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Objectives: A post hoc analysis used pooled STRIVE/ReSTORE trial data to determine outcomes with rezafungin versus caspofungin by Candida species and antifungal susceptibility., Methods: The efficacy and safety of once weekly rezafungin 400/200 mg versus once daily caspofungin 70/50 mg was demonstrated in the randomized, double-blind phase 2 STRIVE (NCT02734862) and phase 3 ReSTORE (NCT03667690) trials involving adults with candidaemia and/or invasive candidiasis. In this analysis, data were pooled for patients with a documented Candida infection within 96 hours of randomization who also received ≥1 dose of study drug. Treatment outcomes were evaluated by Candida species and baseline MICs. Susceptibility was determined using European Committee on Antimicrobial Susceptibility Testing E.Def 7.4 broth microdilution methodology, with Tween 20-supplemented medium for rezafungin., Results: A total of 294 patients were included (rezafungin: N = 139, caspofungin: N = 155). Susceptibility testing at baseline identified three rezafungin non-susceptible isolates. Day 14 global cure rates were numerically similar between groups for C. albicans (rezafungin: 61.0% [36/59], caspofungin: 65.2% [45/69]) and C. tropicalis (rezafungin: 70.4% [19/27], caspofungin: 63.6% [14/22]), but higher with rezafungin than caspofungin for C. glabrata (rezafungin: 71.1% [27/38], caspofungin: 60.0% [21/35]) and C. parapsilosis (rezafungin: 78.6% [11/44], caspofungin: 55.6% [15/27]). Day 30 all-cause mortality rates were numerically similar between groups for C. albicans (rezafungin: 22.0% [13/59], caspofungin: 18.8% [13/69]) and C. glabrata (rezafungin: 15.8% [6/38], caspofungin: 11.4% [4/35]), but higher with caspofungin than rezafungin for C. tropicalis (rezafungin: 18.5% [5/27], caspofungin: 31.8% [2/22]) and C. parapsilosis (rezafungin: 7.1% [1/14], caspofungin: 29.6% [8/27]). Day 5/14 mycological eradication rates were numerically similar between treatments for C. albicans and C. parapsilosis, but higher with rezafungin for C. glabrata and C. tropicalis. Outcomes by Candida species were not associated with treatment-specific MICs., Discussion: Rezafungin appears to be an effective treatment for candidaemia/invasive candidiasis irrespective of baseline Candida species., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: Reported amphotericin B resistance rates for Candida auris vary considerably. This may reflect clinically relevant differences in susceptibility, technical issues with testing, or adoption of a clinical breakpoint that bisects the wild-type population. We compared reference methods and two gradient diffusion strips using a shared C. auris strain collection., Methods: Forty C. auris strains from nine U.S. states and ≥3 clades were included. Fourteen MIC data sets were generated using European Committee on Antimicrobial Susceptibility Testing (EUCAST) E.Def 7.4, Clinical and Laboratory Standards Institute (CLSI) M27Ed4, Etest, and MIC gradient test strip (MTS, Liofilchem) MICs. MICs ≤1 mg/L were classified as susceptible., Results: EUCAST and CLSI amphotericin B MIC testing were robust across the included method variables. The modal MIC was 1 mg/L, distributions unimodal and narrow with similar geometric mean (GM)-MICs (0.745-1.072); however, susceptibility classification varied (0-28% resistance). Gradient diffusion strip testing resulted in wider and bimodal distributions for 8/9 data sets. If adopting, per manufacturer's protocol, double inoculation for the Etest method, the modal MIC increased to 2-4 mg/L and resistance rates to 45-63% versus 25-30% with the single inoculation. The EUCAST, CLSI, Etest, and MTS strip MICs correlated to the optical density of drug-free control EUCAST wells, suggesting that some isolates grew better than others and that this was associated with MIC., Discussion: The EUCAST and CLSI MIC results were in close agreement, whereas the strip test showed wider and bimodal distributions with reader to reader and centre to centre variation. Our study adds to the concern for commercial MIC testing of amphotericin B against C. auris and suggests the current breakpoint leads to random susceptibility classification., (Copyright © 2024 European Society of Clinical Microbiology and Infectious Diseases. All rights reserved.)

As comparative pharmacokinetic/pharmacodynamic (PK/PD) studies of liposomal amphotericin B (L-AMB) against Candida spp. are lacking, we explored L-AMB pharmacodynamics against different Candida species in an in vitro PK/PD dilution model. Eight Candida glabrata , Candida parapsilosis , and Candida krusei isolates (EUCAST/CLSI AMB MIC 0.125-1 mg/L) were studied in the in vitro PK/PD model simulating L-AMB C max = 0.25-64 mg/L and t 1/2 = 9 h. The model was validated with one susceptible and one resistant Candida albicans isolate. The C max /MIC-log 10 CFU/mL reduction from the initial inoculum was analyzed with the E max model, and Monte Carlo analysis was performed for the standard (3 mg/kg with C max = 21.87 ± 12.47 mg/L) and higher (5 mg/kg with C max = 83 ± 35.2 mg/L) L-AMB dose. A ≥1.5 log 10 CFU/mL reduction was found at L-AMB C max = 8 mg/L against C. albicans , C. parapsilosis , and C. krusei isolates (MIC 0.25-0.5 mg/L) whereas L-AMB C max ≥ 32 mg/L was required for C. glabrata isolates. The in vitro PK/PD relationship followed a sigmoidal pattern ( R 2 ≥ 0.85) with a mean C max /MIC required for stasis of 2.1 for C. albicans (close to the in vivo stasis), 24/17 (EUCAST/CLSI) for C. glabrata , 8 for C. parapsilosis , and 10 for C. krusei . The probability of target attainment was ≥99% for C. albicans wild-type (WT) isolates with 3 mg/kg and for wild-type isolates of the other species with 5 mg/kg. L-AMB was four- to eightfold less active against the included non- C . albicans species than C. albicans . A standard 3-mg/kg dose is pharmacodynamically sufficient for C. albicans whereas our data suggest that 5 mg/kg may be recommendable for the included non- C . albicans species., Competing Interests: The authors declare no conflict of interest.

