Development of an agar-based screening method for terbinafine, itraconazole, and amorolfine susceptibility testing of Trichophyton spp.

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.

Predictors for Prolonged Hospital Stay Solely to Complete Intravenous Antifungal Treatment in Patients with Candidemia: Results from the ECMM Candida III Multinational European Observational Cohort Study.

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.

Development of an in vitro pharmacokinetic/pharmacodynamic model in the presence of serum for studying micafungin activity against Candida albicans: a need for revision of CLSI susceptibility breakpoints.

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.

Dual use of antifungals in medicine and agriculture: How do we help prevent resistance developing in human pathogens?

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.

EUCAST Ibrexafungerp MICs and Wild-Type Upper Limits for Contemporary Danish Yeast Isolates.

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.

Invasive candidiasis: investigational drugs in the clinical development pipeline and mechanisms of action.

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.

Emergence of methicillin resistance predates the clinical use of antibiotics.

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 antibiotics1. 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 ß-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.

The Emerging Terbinafine-Resistant Trichophyton Epidemic: What Is the Role of Antifungal Susceptibility Testing?

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.

Photodynamic therapy: A treatment option for terbinafine resistant Trichophyton 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.

Comparative Pharmacodynamics of Echinocandins against Aspergillus fumigatus Using an In Vitro Pharmacokinetic/Pharmacodynamic Model That Correlates with Clinical Response to Caspofungin Therapy: Is There a Place for Dose Optimization?

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 (fCmax) of 0.01 to 16 mg/liter and a half-life (t1/2) of 8 to 22 h. The relationship between the area under the dosing interval time-free drug concentration curve (fAUC0-24)/minimum effective concentration (MEC) and % aberrant mycelium formation was analyzed. PK/PD indices associated with 50 to 99.99% maximal activity (EI50 to EI99.99) 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 EI99 values of 766, 8.8, and 115 fAUC0-24/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 EI99, EI99.9, and EI99.99 values corresponded to 2-, 3-, and 4-log10 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.

Lack of relationship between genotype and virulence in Candida species.

BACKGROUND: The virulence of isolates among different Candida species causing candidemia may play a role in the prognosis of the patients. Furthermore, the potential relationship between genotype and virulence is still unclear and need to be further studied. AIMS: We aim to assess the relationship between genotype and virulence in Candida species using a Galleria mellonella larvae infection model. METHODS: One hundred and ninety-four isolates from 68 clusters (Candida albicans, 114/41; Candida parapsilosis, 74/24; Candida tropicalis, 6/3) were compared against the same number of each species singleton genotypes in terms of survival of G. mellonella larvae. RESULTS: The median of survival and the IQR ranges of clusters and singleton were as follows: C. albicans (2 days, IQR 1.5-2 vs. 2 days, IQR 1-2.25), C. parapsilosis (2 days, IQR 1.5-2.6 vs. 2 days, IQR 2-3.3), and C. tropicalis (1 day, IQR 1-3.5 vs. 2 days, IQR 2-3.5; p<0.05). High intra-cluster variability in terms of median of survival was found regardless the species. CONCLUSIONS: No relationship between genotype and virulence in Candida was observed with the G. mellonella model.

How to: perform antifungal susceptibility testing of microconidia-forming dermatophytes following the new reference EUCAST method E.Def 11.0, exemplified by Trichophyton.

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.

Genotyping Reveals High Clonal Diversity and Widespread Genotypes of Candida Causing Candidemia at Distant Geographical Areas.

