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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 (BP) 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 US states and ≥3 clades were included. Fourteen MIC data sets were generated using EUCAST E.Def 7.4, CLSI M27Ed4, Etest and MTS (Liofilchem) strip MICs. MICs ≤1 mg/L were classified as susceptible., Results: EUCAST and CLSI amphotericin B MIC testing were robust across included method variables. The modal MIC was 1 mg/L, distributions unimodal and narrow with similar 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 OD of drug-free control EUCAST wells suggesting that some isolates grew better than others and that this was associated with MIC., Conclusions: 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 suggest the current breakpoint leads to random susceptibility classification., (Copyright © 2024. Published by Elsevier Ltd.)

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.

During construction work (2017-2019), an increase in Aspergillus flavus infections was noted among pediatric patients, the majority of whom were receiving amphotericin B prophylaxis. Microsatellite genotyping was used to characterize the outbreak. A total of 153 A . flavus isolates of clinical and environmental origin were included. Clinical isolates included 140 from 119 patients. Eight patients were outbreak-related patients, whereas 111 were outbreak-unrelated patients from Danish hospitals (1994-2023). We further included four control strains. Nine A. flavus isolates were from subsequent air sampling in the outbreak ward (2022-2023). Typing followed Rudramurthy et al.(S. M. Rudramurthy, H. A. de Valk, A. Chakrabarti, J. Meis, and C. H. W. Klaassen, PLoS One 6:e16086, 2011, https://doi.org/10.1371/journal.pone.0016086). Minimum spanning tree (MST) and discriminant analysis of principal components (DAPC) were used for cluster analysis. DAPC analysis placed all 153 isolates in five clusters. Microsatellite marker pattern was clearly distinct for one cluster compared to the others. The same cluster was observed in an MST. This cluster included all outbreak isolates, air-sample isolates, and additional patient isolates from the outbreak hospital, previously undisclosed as outbreak related. The highest air prevalence of A. flavus was found in two technical risers of the outbreak ward, which were then sealed. Follow-up air samples were negative for A. flavus . Microsatellite typing defined the outbreak as nosocomial and facilitated the identification of an in-hospital source. Six months of follow-up air sampling was without A. flavus . Outbreak-related/non-related isolates were easily distinguished with DAPC and MST, as the outbreak clone's distinct marker pattern was delineated in both statistical analyses. Thus, it could be a variant of A. flavus , with a niche ability to thrive in the outbreak-hospital environment., Importance: Aspergillus flavus can cause severe infections and hospital outbreaks in immunocompromised individuals. Although lack of isogeneity does not preclude an outbreak, our study underlines the value of microsatellite genotyping in the setting of potential A. flavus outbreaks. Microsatellite genotyping documented an isogenic hospital outbreak with an internal source. This provided the "smoking gun" that prompted the rapid allocation of resources for thorough environmental sampling, the results of which guided immediate and relevant cleaning and source control measures. Consequently, we advise that vulnerable patients should be protected from exposure and that genotyping be included early in potential A. flavus outbreak investigations. Inspection and sampling are recommended at any site where airborne spores might disperse from. This includes rarely accessed areas where air communication to the hospital ward cannot be disregarded., Competing Interests: The authors declare no conflict of interest.

Azole-resistant Aspergillus fumigatus (ARAf) fungi have been found inconsistently in the environment in Denmark since 2010. During 2018-2020, nationwide surveillance of clinical A. fumigatus fungi reported environmental TR 34 /L98H or TR 46 /Y121F/T289A resistance mutations in 3.6% of isolates, prompting environmental sampling for ARAf and azole fungicides and investigation for selection of ARAf in field and microcosmos experiments. ARAf was ubiquitous (20% of 366 samples; 16% TR 34 /L98H- and 4% TR 46 /Y121F/T289A-related mechanisms), constituting 4.2% of 4,538 A. fumigatus isolates. The highest proportions were in flower- and compost-related samples but were not correlated with azole-fungicide application concentrations. Genotyping showed clustering of tandem repeat-related ARAf and overlaps with clinical isolates in Denmark. A. fumigatus fungi grew poorly in the field experiment with no postapplication change in ARAf proportions. However, in microcosmos experiments, a sustained complete (tebuconazole) or partial (prothioconazole) inhibition against wild-type A. fumigatus but not ARAf indicated that, under some conditions, azole fungicides may favor growth of ARAf in soil.

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.)