Since its inception in 2002, the EUCAST Antifungal Susceptibility Testing Subcommittee (AFST) has developed and refined susceptibility testing methods for yeast, moulds and dermatophytes, and established epidemiological cut-off values and breakpoints for antifungals. For yeast, three challenges have been addressed. Interpretation of trailing growth in fluconazole susceptibility testing, which has been proven without impact on efficacy if below the 50% endpoint. Variability in rezafungin MIC testing due to laboratory conditions, which has been solved by the addition of Tween 20 to the growth medium in E.Def 7.4. And third, interpretation of MICs for rare yeast with no breakpoints, where recommendations have been established for MIC-based clinical advice. For moulds, refinements include the validation of spectrophotometer reading for A. fumigatus to facilitate objective MIC determination, and for dermatophytes the establishment of a microdilution method with automated reading and a selective medium to minimise the risk of contaminations. Recent initiatives involve development and validation of agar-based screening assays for detection of potential azole and echinocandin resistance in A. fumigatus and Aspergillus species, respectively, and of terbinafine resistance in Trichophyton species. Moreover, the development of a EUCAST guidance document for molecular resistance testing represents an advancement, particularly for identifying target gene alterations associated with resistance. In summary, EUCAST AFST continues to play a pivotal role in standardizing AFST and facilitating accurate interpretation of susceptibility data for clinical decision-making. Adoption of EUCAST breakpoints for commercial test methods, however, requires thorough validation to ensure concordance with EUCAST reference testing species-specific MIC distributions., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Dermatophytosis is commonly encountered in the dermatological clinics. The main aetiological agents in dermatophytosis of skin and nails in humans are Trichophyton (T.) rubrum, T. mentagrophytes and T. interdigitale (former T. mentagrophytes-complex). Terbinafine therapy is usually effective in eradicating infections due to these species by inhibiting their squalene epoxidase (SQLE) enzyme, but increasing numbers of clinically resistant cases and mutations in the SQLE gene have been documented recently. Resistance to antimycotics is phenotypically determined by antifungal susceptibility testing (AFST). However, AFST is not routinely performed for dermatophytes and no breakpoints classifying isolates as susceptible or resistant are available, making it difficult to interpret the clinical impact of a minimal inhibitory concentration (MIC)., Summary: PubMed was systematically searched for terbinafine susceptibility testing of dermatophytes on October 20, 2020, by two individual researchers. The inclusion criteria were in vitro terbinafine susceptibility testing of Trichophyton (T.) rubrum, T. mentagrophytes and T. interdigitale with the broth microdilution technique. The exclusion criteria were non-English written papers. Outcomes were reported as MIC range, geometric mean, modal MIC and MIC50 and MIC90 in which 50 or 90% of isolates were inhibited, respectively. The reported MICs ranged from <0.001 to >64 mg/L. The huge variation in MIC is partly explained by the heterogeneity of the Trichophyton isolates, where some originated from routine specimens (wild types) whereas others came from non-responding patients with a known SQLE gene mutation. Another reason for the great variation in MIC is the use of different AFST methods where MIC values are not directly comparable. High MICs were reported particularly in isolates with SQLE gene mutation. The following SQLE alterations were reported: F397L, L393F, L393S, H440Y, F393I, F393V, F415I, F415S, F415V, S443P, A448T, L335F/A448T, S395P/A448T, L393S/A448T, Q408L/A448T, F397L/A448T, I121M/V237I and H440Y/F484Y in terbinafine-resistant isolates., (© 2021 S. Karger AG, Basel.)

Background: Antifungal drug resistance in dermatophytes was first reported shortly after the turn of the millennium and has today been reported in Trichophyton and occasionally in Microsporum, but not in Epidermophyton species. Although drug resistance in dermatophytes is not routinely investigated, resistance in Trichophyton spp. is increasingly reported worldwide. The highest rates are observed in India (36% and 68% for terbinafine (MIC ≥4 mg/L) and fluconazole (MICs ≥16 mg/L), respectively), and apparently involve the spread of a unique clade related to the Trichophyton mentagrophytes/Trichophyton interdigitale complex., Objectives: The European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has released a new method (E.Def 11.0) for antifungal susceptibility testing against microconidia-forming dermatophytes including tentative MIC ranges for quality control strains and tentative breakpoints against Trichophyton rubrum and T. interdigitale. Here, the details of the new procedure E.Def 11.0 are described., Sources: This technical note is based on the multicentre validation of the EUCAST dermatophyte antifungal susceptibility testing method, the mould testing method (E.Def 9.3.2) and the updated quality control tables for antifungal susceptibility testing document, v 5.0 (available on the EUCAST website)., Contents: The method is based on the EUCAST microdilution method for moulds but significant differences include: (a) an altered test medium selective for dermatophytes; (b) an altered incubation time and temperature; and (c) a different end-point criterion (spectrophotometric determination) of fungal growth. It can easily be implemented in laboratories already performing EUCAST microdilution methods and has been validated for terbinafine, voriconazole, itraconazole and amorolfine against T. rubrum and T. interdigitale., Implications: This standardized procedure with automated end-point reading will allow broader implementation of susceptibility testing of dermatophytes and so facilitate earlier appropriate therapy. This is important, as resistance is rapidly emerging and largely underdiagnosed., (Copyright © 2020. Published by Elsevier Ltd.)

Rezafungin (formerly CD101) is a novel echinocandin in clinical development. EUCAST epidemiological cutoff values (ECOFFs) have not yet been established. We determined the in vitro activity of rezafungin and comparators against 1,293 Nordic yeast isolates and 122 Indian Candida auris isolates and established single-center wild-type upper limits (WT-UL). The isolates (19 Candida spp. and 13 other yeast species) were identified using Chromagar; matrix-assisted laser desorption ionization-time of flight (MALDI-TOF); and, when needed, internal transcribed spacer sequencing. EUCAST E.Def 7.3.1 susceptibility testing included rezafungin, anidulafungin, micafungin, amphotericin B, and fluconazole. WT-UL were established following EUCAST principles for visual and statistical ECOFF setting. fks target genes were sequenced for rezafungin non-wild-type isolates. EUCAST clinical breakpoints for fungi version 9.0 were adopted for susceptibility classification. Rezafungin had species-specific activity similar to that of anidulafungin and micafungin. On a milligram-per-liter basis, rezafungin was overall less active than anidulafungin and micafungin but equally or more active than fluconazole and amphotericin B against the most common Candida species, except C. parapsilosis We identified 37 (3.1%) rezafungin non-wild-type isolates of C. albicans (1.9%), C. glabrata (3.0%), C. tropicalis (2.7%), C. dubliniensis (2.9%), C. krusei (1.2%), and C. auris (14.8%). Alterations in Fks hot spots were found in 26/26 Nordic and 8/18 non-wild-type C. auris isolates. Rezafungin displayed broad in vitro activity against Candida spp., including C. auris Adopting WT-UL established here, few Nordic strains, but a significant proportion of C. auris isolates, had elevated MICs with mutations in fks target genes that conferred echinocandin cross-resistance. fks1 mutations raised rezafungin MICs notably less than anidulafungin and micafungin MICs in C. auris ., (Copyright © 2020 American Society for Microbiology.)

