy. Nonlinear regression analysis suggested that both the Cmax/MIC and AUC/MIC ratios were strongly predictive of treatment success. Studies were then conducted with 13 additional C. albicans, C. tropicalis, and C. glabrataisolates with various anidulafungin susceptibilities (MICs of anidulafungin for these strains, 0.015 to 2.0 μg/ml) to determine if similar Cmax/MIC and AUC0-24/MIC ratios for these isolates were associated with efficacy. The anidulafungin exposures associated with efficacy were similar among Candidaspecies.

ABSTRACTThe 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 Candidasp. isolates is indicated. We determined the in vitro activities of anidulafungin, caspofungin, and micafungin against 5,346 invasive (bloodstream or sterile-site) isolates of Candidaspp. 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 Candidasp. 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 Candidaspp. Overall, all three echinocandins were very active against Candida: anidulafungin (MIC50, 0.06 µg/ml; MIC90, 2 µg/ml), caspofungin (MIC50, 0.03 µg/ml; MIC90, 0.25 µg/ml), micafungin (MIC50, 0.015 µg/ml; MIC90, 1 µg/ml). More than 99% of isolates were inhibited by =2 µg/ml of all three agents. Results by species (expressed as the percentages of isolates inhibited by =2 µg/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 Candidaisolated from centers worldwide. Our prospective sentinel surveillance reveals no evidence of emerging echinocandin resistance among invasive clinical isolates of Candidaspp.

ABSTRACTA fully automated commercial antifungal susceptibility test system (VITEK 2; bioMe´rieux, Inc., Hazelwood, MO) was compared in three different laboratories with the Clinical and Laboratory Standards Institute (formerly the NCCLS) reference broth microdilution method (BMD) by testing 2 quality control strains, 10 reproducibility strains, and 426 isolates of Candidaspp. against amphotericin B, flucytosine, and voriconazole. Reference BMD MIC endpoints were established after 24 and 48 h of incubation. VITEK 2 system MIC endpoints were determined spectrophotometrically after 9.1 to 27.1 h of incubation (mean, 12 to 14 h). Excellent essential agreement (within 2 dilutions) between the VITEK 2 system and the 24- and 48-h BMD MICs was observed for all three antifungal agents: amphotericin B, 99.1% and 97%, respectively; flucytosine, 99.1% and 98.8%, respectively; and voriconazole, 96.7% and 96%, respectively. Both intra- and interlaboratory agreements were >98% for all three drugs. The overall categorical agreements between the VITEK 2 system and BMD for flucytosine and voriconazole were 98.1 to 98.6% at the 24-h BMD time point and 96.9 to 97.4% at the 48-h BMD time point. The VITEK 2 system reliably detected flucytosine and voriconazole resistance among Candidaspp. and demonstrated excellent quantitative and qualitative agreement with the reference BMD method.

ABSTRACTFluconazole in vitro susceptibility test results for 205,329 yeasts were collected from 134 study sites in 40 countries from June 1997 through December 2005. Data were collected for 147,776 yeast isolates tested with voriconazole from 2001 through 2005. All investigators tested clinical yeast isolates by the CLSI M44-A disk diffusion method. Test plates were automatically read and results recorded with a BIOMIC image analysis system. Species, drug, zone diameter, susceptibility category, and quality control results were collected quarterly. Duplicate (same patient, same species, and same susceptible-resistant biotype profile during any 7-day period) and uncontrolled test results were not analyzed. Overall, 90.1% of all Candidaisolates tested were susceptible (S) to fluconazole; however, 10 of the 22 species identified exhibited decreased susceptibility (<75% S) on the order of that seen with the resistant (R) species C. glabrataand C. krusei. Among 137,487 isolates of Candidaspp. tested against voriconazole, 94.8% were S and 3.1% were R. Less than 30% of fluconazole-resistant isolates of C. albicans, C. glabrata, C. tropicalis, and C. rugosaremained S to voriconazole. The non-Candidayeasts (8,821 isolates) were generally less susceptible to fluconazole than Candidaspp. but, aside from Rhodotorulaspp., remained susceptible to voriconazole. This survey demonstrates the broad spectrum of these azoles against the most common opportunistic yeast pathogens but identifies several less common yeast species with decreased susceptibility to antifungal agents. These organisms may pose a future threat to optimal antifungal therapy and emphasize the importance of prompt and accurate species identification.

