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To summarize the potential clinical usefulness of available Etest echinocandin and amphotericin B ECVs in identifying non-WT-type yeasts. In lieu of breakpoints (predictors of clinical response), Etest echinocandin, triazoles, and amphotericin B ECVs have been recently established for certain Candida spp. Reference BPs are only available for echinocandins and triazoles and some Candida spp. The categorical agreement between Etest and reference MICs was mostly evaluated before adjustment of CLSI triazole/echinocandin BPs to current values or using various amphotericin B cutoffs. Etest anidulafungin ECVs for C. albicans (0.015 μg/mL), C. glabrata (0.03 μg/ml), C. tropicalis (0.03 μg/mL), and C. krusei (0.06 μg/mL) categorized 65/69 (93%) as non-wild-type isolates (harboring Fks1p/Fks2p mutations). The amphotericin B Etest ECV for C. lusitaniae (0.5 μg/mL) classified 8/9 as non-wild-type and 38/39 (97%) as wild-type isolates, respectively. Similar results were reported for C. glabrata, C. parapsilosis, and Cryptococcus neoformans. Further evaluation is warranted, especially for amphotericin B.

International audience; Susceptibility testing is an important tool in the clinical setting; its utility is based on the availability of categorical endpoints, breakpoints (BPs) or epidemiological cutoff values (ECVs/ECOFFs). CLSI and EUCAST have developed antifungal susceptibility testing, BPs and ECVs for some fungal species. Although the Concentration Gradient Strip BioMerieux Etest is useful for routine testing in the clinical laboratory, ECVs are not available for all agent/species; the lack of clinical data precludes development of BPs. We re-evaluated and consolidated Etest data points from three previous studies, and included new data. We defined ECOFFinder Etest ECVs for three sets of species/agent combinations: fluconazole, posaconazole and voriconazole and 8 Candida spp.; amphotericin B and 3 non-prevalent Candida spp.; and caspofungin and 5 Aspergillus spp. The total of Etest MICs from 23 laboratories (Europe, the Americas, South Africa) included (antifungal agent/dependent): 17,242 Candida albicans, 244 C. dubliniensis, 5,129 C. glabrata species complex (SC), 275 C. guilliermondii (Meyerozyma guilliermondii), 1,133 C. krusei (Pichia kudriavzevii), 933 C. kefyr (Kluyveromyces marxianus), 519 C. lusitaniae (Clavispora lusitaniae), 2,947 C. parapsilosis SC, 2,214 C. tropicalis, 3,212 Aspergillus fumigatus, 232 A. flavus, 181 A. niger, and 267 A. terreus SC isolates. Triazole MICs for 66 confirmed non-wild-type (non-WT) Candida isolates were available (ERG11 point mutations). Distributions fulfilling CLSI ECV criteria were pooled and ECOFFinder Etest ECVs were established for triazoles (9 Candida spp.); amphotericin B (3 less-prevalent Candida spp.) and caspofungin (4 Aspergillus spp.). Etest fluconazole ECVs could be good detectors of Candida non-WT isolates (59/61 Non-WT: 4 of 6 species).

We determined the in vitro activity of fenticonazole against 318 vaginitis isolates of Candida and bacterial species and selected 28 isolates for time-kill studies. At concentrations equal to 4× MIC, fenticonazole reached the 99.9% killing endpoint by ∼10 h for Staphylococcus aureus , Streptococcus agalactiae , and Escherichia coli and by ∼17 h for Candida albicans and Candida parapsilosis ; and at concentrations equal to 8× MIC, by ∼19 and ∼20 h for Candida glabrata and Candida tropicalis , respectively.

Although the Sensititre Yeast-One (SYO) and Etest methods are widely utilized, interpretive criteria are not available for triazole susceptibility testing of Candida or Aspergillus species. We collected fluconazole, itraconazole, posaconazole, and voriconazole SYO and Etest MICs from 39 laboratories representing all continents for (method/agent-dependent) 11,171 Candida albicans, 215 C. dubliniensis, 4,418 C. glabrata species complex, 157 C. guilliermondii (Meyerozyma guilliermondii), 676 C. krusei (Pichia kudriavzevii), 298 C. lusitaniae (Clavispora lusitaniae), 911 C. parapsilosis sensu stricto, 3,691 C. parapsilosis species complex, 36 C. metapsilosis, 110 C. orthopsilosis, 1,854 C. tropicalis, 244 Saccharomyces cerevisiae, 1,409 Aspergillus fumigatus, 389 A. flavus, 130 A. nidulans, 233 A. Niger, and 302 A. terreus complex isolates. SYO/Etest MICs for 282 confirmed non-wild-type (non-WT) isolates were included: ERG11 (C. albicans), ERG11 and MRR1 (C. parapsilosis), cyp51A (A. fumigatus), and CDR2 and CDR1 overexpression (C. albicans and C. glabrata, respectively). Interlaboratory modal agreement was superior by SYO for yeast species and by the Etest for Aspergillus spp. Distributions fulfilling CLSI criteria for epidemiological cutoff value (ECV) definition were pooled, and we proposed SYO ECVs for S. cerevisiae and 9 yeast and 3 Aspergillus species and Etest ECVs for 5 yeast and 4 Aspergillus species. The posaconazole SYO ECV of 0.06 g/ml for C. albicans and the Etest itraconazole ECV of 2 g/ml for A. fumigatus were the best predictors of non-WT isolates. These findings support the need for method-dependent ECVs, as, overall, the SYO appears to perform better for susceptibility testing of yeast species and the Etest appears to perform better for susceptibility testing of Aspergillus spp. Further evaluations should be conducted with more Candida mutants. Fil: Espinel-Ingroff, A.. Virginia Commonwealth University Medical Center; Fil: Turnidge, J.. University of Adelaide; Australia Fil: Alastruey-Izquierdo, A.. Centro Nacional de Microbiologia; Fil: Botterel, F.. Mycologie; Fil: Canton, Eduardo. Instituto de Investigación Sanitaria la Fe; Fil: Castro, C.. Hospital Universitario de Valme; Fil: Chen, Y.-C.. National Taiwan University Hospital; Fil: Chen, Y.. Public Health Ontario; Fil: Chryssanthou, E.. Karolinska University Hospital; Fil: Dannaoui, E.. Universite Paris Descartes; Fil: Garcia, Guillermo Manuel. Universidad Nacional del Litoral; Argentina Fil: Gonzalez, G.M.. Universidad Autónoma de Nuevo León; Fil: Govender, N.P.. University of the Witwatersrand; Sudáfrica Fil: Guinea, J.. Hospital General Universitario Gregorio Marañon; Fil: Kidd, S.. National Mycology Reference Centre; Fil: Lackner, M.. Medizinische Universitat Innsbruck; Fil: Lass-Flörl, C.. Medizinische Universitat Innsbruck; Fil: Linares-Sicilia, M.J.. Universidad de Cordoba, Facultad de Medicina; Fil: López-Soria, L.. Hospital Universitario Cruces; Fil: Magobo, R.. Universite Paris Descartes; Fil: Pelaez, T.. Hospital Universitario Central de Asturias; Fil: Quindós, G.. Universidad del Pais Vasco - Euskal Herriko Unibertsitatea, Campus Bizkaia; Fil: Rodriguez-Iglesia, M.A.. Universidad de Cádiz; España Fil: Ruiz, M.A.. Instituto de Investigación Sanitaria Aragón; Fil: Sánchez-Reus, F.. Hospital de la Santa Creu I Sant Pau; Fil: Sanguinetti, M.. Università Cattolica del Sacro Cuore, Rome; Fil: Shields, R.. University of Pittsburgh at Johnstown; Estados Unidos. University of Pittsburgh; Estados Unidos Fil: Szweda, P.. Gdan´ Sk University Of Technology; Fil: Tortorano, A.. Università degli Studi di Milano; Italia Fil: Wengenack, N.L.. Mayo Clinic In Rochester, Minnesota; Fil: Bramati, S.. Azienda Ospedaliera San Gerardo Monza; Fil: Cavanna, C.. Fondazione Irccs Policlinico San Matteo; Fil: DeLuca, C.. Humanitas Research Hospital; Fil: Gelmi, M.. Spedali Civili Di Brescia; Fil: Grancini, A.. Ospedale Maggiore Policlinico Milano; Fil: Lombardi, G.. Ospedale Niguarda, Milan; Fil: Meletiadis, J.. University Of Athens Medical School; Fil: Negri, C.E.. Universidade Federal de Sao Paulo; Fil: Passera, M.. Microbiology Institute; Fil: Peman, J.. Hospital Universitari I Politècnic la Fe; Fil: Prigitano, A.. Università degli Studi di Milano; Italia Fil: Sala, E.. Asst Lariana; Fil: Tejada, M.. Irccs Policlinico San Donato