Rezafungin is a new long-acting echinocandin currently in phase 3 development. Epidemiological cutoff values are necessary for breakpoint setting but have not been established due to unexplained interlaboratory MIC variations observed in a prior multicenter study. Here we investigated if the choice of microtiter plates affected the variability when anidulafungin was included as a comparator. Testing by the EUCAST E.Def 7.3.1 reference method using tissue and cell culture-treated polystyrene plates (TC plates) and untreated polystyrene plates (UT plates) from four manufacturers was performed. Six control strains ( Candida albicans , n  = 3; C. krusei , n  = 2; C. parapsilosis , n  = 1) were tested (520 MICs). Subsequently, 5 or 6 wild-type isolates and 4 or 5 fks mutants of C. albicans , C. glabrata , C. krusei , C. parapsilosis (wild type only), and C. tropicalis were tested (930 MICs). For each strain-plate combination, ≥98% of the repetitive MICs were within 3 dilutions. The rezafungin modal MICs for the collated C. albicans control strain distributions were 0.016 mg/liter across TC plates but 0.03 mg/liter across UT plates, whereas they were 0.004 mg/liter and 0.016 mg/liter, respectively, for anidulafungin. The difference was most pronounced with Falcon plates and was not observed for C. krusei and C. parapsilosis Eleven rezafungin MICs for mutants overlapped with the MICs for wild-type isolates (TC plates, n  = 4; UT plates, n  = 7). For anidulafungin, five overlaps (all UT plates) were observed. Most overlaps (rezafungin, n  = 5; anidulafungin, n  = 3) were caused by fks mutants of C. tropicalis (Fks1, F650F/L) and C. glabrata (Fks2. D666Y; rezafungin, n  = 2; anidulafungin, n  = 1). Interlaboratory variation was low. The use of TC plates resulted in lower MICs, particularly for C. albicans and Falcon plates, ad this was more often the case for anidulafungin than for rezafungin. Adoption of TC plates for EUCAST antifungal susceptibility testing would improve interlaboratory reproducibility and the separation of non-wild-type and wild-type strains., (Copyright © 2019 American Society for Microbiology.)

Background: Reference antifungal susceptibility testing of echinocandins against Aspergillus spp. relies on the determination of the minimal effective concentration, which is difficult to perform, time-consuming and subjective. We developed and evaluated in a multicentre study an agar-based screening method for echinocandin susceptibility testing of Aspergillus spp., Methods: Forty WT isolates [10 Aspergillus fumigatus species complex (SC), 10 Aspergillus flavus SC, 10 Aspergillus terreus SC and 10 Aspergillus niger SC] and 4 non-WT A. fumigatus isolates with or without known fks alterations were used. The optimal test conditions and stability over time were evaluated in preliminary studies monitoring colony growth. Twenty-microlitre aliquots of 1-2 McFarland inocula in 0.1% Tween 20 aqueous solution were added to each well and plates were incubated for 24/48 h at 35 ± 2°C. Subsequently, all isolates were tested blindly at three centres using four-well screening plates, containing anidulafungin, caspofungin, micafungin or no antifungal in each of the four wells, respectively., Results: WT isolates produced fluffy colonies on drug-free agar wells only. The non-WT isolates produced fluffy colonies on echinocandin-containing and control agar wells. Using the echinocandin concentrations of 0.25 mg/L anidulafungin, 1 mg/L caspofungin and 0.125 mg/L micafungin, and the compact (non-fluffy) versus fluffy colony morphology endpoint, all centres successfully discriminated non-WT and WT strains even after 24 h. Among the three echinocandins, anidulafungin produced the clearest endpoints., Conclusions: The four-well plate agar method is suitable for echinocandin susceptibility screening of Aspergillus spp. and can be used to detect echinocandin non-WT isolates., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: Azole-resistant Aspergillus fumigatus is emerging worldwide. Reference susceptibility testing methods are technically demanding and no validated commercial susceptibility tests for moulds currently exist. In this multicentre study a 4-well azole-containing screening agar method was evaluated using clinically relevant isolates., Methods: Forty WT and 39 cyp51A mutant A. fumigatus [G54 (n = 10), M220 (n = 10), TR34/L98H (n = 9) and TR46/Y121F/T289A (n = 10)] were tested individually and as simulated mixed samples (sampling 4 WT and 1 mutant colonies). EUCAST MICs were determined following E.Def 9.3. In-house and commercial 4-well plates containing agars supplemented with 4 mg/L itraconazole, 1 mg/L voriconazole, 0.5 mg/L posaconazole and no antifungal, respectively, were evaluated. Growth was scored (0-3) by two independent observers in three laboratories. Inter-plate, inter-observer, essential and categorical agreement, sensitivity and specificity were calculated., Results: CYP51A genotype and antifungal compound-specific MICs and growth patterns were documented. The inter-observer agreement was excellent with 86%-99% identical scores (range 80%-100%) for both plates. The qualitative agreement (no growth versus growth) was excellent (median 95%-100%, range 87%-100%, overall). The overall sensitivity and specificity for the 4-well plate (no growth versus growth) was 99% (range 97%-100%) and 99% (95%-100%), respectively. The sensitivity for simulated WT/mutant specimens was 94% (range 83%-100%) for the WT-TR34/L98H combination, but 100% for the WT/G54W combination. The performance remained unchanged using only itraconazole- and voriconazole-containing agars, but was lower for the other combinations., Conclusions: Implementation of the 4-well screening plate in routine laboratories will allow easy and reliable detection of the most common azole-resistant A. fumigatus., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In our part of the world invasive fungal infections include invasive yeast infections with Candida as the absolutely dominating pathogen and invasive mould infections with Aspergillus as the main organism. Yeasts are part of our normal micro-flora and invasive infections arise only when barrier leakage or impaired immune function occurs. On the contrary, moulds are ubiquitous in the nature and environment and their conidia inhaled at a daily basis. Hence invasive mould infections typically arise from the airways whereas invasive yeast infections typically enter the bloodstream causing fungaemia. Candida is by far the most common fungal blood stream pathogen; hence this genus has been the main focus of this thesis. As neither the Danish epidemiology nor the susceptibility of fungal pathogens was well described when we initiated our studies we initially wanted to be able to include animal models in our work. Therefore, a comprehensive animal study was undertaken comparing the virulence in a haematogenous mouse model of eight different Candida species including the five most common ones in human infections (C. albicans, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis and in addition three rarer species C. guilliermondii, C. lusitaniae and C. kefyr). We found remarkable differences in the virulence among these species and were able to group the species according to decreasing virulence in three groups I: C. albicans and C. tropicalis, II: C. glabrata, C. lusitaniae and C. kefyr, and III: C. krusei, C. parapsilosis and C. guilliermondii. Apart from being necessary for our subsequent animal experiments exploring in vivo antifungal susceptibility, these findings also helped us understand at least part of the reason for the differences in the epidemiology and the pitfalls associated with the establishment of genus rather than species specific breakpoints. In example, it was less surprising that C. albicans has been the dominant pathogen and associated with a significantly higher mortality than C. parapsilosis and that C. glabrata and C. krusei mainly emerged in the post fluconazole era and in settings with azole selection pressure. Moreover, it was less surprising that infections due to mutant C. albicans isolates with echinocandin MICs of 1-2 mg/l were not good targets for the echinocandins despite the fact that the outcome for infections involving wild type C. parapsilosis for which similar echinocandin MICs were similar was not inferior. This last observation highlights the importance of providing optimal, reproducible and sensitive reference susceptibility testing methods and notably accompanied by appropriate breakpoints that allow a separation and detection of susceptible and resistant isolates against which the commercial tests can be validated. Correct detection of resistant isolates is for obvious reasons crucial in order to avoid inappropriate treatment. And if the test method cannot correctly identify resistant isolates it makes little sense performing susceptibility testing at all. On the other hand misclassification of susceptible isolates as resistant is also an issue as the patient is thereby deprived an appropriate treatment option among the few available. These comments may seem very basic; nevertheless, it has taken a lot of effort and patience to optimise the susceptibility tests, understand the variability issue for caspofungin testing, to provide appropriate breakpoints that reduced misclassifications to a minimum and not the least to facilitate a harmonisation of breakpoints across the Atlantic sea. We initially realised that the CLSI method and echinocandin breakpoint misclassified resistant isolates. This was due to the endorsement of a single susceptibility breakpoint across all Candida species and the three echinocandins and therefore set as high as 2 mg/l in order to include and not bisect the C. parapsilosis population. Through our comprehensive comparisons of echinocandin susceptibility testing using EUCAST, CLSI, Etest, disk diffusion and agardilution with different media with and without the supplementation of bovine serum albumin we provided data that supported the current reference methodologies, provided that drug and species specific breakpoints were selected. Moreover, the issues of caspofungin variability and of overlap between micafungin MICs for wild type and mutant C. glabrata populations were handled and understood. Anidulafungin EUCAST breakpoints are now published and publically available at the www.eucast.org website and anidulafungin testing recommended as a marker for the echinocandin class. Our antifungal EUCAST breakpoint setting approach has been adopted by the CLSI leading to revision and harmonisation of breakpoints for the three echinocandins, fluconazole and voriconazole. Our epidemiological studies developed gradually over the years following our observation of a notably high incidence rate of fungaemia compared to our Nordic neighbours. Initially, we anticipated that our high incidence was at least in part related to the fact that the capture area for our initial studies was skewed with dominance of university hospitals and inclusion of all centres performing solid organ or bone marrow transplantation. However, when the surveillance was extended to the entire country, the high incidence remained a consistent finding and we even demonstrated that the incidence rate is still increasing. Additionally we demonstrated a changing epidemiology as a high and increasing proportion of the cases involved fluconazole resistant isolates and that this proportion also was significantly higher than in the other Nordic countries. This appears to be related to a significantly higher and increasing fluconazole use in Denmark than in the other Nordic countries. Exploring the incidence rate for the individual hospitals and age groups we demonstrated not unexpectedly that the incidence rate was highest at the university centres, but also that whereas the age specific incidence rate was comparable in children and the younger adults with that in the other Nordic countries it was notably higher in the elderly population. This in combination with the fact that it is increasing specifically in the elderly men and that the incidence rates in the Nordic countries were comparable two decades back suggest that host specific factors including antifungal consumption rather than genetic differences in susceptibility to fungaemia account for the differences, and hence that it is possibly modifiable by implementing relevant measures. Hence, it was important to investigate the underlying clinical conditions and diagnostic factors and the outcome in Danish patients with fungaemia. In this study we demonstrated that two thirds of the patients had received abdominal surgery or intensive care treatment prior to the development of the fungaemia, a proportion that is higher than in most other studies. We also demonstrated that unless surveillance cultures are handled with careful attention the detection of non-C. albicans may go unnoticed which imply a risk of inappropriate treatment in cases involving intrinsically resistant species. Finally, we demonstrated the necessity of using a fungal blood culture flask in addition to the conventional aerobic and anaerobic ones if all C. glabrata infections (BACTEC) and all polymicrobial infections (BacT/ALERT) are to be diagnosed. Hence close monitoring with the use of improved diagnostic options (such as frequent BC including a mycosis bottle, surveillance cultures and mannan antigen and antibody screening) of particularly ICU and abdominal surgery patients may help better identify patients with fungaemia and allow early treatment. With respect to treatment and outcome we found that the fluconazole resistant species C. glabrata, C. krusei and S. cerevisiae were significantly more common in patients exposed to at least 7 days of antifungal prophylaxis (mainly fluconazole). We also demonstrated that a significant proportion of the patients initially received inappropriate antifungal treatment and that the outcome was significantly improved when patients with C. glabrata received caspofungin as their first line agent. This has today been incorporated in the Danish and international treatment guidelines. The prevalence of acquired antifungal resistance remained very low throughout the study period, however, we may only have detected the tip of the resistance iceberg due to the study design, where for epidemiological purposes only the initial isolate was included with the lowest antifungal exposure, and as the susceptibility tests and breakpoints were not optimal for the detection of resistance at all centres. Most Danish laboratories either do not susceptibility test or use commercial tests such as the Etest and later the VITEK system. These are FDA approved with the CLSI breakpoints which, as we have shown, have been far too high to reliably detect resistance and which despite having now been revised and harmonised are not yet in formal CLSI print and hence not incorporated in the product inserts for the commercial tests on the market. Finally, even for laboratories aware of these issues challenges are still ahead as the official breakpoints not always lead to a correct classification for MIC endpoints obtained using the commercial systems or as the commercial tests do not include a relevant concentration range for all drug bug combinations. I thus believe, the studies included in this thesis have contributed significantly to the understanding of the interplay between the Candida virulence, epidemiology and susceptibility and the importance of appropriate diagnostics and treatment choice. It is my hope that we thereby have contributed to the improved options and outcome for patients with candidaemia.