Fungal diseases represent a considerable global health concern, affecting >1 billion people annually. In response to this growing challenge, the World Health Organization introduced the pivotal fungal priority pathogens list (FPPL) in late 2022. The FPPL highlights the challenges in estimating the global burden of fungal diseases and antifungal resistance (AFR), as well as limited surveillance capabilities and lack of routine AFR testing. Furthermore, training programs should incorporate sufficient information on fungal diseases, necessitating global advocacy to educate health care professionals and scientists. Established international guidelines and the FPPL are vital in strengthening local guidance on tackling fungal diseases. Future iterations of the FPPL have the potential to refine the list further, addressing its limitations and advancing our collective ability to combat fungal diseases effectively. Napp Pharmaceuticals Limited (Mundipharma UK) organized a workshop with key experts from Northern Europe to discuss the impact of the FPPL on regional clinical practice., Competing Interests: Potential conflicts of interest. M.C.A. has, over the past 5 years, received research grants/contract work (paid to the Statens Serum Institute) from Cidara, F2G, Gilead, and Scynexis and speaker honoraria (personal fees) from Astellas, Chiesi, Gilead, and F2G; she is also the current Chair of EUCAST-AFST. D.A.J. has share options in pulmocide. A.B. has no conflicts of interest. D.D. holds founder shares in F2G Ltd., an antifungal discovery company spun out from the University of Manchester, and share options in TFF Pharma; acts or has recently acted as a consultant to Biosergen, Lifemine Therapeutics, Mucpharm PTY, Pulmatrix, Pulmocide, Rostra Therapeutics, and TFF Pharmaceuticals; has received payments for talks on behalf of Avir, Basilea, BioRad, Gilead, Mundipharma, and Pfizer in the past 3 years; and has been involved in multiple guideline groups, primarily focused on diagnostics and aspergillosis. M.F. has received speaker fees and funding from Gilead Scientific and received funding from the Leverhulme Trust and the Welcome Trust. R.G. has no conflicts of interest. J.M. reports support consulting fees from Amplyx, Basilea, Cidara, F2G, Gilead, Mundipharma, Pfizer, Scynexis, and Takeda; honoraria for lectures from Astellas, Basilea, Gilead, MedScape, Mundipharma, Pfizer, Shionogi, and Takeda; payment for expert testimony from Cidara; and participation on advisory boards with Basilea, Cidara, Pulmocide, Sfunga, and Shionogi. I.M.L. has received lecturer honoraria and has participated on advisory boards with Accelerate, bioMérieux, Biotest, Clinigen, Fresenius Kabi, Gilead, MSD, Mundipharma, Polyphor, and ThermoFisher. V.M. has received speaker honoraria and participated on advisory boards and received research funding from different industry partners including Gilead, Pfizer, Mundipharma, MSD, Novartis, Jazz Pharma, Therakos, Sanofi. T.M. reports consultancy fees from AstraZeneca, Gilead Sciences, Pfizer, and Roche; research grants from Gilead Sciences; and travel support from AstraZeneca, Amgen, and Pfizer. J.P. has given a presentation for Pfizer. R.R. has received lecturer honoraria from Astellas, Mundipharma, Basilea, Gilead, and Pfizer and has participated as a clinical investigator for a Phase 2 clinical trial conducted by Scynexis and as a principal investigator for a Phase 2 clinical trial conducted by F2G. R.W. has no conflicts of interest. N.A. works at Napp Pharmaceuticals Ltd. which is a member of the Mundipharma network of independent associated companies. L.W. performed diagnostic evaluations and received meeting sponsorship from Associates of Cape Cod, Bruker, Dynamiker, and Launch Diagnostics; speaker's fees, expert advice fees, and meeting sponsorship from Gilead; speaker and expert advice fees from Pfizer and Mundipharma; and expert advice fees from F2G., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

SUMMARYFungal infections are on the rise, driven by a growing population at risk and climate change. Currently available antifungals include only five classes, and their utility and efficacy in antifungal treatment are limited by one or more of innate or acquired resistance in some fungi, poor penetration into "sequestered" sites, and agent-specific side effect which require frequent patient reassessment and monitoring. Agents with novel mechanisms, favorable pharmacokinetic (PK) profiles including good oral bioavailability, and fungicidal mechanism(s) are urgently needed. Here, we provide a comprehensive review of novel antifungal agents, with both improved known mechanisms of actions and new antifungal classes, currently in clinical development for treating invasive yeast, mold (filamentous fungi), Pneumocystis jirovecii infections, and dimorphic fungi (endemic mycoses). We further focus on inhaled antifungals and the role of immunotherapy in tackling fungal infections, and the specific PK/pharmacodynamic profiles, tissue distributions as well as drug-drug interactions of novel antifungals. Finally, we review antifungal resistance mechanisms, the role of use of antifungal pesticides in agriculture as drivers of drug resistance, and detail detection methods for antifungal resistance.

Resistance in dermatophytes is an emerging global public health issue. We, therefore, developed an agar-based method for screening Trichophyton spp. susceptibility to terbinafine (TRB), itraconazole (ITC), and amorolfine (AMF) and validated it using molecularly characterized isolates. Α total of 40 Trichophyton spp. isolates, 28 TRB wild type (WT) (13 T. rubrum , 10 T. mentagrophytes , 5 T. interdigitale ) and 12 TRB non-WT (7 T. rubrum , 5 T. indotineae ) with different alterations in the squalene epoxidase (SQLE) gene, were used. The optimal test conditions (inoculum and drug concentrations, incubation time, and temperature) and stability over time were evaluated. The method was then applied for 86 WT Trichophyton spp. clinical isolates (68 T. rubrum , 7 T. interdigitale , 6 T. tonsurans , 5 T. mentagrophytes ) and 4 non-WT T. indotineae . Optimal growth of drug-free controls was observed using an inoculum of 20 µL 0.5 McFarland after 5-7 days of incubation at 30°C. The optimal concentrations that prevented the growth of WT isolates were 0.016 mg/L of TRB, 1 mg/L of ITC, and 0.25 mg/L of AMF, whereas 0.125 mg/L of TRB was used for the detection of Trichophyton strong SQLE mutants (MIC ≥0.25 mg/L). The agar plates were stable up to 4 months. Inter-observer and inter-experimental agreement were 100%, and the method successfully detected TRB non-WT Trichophyton spp. strains showing 100% agreement with the reference EUCAST methodology. An agar-based method was developed for screening Trichophyton spp. in order to detect TRB non-WT weak and strong mutant isolates facilitating their detection in non-expert routine diagnostic laboratories., Competing Interests: The authors declare no conflict of interest.