Method-dependent Etest epidemiological cutoff values (ECVs) are not available for susceptibility testing of either Candida or Aspergillus species with amphotericin B or echinocandins. In addition, reference caspofungin MICs for Candida spp. are unreliable. Candida and Aspergillus species wild-type (WT) Etest MIC distributions (microorganisms in a species-drug combination with no detectable phenotypic resistance) were established for 4,341 Candida albicans , 113 C. dubliniensis , 1,683 C. glabrata species complex (SC), 709 C. krusei , 767 C. parapsilosis SC, 796 C. tropicalis , 1,637 Aspergillus fumigatus SC, 238 A. flavus SC, 321 A. niger SC, and 247 A. terreus SC isolates. Etest MICs from 15 laboratories (in Argentina, Europe, Mexico, South Africa, and the United States) were pooled to establish Etest ECVs. Anidulafungin, caspofungin, micafungin, and amphotericin B ECVs (in micrograms per milliliter) encompassing ≥97.5% of the statistically modeled population were 0.016, 0.5, 0.03, and 1 for C. albicans ; 0.03, 1, 0.03, and 2 for C. glabrata SC; 0.06, 1, 0.25, and 4 for C. krusei ; 8, 4, 2, and 2 for C. parapsilosis SC; and 0.03, 1, 0.12, and 2 for C. tropicalis . The amphotericin B ECV was 0.25 μg/ml for C. dubliniensis and 2, 8, 2, and 16 μg/ml for the complexes of A. fumigatus , A. flavus , A. niger , and A. terreus , respectively. While anidulafungin Etest ECVs classified 92% of the Candida fks mutants evaluated as non-WT, the performance was lower for caspofungin (75%) and micafungin (84%) cutoffs. Finally, although anidulafungin (as an echinocandin surrogate susceptibility marker) and amphotericin B ECVs should identify Candida and Aspergillus isolates with reduced susceptibility to these agents using the Etest, these ECVs will not categorize a fungal isolate as susceptible or resistant, as breakpoints do.

The role of antimicrobial susceptibility testing is to aid in selecting the best agent for the treatment of bacterial and fungal diseases. This has been best achieved by the setting of breakpoints by Clinical Laboratory Standards Institute (CLSI) for prevalent Candida spp. versus anidulafungin, caspofungin, micafungin, fluconazole, and voriconazole. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) also has set breakpoints for prevalent and common Candida and Aspergillus species versus amphotericin B, itraconazole, and posaconazole. Recently, another interpretive category, the epidemiological cut off value, could aid in the early identification of strains with acquired resistance mechanisms. CLSI has postulated that epidemiological cut off values may, with due caution, aid physicians in managing mycosis by species where breakpoints are not available. This review provides (1) the criteria and statistical approach to establishing and estimating epidemiological cut off values (ECVs), (2) the role of the epidemiological cut off value in establishing breakpoints, (3) the potential role of epidemiological cut off values in clinical practice, (4) and the wide range of CLSI-based epidemiological cut off values reported in the literature as well as EUCAST and Sensititre Yeast One-ECVs. Additionally, we provide MIC/MEC (minimal inhibitory concentrations/minimum effective concentrations) ranges/modes of each pooled distribution used for epidemiological cut off value calculation. We focus on the epidemiological cut off value, the new interpretive endpoint that will identify the non-wild type strains (defined as potentially harboring resistance mechanisms). However, we emphasize that epidemiological cut off values will not categorize a fungal isolate as susceptible or resistant as breakpoints do, because the former do not account for the pharmacology of the antifungal agent or the findings from clinical outcome studies.

Estimating epidemiological cutoff endpoints (ECVs/ECOFFS) may be hindered by the overlap of MICs for mutant and nonmutant strains (strains harboring or not harboring mutations, respectively). Posaconazole MIC distributions for the Aspergillus fumigatus species complex were collected from 26 laboratories (in Australia, Canada, Europe, India, South and North America, and Taiwan) and published studies. Distributions that fulfilled CLSI criteria were pooled and ECVs were estimated. The sensitivity of three ECV analytical techniques (the ECOFFinder, normalized resistance interpretation [NRI], derivatization methods) to the inclusion of MICs for mutants was examined for three susceptibility testing methods (the CLSI, EUCAST, and Etest methods). The totals of posaconazole MICs for nonmutant isolates (isolates with no known cyp51A mutations) and mutant A. fumigatus isolates were as follows: by the CLSI method, 2,223 and 274, respectively; by the EUCAST method, 556 and 52, respectively; and by Etest, 1,365 and 29, respectively. MICs for 381 isolates with unknown mutational status were also evaluated with the Sensititre YeastOne system (SYO). We observed an overlap in posaconazole MICs among nonmutants and cyp51A mutants. At the commonly chosen percentage of the modeled wild-type population (97.5%), almost all ECVs remained the same when the MICs for nonmutant and mutant distributions were merged: ECOFFinder ECVs, 0.5 μg/ml for the CLSI method and 0.25 μg/ml for the EUCAST method and Etest; NRI ECVs, 0.5 μg/ml for all three methods. However, the ECOFFinder ECV for 95% of the nonmutant population by the CLSI method was 0.25 μg/ml. The tentative ECOFFinder ECV with SYO was 0.06 μg/ml (data from 3/8 laboratories). Derivatization ECVs with or without mutant inclusion were either 0.25 μg/ml (CLSI, EUCAST, Etest) or 0.06 μg/ml (SYO). It appears that ECV analytical techniques may not be vulnerable to overlap between presumptive wild-type isolates and cyp51A mutants when up to 11.6% of the estimated wild-type population includes mutants.

Contains fulltext : 196638.pdf (Publisher’s version ) (Open Access) Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the in vivo efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the in vitro and in vivo performance of anti-infective coatings and materials to prevent fungal biofilm-based infections.

The U.S. Clinical and Laboratory Standards Institute (CLSI) and the European Committee of Antimicrobial Susceptibility Testing (AFST-EUCAST) have developed broth microdilution methodologies for testing yeasts and filamentous fungi (molds). The mission of these methodologies is to identify in vitro antifungal resistance, which is accomplished by the use of either clinical breakpoints (CBPs), or to a lesser degree, epidemiologic cutoff values (ECVs). The newly adjusted and species-specific CLSI CBPs for Candida spp. versus fluconazole and voriconazole have ameliorated some of the differences between the two methodologies. In the absence of CBPs for mold testing, CLSI ECVs are available for six Aspergillus species versus the triazoles, caspofungin and amphotericin B. Recently, breakpoints were developed by the EUCAST for certain Aspergillus spp. versus amphotercin B, itraconazole and posaconazole, which to some extent are comparable to ECVs. We summarize these latest accomplishments, which have made possible the harmonization of some susceptibility cutoffs, if not methodologies for some agent/species combinations.