The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has revised the EDef 7.1 document on the method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for fermentative yeasts. Changes are: dimethylsulphoxide is now the recommended solvent for caspofungin, micafungin and fluconazole; the shelf-life of plates containing the echinocandins prepared from stock solutions in dimethylsulphoxide is extended to 6 months at -80°C; testing of amphotericin and Cryptococcus has been incorporated; and minimum inhibitory concentration ranges for quality control strains and anidulafungin are included., (© 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.)

Disk diffusion testing has recently been standardized by the CLSI, and susceptibility breakpoints have been established for several antifungal compounds. For caspofungin, 5-μg disks are approved, and for micafungin, 10-μg disks are under evaluation. We evaluated the performances of caspofungin and micafungin disk testing using a panel of Candida isolates with and without known FKS echinocandin resistance mechanisms. Disk diffusion and microdilution assays were performed strictly according to CLSI documents M44-A2 and M27-A3. Eighty-nine clinical Candida isolates were included: Candida albicans (20 isolates/10 mutants), C. glabrata (19 isolates/10 mutants), C. dubliniensis (2 isolates/1 mutant), C. krusei (16 isolates/3 mutants), C. parapsilosis (14 isolates/0 mutants), and C. tropicalis (18 isolates/4 mutants). Quality control strains were C. parapsilosis ATCC 22019 and C. krusei ATCC 6258. The correlations between zone diameters and MIC results were good for both compounds, with identical susceptibility classifications for 93.3% of the isolates by applying the current CLSI breakpoints. However, the numbers of fks hot spot mutant isolates misclassified as being susceptible (S) (very major errors [VMEs]) were high (61% for caspofungin [S, ≥11 mm] and 93% for micafungin [S, ≥14 mm]). Changing the disk diffusion breakpoint to S at ≥22 mm significantly improved the discrimination. For caspofungin, 1 VME was detected (a C. tropicalis isolate with an F76S substitution) (3.5%), and for micafungin, 10 VMEs were detected, the majority of which were for C. glabrata (8/10). The broadest separation between zone diameter ranges for wild-type (WT) and mutant isolates was seen for caspofungin (6 to 12 mm versus -4 to 7 mm). In conclusion, caspofungin disk diffusion testing with a modified breakpoint led to excellent separation between WT and mutant isolates for all Candida species.