Background: To date, azoles represent the only viable option for oral treatment of invasive Candida infections, while rates of azole resistance among non-albicans Candida spp. continue to increase. The objective of this sub-analysis of the European multicenter observational cohort study Candida III was to describe demographical and clinical characteristics of the cohort requiring prolonged hospitalization solely to complete intravenous (iv) antifungal treatment (AF Tx)., Methods: Each participating hospital (number of eligible hospitals per country determined by population size) included the first ~ 10 blood culture proven adult candidemia cases occurring consecutively after July 1st, 2018, and treating physicians answered the question on whether hospital stay was prolonged only for completion of intravenous antifungal therapy. Descriptive analyses as well as binary logistic regression was used to assess for predictors of prolonged hospitalization solely to complete iv AF Tx., Findings: Hospital stay was prolonged solely for the completion of iv AF Tx in 16% (100/621) of candidemia cases by a median of 16 days (IQR 8 - 28). In the multivariable model, initial echinocandin treatment was a positive predictor for prolonged hospitalization to complete iv AF Tx (aOR 2.87, 95% CI 1.55 - 5.32, p < 0.001), while (i) neutropenia, (ii) intensive care unit admission, (iii) catheter related candidemia, (iv) total parenteral nutrition, and (v) C. parapsilosis as causative pathogen were found to be negative predictors (aOR 0.22 - 0.45; p < 0.03)., Interpretation: Hospital stays were prolonged due to need of iv AF Tx in 16% of patients with candidemia. Those patients were more likely to receive echinocandins as initial treatment and were less severely ill and less likely infected with C. parapsilosis., (© 2023. The Author(s).)

The rapid pace of name changes of medically important fungi is creating challenges for clinical laboratories and clinicians involved in patient care. We describe two sources of name change which have different drivers, at the species versus the genus level. Some suggestions are made here to reduce the number of name changes. We urge taxonomists to provide diagnostic markers of taxonomic novelties. Given the instability of phylogenetic trees due to variable taxon sampling, we advocate to maintain genera at the largest possible size. Reporting of identified species in complexes or series should where possible comprise both the name of the overarching species and that of the molecular sibling, often cryptic species. Because the use of different names for the same species will be unavoidable for many years to come, an open access online database of the names of all medically important fungi, with proper nomenclatural designation and synonymy, is essential. We further recommend that while taxonomic discovery continues, the adaptation of new name changes by clinical laboratories and clinicians be reviewed routinely by a standing committee for validation and stability over time, with reference to an open access database, wherein reasons for changes are listed in a transparent way., Competing Interests: The authors declare no conflict of interest.

The optimal prevention strategy for invasive aspergillosis (IA) in lung transplant recipients (LTXr) is unknown. In 2016, the Danish guidelines were changed from universal to targeted IA prophylaxis. Previously, we found higher rates of adverse events in the universal prophylaxis period. In a Danish nationwide study including LTXr, for 2010-2019, we compared IA rates in time periods with universal vs. targeted prophylaxis and during person-time with vs. person-time without antifungal prophylaxis. IA hazard rates were analyzed in multivariable Cox models with adjustment for time after LTX. Among 295 LTXr, antifungal prophylaxis was initiated in 183/193 and 6/102 during the universal and targeted period, respectively. During the universal period, 62% discontinued prophylaxis prematurely. The median time on prophylaxis was 37 days (IQR 11-84). IA was diagnosed in 27/193 (14%) vs. 15/102 (15%) LTXr in the universal vs. targeted period, with an adjusted hazard ratio (aHR) of 0.94 (95% CI 0.49-1.82). The aHR of IA during person-time with vs. person-time without antifungal prophylaxis was 0.36 (95% CI 0.12-1.02). No difference in IA was found during periods with universal vs. targeted prophylaxis. Prophylaxis was protective of IA when taken. Targeted prophylaxis may be preferred over universal due to comparable IA rates and lower rates of adverse events.

Cytomegalovirus (CMV) and invasive aspergillosis (IA) cause morbidity among lung transplant recipients (LTXr). Early diagnosis and treatment could improve outcomes. We examined rates of CMV after IA and vice versa to assess whether screening for one infection is warranted after detecting the other. All Danish LTXr, 2010-2019, were followed for IA and CMV for 2 years after transplantation. IA was defined using ISHLT criteria. Adjusted incidence rate ratios (aIRR) were estimated by Poisson regression adjusted for time after transplantation. We included 295 LTXr, among whom CMV and IA were diagnosed in 128 (43%) and 48 (16%). The risk of CMV was high the first 3 months after IA, IR 98/100 person-years of follow-up (95% CI 47-206). The risk of IA was significantly increased in the first 3 months after CMV, aIRR 2.91 (95% CI 1.32-6.44). Numbers needed to screen to diagnose one case of CMV after IA, and one case of IA after CMV was approximately seven and eight, respectively. Systematic screening for CMV following diagnosis of IA, and vice versa, may improve timeliness of diagnosis and outcomes for LTXr., (© 2023 The Authors. APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and Immunology.)