Objectives: Candida lusitaniae fungaemia, although infrequent (1%), is more common in immunocompromised patients than Candida albicans. Although infections produced by Candida spp. are therapeutic targets for treatment with echinocandins, little information is available regarding their killing kinetics against C. lusitaniae. The objectives of this study were to determine the killing kinetics of anidulafungin, micafungin and caspofungin against four blood isolates of C. lusitaniae by time –kill methodology. Methods: Time–kill studies were performed in RMPI 1640 medium (5 mL, inoculum � 10 5 cfu/mL). The number of cfu/mL was determined at 0, 2, 4, 6 and 24 h. The anidulafungin concentrations assayed were 0.03, 0.12, 0.5, 2 and 8 mg/L, while micafungin and caspofungin concentrations were 0.25, 1, 4, 16 and 32 mg/L. Results: MIC ranges were 0.03–1 mg/L (anidulafungin), 0.016–0.06 mg/L (micafungin) and 0.03–1 mg/L (caspofungin). The mean maximum log decrease in cfu/mL was reached with 2 mg/L anidulafungin (1.85+0.4 log), 32 mg/L caspofungin (5.5+0.2 log) and 32 mg/L micafungin (2.65+1.9 log). Only caspofungin and micafungin reached the fungicidal endpoint (99.9% growth reduction or a 3 log decrease) with 32 mg/L at 22.8 h (caspofungin) and 26.5 h (micafungin). Analysis of variance showed significant differences in killing activity among isolates, but not among concentrations reached in serum or echinocandins. Conclusions: Anidulafungin and micafungin exhibit greater killing rates than caspofungin. Caspofungin was the only echinocandin that reached the fungicidal endpoint before 24 h, but at drug concentrations (≥16 mg/L) not usually reached in serum. The echinocandin killing rate was isolate dependent and concentration independent.

The CLSI epidemiological cutoff values (ECVs) of antifungal agents are available for various Candida spp., Aspergillus spp., and the Mucorales. However, those categorical endpoints have not been established for Fusarium spp., mostly due to the difficulties associated with collecting sufficient CLSI MICs for clinical isolates identified according to the currently recommended molecular DNA-PCR-based identification methodologies. CLSI MIC distributions were established for 53 Fusarium dimerum species complex (SC), 10 F. fujikuroi , 82 F. proliferatum , 20 F. incarnatum-F. equiseti SC, 226 F. oxysporum SC, 608 F. solani SC, and 151 F. verticillioides isolates originating in 17 laboratories (in Argentina, Australia, Brazil, Canada, Europe, Mexico, and the United States). According to the CLSI guidelines for ECV setting, ECVs encompassing ≥97.5% of pooled statistically modeled MIC distributions were as follows: for amphotericin B, 4 μg/ml ( F. verticillioides ) and 8 μg/ml ( F. oxysporum SC and F. solani SC); for posaconazole, 2 μg/ml ( F. verticillioides ), 8 μg/ml ( F. oxysporum SC), and 32 μg/ml ( F. solani SC); for voriconazole, 4 μg/ml ( F. verticillioides ), 16 μg/ml ( F. oxysporum SC), and 32 μg/ml ( F. solani SC); and for itraconazole, 32 μg/ml ( F. oxysporum SC and F. solani SC). Insufficient data precluded ECV definition for the other species. Although these ECVs could aid in detecting non-wild-type isolates with reduced susceptibility to the agents evaluated, the relationship between molecular mechanisms of resistance (gene mutations) and MICs still needs to be investigated for Fusarium spp.

Methods developed for testing filamentous fungi (molds) include standardized broth microdilution (Clinical and Laboratory Standards Institute [CLSI] and European Committee for Antimicrobial Susceptibility Testing [AFST-EUCAST]) methods and disk diffusion (CLSI) methods. Quality control limits also are available from CLSI for MIC (minimal inhibitory concentration), MEC (minimal effective concentration), and zone diameters. Although clinical breakpoints based on correlations of in vitro results with clinical outcome have not been established, epidemiologic cutoff values have been defined for six Aspergillus species and the triazoles, caspofungin, and amphotericin B. The link between resistance molecular mechanisms, elevated MICs, and clinical treatment failure has also been documented, especially for Aspergillus and the triazoles. Other insights into the potential clinical value of high MICs have also been reported. Various commercial methods (e.g., YeastOne, Etest, and Neo-Sensitabs) have been evaluated in comparison with reference methods. This review summarizes and discusses these developments.

Micafungin Etest and Clinical and Laboratory Standards Institute (CLSI) MICs were compared for 337 Candida spp. isolates. The performance of Etest for testing the susceptibilities of Candida spp. to micafungin was evaluated by the assessment of both categorical (CA) and essential (EA) agreements. The CA was evaluated 2 ways: (i) by the ability of Etest to separate resistant (nontreatable) from susceptible (treatable) isolates by using the newly adjusted species-specific micafungin clinical breakpoints (CBPs) that are available for most of the common species tested and (ii) by the ability to separate wild type (WT) from non-WT isolates or those harboring FKS mutations (with reduced echinocandin susceptibility) by using micafungin epidemiologic cutoff values (ECVs). Etest and CLSI MICs were in EA when the MICs were within 2 log(2) dilutions. Based on agreement percentages, our data indicated that Etest is suitable to test micafungin for most of the Candida species evaluated (overall EA 94.7%; overall CA according to CBPs 97.2% and according to ECVs 97.3%). However, the number of resistant isolates was small, so further evaluations are needed with a higher number of such isolates including more resistant or those with known mechanisms of resistance (non-WT).

The echinocandins anidulafungin, caspofungin, and micafungin have a broad and similar spectrum of in vitro and in vivo activity against most Candida spp. Minimal inhibitory concentrations (MICs) for Candida spp. are usually below 1 μg/mL for most isolates. The exceptions are Candida parapsilosis and C. guilliermondii. Species-specific clinical breakpoints (CBPs) and epidemiologic cutoff values (ECVs) have been proposed by the Clinical and Laboratory Standards Institute (CLSI) for the eight most common Candida spp. versus each echinocandin; these values are useful to detect in vitro antifungal resistance (CBPs) and to identify isolates harboring fks mutations or having reduced susceptibility (ECVs). This paper presents a review of the literature (2006–2010) regarding the in vitro activity similarities or differences among the three echinocandins against Candida spp.; different parameters or measurements of in vitro potency were evaluated. The focus of the review is the non-Candida albicans species.

Background Both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) have MIC clinical breakpoints (CBPs) for fluconazole (FLU) and Candida . EUCAST CBPs are species-specific, and apply only to C. albicans , C. tropicalis and C. parapsilosis , while CLSI CBPs apply to all species. We reassessed the CLSI CBPs for FLU and Candida in light of recent data. Methods We examined (1) molecular mechanisms of resistance and cross-resistance profiles, (2) wild-type (WT) MICs and epidemiological cutoff values (ECVs) for FLU and major Candida species by both CLSI and EUCAST methods, (3) determination of essential (EA) and categorical agreement (CA) between CLSI and EUCAST methods, (4) correlation of MICs with outcomes from previously published data using CLSI and EUCAST methods, and (5) pharmacokinetic and pharmacodynamic considerations. We applied these findings to propose new species-specific CLSI CBPs for FLU and Candida . Results WT distributions from large collections of Candida revealed similar ECVs by both CLSI and EUCAST methods (0.5–1mcg/ml for C. albicans , 2mcg/ml for C. parapsilosis and C. tropicalis , 32mcg/ml for C. glabrata , and 64–128 for C. krusei ). Comparison of CLSI and EUCAST MICs reveal EA and CA of 95% and 96%, respectively. Datasets correlating CLSI and EUCAST FLU MICs with outcomes revealed decreased response rates when MICs were >4mcg/ml for C. albicans , C. tropicalis and C. parapsilosis , and >16mcg/ml for C. glabrata . Conclusions Adjusted CLSI CBPs for FLU and C. albicans , C. parapsilosis , C. tropicalis ( S , ≤2mcg/ml; SDD, 4mcg/ml; R , ≥8mcg/ml), and C. glabrata (SDD, ≤32mcg/ml; R , ≥64mcg/ml) should be more sensitive for detecting emerging resistance among common Candida species and provide consistency with EUCAST CBPs.