The objectives of this study were to gain further insight on Candida genotype distribution and percentage of clustered isolates between hospitals and to identify potential clusters involving different hospitals and cities. We aim to genotype Candida spp. isolates causing candidemia in patients admitted to 16 hospitals in Spain, Italy, Denmark, and Brazil. Eight hundred and eighty-four isolates (Candida albicans, n = 534; C. parapsilosis, n = 282; and C. tropicalis, n = 68) were genotyped using species-specific microsatellite markers. CDC3, EF3, HIS3, CAI, CAIII, and CAVI were used for C. albicans, Ctrm1, Ctrm10, Ctrm12, Ctrm21, Ctrm24, and Ctrm28 for C. tropicalis, and CP1, CP4a, CP6, and B for C. parapsilosis. Genotypes were classified as singletons (genotype only found once) or clusters (same genotype infecting two or more patients). Clusters were defined as intra-hospital (involving patients admitted to a single hospital), intra-ward (involving patients admitted to the same hospital ward) or widespread (involving patients admitted to different hospitals). The percentage of clusters and the proportion of patients involved in clusters among species, genotypic diversity and distribution of genetic diversity were assessed. Seven hundred and twenty-three genotypes were detected, 78 (11%) being clusters, most of which (57.7%; n = 45/78) were intra-hospital clusters including intra-ward ones (42.2%; n = 19/45). The proportion of clusters was not statistically different between species, but the percentage of patients in clusters varied among hospitals. A number of genotypes (7.2%; 52/723) were widespread (found at different hospitals), comprising 66.7% (52/78) of clusters, and involved patients at hospitals in the same city (n = 21) or in different cities (n = 31). Only one C. parapsilosis cluster was a widespread genotype found in all four countries. Around 11% of C. albicans and C. parapsilosis isolates causing candidemia are clusters that may result from patient-to-patient transmission, widespread genotypes commonly found in unrelated patients, or insufficient microsatellite typing genetic discrimination.

Photodynamic therapy treatment of superficial fungal infections: A systematic review.

BACKGROUND: Fungal infections in skin, hair and nails affect up to 25 % of the global population. Conventional antifungal treatment is effective but due to resistance, treatment failure, drug interactions, and treatment related toxicity, there is a need for alternative treatments. Photodynamic therapy (PDT) has shown antimicrobial properties and is used increasingly for fungal infections. This review investigates the reported efficacy and side effects of PDT of superficial mycoses. METHODS: Pubmed and Embase were searched 26-01-2020 for "superficial fungal infections" and "photodynamic therapy" in "Human subjects" using a predefined search string. Criteria for inclusion were: clinical trials and cases involving PDT-treated patients with primary fungal infections in skin, hair and nails. Criteria for exclusion were: languages other than English, animal models, in vitro trials, secondary fungal infections, reviews and guidelines. RESULTS: 541 records were identified and 34 papers fulfilled the criteria. PDT of onychomycosis (n = 380 patients) found treatment with methylene blue (MB) photosensitizer (PS) more efficacious with complete cure rates of 70 %-80 % than 5-aminolevulinic acid (ALA)-PDT (mycological cure rates of 17 %-57 %) and methyl aminolevulinate (MAL)-PDT (mycological cure rate of 32 %). Other PDT-treated fungal diseases included (n = 55): foot infections (n = 19), tinea cruris (n = 10), scalp infections (n = 2), Malassezia infections (n = 9) and subcutaneous fungal infections (n = 15) achieved promising effect. CONCLUSION: PDT-treatment of superficial mycoses can be efficacious as salvage therapy. In the light of increasing resistance and few licensed treatment alternatives, larger randomized controlled trials investigations and optimization of the PDT-treatment protocol are warranted to evaluate PDT's potential as a future antifungal treatment.

Candidemia Candida albicans clusters have higher tendency to form biofilms than singleton genotypes†.

The capacity of Candida spp. to form biofilms allows them to attach either to living or inert surfaces, promoting their persistence in hospital environments. In a previous study, we reported strain-to-strain variations in Candida spp. biofilm development, suggesting that some genotypes may be greater biofilm formers than others. In this study, we hypothesize that isolates pertaining to clusters may be found more frequently in the environment due to their ability to form biofilms compared to singleton genotypes. Two hundred and thirty-nine Candida spp. isolates (78 clusters) from candidemia patients admitted to 16 hospitals located in different cities and countries-and the same number of singleton genotypes used as controls-were tested in terms of biofilm formation using the crystal violet and the XTT reduction assays. Candida albicans clusters showed higher biofilm formation in comparison to singleton genotypes (P < .01). The biofilms formed by intra-hospital C. albicans clusters showed higher metabolic activity (P < .05). Furthermore, marked variability was found among species and type of cluster. We observed that the higher the number of isolates, the higher the variability of biofilm production by isolates within the cluster, suggesting that the production of biofilm by isolates of the same genotype is quite diverse and does not depend on the type of cluster studied. In conclusion, candidemia Candida spp. clusters-particularly in the case of C. albicans-show significantly more biomass production and metabolic activity than singleton genotypes.