Background: The CLSI breakpoint for micafungin and Candida albicans is 0.25 mg/L, higher than the CLSI epidemiological cut-off value (0.03 mg/L) whereas the EUCAST values are identical (0.016 mg/L). We developed a novel in vitro dialysis-diffusion pharmacokinetic/pharmacodynamic (PK/PD) model, confirmed correlation to in vivo outcome and studied micafungin pharmacodynamics against Canida albicans., Methods: Four C. albicans isolates, including a weak (F641L) and a strong (R647G) fks1 mutants, were studied using a 104 cfu/mL inoculum and RPMI medium with and without 10% pooled human serum. The exposure-effect relationship fAUC0-24/MIC was described for CLSI and EUCAST methodology. Monte Carlo simulation analysis included standard (100 mg i.v.) and higher (150-300 mg) doses q24h to determine the corresponding probability of target attainment (PTA)., Results: The in vitro PK/PD targets for stasis/1-log kill were 36/57 fAUC0-24/MIC in absence and 2.8/9.2 fAUC0-24/MIC in the presence of serum, and similar for wild-type and fks mutant isolates. The PTAs for both PK/PD targets were high (>95%) for EUCAST susceptible isolates but not for CLSI susceptible non-wild-type isolates (CLSI MICs 0.06-0.25 mg/L). 300 mg q24h was needed to attain PK/PD targets for non-wild-type isolates with CLSI MICs 0.06-0.125 mg/L and EUCAST MICs 0.03-0.06 mg/L., Conclusion: The in vitro 1-log kill effect corresponded to stasis in animal model and mycological response in patients with invasive candidiasis, thereby validating the model for studying pharmacodynamics of echinocandins in vitro. EUCAST breakpoints were well supported by our findings but our data questions whether the current CLSI breakpoint, which is higher than the epidemiological cut-off values, is appropriate., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Rezafungin is a novel, once-weekly echinocandin. EUCAST rezafungin MIC testing has been associated with a good separation of WT and target gene mutant isolates in single-centre studies, but an unacceptable inter-laboratory MIC variation has prevented EUCAST breakpoint setting. This has been attributed to non-specific binding to surfaces across microtitre plates, pipettes, reservoirs, etc. used, as previously encountered for some antibiotics., Objectives: To investigate use of a surfactant to mitigate non-specific binding of rezafungin in EUCAST E.Def 7.3 MIC testing., Methods: Surfactants including Tween 20 (T20), Tween 80 (T80) and Triton X-100 (TX100) were evaluated for stand-alone or synergistic antifungal activity via checkerboard assays in combination with rezafungin. Subsequent T20 studies defined an optimized assay concentration, validated in up to four microtitre plate types for WT and fks mutant Candida strains (seven species total) and the six-strain EUCAST Candida quality control (QC) panel. Lastly, T20 inter-manufacturer variability, thermostability and best handling practices were investigated., Results: T20 and T80 performed equivalently, with characteristics slightly preferable to TX100. Due to existing use in EUCAST mould susceptibility testing, T20 was pursued. An optimized concentration of 0.002% T20 normalized rezafungin MIC values across plate types for all Candida spp. evaluated, maintained differentiation of WT versus fks mutants and generated robust QC ranges. Additionally, T20 performance was consistent across manufacturers and temperatures. T20 can be reliably transferred utilizing a syringe, wide-orifice pipette tip and/or by mass., Conclusions: Supplementation of RPMI (Roswell Park Memorial Institute) 1640 medium with 0.002% T20 generated a highly reproducible EUCAST yeast MIC methodology for rezafungin., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Rezafungin EUCAST MIC testing has been associated with notable inter-laboratory variation, which prevented ECOFF setting for C. albicans. We assessed in vitro susceptibility and reproducibility for a modified EUCAST methodology and established associated wild-type upper limits (WT-ULs)., Methods: MICs against 150 clinical Candida isolates (six species), molecularly characterized fks mutants (n = 13), and QC strains (n = 6) were determined at six laboratories according to E.Def 7.3 but using Tween 20 supplemented medium. WT-ULs were determined using the derivatization method, the ECOFFinder programme and visual inspection. Consensus WT-ULs were determined., Results: The laboratory- and species-specific MIC distributions were Gaussian with >99.5% MICs within four 2-fold dilutions except for C. parapsilosis (92.8%). The following consensus WT-UL were determined: C. albicans 0.008 mg/L; C. dubliniensis and C. glabrata 0.016 mg/L; C. krusei and C. tropicalis 0.03 mg/L; and C. parapsilosis 4 mg/L. Adopting these WT-UL, six clinical isolates were non-wild-type, five of which harboured Fks alterations. For 11/13 mutants, all 670 MICs were categorized as non-wild-type whereas MICs for C. glabrata Fks2 D666Y and C. tropicalis Fks1 R656R/G overlapped with the corresponding wild-type distributions. Repeat testing of six reference strains yielded 98.3%-100% of MICs within three 2-fold dilutions except for C. albicans CNM-CL-F8555 (96%) and C. parapsilosis ATCC 22019 (93.3%)., Conclusions: The modified EUCAST method significantly improved inter-laboratory variation, identified wild-type populations and allowed perfect separation of wild-type and fks mutants except for two isolates harbouring weak mutations. These consensus WT-UL have been accepted as ECOFFs and will be used for rezafungin breakpoint setting., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Invasive fungal infections in lung transplant (LTX) recipients cause substantial morbidity, but the best strategy for prevention has not yet been determined. We evaluated adherence to and rates of adverse events of universal versus targeted prophylaxis. Methods: All LTX recipients in the Danish National LTX Centre (2010−2019) were included. Before July 2016, universal voriconazole prophylaxis was used. After July 2016, only high-risk patients received targeted prophylaxis with posaconazole and inhaled amphotericin B. Proportions of triazole discontinuation, side-effects, off-target calcineurin-inhibitor (CNI) levels, and acute rejection were compared between the two periods. Results: Universal and targeted prophylaxis was initiated in 183/193 and 6/102 patients, respectively. Only 37% completed > 9 of the intended 12 weeks of voriconazole; 72% of discontinuations were due to hepatotoxicity. In the universal vs. targeted prophylaxis period, 89% vs. 72% (p < 0.001) patients had low CNI episodes, and 37% vs. 1% (p < 0.001) of these were associated with discontinuation of triazole; 40% vs. 14% (p < 0.001) had acute rejection; and 23% vs. 3% (p < 0.001) had acute rejection associated with low CNI episodes. Conclusions: Universal voriconazole prophylaxis was associated with high rates of discontinuation, mainly caused by hepatotoxicity. In comparison to the targeted posaconazole period, more patients had low CNI levels and acute rejection in the universal voriconazole period.