Failure to respond to antifungal therapy could be due to in vitro resistance (intrinsic or developed during therapy) or clinical resistance. In vitro resistance is mostly due to genetic mutations (resistance mechanisms), and it is associated with high minimal inhibitory concentrations (MICs), minimal effective concentrations (MECs), and/or clinical failure. Clinical breakpoints (CBPs) and/or epidemiologic cutoff values (ECVs) are useful to detect the in vitro antifungal resistance when isolates are tested by standardized methods. ECVs are available from the Clinical and Laboratory Standards Institute (CLSI) for Candida spp. versus echinocandins (anidulafungin, caspofungin, and micafungin) and triazoles (fluconazole, posaconazole, and voriconazole). Lately, the CLSI has adjusted to species-specific CBPs for Candida spp. versus fluconazole, similar to those of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and versus echinocandins. However, the available voriconazole EUCAST and CLSI CBPs differ. In the absence of CBPs, EUCAST and CLSI assigned ECVs for various Aspergillus spp. and triazoles. This article reviews emerging resistance, laboratory detection, and clinical relevance as reported in the literature in the past 3 to 4 years.

Antifungal susceptibility testing of Aspergillus species 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 A spergillus fumigatus with 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. fumigatus for 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. fumigatus for 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. fumigatus and 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. fumigatus will be useful in resistance surveillance and is an important step toward the development of clinical breakpoints.

The Clinical and Laboratory Standards Institute (CLSI) has standardized broth microdilution and disk diffusion methodology for testing filamentous fungi (molds) that cause invasive disease. Quality control MIC (minimal inhibitory concentration) and MEC (minimal effective concentration; echinocandins only) limits are also available in the recently published CLSI M38-A2 document. Although breakpoints based on correlations of in vitro results and clinical outcome have not been established, MIC or MEC and zone diameter categories for five antifungal agents and various mold species, as well as epidemiologic cutoffs for Aspergillus fumigatus versus the triazoles, have been recently documented. Some insights of the potential clinical value of reference methods also have been reported. During the past few years, the potential utility of various commercial methods has been evaluated by comparing them with reference methodology. This review summarizes and discusses the advantages and disadvantages of these developments.

We evaluated the in vitro activities of anidulafungin, micafungin, and caspofungin againstCandida kruseiby determining MIC and minimum fungicidal concentration (MFC) measurements and by the time-kill method. The geometric mean (GM)-MIC/GM-MFC values were 0.1/0.34, 0.25/0.44, and 1/2.29, respectively. The mean times to reach 99.9% growth reduction were 19.1 ± 18.2 h (mean ± standard deviation) for 2 mg/liter anidulafungin, 37.4 ± 8.8 h for 2 mg/liter caspofungin, and 30.7 ± 12.2 h for 1 mg/liter micafungin. Anidulafungin exhibited the highest time-kill rate, followed by micafungin. The three echinocandins showed fungicidal activity at concentrations reached in serum.

The susceptibility of 1763 yeast isolates (from 22 species and seven genera) was tested using Clinical and Laboratory Standards Institute M27-A2 microdilution methodology. Candida spp. predominated (97.1%), mainly C. albicans (51.4%), C. glabrata (16.4%) and C. tropicalis (13.7%), followed by Trichosporon spp. (1.1%) and Cryptococcus neoformans (1.0%). Most isolates came from blood/catheters (72.0%) or the oesophagus/oropharynx (11.3%). The voriconazole, itraconazole, fluconazole and amphotericin B MIC90 values (minimum inhibitory concentration for 90% of the isolates) for all isolates were 1.0, 2.0, 64 and 1.0 microg/mL, respectively. Voriconazole MICs correlated with those for fluconazole (r = 0.91) and itraconazole (r = 0.90). Only 109 isolates (6.2%) had voriconazole MICs > or = 4.0 microg/mL; all were C. albicans, C. glabrata or C. tropicalis resistant to itraconazole (and most to fluconazole). Isolates from 22 patients with amphotericin MICs > or = 2.0 microg/mL (range 2.0-16.0 microg/mL) were also cross-resistant to one or more of the triazoles. Patients (n = 34) with voriconazole-resistant isolates showed a 56% response to voriconazole therapy, and those patients (n = 261) with susceptible isolates showed a 71% response. Twenty-three voriconazole-treated patients had baseline resistant isolates, in eight patients voriconazole resistance developed during therapy and in three patients a different resistant species arose during therapy. Thus, voriconazole MICs correlate with those of fluconazole and itraconazole and may predict clinical outcome.

Failure to respond to antifungal therapy could be due to in vitro resistance (intrinsic or developed during therapy) or clinical resistance; the latter is associated with numerous factors related to the host, the antifungal agent, or the infecting isolate. Recently, a susceptible MIC breakpoint ( 2 microg/ml. In some of these cases, clinical failure was associated with the genetic mutations described below. Azole and flucytosine breakpoints, and the echinocandin susceptible breakpoint, are useful when isolates are tested by CLSI standardized methods; breakpoints are also available by the EUCAST method. More recently, in vitro resistant MIC breakpoints have been assigned for filamentous fungi (moulds) vs. five antifungal agents, but these categories are not based on correlations of in vitro with in vivo response to therapy. However, itraconazole (Janssen), amphotericin B (Bristol-Myers) and voriconazole (Pfizer) clinical failures in aspergillosis have been correlated with MICs > 2 microg/ml. This article provides a review of reported resistance molecular mechanisms to antifungal agents since 2005; previous related reviews are also listed.

ATB Fungus 2 and SensititreYeastOne are commercial methods for antifungal susceptibility testing of yeasts. The agreement between these two methods was assessed with a total of 133 Candida strains (60 Candida albicans, 18 Candida dubliniensis, 29 Candida glabrata, and 26 Candida krusei). MIC endpoints were established after 24 h of incubation at 36 ± 1 oC by each method. Intra-laboratory reproducibility of both methods was excellent (� 99%). Overall agreement between ATB Fungus 2 and Sensititre YeastOne 3 MICs (within 2 dilutions) was 91.2-97.7% for amphotericin B, 5-fluorocytosine and itraconazole, and 82.7% for fluconazole. The categorical agreement when ATB Fungus 2 results were compared to those by SensititreYeastOne 3 was 93.2-98.5% for 5-fluorocytosine and amphotericin B, but lower for the triazoles (72.9-75.9%). This easy to perform method could be an alternative for routine use in the clinical microbiology laboratory for susceptibility testing of common Candida spp. Summary

INTRODUCTION Fungal pathogens from the voriconazole trials were identified and tested for susceptibility at two reference laboratories. METHODS MICs were measured using CLSI M38-A 48 h microdilution methodology. RESULTS Moulds from 29 genera and 38 species were isolated from 18 countries. Aspergillus spp. predominated (69%), followed by Scedosporium spp. (11.5%). Aspergillus fumigatus (292/590, 49.5%) was the most common species, followed by Scediosporium apiospermum (9.7%) and Aspergillus terreus (7.3%). The bronchi, lungs and sinuses yielded 45% of the isolates (57% of aspergilli), with 24% from the oropharynx/oesophagus. Other sites included blood/catheter (7.3%) and CNS (5.2%). MIC90s of itraconazole and voriconazole for Aspergillus spp. were the same (0.5 mg/L), but 17 Aspergillus isolates were itraconazole-resistant (MICs > or = 1-16 mg/L). Additionally, in 31 A. fumigatus and 23 A. terreus isolates, amphotericin MICs were > or = 2.0 mg/L. Voriconazole MICs exceeded 4 mg/L in only 5.8% (34/590) of the isolates, including one A. fumigatus (8.0 mg/L), 9/11 Scedosporium prolificans, 10/13 Fusarium solani and all 9 Zygomycetes. Most were also not susceptible to itraconazole or amphotericin B. A notable increase in MIC (more than two doubling dilutions) during voriconazole therapy was seen for one A. fumigatus isolate. The response rate of voriconazole-treated patients with isolate MICs > or = 4.0 mg/L was 38% when compared with 52% for those with MICs < 4.0 mg/L. CONCLUSIONS Voriconazole shows activity, in vitro, similar to that of itraconazole against a wide range of moulds. It is also active against some isolates not susceptible to itraconazole or amphotericin B, but not the Zygomycetes. The relationship between voriconazole MIC and clinical outcome requires further study.