ABSTRACTA fully automated commercial antifungal susceptibility test system (VITEK 2 yeast susceptibility test; bioMerieux, Inc., Hazelwood, Mo.) was compared in three different laboratories with Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution (BMD) method by testing two quality control strains and a total of 426 isolates of Candidaspp. (103 to 135 clinical isolates in each laboratory plus 80 challenge isolates in one laboratory) against fluconazole. Reference BMD MIC endpoints were established after 24 and 48 h of incubation. VITEK 2 endpoints were determined spectrophotometrically after 10 to 26 h of incubation (mean, 13 h). Excellent essential agreement (within two dilutions) between the VITEK 2 and the 24- and 48-h BMD MICs was observed. The overall agreement values were 97.9 and 93.7%, respectively. Both intra- and interlaboratory agreement was 100%. The overall categorical agreement between VITEK 2 and BMD was 97.2% at the 24-h BMD time point and 88.3% at the 48-h BMD time point. Decreased categorical agreement at 48 h was attributed to trailing growth observed with Candida glabrata. The VITEK 2 system reliably detected fluconazole resistance among Candidaspp. and demonstrated excellent quantitative and qualitative agreement with the reference BMD method.

ABSTRACTClinical 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 Candidaspp., Aspergillusspp., and other opportunistic fungal pathogens. The present study addresses the use of fluconazole as a surrogate marker to predict the susceptibility of Candidaspp. to voriconazole. Reference broth microdilution MIC results for 13,338 strains of Candidaspp. isolated from more than 200 medical centers worldwide were used. Voriconazole MICs and interpretive categories (susceptible, =1 µg/ml; susceptible dose dependent, 2 µg/ml; resistant, =4 µg/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 =32 µg/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 Candidaspp. can reliably use these results as surrogate markers until commercial FDA-approved voriconazole susceptibility tests become available.

SUMMARYInvasive candidiasis (IC) is a leading cause of mycosis-associated mortality in the United States. We examined data from the National Center for Health Statistics and reviewed recent literature in order to update the epidemiology of IC. IC-associated mortality has remained stable, at approximately 0.4 deaths per 100,000 population, since 1997, while mortality associated with invasive aspergillosis has continued to decline. Candida albicansremains the predominant cause of IC, accounting for over half of all cases, but Candida glabratahas emerged as the second most common cause of IC in the United States, and several less common Candidaspecies may be emerging, some of which can exhibit resistance to triazoles and/or amphotericin B. Crude and attributable rates of mortality due to IC remain unacceptably high and unchanged for the past 2 decades. Nonpharmacologic preventive strategies should be emphasized, including hand hygiene; appropriate use, placement, and care of central venous catheters; and prudent use of antimicrobial therapy. Given that delays in appropriate antifungal therapy are associated with increased mortality, improved use of early empirical, preemptive, and prophylactic therapies should also help reduce IC-associated mortality. Several studies have now identified important variables that can be used to predict risk of IC and to help guide preventive strategies such as antifungal prophylaxis and early empirical therapy. However, improved non-culture-based diagnostics are needed to expand the potential for preemptive (or early directed) therapy. Further research to improve diagnostic, preventive, and therapeutic strategies is necessary to reduce the considerable morbidity and mortality associated with IC.

ABSTRACTAlthough a rare cause of invasive candidiasis, Candida guilliermondiihas been reported to exhibit decreased susceptibility to antifungal agents. Aside from case reports and small surveys, there is little information regarding the epidemiology and antifungal susceptibility profile of C. guilliermondii. We report geographic and temporal trends in the isolation and antifungal susceptibilities of 1,029 C. guilliermondiiclinical isolates collected from 127 medical centers as part of the ARTEMIS DISK Antifungal Surveillance Program. In addition, we report the in vitro susceptibility of 132 bloodstream isolates of C. guilliermondiito caspofungin. C. guilliermondiirepresented 1.4% of the 75,761 isolates collected from 2001 to 2003 and was most common among isolates from Latin America (3.7% versus 0.6 to 1.1%). Decreased susceptibility to fluconazole was noted (75% susceptible; range, 68 to 77% across regions), and voriconazole was more active in vitro against C. guilliermondiithan fluconazole (91% susceptible; range, 88 to 93% across regions). Fluconazole was least active against isolates from dermatology (58%) and surgical (69%) services and against isolates associated with skin and soft tissue infection (68%, compared to 85% susceptible for bloodstream isolates). There was no evidence of increasing azole resistance over time among C. guilliermondiiisolates tested from 2001 to 2003. Of 132 bloodstream isolates of C. guilliermondiitested against caspofungin, most were inhibited by =2 µg/ml (96%; MIC50/MIC90, 0.5/1.0 µg/ml). C. guilliermondii, a species that exhibits reduced susceptibility to fluconazole, is the sixth most frequently isolated Candidaspecies from this large survey and may be an emerging pathogen in Latin America.