Azole resistance in Aspergillus fumigatus is a One Health resistance threat, where azole fungicide exposure compromises the efficacy of medical azoles. The use of the recently authorized fungicide ipflufenoquin, which shares its mode-of-action with a new antifungal olorofim, underscores the need for risk assessment for dual use of antifungals., Competing Interests: Declaration of Competing Interest Paul E Verweij declares grants from F2G and Gilead Sciences and payments for lectures and advisory boards by F2G, Gilead Sciences, Pfizer and Mundipharma. All payments to institute. Maiken C Arendrup, Ana Alastruey-Izquierdo, Jeremy A W Gold, Shawn R Lockhart, and Tom Chiller declare no competing interests. P Lewis White declares grants from F2G, CERBA, IHMA, payments for lectures and advisory boards by F2G, Gilead Sciences, and Pfizer, and support for travel from OLM., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ibrexafungerp is a novel triterpenoid antifungal that inhibits glucan synthase and thus fungal cell wall synthesis. We examined the in vitro activity against contemporary clinical yeast, investigated inter-laboratory and intra-laboratory variability, suggested wild-type upper-limit values (WT-UL), and compared in vitro activity of ibrexafungerp to five licensed antifungals. Susceptibility to ibrexafungerp and comparators was investigated prospectively for 1965 isolates (11,790 MICs) and repetitively for three QC strains (1764 MICs) following the EUCAST E.Def 7.3.2 method. Elevated ibrexafungerp/echinocandin MICs prompted FKS sequencing. Published ibrexafungerp EUCAST MIC-distributions were retrieved and aggregated for WT-UL determinations following EUCAST principles. Ibrexafungerp MICs were ≤2 mg/L except against C. pararugosa, Cryptococcus and some rare yeasts. Modal MICs (mg/L) were 0.06/0.125/0.25/0.5/0.5/0.5/0.5/1/2 for C. albicans / C. dubliniensis / C. glabrata / C. krusei / C. parapsilosis / C. tropicalis / S. cerevisiae / C. guilliermondii / C. lusitaniae and aligned within ±1 dilution with published values. The MIC ranges for QC strains were: 0.06-0.25/0.5-1/0.125-0.5 for CNM-CL-F8555/ATCC6258/ATCC22019. The WT-UL (mg/L) were: 0.25/0.5/1/1/2 for C. albicans / C. glabrata / C. krusei / C. parapsilosis / C. tropicalis . Adopting these, non-wild-type rates were 0.3%/0.6%/0%/8%/3% for C. albicans / C. glabrata / C. krusei / C. parapsilosis / C. tropicalis and overall lower than for comparators except amphotericin B. Five/six non-wild-type C. albicans / C. glabrata were echinocandin and Fks non-wild-type (F641S, F659del or F659L). Eight C. parapsilosis and three C. tropicalis non-wild-type isolates were echinocandin and Fks wild-type. Partial inhibition near 50% in the supra-MIC range may explain variable MICs. Ibrexafungerp EUCAST MIC testing is robust, although the significance of paradoxical growth for some species requires further investigation. The spectrum is broad and will provide an oral option for the growing population with azole refractory infection.

Introduction: The 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 Covered: The 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 Opinion: Ibrexafungerp 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.

The increasing incidence and changing epidemiology of invasive fungal infections continue to present many challenges to their effective management. The repertoire of antifungal drugs available for treatment is still limited although there are new antifungals on the horizon. Successful treatment of invasive mycoses is dependent on a mix of pathogen-, host- and antifungal drug-related factors. Laboratories need to be adept at detection of fungal pathogens in clinical samples in order to effectively guide treatment by identifying isolates with acquired drug resistance. While there are international guidelines on how to conduct in vitro antifungal susceptibility testing, these are not performed as widely as for bacterial pathogens. Furthermore, fungi generally are recovered in cultures more slowly than bacteria, and often cannot be cultured in the laboratory. Therefore, non-culture-based methods, including molecular tests, to detect fungi in clinical specimens are increasingly important in patient management and are becoming more reliable as technology improves. Molecular methods can also be used for detection of target gene mutations or other mechanisms that predict antifungal drug resistance. This review addresses acquired antifungal drug resistance in the principal human fungal pathogens and describes known resistance mechanisms and what in-house and commercial tools are available for their detection. It is emphasized that this approach should be complementary to culture-based susceptibility testing, given the range of mutations, resistance mechanisms and target genes that may be present in clinical isolates, but may not be included in current molecular assays., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Background: Treatment of tinea pedis and onychomycosis is complicated by high rates of reinfection and the emergence of terbinafine-resistant strains of Trichophyton spp. Effective disinfection of contaminated socks is an important measure. Appropriate washing reduces the risk of reinfection and is paramount in treating tinea pedis and onychomycosis., Objectives: The aim of this study was to describe the effect of commonplace disinfection methods using socks pieces inoculated with terbinafine-resistant or terbinafine-susceptible isolates of Trichophyton spp., Methods: Sock pieces were inoculated with seven terbinafine-resistant isolates of Trichophyton spp. with known mutations in the SQLE-gene (T. rubrum (n = 3), T. interdigitale (n = 1) and T. indotineae (n = 3)) and six terbinafine-susceptible isolates of Trichophyton spp. (T. rubrum (n = 3) and T. interdigitale (n = 3)). Methods of disinfection included soaking in a quaternary ammonium (QAC) detergent (0.5, 2 and 24 h), freezing at -20°C (0.5, 12 and 24 h), domestic and steam washing (both at 40°C with detergent). Sock pieces were cultured for 4 weeks following disinfection. The primary end point was no growth at the end of week 4., Results: Soaking in a QAC-detergent for 24 h procured at disinfectant rate of 100% (13/13), whilst soaking in 0.5 and 2 h had a disinfectant rate of 46.2% (6/13) and 84.6% (11/13), respectively. Domestic washing (40°C with detergent) produced a disinfectant rate of 7.7% (1/13). Freezing at -20°C (0.5, 12 and 24 h) and steam washing (40°C with detergent) had no disinfectant properties., Conclusions: Soaking in a QAC-detergent for 24 h effectively disinfected sock pieces contaminated with dermatophytes., (© 2022 Wiley-VCH GmbH.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This is a case report of the first two cases of Candida auris in Denmark. Patient 1 was known to be colonized with C. auris when transferred from a foreign hospital to a Danish hospital. The patient was isolated during the entire hospitalization and the room was thoroughly cleaned after discharge. Patient 2 who had no travel history spent five hours in the room of Patient 1 after disinfection. One month later, C. auris was found in the blood of Patient 2. Transmission from Patient 1 to Patient 2 must be suspected.