A multicenter study was conducted to assess the accuracy of the ISO standard 20776-1 and the serial 2-fold dilution procedures for antifungal susceptibility testing. Fluconazole trays can be accurately prepared by following ISO and serial dilution schemes. However, itraconazole trays showed a significant lack of reproducibility that was independent of which method was followed.

Susceptibility testing of fungi and development of interpretative breakpoints has become increasingly important due to the growing incidence of invasive fungal infections, the number and classes of antifungals, and the emerging reports of acquired resistance. The subcommittee on antifungal susceptibility testing of the European Committee on Antibiotic Susceptibility Testing (EUCAST) has developed standards for susceptibility testing of fermentative yeasts and molds as well as proposing breakpoints for fluconazole and voriconazole against Candida. The aim of this work is to describe the EUCAST process of setting breakpoints for antifungals. Five aspects are evaluated during the process of developing breakpoints: 1) the most common dosage used in each European country, 2) the definition of the wild-type population for each target microorganism at the species level and the determination of epidemiological cutoffs, 3) the drug's pharmacokinetics and 4) pharmacodynamics, including Monte Carlo simulations, and 5) the correlation of MICs with clinical outcome of patients treated with the compound. When insufficient data are available (e.g., due to lack of information on the clinical outcome of infections caused by isolates with an elevated MIC), epidemiological cutoff values, rather than breakpoints, are recommended until the necessary information becomes available., (Copyright 2010 Prous Science, S.A.U. or its licensors. All rights reserved.)

This study compared nine susceptibility testing methods and 12 endpoints for anidulafungin, caspofungin, and micafungin with the same collection of blinded FKS hot spot mutant (n = 29) and wild-type isolates (n = 94). The susceptibility tests included EUCAST Edef 7.1, agar dilution, Etest, and disk diffusion with RPMI-1640 plus 2% glucose (2G) and IsoSensitest-2G media and CLSI M27A-3. Microdilution plates were read after 24 and 48 h. The following test parameters were evaluated: fks hot spot mutants overlapping the wild-type distribution, distance between the two populations, number of very major errors (VMEs; fks mutants misclassified as susceptible), and major errors (MEs; wild-type isolates classified as resistant) using a wild-type-upper-limit value (WT-UL) (two twofold-dilutions higher than the MIC(50)) as the susceptibility breakpoint. The methods with the lowest number of errors (given as VMEs/MEs) across the three echinocandins were CLSI (12%/1%), agar dilution with RPMI-2G medium (14%/0%), and Etest with RPMI-2G medium (8%/3%). The fewest errors overall were observed for anidulafungin (4%/1% for EUCAST, 4%/3% for CLSI, and 3%/9% for Etest with RPMI-2G). For micafungin, VME rates of 10 to 71% were observed. For caspofungin, agar dilution with either medium was superior (VMEs/MEs of 0%/1%), while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal (VMEs of 7%, 10%, and 10%, respectively). Applying the CLSI breakpoint (S

The fluconazole MIC distributions for Candida glabrata from testing 34 different clinical isolates and performing 51 tests on a single isolate mirrored each other. Since what is perceived as biological variation in isolates without resistance mechanisms is mainly methodological variation, breakpoints which divide this distribution not only lack a sound biological basis but also result in poor reproducibility of susceptibility characterization. This makes 2, 4, 8, and possibly 16 microg/ml unsuitable breakpoints for C. glabrata and fluconazole.

IntroductionThe epidemiology of invasive Candida infections is evolving. Infections caused by non-albicans Candida spp. are increasing; however, the antifungal pipeline is more promising than ever and is enriched with repurposed drugs and agents that have new mechanisms of action. Despite progress, unmet needs in the treatment of invasive candidiasis remain, and there are still too few antifungals that can be administered orally or that have CNS penetration.Areas coveredThe authors shed light on those antifungal agents active against Candida that are in early- and late-stage clinical development. Mechanisms of action and key pharmacokinetic and pharmacodynamic properties are discussed. Insights are offered on the potential future roles of the investigational agents MAT-2203, oteseconazole, ATI-2307, VL-2397, NP-339, and the repurposed drug miltefosine.Expert opinionIbrexafungerp and fosmanogepix have novel mechanisms of action and will provide effective options for the treatment of Candida infections (including those caused by multiresistant Candida spp). Rezafungin, an echinocandin with an extended half-life allowing for once weekly administration, will be particularly valuable for outpatient treatment and prophylaxis. Despite this, there is an urgent need to garner clinical data on investigational drugs, especially in the current rise of azole-resistant and multidrug-resistant Candida spp.

Background: Increased fluconazole and echinocandin resistance in Candida glabrata requires prompt detection in routine settings. A phenotypic test based on the EUCAST E.DEF 7.3.2 protocol was developed for the detection of fluconazole- and anidulafungin-resistant isolates utilizing the colorimetric dye XTT.Methods: Thirty-one clinical C. glabrata isolates, 11 anidulafungin resistant and 14 fluconazole resistant, were tested. After optimization studies, 0.5-2.5 × 105 cfu/mL of each isolate in RPMI 1640 + 2% d-glucose medium containing 100 mg/L XTT + 0.78 μΜ menadione and 0.06 mg/L anidulafungin (S breakpoint) or 16 mg/L fluconazole (I breakpoint) in 96-well flat-bottom microtitration plates were incubated at 37°C for 18 h; we also included drug-free wells. XTT absorbance was measured at 450 nm every 15 min. Differences between the drug-free and the drug-treated wells were assessed using Student's t-test at different timepoints. ROC curves were used in order to identify the best timepoint and cut-off.Results: The XTT absorbance differences between fluconazole-containing and drug-free wells were significantly lower for the resistant isolates compared with susceptible increased exposure isolates (0.08 ± 0.05 versus 0.25 ± 0.06, respectively, P = 0.005) at 7.5 h, with a difference of <0.157 corresponding to 100% sensitivity and 94% specificity for detection of resistance. The XTT absorbance differences between anidulafungin-containing and drug-free wells were significantly lower for the resistant isolates compared with susceptible isolates (0.08 ± 0.07 versus 0.200 ± 0.03, respectively, P < 0.001) at 5 h, with a difference of <0.145 corresponding to 91% sensitivity and 100% specificity, irrespective of underlying mutations.Conclusions: A simple, cheap and fast phenotypic test was developed for detection of fluconazole- and anidulafungin-resistant C. glabrata isolates. [ABSTRACT FROM AUTHOR]