ABSTRACTCandida rugosais a fungus that appears to be emerging as a cause of infection in some geographic regions. We utilized the extensive database of the ARTEMIS DISK Antifungal Surveillance Program to describe the geographic and temporal trends in the isolation of C. rugosafrom clinical specimens and the in vitro susceptibilities of 452 isolates to fluconazole and voriconazole. C. rugosaaccounted for 0.4% of 134,715 isolates of Candida, and the frequency of isolation increased from 0.03% to 0.4% over the 6.5-year study period (1997 to 2003). C. rugosawas most common in the Latin American region (2.7% versus 0.1 to 0.4%). Decreased susceptibility to fluconazole (40.5% susceptible) was observed in all geographic regions; however, isolates from Europe and North America were much more susceptible (97 to 100%) to voriconazole than those from other geographic regions (55.8 to 58.8%). C. rugosawas most often isolated from blood and urine in patients hospitalized at the Medical and Surgical inpatient services. Notably, bloodstream isolates were the least susceptible to both fluconazole and voriconazole. C. rugosashould be considered, along with the established pathogens Candida kruseiand Candida glabrata, as a species of Candidawith reduced susceptibility to the azole antifungal agents.

ABSTRACTMicafungin 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 Candidasp. isolates is indicated. We determined the in vitro activity of micafungin against 2,656 invasive (bloodstream or sterile site) unique patient isolates of Candidaspp. 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 Candidasp. 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 Candidaspecies. Overall, micafungin was very active against Candida(MIC50/MIC at which 90% of the isolates tested are inhibited [MIC90], 0.015/1 µg/ml; 96% inhibited at a MIC of =1 µg/ml, 100% inhibited at a MIC of =2 µg/ml) and comparable to caspofungin (MIC50/MIC90, 0.03/0.25 µg/ml; 99% inhibited at a MIC of =2 µg/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 Candidaspp., 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 Candidafrom centers worldwide.

Developing interpretive breakpoints for any given organism-drug combination requires integration of the MIC distribution, pharmacokinetic and pharmacodynamic parameters, and the relationship between in vitro activity and outcome from both in vivo and clinical studies. Previously, the Subcommittee for Antifungal Testing of the Clinical and Laboratory Standards Institute (CLSI [formerly National Committee for Clinical Laboratory Standards]) proposed MIC interpretive breakpoints for fluconazole and Candida spp. These breakpoints were considered to be somewhat weak, because the clinical data supporting them came largely from mucosal infections and there were very few infections involving strains with elevated fluconazole MICs. We readdress the issue of fluconazole breakpoints for Candida by using published clinical and microbiologic data to provide further validation of the breakpoints proposed by the CLSI in 1997. We also address interpretive breakpoints for agar disk diffusion testing of fluconazole. The MIC distribution for fluconazole was determined with a collection of 13,338 clinical isolates. The overall MIC at which 90% of the isolates were inhibited was 8 µg/ml: 91% were susceptible (S) at a MIC of ≤8 µg/ml and 3% were resistant (R) (MIC ≥ 64 µg/ml). Similar results were obtained for 2,190 isolates from randomized clinical trials. Analysis of available data for 1,295 patient-episode-isolate events (692 represented mucosal infections and 603 represented invasive infections) from 12 published clinical studies demonstrated an overall success rate of 77%, including 85% for those episodes in which the fluconazole MIC was ≤8 µg/ml, 67% for those episodes in which the MIC was 16 to 32 µg/ml, and 42% for those episodes with resistant (MIC ≥ 64 µg/ml) isolates. Pharmacodynamic analysis demonstrated a strong relationship between MIC, fluconazole dose, and outcome. A dose/MIC ratio of ∼25 was supportive of the following susceptibility breakpoints for fluconazole and Candida spp.: S, MIC ≤ 8 µg/ml; susceptible-dose dependent (SDD), MIC = 16 to 32 µg/ml; R, MIC ≥ 64 µg/ml. The corresponding disk test breakpoints are as follows: S, ≥19 mm; SDD, 15 to 18 mm; R, ≤14 mm.