Background: Azole resistance complicates treatment of patients with invasive aspergillosis with an increased mortality. Azole resistance in Aspergillus fumigatus is a growing problem and associated with human and environmental azole use. Denmark has a considerable and highly efficient agricultural sector. Following reports on environmental azole resistance in A. fumigatus from Danish patients, the ministry of health requested a prospective national surveillance of azole-resistant A. fumigatus and particularly that of environmental origin., Objectives: To present the data from the first 2 years of the surveillance programme., Methods: Unique isolates regarded as clinically relevant and any A. fumigatus isolated on a preferred weekday (background samples) were included. EUCAST susceptibility testing was performed and azole-resistant isolates underwent cyp51A gene sequencing., Results: The azole resistance prevalence was 6.1% (66/1083) at patient level. The TR 34 /L98H prevalence was 3.6% (39/1083) and included the variants TR 34 /L98H, TR 34 3 /L98H and TR 34 /L98H/S297T/F495I. Resistance caused by other Cyp51A variants accounted for 1.3% (14/1083) and included G54R, P216S, F219L, G54W, M220I, M220K, M220R, G432S, G448S and Y121F alterations. Non-Cyp51A-mediated resistance accounted for 1.2% (13/1083). Proportionally, TR 34 /L98H, other Cyp51A variants and non-Cyp51A-mediated resistance accounted for 59.1% (39/66), 21.2% (14/66) and 19.7% (13/66), respectively, of all resistance. Azole resistance was detected in all five regions in Denmark, and TR 34 /L98H specifically, in four of five regions during the surveillance period., Conclusion: The azole resistance prevalence does not lead to a change in the initial treatment of aspergillosis at this point, but causes concern and leads to therapeutic challenges in the affected patients., (© 2022 The Authors. Mycoses published by Wiley-VCH GmbH.)

Candida parapsilosis is the second most common cause of candidemia in some geographical areas and in children in particular. Yet, the proportion among children varies, for example, from 10.4% in Denmark to 24.7% in Tehran, Iran. As this species is also known to cause hospital outbreaks, we explored if the relatively high number of C. parapsilosis pediatric cases in Tehran could in part be explained by undiscovered clonal outbreaks. Among 56 C. parapsilosis complex isolates, 50 C. parapsilosis were genotyped by Amplified Fragment Length Polymorphism (AFLP) fingerprinting and microsatellite typing and analyzed for nucleotide polymorphisms by FKS1 and ERG11 sequencing. AFLP fingerprinting grouped Iranian isolates in two main clusters. Microsatellite typing separated the isolates into five clonal lineages, of which four were shared with Danish isolates, and with no correlation to the AFLP patterns. ERG11 and FKS1 sequencing revealed few polymorphisms in ERG11 leading to amino-acid substitutions (D133Y, Q250K, I302T, and R398I), with no influence on azole-susceptibilities. Collectively, this study demonstrated that there were no clonal outbreaks at the Iranian pediatric ward. Although possible transmission of a diverse C. parapsilosis community within the hospital cannot be ruled out, the study also emphasizes the necessity of applying appropriately discriminatory methods for outbreak investigation.

The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics 1 . Here we show that particular lineages of methicillin-resistant Staphylococcus aureus-a notorious human pathogen-appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two β-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development., (© 2022. The Author(s).)

Terbinafine resistance in Trichophyton species has emerged and appears to be increasing. A new EUCAST susceptibility testing method and tentative ECOFFs were recently proposed for Trichophyton . Terbinafine resistance and target gene mutations were detected in 16 Danish isolates in 2013-2018. In this study, samples/isolates submitted for dermatophyte susceptibility testing 2019-2020 were examined. Species identification (ITS sequencing for T. mentagrophytes/T. interdigitale species complex (SC) isolates), EUCAST MICs and squalene epoxidase (SQLE) profiles were obtained. Sixty-three isolates from 59 patients were included. T. rubrum accounted for 81% and T. mentagrophytes/T. interdigitale SC for 19%. Approximately 60% of T. rubrum and T. mentagrophytes/interdigitale SC isolates were terbinafine non-wildtype and/or had known/novel SQLE mutations with possible implications for terbinafine MICs. All infections with terbinafine-resistant T. mentagrophytes/interdigitale SC isolates were caused by Trichophyton indotineae . Compared to 2013-2018, the number of patients with terbinafine-resistant Trichophyton isolates increased. For T. rubrum , this is partly explained by an increase in number of requests for susceptibility testing. Terbinafine-resistant T. indotineae was first detected in 2018, but accounted for 19% of resistance (4 of 21 patients) in 2020. In conclusion, terbinafine resistance is an emerging problem in Denmark. Population based studies are warranted and susceptibility testing is highly relevant in non-responding cases.

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.

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.)