Objectives: Current reference susceptibility testing methods of Aspergillus require visual reading, which is subjective and necessitates experienced staff. We compared spectrophotometric and visual MIC reading of EUCAST E.Def 9.3.2 susceptibility testing of Aspergillus fumigatus for a large collection of isolates with different azole resistance mechanisms.Methods: A. fumigatus (n = 200) were examined, including 62 WT and 138 non-WT with the following alterations: TR34/L98H (n = 57), TR46/Y121F/T289A (n = 54) or single point mutations (n = 27). EUCAST E.Def 9.3.2 susceptibility testing was performed for amphotericin B, itraconazole, voriconazole, posaconazole and isavuconazole. MICs were determined after 48 h of incubation visually and spectrophotometrically, as the lowest concentration corresponding to a 1%, 3%, 5%, 10% or 15% OD increase above the background OD. The best spectrophotometric endpoint (SPE) was identified based on the highest essential agreement (EA; ±1 two-fold dilution) and categorical agreement (CA) and fewer very major errors (VMEs) and major errors (MEs).Results: Τhe best SPEs were 5% and 10% for all drugs. The best agreement between visual and spectrophotometric MICs was found with the 10% growth endpoint, which resulted in identical median MICs with 90% of differences being ≤1 two-fold and higher EA (91%-100%) and CA (100%) and no VMEs and MEs compared with the 5% endpoint (77%-100%, 96%-98%, 0% and 0%-4%, respectively).Conclusions: Spectrophotometric MIC reading can be used for A. fumigatus susceptibility testing and for detecting azole resistance. A visual inspection of the plate should be performed to confirm equal inoculation, absence of well contamination and proper growth, and to identify potential uncommon phenotypes or subpopulations. [ABSTRACT FROM AUTHOR]

Background: Since the ISO standard 20776-1 and serial dilution procedures were compared in 2010 for fluconazole and itraconazole, several new antifungals that are hydrophobic and highly potent have been introduced.Objectives: To investigate the impact of the number of tip changes during serial dilution, and ISO and serial dilution for nine antifungals.Methods: EUCAST E.Def 7.3.2 with serial (0-10 tip changes) and ISO dilution. Candida parapsilosis ATCC 22019, Candida albicans ATCC 64548, C. albicans CNM CL-F8555, Candida krusei ATCC 6258, Aspergillus flavus ATCC 204304 and clinical isolates (n = 5) of C. albicans, Candida dubliniensis, Candida glabrata, C. krusei, A. flavus and Aspergillus terreus were included. GM MICs were compared for ISO and serial dilution and with QC values where available.Results: Increasing the number of tip changes (0/1/2/10 times) during serial dilution for plate preparation increased the MICs 1 to >2 dilutions for amphotericin B, anidulafungin, micafungin, fluconazole, voriconazole and isavuconazole against C. albicans ATCC 64548 but only isavuconazole MICs against C. parapsilosis ATCC 22019 (3 dilutions). ISO and serial dilution (two tip changes) were compared for eight compounds and four Candida QC strains (352 MICs). Six/41 GM MIC pairs deviated with 1-1.8 dilution (14.6%). Comparing the GM MIC with the QC values, the ISO method GM MIC was closest to the target in 30.8%, the serial dilution in 34.6% and the methods identical in 34.6% of the cases. Finally, ISO and serial dilution MICs were compared for clinical isolates (920 MICs). Five/23 GM MIC pairs (21.7%) deviated 1.0-1.1 dilutions.Conclusions: The ISO and serial dilution (two tip changes) method were in acceptable agreement and thus equally applicable for EUCAST testing. [ABSTRACT FROM AUTHOR]

Background: EUCAST recently revised the definition of the 'I' category from 'intermediate' to 'susceptible, increased exposure'. Consequently, all current antifungal breakpoints have been reviewed and revised breakpoints (v 10.0) have been released.Objectives: We investigated isavuconazole and comparator MICs (mg/L) against contemporary moulds and the consequences of the breakpoint revision for susceptibility classification.Methods: Six hundred and ninety-six Aspergillus and 46 other moulds were included. EUCAST E.Def 10.1 azole resistance screening was performed for Aspergillus fumigatus and E.Def 9.3.1 testing of non-susceptible A. fumigatus and other moulds. Most non-wildtype/resistant isolates underwent cyp51A sequencing.Results: Isavuconazole MIC50/MIC90s were ≤1/≤2 mg/L for Aspergillus flavus, A. fumigatus and Aspergillus nidulans versus 2/4 mg/L for Aspergillus niger and 2/16 mg/L for Aspergillus terreus. For the remaining moulds, MICs were highest for Fusarium (16 to >16 mg/L), lowest for dermatophytes (0.06-0.5 mg/L) and in between for Mucorales and others (1 to >16 mg/L). A very strong isavuconazole-voriconazole MIC correlation was found for A. fumigatus (Pearson r = 0.888) and itraconazole-posaconazole correlation for A. fumigatus (r = 0.905) and A. terreus (r = 0.848). For A. fumigatus, the revised breakpoints lowered isavuconazole resistance (22.6% to 7.7%, P < 0.0001) and increased voriconazole resistance (3.8% to 6.7%, P = 0.025), resulting in similar resistance rates across the four azoles (range: 6.7%-7.7%). For A. terreus, isavuconazole resistance remained unchanged (81.3%) and higher than itraconazole (43.8%, P = 0.004) and posaconazole (53.1%, P = 0.03) resistance. Azole cross-resistance was found in 24/24, 13/20 and 4/90 isolates, and Cyp51A alterations in 16/18, 1/7 and 2/4 sequenced isolates with isavuconazole MICs of >4, 4 and 2 mg/L, respectively.Conclusions: Isavuconazole displays broad anti-mould activity. The revised breakpoints result in fewer misclassifications of wildtype isolates without compromising detection of resistant mutants. [ABSTRACT FROM AUTHOR]