SUMMARYDeveloping interpretive breakpoints for any given organism-drug combination requires integration of the MIC distribution, pharmacokinetic and pharmacodynamic parameters, and the relationship between in vitro activity and outcome from both in vivo and clinical studies. Previously, the Subcommittee for Antifungal Testing of the Clinical and Laboratory Standards Institute (CLSI [formerly National Committee for Clinical Laboratory Standards]) proposed MIC interpretive breakpoints for fluconazole and Candidaspp. These breakpoints were considered to be somewhat weak, because the clinical data supporting them came largely from mucosal infections and there were very few infections involving strains with elevated fluconazole MICs. We readdress the issue of fluconazole breakpoints for Candidaby using published clinical and microbiologic data to provide further validation of the breakpoints proposed by the CLSI in 1997. We also address interpretive breakpoints for agar disk diffusion testing of fluconazole. The MIC distribution for fluconazole was determined with a collection of 13,338 clinical isolates. The overall MIC at which 90% of the isolates were inhibited was 8 µg/ml: 91% were susceptible (S) at a MIC of =8 µg/ml and 3% were resistant (R) (MIC = 64 µg/ml). Similar results were obtained for 2,190 isolates from randomized clinical trials. Analysis of available data for 1,295 patient-episode-isolate events (692 represented mucosal infections and 603 represented invasive infections) from 12 published clinical studies demonstrated an overall success rate of 77%, including 85% for those episodes in which the fluconazole MIC was =8 µg/ml, 67% for those episodes in which the MIC was 16 to 32 µg/ml, and 42% for those episodes with resistant (MIC = 64 µg/ml) isolates. Pharmacodynamic analysis demonstrated a strong relationship between MIC, fluconazole dose, and outcome. A dose/MIC ratio of ~25 was supportive of the following susceptibility breakpoints for fluconazole and Candidaspp.: S, MIC = 8 µg/ml; susceptible-dose dependent (SDD), MIC = 16 to 32 µg/ml; R, MIC = 64 µg/ml. The corresponding disk test breakpoints are as follows: S, =19 mm; SDD, 15 to 18 mm; R, =14 mm.

ABSTRACTDeveloping interpretive breakpoints for any given organism-drug combination requires integration of the MIC distribution, pharmacokinetic and pharmacodynamic parameters, and the relationship between the in vitro activity and outcome from both in vivo and clinical studies. Using data generated by standardized broth microdilution and disk diffusion test methods, the Antifungal Susceptibility Subcommittee of the Clinical and Laboratory Standards Institute has now proposed interpretive breakpoints for voriconazole and Candidaspecies. The MIC distribution for voriconazole was determined using a collection of 8,702 clinical isolates. The overall MIC90was 0.25 µg/ml and 99% of the isolates were inhibited at =1 µg/ml of voriconazole. Similar results were obtained for 1,681 Candidaisolates (16 species) from the phase III clinical trials. Analysis of the available data for 249 patients from six phase III voriconazole clinical trials demonstrated a statistically significant correlation (P= 0.021) between MIC and investigator end-of-treatment assessment of outcome. Consistent with parallel pharmacodynamic analyses, these data support the following MIC breakpoints for voriconazole and Candidaspecies: susceptible (S), =1 µg/ml; susceptible dose dependent (SDD), 2 µg/ml; and resistant (R), =4 µg/ml. The corresponding disk test breakpoints are as follows: S, =17 mm; SDD, 14 to 16 mm; and R, =13 mm.