Background In previous studies, itraconazole was revealed to be an effective therapy and was considered to be the gold standard treatment for mild-to-moderate acute and chronic clinical forms of paracoccidioidomycosis. A pilot study was conducted to investigate the efficacy, safety, and tolerability of voriconazole for the long-term treatment of acute or chronic paracoccidioidomycosis, with itraconazole as the control treatment. Methods A randomized, open-label study was conducted at 3 Brazilian tertiary care hospitals. Patients were randomized (at a 2 : 1 ratio) to receive oral therapy with voriconazole or itraconazole for 6 months. Patients receiving >or=1 dose of study drug were evaluated for safety; patients with confirmed paracoccidioidomycosis who completed >or=6 months of therapy (treatment-evaluable patients) were evaluated for treatment efficacy. Satisfactory global response was assessed at the end of treatment. Results Fifty-three patients were evaluated for treatment safety (35 received voriconazole, and 18 received itraconazole). Both drugs were well tolerated. The most common treatment-related adverse events in the voriconazole group included abnormal vision, chromatopsia, rash, and headache; the most common treatment-related adverse events in the itraconazole group included bradycardia, diarrhea, and headache. Liver function test values were slightly higher in patients receiving voriconazole than in those receiving itraconazole; 2 patients in the voriconazole group were withdrawn from treatment because of increased liver function test values. In the intent-to-treat populations, the satisfactory response rate (i.e., complete or partial global response) was 88.6% among the voriconazole group and 94.4% among the itraconazole group. The response rate among treatment-evaluable patients was 100% for both treatment groups; no relapses were observed after 8 weeks of follow-up. Conclusions This is, to our knowledge, the first study to demonstrate that voriconazole is as well tolerated and effective as itraconazole for the long-term treatment of paracoccidioidomycosis.

Objectives To evaluate the suitability of disc diffusion (DD) assay for testing posaconazole activity and to corroborate its activity against recently isolated yeasts by the CLSI reference microdilution M27-A2 method. Methods A total of 224 yeast isolates (7 species with 52 to 11 isolates each, and 15 species with 1 to 6 isolates) were evaluated, 125 were recent bloodstream isolates, 30 isolates from other sources and six ATCC isolates that included amphotericin B-resistant Candida albicans ATCC 200955, Candida lusitaniae (ATCC 200950, 200951, 200952 and 200953) and amphotericin B- and itraconazole-resistant Candida tropicalis ATCC 200956. MICs were determined at 24 and 48 h by following the CLSI guidelines, document M27-A2. DD testing was performed by following CLSI M44-A document with 5 microg posaconazole discs. Inhibition zone diameters were measured at the transition point at which growth decreased at both 24 and 48 h. Results DD showed very good reproducibility, with coefficient of variability median value 4.56. Posaconazole demonstrated good in vitro activity against all clinical isolates, including the emerging species and amphotericin B-resistant ATCC isolates except for C. tropicalis ATCC 200956 (posaconazole MIC >or= 16 mg/L). Only 1.5% and 4.1% of isolates were inhibited by >2 mg/L posaconazole at 24 and 48 h. Good correlation was obtained between methods (R = 0.763 at 24 h and 0.602 at 48 h). DD detected posaconazole-resistant isolates (MIC > 2 mg/L). Conclusions DD could be an alternative to the microdilution reference method, as no major discrepancies were detected.

Worldwide, the most commonly used technique for antibacterial susceptibility testing is the disk diffusion test. However, disk diffusion testing of antifungal agents has been slow to develop despite the fact that early studies with fluconazole disks showed promise for testing Candida spp. To make antifungal susceptibility testing more readily available to clinical microbiology laboratories, the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) has proposed a standardized disk diffusion method for susceptibility testing of Candida spp. Although the Subcommittee has established quality control disk parameters for fluconazole, voriconazole, and posaconazole, zone interpretive criteria (breakpoints) are only available for fluconazole and voriconazole. Therefore, the clinical relevance for any other (than fluconazole and voriconazole) drug-organism combinations is uncertain. The design of the disk diffusion method for Candida spp. is similar to that for bacteria using the same medium (Mueller-Hinton agar), but supplemented with glucose and methylene blue dye. Other specific testing guidelines are summarized below. A commercial method, similar to the CLSI M44-A method, that uses a 9-mm tablet instead of the 6-mm disk is also available in Europe.

The purpose of this study was to correlate inhibition zone diameters, in millimeters (agar diffusion disk method), with the broth dilution MICs or minimum effective concentrations (MECs) (CLSI M38-A method) of five antifungal agents to identify optimal testing guidelines for disk mold testing. The following disk diffusion testing parameters were evaluated for 555 isolates of the molds Absidia corymbifera , Aspergillus sp. (five species), Alternaria sp., Bipolaris spicifera , Fusarium sp. (three species), Mucor sp. (two species), Paecilomyces lilacinus , Rhizopus sp. (two species), and Scedosporium sp. (two species): (i) two media (supplemented Mueller-Hinton agar [2% dextrose and 0.5 μg/ml methylene blue] and plain Mueller-Hinton [MH] agar), (ii) three incubation times (16 to 24, 48, and 72 h), and (iii) seven disks (amphotericin B and itraconazole 10-μg disks, voriconazole 1- and 10-μg disks, two sources of caspofungin 5-μg disks [BBL and Oxoid], and posaconazole 5-μg disks). MH agar supported better growth of all of the species tested (24 to 48 h). The reproducibility of zone diameters and their correlation with either MICs or MECs (caspofungin) were superior on MH agar (91 to 100% versus 82 to 100%; R , 0.71 to 0.93 versus 0.53 to 0.96 for four of the five agents). Based on these results, the optimal testing conditions for mold disk diffusion testing were (i) plain MH agar; (ii) incubation times of 16 to 24 h (zygomycetes), 24 h ( Aspergillus fumigatus , A. flavus , and A. niger ), and 48 h (other species); and (iii) the posaconazole 5-μg disk, voriconazole 1-μg disk, itraconazole 10-μg disk (for all except zygomycetes), BBL caspofungin 5-μg disk, and amphotericin B 10-μg (zygomycetes only).