Objectives: Terbinafine resistance is increasingly reported in Trichophyton, rendering susceptibility testing particularly important in non-responding cases. We performed a multicentre evaluation of six EUCAST-based methods.Methods: Ten laboratories susceptibility tested terbinafine, itraconazole, voriconazole and amorolfine against a blinded panel of 38 terbinafine WT and target gene mutant isolates. E.Def 9.3.1 modifications included: medium with/without addition of chloramphenicol and cycloheximide (CC), incubation at 25°C to 28°C for 5-7 days and three MIC endpoints [visually and spectrophotometrically (90%/50% inhibition)], generating 7829 MICs. Quality control (QC) strains were Aspergillus flavus ATCC 204304 and CNM-CM1813. Eyeball, ECOFFinder (where ECOFF stands for epidemiological cut-off) and derivatization WT upper limits (WT-ULs), very major errors (VMEs; mutants with MICs ≤WT-ULs) and major errors (MEs; WT isolates with MICs >WT-ULs) were determined.Results: MICs fell within the QC ranges for ATCC 204304/CNM-CM1813 for 100%/96% (voriconazole) and 84%/84% (itraconazole), respectively. Terbinafine MICs fell within 0.25-1 mg/L for 96%/92%, suggesting high reproducibility. Across the six methods, the number of terbinafine MEs varied from 2 to 4 (2.6%-5.2%) for Trichophyton rubrum and from 0 to 2 (0%-2.0%) for Trichophyton interdigitale. Modes for WT and mutant populations were at least seven 2-fold dilutions apart in all cases. Excluding one I121M/V237I T. rubrum mutant and two mixed WT/mutant T. interdigitale specimens, the numbers of VMEs were as follows: T. rubrum: CC visual, 1/67 (1.5%); CC spectrophotometric 90% inhibition, 3/59 (5.1%); and CC spectrophotometric 50% inhibition, 1/67 (1.5%); and T. interdigitale: none. Voriconazole and amorolfine MICs were quite uniform, but trailing growth complicated determination of itraconazole visual and spectrophotometric 90% inhibition MIC.Conclusions: Although none of the laboratories was experienced in dermatophyte testing, error rates were low. We recommend the CC spectrophotometric 50% inhibition method and provide QC ranges and WT-ULs for WT/non-WT classification. [ABSTRACT FROM AUTHOR]

Background: The determination of the minimal effective concentration (MEC) of echinocandins against Aspergillus species is subjective, time consuming and has been associated with very major errors.Methods: The MECs/MICs of 40 WT [10 each of Aspergillus fumigatus species complex (SC), Aspergillus flavus SC, Aspergillus terreus SC and Aspergillus niger SC] and 4 non-WT A. fumigatus isolates were determined with EUCAST E.Def 9.3.1 read microscopically, macroscopically, spectrophotometrically and colorimetrically in three centres. The optimal conditions for spectrophotometric (single- versus multi-point readings) and colorimetric (XTT/menadione concentration and stability, incubation time) methods were evaluated in preliminary studies using different cut-offs for the determination of macroscopic, spectrophotometric and colorimetric MIC endpoints compared with the microscopically determined MEC. Inter-centre and inter-method essential (within one 2-fold dilution) agreement (EA) and categorical agreement (CA) were determined.Results: Both macroscopic and spectrophotometric endpoint readings showed poor inter-centre EA (53%-66%) and low CA (41%-88%) in distinguishing WT from non-WT A. fumigatus SC isolates, while significant differences compared with the microscopic MECs were observed for all echinocandins (EA 6%-54%). For the colorimetric method, the optimal conditions were 400 mg/L XTT/6.25 μΜ menadione, incubation for 1-2 h until the drug-free control reached an absorbance at 450/630 nm of >0.8 and use of 50% inhibition of XTT conversion as a cut-off for all species and echinocandins. All non-WT isolates had high XTT MICs >1 mg/L, whereas the overall inter-centre EA and CA were 72%-89% and 100%, respectively.Conclusions: The XTT colorimetric assay improved the antifungal susceptibility testing of echinocandins against Aspergillus spp., reliably detecting non-WT isolates. [ABSTRACT FROM AUTHOR]

Objectives: Azole-resistant Aspergillus fumigatus is emerging worldwide. Reference susceptibility testing methods are technically demanding and no validated commercial susceptibility tests for moulds currently exist. In this multicentre study a 4-well azole-containing screening agar method was evaluated using clinically relevant isolates.Methods: Forty WT and 39 cyp51A mutant A. fumigatus [G54 (n = 10), M220 (n = 10), TR34/L98H (n = 9) and TR46/Y121F/T289A (n = 10)] were tested individually and as simulated mixed samples (sampling 4 WT and 1 mutant colonies). EUCAST MICs were determined following E.Def 9.3. In-house and commercial 4-well plates containing agars supplemented with 4 mg/L itraconazole, 1 mg/L voriconazole, 0.5 mg/L posaconazole and no antifungal, respectively, were evaluated. Growth was scored (0-3) by two independent observers in three laboratories. Inter-plate, inter-observer, essential and categorical agreement, sensitivity and specificity were calculated.Results: CYP51A genotype and antifungal compound-specific MICs and growth patterns were documented. The inter-observer agreement was excellent with 86%-99% identical scores (range 80%-100%) for both plates. The qualitative agreement (no growth versus growth) was excellent (median 95%-100%, range 87%-100%, overall). The overall sensitivity and specificity for the 4-well plate (no growth versus growth) was 99% (range 97%-100%) and 99% (95%-100%), respectively. The sensitivity for simulated WT/mutant specimens was 94% (range 83%-100%) for the WT-TR34/L98H combination, but 100% for the WT/G54W combination. The performance remained unchanged using only itraconazole- and voriconazole-containing agars, but was lower for the other combinations.Conclusions: Implementation of the 4-well screening plate in routine laboratories will allow easy and reliable detection of the most common azole-resistant A. fumigatus. [ABSTRACT FROM AUTHOR]

Background: Voriconazole exhibits in vitro activity against Candida glabrata and Candida krusei (EUCAST/CLSI epidemiological cut-off values 1/0.25 and 1/0.5 mg/L, respectively). Yet, EUCAST found insufficient evidence to set breakpoints for these species. We explored voriconazole pharmacodynamics (PD) in an in vitro dynamic model simulating human pharmacokinetics (PK).Methods: Four C. glabrata and three C. krusei isolates (voriconazole EUCAST and CLSI MICs of 0.03-2 mg/L) were tested in the PK/PD model simulating voriconazole exposures (t½ ∼6 h q12h dosing for 3 days). PK/PD breakpoints were determined calculating the PTA for exposure indices fAUC0-24/MIC associated with half-maximal activity (EI50) using Monte Carlo simulation analysis.Results: Fungal load increased from 3.60±0.35 to 8.41±0.24 log10 cfu/mL in the drug-free control, with a maximum effect of ∼1 log10 kill of C. glabrata and C. krusei isolates with MICs of 0.06 and 0.25 mg/L, respectively, at high drug exposures. The 72 h log10 cfu/mL change versus fAUC0-24/MIC relationship followed a sigmoid curve for C. glabrata (R2=0.85-0.87) and C. krusei (R2=0.56-0.76) with EI50 of 49 (32-76) and 52 (33-78) fAUC/MIC for EUCAST and 55 (31-96) and 80 (42-152) fAUC/MIC for CLSI, respectively. The PTAs for C. glabrata and C. krusei isolates with EUCAST/CLSI MICs ≤0.125/≤0.06 mg/L were >95%. Isolates with EUCAST/CLSI MICs of 0.25-1/0.125-0.5 would require trough levels 1-4 mg/L; isolates with higher MICs would not attain the corresponding PK/PD targets without reaching toxicity.Conclusions: The in vitro PK/PD breakpoints for C. glabrata and C. krusei for EUCAST (0.125 mg/L) and CLSI (0.06 mg/L) bisected the WT populations. Trough levels of >4 mg/L, which are not clinically feasible, are necessary for efficacy against WT isolates. [ABSTRACT FROM AUTHOR]