Since the last revision in 2015, the taxonomy of section Flavipedes evolved rapidly along with the availability of new species delimitation techniques. This study aims to re-evaluate the species boundaries of section Flavipedes members using modern delimitation methods applied to an extended set of strains (n = 90) collected from various environments. The analysis used DNA sequences of three house-keeping genes ( benA , CaM , RPB2 ) and consisted of two steps: application of several single-locus (GMYC, bGMYC, PTP, bPTP) and multi-locus (STACEY) species delimitation methods to sort the isolates into putative species, which were subsequently validated using DELINEATE software that was applied for the first time in fungal taxonomy. As a result, four new species are introduced, i.e. A. alboluteus , A. alboviridis , A. inusitatus and A. lanuginosus , and A. capensis is synonymized with A. iizukae . Phenotypic analyses were performed for the new species and their relatives, and the results showed that the growth parameters at different temperatures and colonies characteristics were useful for differentiation of these taxa. The revised section harbors 18 species, most of them are known from soil. However, the most common species from the section are ecologically diverse, occurring in the indoor environment (six species), clinical samples (five species), food and feed (four species), droppings (four species) and other less common substrates/environments. Due to the occurrence of section Flavipedes species in the clinical material/hospital environment, we also evaluated the susceptibility of 67 strains to six antifungals (amphotericin B, itraconazole, posaconazole, voriconazole, isavuconazole, terbinafine) using the reference EUCAST method. These results showed some potentially clinically relevant differences in susceptibility between species. For example, MICs higher than those observed for A. fumigatus wild-type were found for both triazoles and amphotericin B for A. ardalensis, A. iizukae, and A. spelaeus whereas A. lanuginosus, A. luppiae, A. movilensis, A. neoflavipes, A. olivimuriae and A. suttoniae were comparable to or more susceptible as A. fumigatus . Finally, terbinafine was in vitro active against all species except A. alboviridis ., (© 2021 THE AUTHORS.)

Liposomal amphotericin-B (L-AmB) prophylaxis is used in children with leukemia when azoles are contraindicated, but its effect is debated. We reviewed cases of invasive aspergillosis despite L-AmB 2.5 mg/kg twice weekly in children with high-risk leukemia during 2012-2019. Ten (16%) of 62 children had proven or probable aspergillosis. Thus, L-AmB prophylaxis offered insufficient protection for Aspergillus, in particular for Aspergillus flavus., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

As part of a national surveillance programme initiated in 2004, fungal blood isolates from 2016-2018 underwent species identification and EUCAST susceptibility testing. The epidemiology was described and compared to data from previous years. In 2016-2018, 1454 unique isolates were included. The fungaemia rate was 8.13/100,000 inhabitants compared to 8.64, 9.03, and 8.38 in 2004-2007, 2008-2011, and 2012-2015, respectively. Half of the cases (52.8%) involved patients 60-79 years old and the incidence was highest in males ≥70 years old. Candida albicans accounted for 42.1% of all isolates and Candida glabrata for 32.1%. C. albicans was more frequent in males ( p = 0.03) and C. glabrata in females ( p = 0.03). During the four periods, the proportion of C. albicans decreased ( p < 0.001), and C. glabrata increased ( p < 0.001). Consequently, fluconazole susceptibility gradually decreased from 68.5% to 59.0% ( p < 0.001). Acquired fluconazole resistance was found in 4.6% Candida isolates in 2016-2018. Acquired echinocandin resistance increased during the four periods 0.0%, 0.6%, 1.7% to 1.5% ( p < 0.0001). Sixteen echinocandin-resistant isolates from 2016-2018 harboured well-known FKS resistance-mutations and one echinocandin-resistant C. albicans had an FKS mutation outside the hotspot (P1354P/S) of unknown importance. In C. glabrata specifically, echinocandin resistance was detected in 12/460 (2.6%) in 2016-2018 whereas multidrug-class resistance was rare (1/460 isolates (0.2%)). Since the increase in incidence during 2004-2011, the incidence has stabilised. In contrast, the species distribution has changed gradually over the 15 years, with increased C. glabrata at the expense of C. albicans . The consequent decreased fluconazole susceptibility and the emergence of acquired echinocandin resistance complicates the management of fungaemia and calls for antifungal drug development.

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., (© The Author(s) 2021. 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.)

Posaconazole is more active than fluconazole against Candida albicans in vitro and is approved for the treatment of oropharyngeal candidiasis but not for that of invasive candidiasis (IC). Here, we explored the efficacy of posaconazole against C. albicans in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model of IC and determined the probability of pharmacodynamic target attainment for the oral solution and intravenous (i.v.)/tablet formulations. Three clinical C. albicans isolates (posaconazole MICs, 0.008 to 0.25 mg/liter) were studied in the in vitro PK/PD dilution model simulating steady-state posaconazole PK. The in vitro exposure-effect relationship, area under the 24-h free drug concentration curve ( f AUC 0-24 )/MIC, was described and compared with in vivo outcome in animals with IC. PK/PD susceptibility breakpoints and trough levels required for optimal treatment were determined for EUCAST and CLSI 24-h/48-h (CLSI24h/CLSI48h) methods using the f AUC 0-24 /MIC associated with half-maximal activity (EI 50 ) and Monte Carlo simulation analysis for oral solution (400 mg every 12 hours [q12h]) and i.v./tablet formulations (300 mg q24h). The in vitro mean (95% confidence interval [CI]) EI 50 was 330 (183 to 597) f AUC 0-24 /MIC for CLSI24h and 169 (92 to 310) for EUCAST/CLSI48h methods, which are close to the near-stasis in vivo effect. The probability of target attainment for EI 50 was estimated; for the wild-type isolates (MIC ≤ 0.06 mg/liter), it was low for the oral solution and higher than 95% for the i.v./tablet formulations for the EUCAST/CLSI48h methods but not for the CLSI 24-h method. Non-wild-type isolates with EUCAST/CLSI48h MICs of 0.125 and 0.25 mg/liter would require trough levels of >1.2 and >2.4 mg/liter, respectively. Posaconazole i.v./tablet formulations may have a role in the therapy of invasive infections by wild-type C. albicans isolates, provided that a steady state is reached quickly. A PK/PD susceptibility breakpoint at the epidemiological cutoff (ECV/ECOFF) of 0.06 mg/liter was determined., (Copyright © 2021 American Society for Microbiology.)