We compared the Neo-Sensitabs tablet assay to both reference M27-A2 broth microdilution and M44-A disk diffusion methods for testing susceptibilities of 110 isolates of Candida spp. and Cryptococcus neoformans to amphotericin B, caspofungin, fluconazole, itraconazole, and voriconazole. Neo-Sensitabs assay inhibition zone diameters in millimeters on three agars (Mueller-Hinton agar supplemented with 2% dextrose and 0.5 μg/ml methylene blue [MGM], Shadomy [SHA], and RPMI 1640 [RPMI, 2% dextrose]) were obtained at 24 to 72 h. The correlation coefficient of Neo-Sensitabs results with MICs was similar to that of the disk method for most of the five agents on MGM ( R , 0.80 to 0.89 versus 0.76 to 0.89, respectively). Overall, superior correlation was observed at 24 h for most agents. The exception was amphotericin B ( R values of 0.68 and 0.5 for disk and tablet, respectively, at 48 h versus 0.68 and 0.48, respectively, at 24 h). In general, Neo-Sensitabs results were less consistent on SHA and RPMI agars. Although agreement by breakpoint category of Neo-Sensitabs and disk results with CLSI method M27-A2 was also similar on MGM (92.7 to 98.2% versus 95.5 to 100%, respectively), the Neo-Sensitabs method failed to identify two of the six isolates with high amphotericin B MICs. These data suggest the potential value of the Neo-Sensitabs assay for testing at least four of the five agents against yeasts evaluated in the clinical laboratory.

Epidemiological cutoff values (ECVs) of isavuconazole are not available for Cryptococcus spp. The isavuconazole ECVs based on wild-type (WT) MIC distributions for 438 Cryptococcus neoformans nongenotyped isolates, 870 isolates of genotype VNI, and 406 Cryptococcus gattii isolates from six laboratories and different geographical areas were 0.06, 0.12, and 0.25 μg/ml, respectively. These ECVs may aid in detecting non-WT isolates with reduced susceptibilities to isavuconazole.

Clinical breakpoints (CBPs) have not been established for the Mucorales and any antifungal agent. In lieu of CBPs, epidemiologic cutoff values (ECVs) are proposed for amphotericin B, posaconazole, and itraconazole and four Mucorales species. Wild-type (WT) MIC distributions (organisms in a species-drug combination with no detectable acquired resistance mechanisms) were defined with available pooled CLSI MICs from 14 laboratories (Argentina, Australia, Canada, Europe, India, Mexico, and the United States) as follows: 10 Apophysomyces variabilis, 32 Cunninghamella bertholletiae, 136 Lichtheimia corymbifera, 10 Mucor indicus, 123 M. circinelloides, 19 M. ramosissimus, 349 Rhizopus arrhizus, 146 R. microsporus, 33 Rhizomucor pusillus, and 36 Syncephalastrum racemosum isolates. CLSI broth microdilution MICs were aggregated for the analyses. ECVs comprising ≥95% and ≥97.5% of the modeled populations were as follows: amphotericin B ECVs for L. corymbifera were 1 and 2 μg/ml, those for M. circinelloides were 1 and 2 μg/ml, those for R. arrhizus were 2 and 4 μg/ml, and those for R. microsporus were 2 and 2 μg/ml, respectively; posaconazole ECVs for L. corymbifera were 1 and 2, those for M. circinelloides were 4 and 4, those for R. arrhizus were 1 and 2, and those for R. microsporus were 1 and 2, respectively; both itraconazole ECVs for R. arrhizus were 2 μg/ml. ECVs may aid in detecting emerging resistance or isolates with reduced susceptibility (non-WT MICs) to the agents evaluated., Facultad de Ciencias Veterinarias

Neither breakpoints (BPs) nor epidemiological cutoff values (ECVs) have been established for Candida spp. with anidulafungin, caspofungin, and micafungin when using the Sensititre YeastOne (SYO) broth dilution colorimetric method. In addition, reference caspofungin MICs have so far proven to be unreliable. Candida species wild-type (WT) MIC distributions (for microorganisms in a species/drug combination with no detectable phenotypic resistance) were established for 6,007 Candida albicans , 186 C. dubliniensis , 3,188 C. glabrata complex, 119 C. guilliermondii , 493 C. krusei , 205 C. lusitaniae , 3,136 C. parapsilosis complex, and 1,016 C. tropicalis isolates. SYO MIC data gathered from 38 laboratories in Australia, Canada, Europe, Mexico, New Zealand, South Africa, and the United States were pooled to statistically define SYO ECVs. ECVs for anidulafungin, caspofungin, and micafungin encompassing ≥97.5% of the statistically modeled population were, respectively, 0.12, 0.25, and 0.06 μg/ml for C. albicans , 0.12, 0.25, and 0.03 μg/ml for C. glabrata complex, 4, 2, and 4 μg/ml for C. parapsilosis complex, 0.5, 0.25, and 0.06 μg/ml for C. tropicalis , 0.25, 1, and 0.25 μg/ml for C. krusei , 0.25, 1, and 0.12 μg/ml for C. lusitaniae , 4, 2, and 2 μg/ml for C. guilliermondii , and 0.25, 0.25, and 0.12 μg/ml for C. dubliniensis . Species-specific SYO ECVs for anidulafungin, caspofungin, and micafungin correctly classified 72 (88.9%), 74 (91.4%), 76 (93.8%), respectively, of 81 Candida isolates with identified fks mutations. SYO ECVs may aid in detecting non-WT isolates with reduced susceptibility to anidulafungin, micafungin, and especially caspofungin, since testing the susceptibilities of Candida spp. to caspofungin by reference methodologies is not recommended.

The Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards, or NCCLS) M38-A standard for the susceptibility testing of filamentous fungi does not specifically address the testing of dermatophytes. In 2003, a multicenter study investigated the reproducibility of the microdilution method developed at the Center for Medical Mycology, Cleveland, Ohio, for testing the susceptibility of dermatophytes. Data from that study supported the introduction of this method for testing dermatophytes in the future version of the CLSI M38-A standard. In order for the method to be accepted by CLSI, appropriate quality control isolates needed to be identified. To that end, an interlaboratory study, involving the original six laboratories plus two additional sites, was conducted to evaluate potential candidates for quality control isolates. These candidate strains included five Trichophyton rubrum strains known to have elevated MICs to terbinafine and five Trichophyton mentagrophytes strains. Antifungal agents tested included ciclopirox, fluconazole, griseofulvin, itraconazole, posaconazole, terbinafine, and voriconazole. Based on the data generated, two quality control isolates, one T. rubrum isolate and one T. mentagrophytes isolate, were identified and submitted to the American Type Culture Collection (ATCC) for inclusion as reference strains. Ranges encompassing 95.2 to 97.9% of all data points for all seven drugs were established.

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 MIC 90 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.

Although standard conditions are available for testing the susceptibilities of filamentous fungi to antifungal agents by the Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) broth microdilution assay, quality control (QC) MIC limits have not been established for any mold-agent combination. This multicenter (eight-center) study documented the reproducibility of tests for one isolate of Paecilomyces variotii ATCC MYA-3630 and 11 other mold isolates (three isolates of Aspergillus fumigatus ; two isolates of A. terreus ; one isolate each of A. flavus , A. nidulans , Fusarium moniliforme , and F. solani ; and two isolates of Scedosporium apiospermum ) by the CLSI reference broth microdilution method (M38-A document). Control limits (amphotericin B, 1 to 4 μg/ml; itraconazole, 0.06 to 0.5 μg/ml; posaconazole, 0.03 to 0.25 μg/ml; voriconazole, 0.015 to 0.12 μg/ml) for the selected QC P. variotii ATCC MYA-3630 were established by the analysis of replicate MIC results. Reference isolates and corresponding MIC ranges were also established for 6 of the 12 molds evaluated. MIC limits were not proposed for the other five molds tested due to low testing reproducibility for these isolates.