Emerging Terbinafine Resistance in Trichophyton: Clinical Characteristics, Squalene Epoxidase Gene Mutations, and a Reliable EUCAST Method for Detection.

In recent years, cases involving terbinafine-resistant Trichophyton isolates have been reported increasingly, particularly in India. We present 14 cases of terbinafine treatment failure in Trichophyton-infected Danish patients due to acquired resistance. Patients infected with Trichophyton rubrum (n = 12) or Trichophyton interdigitale (n = 2) with elevated terbinafine MICs during 2013-2018 were included. Antifungal susceptibility testing (AFST) was performed following a modified EUCAST E.Def 9.3.1 method (5 days of incubation) with or without cycloheximide and chloramphenicol (CC) supplementation of the growth medium. The squalene epoxidase (SE) target gene was sequenced, and 3-dimensional enzyme homology modeling was performed. Most patients (12/14 [86%]) were male. The mean age was 53.5 years (range, 11 to 77 years). The mean duration of infections was 4.8 years at the time of resistance detection. Prior systemic terbinafine treatment was documented for all patients, and topical therapy for 62% (information was missing in one case). Overall, nine isolates (64%) displayed high terbinafine resistance (MICs, 4 to >8 mg/liter), while two (14%) displayed moderate (MICs, 1 to 2 mg/liter) and three (21%) displayed low (MICs, 0.125 to 0.25 mg/liter) terbinafine resistance compared with control isolates. MICs generated with or without CC supplementation were similar, but CC prevented contamination. Known and novel SE amino acid substitutions (F397L, L393F, L393S, F415S, H440Y F484Y, and I121M V237I) were detected in resistant but not control isolates. Three-dimensional homology modeling suggested a role of the novel I121M and V237I alterations. Terbinafine resistance has been detected in Denmark using a modified EUCAST method, which facilitated susceptibility testing of dermatophytes. Action is needed for this emerging public health problem.

Relevance of heterokaryosis for adaptation and azole-resistance development in Aspergillus fumigatus.

Aspergillus fumigatus causes a range of diseases in humans, some of which are characterized by fungal persistence. Aspergillus fumigatus, being a generalist saprotroph, may initially establish lung colonization due to its physiological versatility and subsequently adapt through genetic changes to the human lung environment and antifungal treatments. Human lung-adapted genotypes can arise by spontaneous mutation and/or recombination and subsequent selection of the fittest genotypes. Sexual and asexual spores are considered crucial contributors to the genetic diversity and adaptive potential of aspergilli by recombination and mutation supply, respectively. However, in certain Aspergillus diseases, such as cystic fibrosis and chronic pulmonary aspergillosis, A. fumigatus may not sporulate but persist as a network of fungal mycelium. During azole therapy, such mycelia may develop patient-acquired resistance and become heterokaryotic by mutations in one of the nuclei. We investigated the relevance of heterokaryosis for azole-resistance development in A. fumigatus. We found evidence for heterokaryosis of A. fumigatus in patients with chronic Aspergillus diseases. Mycelium from patient-tissue biopsies segregated different homokaryons, from which heterokaryons could be reconstructed. Whereas all variant homokaryons recovered from the same patient were capable of forming a heterokaryon, those from different patients were heterokaryon-incompatible. We furthermore compared heterokaryons and heterozygous diploids constructed from environmental isolates with different levels of azole resistance. When exposed to azole, the heterokaryons revealed remarkable shifts in their nuclear ratio, and the resistance level of heterokaryons exceeded that of the corresponding heterozygous diploids.

In Vivo Selection of a Unique Tandem Repeat Mediated Azole Resistance Mechanism (TR120) in Aspergillus fumigatus cyp51A, Denmark.

We report a fatal aspergillosis case in which STRAf typing and whole-genome sequencing substantiated in vivo emergence of an azole-resistant Aspergillus fumigatus with a 120-bp tandem repeat in the promoter region of cyp51A. This event, previously restricted to the environment, challenges current understanding of azole resistance development in A. fumigatus.

Methodologies for in vitro and in vivo evaluation of efficacy of antifungal and antibiofilm agents and surface coatings against fungal biofilms.

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