Echinocandins have been used as primary therapy of invasive aspergillosis (IA), with suboptimal results at standard dosing. Here, we explored the efficacy of dose escalation in a validated in vitro pharmacokinetic/pharmacodynamic (PK/PD) model. Six echinocandin wild-type (WT) and three non-WT A. fumigatus isolates were tested in an in vitro PK/PD model simulating anidulafungin, caspofungin, and micafungin exposures with a free drug maximum concentration ( f C max ) of 0.01 to 16 mg/liter and a half-life ( t 1/2 ) of 8 to 22 h. The relationship between the area under the dosing interval time-free drug concentration curve ( f AUC 0-24 )/minimum effective concentration (MEC) and % aberrant mycelium formation was analyzed. PK/PD indices associated with 50 to 99.99% maximal activity (EI 50 to EI 99.99 ) were correlated with the clinical outcome of a 50-mg/day standard dose of caspofungin. The probability of target attainment (PTA) was calculated for different dosing regimens of each echinocandin via Monte Carlo analysis. A sigmoidal PK/PD relationship was found for WT isolates with EI 99 values of 766, 8.8, and 115 f AUC 0-24 /CLSI MEC for anidulafungin, caspofungin, and micafungin, respectively. No aberrant mycelia were observed for non-WT isolates, irrespective of their MEC and drug exposure. The EI 99 , EI 99.9 , and EI 99.99 values corresponded to 2-, 3-, and 4-log 10 formation of aberrant mycelia and correlated with survival, favorable, and complete response rates to caspofungin primary therapy in patients with IA. A very low PTA (<13%) was found for the standard doses of all echinocandins, whereas a PTA of ≥90% was found with 100 and 150 mg/day of caspofungin and 1,400 mg/day micafungin against WT isolates. For anidulafungin, the PTA for 1,500 mg/day was 10%. Among the three echinocandins, only caspofungin at 2 or 3 times the licensed dosing was associated with a high PTA. Caspofungin dose escalation might deserve clinical validation., (Copyright © 2021 American Society for Microbiology.)

Azole resistance is an emerging problem in patients with aspergillosis. The role of fungicides for resistance development and occurrence is not fully elucidated. EUCAST reference MICs of 17 fungicides (11 azoles and 6 others), five azole fungicide metabolites and four medical triazoles were examined against two reference and 28 clinical isolates of A. fumigatus , A. flavus and A. terreus with ( n = 12) and without ( n = 16) resistance mutations. Eight/11 azole fungicides were active against wild-type A. fumigatus , A. flavus and A. terreus , including four (metconazole, prothioconazole-desthio, prochloraz and imazalil) with low MIC 50 (≤2 mg/L) against all three species and epoxiconazole, propiconazole, tebuconazole and difenoconazole also against wild-type A. terreus . Mefentrifluconazole, azole metabolites and non-azole fungicides MICs were >16 mg/L against A. fumigatus although partial growth inhibition was found with mefentrifluconazole. Moreover, mefentrifluconazole and axozystrobin were active against wild-type A. terreus . Increased MICs (≥3 dilutions) were found for TR 34 /L98H, TR 34 (3) /L98H, TR 46 /Y121F/T289A and G432S compared to wild-type A. fumigatus for epoxiconazole, propiconazole, tebuconazole, difenoconazole, prochloraz, imazalil and metconazole (except G432S), and for prothioconazole-desthio against TR 46 /Y121F/T289A, specifically. Increased MICs were found in A. fumigatus harbouring G54R, M220K and M220R alterations for five, one and one azole fungicides, respectively, compared to MICs against wild-type A. fumigatus . Similarly, increased MICs wer found for A. terreus with G51A, M217I and Y491H alterations for five, six and two azole fungicides, respectively. Azole fungicides showed activity against wild-type A. fumigatus , A. terreus and A. flavus , but not against all mutant isolates, suggesting the environmental route of azole resistance may have a role for all three species.

Background: Terbinafine is a first-line agent against Trichophyton-infections. However, treatment failure and resistance due to squalene epoxidase (SQLE) alterations are increasingly being reported. Photodynamic therapy (PDT) is based on combining a photosensitizer, light and oxygen to create photo-activated reactive oxygen species. It has demonstrated in vitro and in vivo activity against various microorganisms including dermatophytes. We investigated if PDT is equally effective against terbinafine resistant and susceptible strains., Methods: Minimum inhibitory concentrations (MIC) of methylene blue (MB)-PDT against wildtype and resistant Trichophyton rubrum and Trichophyton interdigitale were determined in duplicate in microtitre plates following EUCAST E.Def 11.0 reference methodology. Included mutants harboured F397L, L393F, L393S, F415S or F397I SQLE-alterations. Illumination with red diode light was performed after <3 min, 30 min and 3 h of incubation, respectively, and plates were cultured at 25 °C for 5 days. Geometric mean MICs and MIC ranges were calculated for each isolate., Results: MB-PDT led to complete inhibition of all isolates at geometric mean concentrations of 1-16 mg/L. Efficacy was independent of incubation time prior to illumination, terbinafine susceptibility (MICs ≤0.004-4 mg/L) and presence of SQLE mutations. However, the MB-PDT MIC was slightly elevated (MB: 2-8 mg/L and 8-16 mg/L) in isolates from two pigmented cultures of Trichophyton interdigitale (one wildtype and one harbouring L393F) with a darker color when compared to unpigmented cultures (MB: 0.5-4 mg/L)., Conclusion: Terbinafine resistant and susceptible strains are equally susceptible to MB-PDT. Lower efficacy was observed against dark coloured isolates which we speculate may be due to melanisation interfering with photo-activation due to preferential light absorption., (Copyright © 2020. Published by Elsevier B.V.)

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 fAUC 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 (fAUC 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)., (Copyright © 2021 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

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.)