A cytometric approach to determine the susceptibilities of Candida spp. and Cryptococcus neoformans to voriconazole, itraconazole, and caspofungin is described. A total of 63 clinical isolates with different susceptibility patterns were exposed for 1, 2, 4, and 6 h to serial concentrations of each antifungal agent, followed by staining with two fluorescent probes: propidium iodide (PI) and FUN-1. FUN-1 was able to identify the susceptibility patterns of the assayed strains to the three agents after 1 h. PI penetrated a maximum of 50% of the cells treated with PI, at the highest concentration of caspofungin, 16 μg/ml, after 6 h of incubation (this percentage varied with the strain and was drug concentration and time of incubation dependent) and did not stain cells treated with high concentrations of either azole after 6 h. The use of FUN-1 appears to be an excellent fast and reliable alternative to the classical dilution method for determining the susceptibility of Candida spp. and C. neoformans to these three antifungal agents.

The in vitro activities of fluconazole or voriconazole plus terbinafine were evaluated against 20 Candida isolates by the checkerboard, time-kill, and Etest methods. Synergism ( C. albicans , C. glabrata , and C. tropicalis ) and indifference ( C. krusei ) were observed. Correlation among methods was good. The Etest is a suitable method to determine drug interactions.

A commercially prepared dried colorimetric microdilution panel (Sensititre YeastOne, TREK Diagnostic Systems, Cleveland, Ohio) was compared in three different laboratories with the National Committee for Clinical Laboratory Standards (NCCLS) reference microdilution method by testing two quality control strains and 300 clinical isolates of Candida spp. against fluconazole, voriconazole, posaconazole, and ravuconazole. Reference MIC endpoints were established after 48 h of incubation and YeastOne colorimetric endpoints were established after 24 h of incubation. YeastOne endpoints were determined to be the lowest concentration at which the color in the well changed from red (indicating growth) to purple (indicating growth inhibition) or blue (indicating no growth). Excellent agreement (within two dilutions) between the reference and colorimetric MICs was observed. Overall agreement was 95.4%. Agreement ranged from 92.3% with posaconazole to 98.0% with fluconazole. The YeastOne colorimetric method appears to be comparable to the NCCLS reference method for testing the susceptibility of Candida spp to the new triazoles voriconazole, posaconazole, and ravuconazole.

In a previous study tolerance to amphotericin B (AMB) was found among Candida parapsilosis and C. dubliniensis strains by seeding the whole volumes of wells used for MIC determinations, and minimum fungicidal concentrations (MFC) for non- C. albicans Candida strains were demonstrated to be above the levels safely achievable in serum. As an extension of that study, we performed time-kill assays with 26 blood culture isolates (6 C. albicans , 5 C. parapsilosis , 5 C. krusei , 4 C. glabrata , 3 C. lusitaniae , and 3 C. tropicalis isolates), 3 oropharyngeal C. dubliniensis isolates, 3 AMB-susceptible isolates (ATCC 90028, ATCC 22019, ATCC 6254), and 6 AMB-resistant isolates (ATCC 200955, ATCC 200956, ATCC 200950, ATCC 200951, ATCC 200952, ATCC 200953) using RPMI 1640 medium and 0.12 to 32 μg of AMB per ml and determined the numbers of CFU per milliliter at 0, 2, 4, 8, 12, 24, and 48 h. MFCs and time-kill patterns were species specific (MFCs, ≤1 μg/ml for all C. dubliniensis and C. albicans isolates except AMB-resistant strain ATCC 200955; MFCs, 2 to >16 μg/ml for the other isolates). The times required to reach the fungicidal endpoint (99.9% killing) at four times the MIC were 2 h for C. albicans and C. dubliniensis , 16 h for C. glabrata , 24 h for C. parapsilosis and C. lusitaniae , and ≥40 h for C. tropicalis and C. krusei . The killing rate increased as the AMB concentration was increased up to 2 μg/ml. The highest killing rates were achieved for C. albicans , C. dubliniensis , and C. lusitaniae , while viable C. tropicalis, C. krusei , and C. parapsilosis cells were present after 48 h (MICs, ≤2 μg/ml) when AMB was used at 2 μg/ml. Time-kill curves and MFCs can detect viable cells after 48 h when AMB is used at ≥2 μg/ml. The failure of AMB treatment could be due to its poor killing activity against some species at the concentrations reached in patients' serum.

Purpose of review As new antifungal agents were introduced for the treatment of fungal infections, reliable methods were developed or adapted for the in-vitro susceptibility testing of yeasts and moulds (filamentous fungi). This paper reviews the available methods for antifungal susceptibility testing of moulds as well as the scant data that have been published since 2002 regarding the clinical implications of in-vitro testing. Recent findings Caspofungin and voriconazole have been recently licensed for treatment of certain mould infections that are usually refractory to treatment with established agents. The introduction of new agents and the frequent reports of mould resistance have underscored the role of the laboratory in patient management and initiated the study of mechanisms of resistance in moulds. In 2002, the US National Committee for Clinical Laboratory Standards M38-P document moved to the approved level of development (M38-A document). Although combination therapy and synergistic studies with the new agents have been documented, very little information is available on in-vitro/in-vivo correlations for moulds. Summary The ultimate goal of in-vitro testing is the prediction of the clinical outcome of therapy. The use of National Committee for Clinical Laboratory Standards procedures has led to increased interlaboratory agreement of minimum inhibitory concentrations. Reproducibility of minimum inhibitory concentrations for yeasts has facilitated the establishment of interpretive breakpoints for fluconazole and itraconazole versus Candida spp., but breakpoints are not available for any antifungal against mould species. Although some insights have been documented regarding the potential utility of in-vitro testing for moulds, further documentation of in-vitro versus in-vivo data is needed.

Aspergillus terreus is an uncommon but emerging fungal pathogen, which causes lethal infections that are often refractory to amphotericin B (AmB). In comparison to Aspergillus fumigatus, A. terreus was resistant to the in vitro fungicidal effects of safely achievable concentrations of AmB. These in vitro findings correlated directly with resistance of A. terreus to AmB in experimental invasive pulmonary aspergillosis. Residual fungal pulmonary burden and galactomannan antigenemia demonstrated persistent infection, despite therapy with deoxycholate AmB or liposomal AmB. By comparison, posaconazole and itraconazole resolved GM antigenemia, reduced residual fungal burden, and improved survival. There were no differences in phagocytic host response to A. terreus versus A. fumigatus; however, the rate of conidial germination of A. terreus was slower. The strain of A. terreus with the highest minimum inhibitory and minimum lethal concentration of AmB also had the lowest membrane ergosterol content. The hyphae of A. terreus in vivo displayed distinctive aleurioconidia, which may be a practical microscopic feature for rapid preliminary diagnosis.

Standard conditions are not available for evaluating the minimum fungicidal concentrations (MFCs) of antifungal agents. This multicenter collaborative study investigated the reproducibility in three laboratories of itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B MFCs for 15 selected isolates of Aspergillus spp. After MIC determinations for the 15 isolates in each center by the NCCLS M38-A broth microdilution method with four media, standard RPMI 1640 (RPMI), RPMI with 2% dextrose, antibiotic medium 3 (M3), and M3 with 2% dextrose, MFCs were determined for each isolate-medium-drug combination. MFCs were defined as the lowest drug dilutions that yielded Aspergillus spp. and warrant consideration for introduction into future versions of the M38 document. The role of the MFC under these standardized testing conditions as a predictor of clinical outcome needs to be established in clinical trials.