ABSTRACTCaspofungin is being used increasingly as therapy for invasive candidiasis. Prospective sentinel surveillance for emergence of in vitro resistance to caspofungin among invasive Candidaspp. isolates is indicated. We determined the in vitro activity of caspofungin against 8,197 invasive (bloodstream or sterile-site) unique patient isolates of Candidacollected from 91 medical centers worldwide from 1 January 2001 to 31 December 2004. We performed antifungal susceptibility testing according to the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) M27-A2 method and used a 24-h prominent inhibition endpoint for determination of the MIC. Of 8,197 invasive Candidaspp. isolates, species distribution was as follows: 54% Candida albicans, 14% C. glabrata, 14% C. parapsilosis, 11% C. tropicalis, 3% C. krusei, and 4% other Candidaspp. Overall, caspofungin was very active against Candida(MIC50/MIC90, 0.03/0.25 µg/ml; 98.2% were inhibited at a MIC of =0.5 µg/ml and 99.7% were inhibited at a MIC of =1 µg/ml). Results by species (expressed as MIC50/MIC90and the percentage inhibited at =1 µg/ml) were as follows: C. albicans, 0.03/0.06, 99.9; C. glabrata, 0.03/0.06, 99.9; C. parapsilosis, 0.5/0.5, 99.0; C. tropicalis, 0.03/0.06, 99.7; C. krusei, 0.12/0.5, 99.0; and C. guilliermondii, 0.5/1, 94.4. Of the 25 isolates with caspofungin MICs of >1 µg/ml, 12 isolates were C. parapsilosis, 6 isolates were C. guilliermondii, 2 isolates were C. rugosa, and 1 isolate each was C. albicans, C. glabrata, C. krusei, C. lusitaniae, and C. tropicalis. There was no significant change in caspofungin activity over the 4-year study period. Likewise, there was no difference in activity by geographic region. Caspofungin has excellent in vitro activity against invasive clinical isolates of Candidafrom centers worldwide. Our prospective sentinel surveillance reveals no evidence of emerging caspofungin resistance among invasive clinical isolates of Candida.

Developing interpretive breakpoints for any given organism-drug combination requires integration of the MIC distribution, pharmacokinetic and pharmacodynamic parameters, and the relationship between the in vitro activity and outcome from both in vivo and clinical studies. Using data generated by standardized broth microdilution and disk diffusion test methods, the Antifungal Susceptibility Subcommittee of the Clinical and Laboratory Standards Institute has now proposed interpretive breakpoints for voriconazole and Candida species. The MIC distribution for voriconazole was determined using a collection of 8,702 clinical isolates. The overall MIC90 was 0.25 µg/ml and 99% of the isolates were inhibited at ≤1 µg/ml of voriconazole. Similar results were obtained for 1,681 Candida isolates (16 species) from the phase III clinical trials. Analysis of the available data for 249 patients from six phase III voriconazole clinical trials demonstrated a statistically significant correlation (P = 0.021) between MIC and investigator end-of-treatment assessment of outcome. Consistent with parallel pharmacodynamic analyses, these data support the following MIC breakpoints for voriconazole and Candida species: susceptible (S), ≤1 µg/ml; susceptible dose dependent (SDD), 2 µg/ml; and resistant (R), ≥4 µg/ml. The corresponding disk test breakpoints are as follows: S, ≥17 mm; SDD, 14 to 16 mm; and R, ≤13 mm.

Caspofungin is being used increasingly as therapy for invasive candidiasis. Prospective sentinel surveillance for emergence of in vitro resistance to caspofungin among invasive Candida spp. isolates is indicated. We determined the in vitro activity of caspofungin against 8,197 invasive (bloodstream or sterile-site) unique patient isolates of Candida collected from 91 medical centers worldwide from 1 January 2001 to 31 December 2004. We performed antifungal susceptibility testing according to the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) M27-A2 method and used a 24-h prominent inhibition endpoint for determination of the MIC. Of 8,197 invasive Candida spp. isolates, species distribution was as follows: 54% Candida albicans, 14% C. glabrata, 14% C. parapsilosis, 11% C. tropicalis, 3% C. krusei, and 4% other Candida spp. Overall, caspofungin was very active against Candida (MIC50/MIC90, 0.03/0.25 µg/ml; 98.2% were inhibited at a MIC of ≤0.5 µg/ml and 99.7% were inhibited at a MIC of ≤1 µg/ml). Results by species (expressed as MIC50/MIC90 and the percentage inhibited at ≤1 µg/ml) were as follows: C. albicans, 0.03/0.06, 99.9; C. glabrata, 0.03/0.06, 99.9; C. parapsilosis, 0.5/0.5, 99.0; C. tropicalis, 0.03/0.06, 99.7; C. krusei, 0.12/0.5, 99.0; and C. guilliermondii, 0.5/1, 94.4. Of the 25 isolates with caspofungin MICs of >1 µg/ml, 12 isolates were C. parapsilosis, 6 isolates were C. guilliermondii, 2 isolates were C. rugosa, and 1 isolate each was C. albicans, C. glabrata, C. krusei, C. lusitaniae, and C. tropicalis. There was no significant change in caspofungin activity over the 4-year study period. Likewise, there was no difference in activity by geographic region. Caspofungin has excellent in vitro activity against invasive clinical isolates of Candida from centers worldwide. Our prospective sentinel surveillance reveals no evidence of emerging caspofungin resistance among invasive clinical isolates of CANDIDA:

ABSTRACTFluconazole in vitro susceptibility test results for 140,767 yeasts were collected from 127 participating investigators in 39 countries from June 1997 through December 2003. Data were collected on 79,343 yeast isolates tested with voriconazole from 2001 through 2003. All investigators tested clinical yeast isolates by the CLSI (formerly NCCLS) M44-A disk diffusion method. Test plates were automatically read and results were recorded with the BIOMIC Vision Image Analysis System. Species, drug, zone diameter, susceptibility category, and quality control results were collected quarterly via e-mail for analysis. Duplicate (the same patient, same species, and same susceptible-resistant biotype profile during any 7-day period) and uncontrolled test results were not analyzed. The 10 most common species of yeasts all showed less resistance to voriconazole than to fluconazole. Candida kruseishowed the largest difference, with over 70% resistance to fluconazole and less than 8% to voriconazole. All species of yeasts tested were more susceptible to voriconazole than to fluconazole, assuming proposed interpretive breakpoints of =17 mm (susceptible) and= 13 mm (resistant) for voriconazole. MICs reported in this study were determined from the zone diameter in millimeters from the continuous agar gradient around each disk, which was calibrated with MICs determined from the standard CLSI M27-A2 broth dilution method by balanced-weight regression analysis. The results from this investigation demonstrate the broad spectrum of the azoles for most of the opportunistic yeast pathogens but also highlight several areas where resistance may be progressing and/or where previously rare species may be“ emerging.”

ABSTRACTThe accuracy of antifungal susceptibility testing is important for reliable resistance surveillance and for the clinical management of patients with serious infections. Our primary objective was to compare the results of voriconazole disk diffusion testing of Candidaspp. performed by centers participating in the ARTEMIS program with disk diffusion and MIC results obtained by the central reference laboratory. A total of 2,934 isolates of Candidaspp. were tested by CLSI disk diffusion and reference broth microdilution methods in the central reference laboratory. These results were compared to the results of disk diffusion testing performed in the 54 participating centers. All tests were performed and interpreted following CLSI recommendations, as follows: susceptible (S), MIC of =1 µg/ml (=17 mm); susceptible dose dependent (SDD), MIC of 2 µg/ml (14 to 16 mm); and resistant (R), MIC of =4 µg/ml (=13 mm). The overall categorical agreement between participant disk diffusion test results and reference laboratory MIC results was 94.1%, with 0.1% very major errors (VME) and 3.4% major errors (ME). The categorical agreement between disk diffusion test results obtained in the reference laboratory and the MIC test results was 99.0%. Likewise, good agreement was observed between participant disk diffusion test results and reference laboratory disk diffusion test results, with an agreement of 93.8%, 0.2% VME, and 3.4% ME. The disk diffusion test was reliable for detecting those isolates of Candidaspp. that were characterized as resistant (MIC of =4 µg/ml) by MIC testing. External quality assurance data obtained by surveillance programs such as the ARTEMIS Global Antifungal Surveillance Program ensure the generation of useful surveillance data and result in the continued improvement of antifungal susceptibility testing practices.

ABSTRACTThe in vitro activity of itraconazole was determined against 7,299 isolates of Candidaspp., 1,615 isolates of Cryptococcus neoformans, and 445 isolates of Aspergillusspp. obtained from over 200 medical centers worldwide. Itraconazole was active against all Candidaspp. (96% of MICs were =1 µg/ml) with the exception of C. glabrata(77% of MICs were =1 µg/ml). Itraconazole inhibited 94% of C. kruseiand 84% of other fluconazole-resistant Candidaspecies, exclusive of C. glabrata, at a MIC of =1 µg/ml. Itraconazole was not active against fluconazole-resistant isolates of C. glabrata. Only modest activity was seen against C. neoformans(80% of MICs were =1 µg/ml); however, itraconazole showed excellent activity against Aspergillusspp. (94% of MICs were =1 µg/ml). These results provide an update on the antifungal activity of itraconazole against major opportunistic fungal pathogens. In light of the new intravenous formulation of itraconazole these data suggest that this agent remains a viable systemically active antifungal agent.