Invasive Candida infections remain an important cause of morbidity and mortality, especially in hospitalized and immunocompromised or critically ill patients. A limited number of antifungal agents from only a few drug classes are available to treat patients with these serious infections. Resistance can be either intrinsic or acquired. Resistance mechanisms are not exchanged between Candida; thus, acquired resistance either emerges in response to an antifungal selection pressure in the individual patient or, more rarely, occur due to horizontal transmission of resistant strains between patients. Although multidrug resistance is uncommon, increasing reports of multidrug resistance to the azoles, echinocandins, and polyenes have occurred in several Candida species, most notably Candida glabrata and more recently Candida auris. Drivers are overall antifungal use, subtherapeutic drug levels at sites of infection/colonization, drug sequestration in the biofilm matrix, and, in the setting of outbreaks, suboptimal infection control. Moreover, recent research suggests that DNA mismatch repair gene mutations may facilitate acquisition of resistance mutations in C. glabrata specifically. Diagnosis of antifungal-resistant Candida infections is critical to the successful management of patients with these infections. Reduction of unnecessary use of antifungals via antifungal stewardship is critical to limit multidrug resistance emergence. [ABSTRACT FROM AUTHOR]

Three methods were compared for the susceptibility testing of yeast isolates to fluconazole and amphotericin B: two fagar diffusion methods (Etest and a tablet diffusion test) and the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method. Given as MIC(50)s (range), fluconazole endpoints were: for the 24 h broth microdilution test, 0.25 mg/L (0.06-32 mg/L); for the Etest, 0.38 mg/L (0.064-24 mg/L); and for the NCCLS broth microdilution test, 2 mg/L (0.06->or=64 mg/L). With breakpoints of <3 mg/L for susceptible and >16 mg/L for resistant, the Etest and the 24 h microdilution test classified the isolates in agreement with the classification obtained by the NCCLS method. Results obtained by Etest were in closer NCCLS method than those obtained with the tablet test. Amphotericin B endpoints were lower for the 24 h microdilution and Etests than MICs obtained by the NCCLS broth microdilution method. Reproducibility was high for all tests; however, disadvantages of both diffusion tests were microcolonies in the inhibition zone and dependence on stringent standardization of inoculum. [ABSTRACT FROM AUTHOR]

EUCAST has established clinical breakpoints for the six most common Candida species and Cryptococcus neoformans but not for less common yeasts because sufficient evidence is lacking. Consequently, the question "How to interpret the MIC?" for other yeasts often arises. We propose a pragmatic classification for amphotericin B, anidulafungin, fluconazole, and voriconazole MICs against 30 different rare yeasts. This classification takes advantage of MIC data for more than 4000 isolates generated in the EUCAST Development Laboratory for Fungi validated by alignment to published EUCAST MIC data. The classification relies on the following two important assumptions: first, that when isolates are genetically related, pathogenicity and intrinsic susceptibility patterns may be similar; and second, that even if species are not phylogenetically related, the rare yeasts will likely respond to therapy, provided the MIC is comparable to that against wild-type isolates of more prevalent susceptible species because rare yeasts are most likely "rare" due to a lower pathogenicity. In addition, the treatment recommendations available in the current guidelines based on the in vivo efficacy data and clinical experience are taken into consideration. Needless to say, it is of utmost importance (a) to ascertain that the species identification is correct (using MALDI-TOF or sequencing), and (b) to re-test the isolate once or twice to confirm that the MIC is representative for the isolate (because of the inherent variability in MIC determinations). We hope this pragmatic guidance is helpful until evidence-based EUCAST breakpoints can be formally established. [ABSTRACT FROM AUTHOR]

• Posaconazole was found to have strong fungicidal activity against all tested Candida krusei isolates. • Posaconazole only had fungicidal activity against the most susceptible Candida glabrata isolate. • The pharmacokinetic/pharmacodynamic targets for C. krusei were 10-fold lower than those of C. glabrata. • The probability of target attainment (PTA) was low for wild-type C. glabrata. • High PTA was found for wild-type C. krusei with intravenous and tablet formulations of posaconazole. Posaconazole exhibits in-vitro activity against Candida glabrata and Candida krusei. Epidemiological cut-off values set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) are 1/1 and 0.5/0.5 mg/L, respectively, but clinical breakpoints have not been established to date. This study explored the pharmacodynamics (PD) of posaconazole in a validated one-compartment in-vitro pharmacokinetic (PK)/PD model, and determined the probability of PK/PD target attainment (PTA) for the available formulations. Five C. glabrata and three C. krusei isolates with posaconazole minimum inhibitory concentrations (MICs) of 0.06–2 and 0.03–0.25 mg/L, respectively, were tested in the PK/PD model simulating different time–concentration profiles of posaconazole. The exposure–effect relationship f AUC 0–24 /MIC was described for EUCAST/CLSI methods, and PTA was calculated in order to determine PK/PD susceptibility breakpoints for oral solution (400 mg q12h), and intravenous (i.v.)/tablet formulations (300 mg q24h). Fungicidal activity (~2log kill) was found against the most susceptible C. glabrata isolate alone, and against all three C. krusei isolates. The corresponding EUCAST/CLSI PK/PD targets (f AUC 0–24 /MIC) were 102/79 for C. glabrata and 12/8 for C. krusei. Mean PTA was high (>95%) for C. glabrata isolates with EUCAST/CLSI MICs ≤0.03/≤0.03 mg/L for oral solution and ≤0.125/≤0.125 mg/L for i.v. and tablet formulations for the wild-type population. For C. krusei isolates, mean PTA was high (>95%) for EUCAST/CLSI MICs ≤0.25/≤0.5 mg/L for oral solution and ≤1/≤2 mg/L for i.v. and tablet formulations for the wild-type population. The use of posaconazole to treat C. glabrata infections is questionable. Intravenous and tablet formulations may be therapeutic options for the treatment of C. krusei infections, and oral exposure can be optimized with therapeutic drug monitoring (trough levels >0.6–0.9 mg/L). [ABSTRACT FROM AUTHOR]

Besides inherent differences in in vitro susceptibilities, clinically-relevant yeast species may acquire resistance upon exposure to most antifungal drugs used in the clinic. In recent years, major fundamental research studies have been conducted to improve our understanding of the molecular basis of antifungal resistance. This topic is of major interest as antifungal resistance in yeast is clearly evolving and is correlated with clinical failure. This minireview is an overview of the most recent findings about key molecular mechanisms evolving in human pathogenic yeasts, particularly Candida spp., in the context of antifungal drug resistance. Also included are the methods currently available for in vitro antifungal susceptibility testing and for molecular detection of mutations associated with resistance. Finally, the genetic drivers of antifungal resistance are discussed in light of the spectra of multidrug resistance as observed in Candida glabrata . [ABSTRACT FROM AUTHOR]