NCCLS document M38-P describes standard parameters for testing the fungistatic activities (MICs) of established agents against filamentous fungi (molds). This study evaluated the in vitro susceptibilities of 15 Aspergillus flavus isolates, 62 A. fumigatus isolates, and 10 isolates each of A. niger , A. nidulans , and A. terreus to voriconazole, posaconazole, itraconazole, and amphotericin B by the E-test and NCCLS M38-P microdilution methods. The agreement (within 3 dilutions) between methods for voriconazole was independent of the E-test incubation time (93.3 to 100% for four of five species at both incubation times). In contrast, with amphotericin B, itraconazole, and posaconazole, E-test results were more dependent on the incubation time for certain species. For A. fumigatus, posaconazole E-test MICs had better concordance with reference values after 48 h (95.2%) than after 24 h (90%), while the highest agreement for itraconazole MICs was after 24 h (90.3 versus 74.2%) of incubation. Better agreement between the methods was also obtained with 24-h E-test amphotericin B MICs for A. flavus (73.3 versus 26.7%) and A. fumigatus (96.7 versus 64.5%). E-test MICs of the four agents had the lowest percentages of agreement with reference values for A. nidulans (60 to 80%). For isolates for which high MICs were obtained for the four agents by the reference method, high MICs were also obtained by E-test at both 24 and 48 h. The utility of in vitro results of either the E-test or the NCCLS broth microdilution (M38-P) method for Aspergillus spp. needs to be established in clinical trials.

Clinical and Laboratory Standards Institute (CLSI) conditions for testing the susceptibilities of pathogenicSporothrixspecies to antifungal agents are based on a collaborative study that evaluated five clinically relevant isolates ofSporothrixschenckii sensu latoand some antifungal agents. With the advent of molecular identification, there are two basic needs: to confirm the suitability of these testing conditions for all agents andSporothrixspecies and to establish species-specific epidemiologic cutoff values (ECVs) or breakpoints (BPs) for the species. We collected available CLSI MICs/minimal effective concentrations (MECs) of amphotericin B, five triazoles, terbinafine, flucytosine, and caspofungin for 301Sporothrix schenckii sensu stricto, 486S. brasiliensis, 75S. globosa, and 13S. mexicanamolecularly identified isolates. Data were obtained in 17 independent laboratories (Australia, Europe, India, South Africa, and South and North America) using conidial inoculum suspensions and 48 to 72 h of incubation at 35°C. Sufficient and suitable data (modal MICs within 2-fold concentrations) allowed the proposal of the following ECVs forS. schenckiiandS. brasiliensis, respectively: amphotericin B, 4 and 4 μg/ml; itraconazole, 2 and 2 μg/ml; posaconazole, 2 and 2 μg/ml; and voriconazole, 64 and 32 μg/ml. Ketoconazole and terbinafine ECVs forS. brasiliensiswere 2 and 0.12 μg/ml, respectively. Insufficient or unsuitable data precluded the calculation of ketoconazole and terbinafine (or any other antifungal agent) ECVs forS. schenckii, as well as ECVs forS. globosaandS. mexicana. These ECVs could aid the clinician in identifying potentially resistant isolates (non-wild type) less likely to respond to therapy.

Since epidemiological cutoff values (ECVs) using CLSI MICs from multiple laboratories are not available for Candida spp. 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. dubliniensis isolates. 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 fks mutations) and 51 isolates with fks mutations, the species-specific ECVs for anidulafungin and micafungin correctly classified 47 (92.2%) and 51 (100%) of the fks mutants, respectively, as non-WT strains. These ECVs may aid in detecting non-WT isolates with reduced susceptibility to anidulafungin and micafungin due to fks mutations.

The in vitro susceptibilities of 183 clinical yeast isolates to sertaconazole (STZ) were compared to their susceptibilities to clotrimazole (CTZ), econazole (ECZ), ketoconazole (KTZ), miconazole (MNZ), fluconazole (FLZ), itraconazole (ITZ), tioconazole (TCZ), amphotericin B (AMB) and flucytosine (5FC) by using a commercial agar diffusion method. Strains were isolated from vaginal and other superficial clinical samples (18 species of Candida and five strains belonging to other yeast genera). Only one strain (0.5%) was resistant to STZ out of 87.4% of susceptible strains (n=160). The percentage of susceptible strains was higher than those obtained with the other agents evaluated and the percentage of resistant strains was lower than for most of the other antifungals. The pattern of susceptibility of C. albicans to STZ, TCZ, ITZ and CLZ was similar and superior to the pattern of susceptibility of this species to MNZ, ECZ, FLZ, 5FC and KTZ. C. dubliniensis was more susceptible to STZ, MNZ, MNZ, FLZ, ITZ, CLZ than to TCZ, ECZ, 5FC, AMB or KTZ. Ten susceptible strains to STZ were resistant to FLZ and one strain was resistant to ITZ. The overall antifungal activity of STZ in vitro against a wide range of clinically important yeasts from vaginal and cutaneous samples indicates the therapeutic potential of this agent for the treatment of infections caused by these fungi. However, the activity of STZ and the clinical value of in vitro data need to be verified in human clinical trials.

Voriconazole (VfendTM) is a new triazole that currently is undergoing phase III clinical trials. This review summarizes the published data obtained by NCCLS methods on the in vitro antifungal activity of voriconazole in comparison to itraconazole, amphotericin B, fluconazole, ketoconazole and flucytosine. Voriconazole had fungistatic activity against most yeasts and yeastlike species (minimum inhibitory concentrations [MICs] 4 microg/ml, respectively. For four of the six Aspergillus spp. evaluated, voriconazole MICs ( or = 8 microg/ml). Only amphotericin B had good fungistatic activity against the Zygomycetes species (voriconazole MICs ranged from 2 to > 32 microg/ml). Voriconazole showed excellent in vitro activity (MICs < 0.03 to 1.0 microg/ml) against most of the 50 species of dematiaceous fungi tested, but the activity of all the agents was poor against most isolates of Scedosporium prolificans and Phaeoacremonium parasiticum (Phialophora parasitica). Voriconazole had fungicidal activity against most Aspergillus spp., B. dermatitidis, and some dematiaceous fungi. In vitro/in vivo correlations should aid in the interpretation of these results.

As new antifungal agents are introduced for the treatment of infections caused by yeasts and filamentous fungi (moulds), it is important that reliable methods are available for the in vitro testing of both new and established agents. The ultimate goal of in vitro testing is the prediction of the clinical outcome of therapy. The use of the M27-A procedures that were developed by the US National Committee for Clinical Laboratory Standards (NCCLS) has led to increased interlaboratory agreement of minimum inhibitory concentrations (MICs) for yeasts and has facilitated the establishment of interpretive breakpoints for fluconazole and itraconazole. The clinical relevance and limitations of these breakpoints are discussed elsewhere. The focus of this paper is to review the advantages and disadvantages of the available methods for antifungal susceptibility testing of yeasts and moulds as well as the clinical implications of in vitro antifungal resistance.

Posaconazole MICs for 50 Aspergillus fumigatus isolates with distinct genotypes were determined by three methods. MICs were ≥0.5 μg/ml for 5, 11, and 15 strains by the CLSI reference M38-A2, Etest (48-h), and oxygen consumption methods, respectively. The levels of categorical agreement between the results obtained by the CLSI method and those obtained by the oxygen consumption and Etest methods were 80 and 84%, respectively.

Contains fulltext : 53309.pdf (Publisher’s version ) (Closed access) The European Committee on Antimicrobial Susceptibility Testing (EUCAST) Subcommittee on Antifungal Susceptibility Testing recently published a standard for determining the susceptibility of fermentative yeasts to antifungals. From the beginning, the EUCAST and its North American counterpart, the CLSI, decided to work together in order to establish common standards. As part of this exercise, the susceptibility of a set of 475 yeast isolates was tested by both standards. The intraclass correlation coefficient and the equations defining the linear regression between both methods were estimated. Both methods produced very similar results, with an intraclass correlation coefficient of 0.954 (0.945 to 0.962), although linear regression analysis shows that the EUCAST standard resulted in slightly lower MICs. There were only eight isolates showing at least four twofold dilution MIC differences between both standards. After 24 h of incubation, the MICs obtained by the CLSI method were equivalent to those obtained by the EUCAST standard. In summary, both methods produce very similar MICs, indicating that methodology does not pose any obstacle to obtaining uniform standards for antifungal susceptibility testing of yeasts.