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

  • Maiken C. Arendrup
    Correspondence
    Corresponding author: Maiken C. Arendrup, Unit for Mycology building 43/317, Statens Serum Institut, Artillerivej 5, DK-2300, Copenhagen S, Denmark.
    Affiliations
    Unit of Mycology, Department of Microbiological Surveillance and Research, Statens Serum Institut, Copenhagen, Denmark

    Department of Clinical Microbiology, University Hospital Rigshospitalet, Copenhagen, Denmark

    Department of Clinical Medicine, University of Copenhagen, Denmark
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  • Gunnar Kahlmeter
    Affiliations
    The EUCAST Development Laboratory, Clinical Microbiology, Växjö, Sweden
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  • Author Footnotes
    † Jesus Guinea and Joseph Meletiadis contributed equally and share the last author position.
    Jesus Guinea
    Footnotes
    † Jesus Guinea and Joseph Meletiadis contributed equally and share the last author position.
    Affiliations
    Clinical Microbiology and Infectious Diseases Department, Hospital General Universitario Gregorio Marañón, Madrid, Spain

    CIBER de Enfermedades Respiratorias-CIBERES (CB06/06/0058), Madrid, Spain

    Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
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  • Author Footnotes
    † Jesus Guinea and Joseph Meletiadis contributed equally and share the last author position.
    Joseph Meletiadis
    Footnotes
    † Jesus Guinea and Joseph Meletiadis contributed equally and share the last author position.
    Affiliations
    Clinical Microbiology Laboratory, Attikon University Hospital, National and Kapodistrian University of Athens, Athens, Greece

    Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rotterdam, the Netherlands
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  • the Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST)
  • Author Footnotes
    † Jesus Guinea and Joseph Meletiadis contributed equally and share the last author position.
Published:September 08, 2020DOI:https://doi.org/10.1016/j.cmi.2020.08.042

      Abstract

      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.

      Keywords

      Introduction

      Antifungal susceptibility tests are performed for fungi causing disease, especially when infections are invasive, relapsing or failing therapy, when inherent or acquired resistance is a possibility, or when susceptibility cannot reliably be predicted from the species identification alone. Antifungal susceptibility testing (AFST) is also important in resistance surveillance, in epidemiological studies and for comparison of the in vitro activity of new and existing agents.
      Dilution methods are used to establish the MICs of antimicrobial agents. These are the reference methods for antimicrobial susceptibility testing and are mainly used: to (a) establish the activity of new antimicrobial agents; (b) confirm the susceptibility of organisms that give equivocal results in other test formats (such as commercial susceptibility tests); and (c) determine the susceptibility of organisms where other test formats may be unreliable or not yet validated (which is still a common scenario for susceptibility testing of fungi and dermatophytes in particular). In dilution tests, fungi are tested for their ability to produce sufficient growth in microplate wells of broth culture medium containing serial dilutions of the antimicrobial agents (broth microdilution).
      The antifungal MIC is defined as the lowest concentration, in mg/L, of an agent that inhibits the growth of a fungus. The MIC informs about the susceptibility or resistance of the organism to the antifungal agent, which can help in treatment decisions.
      Dermatophyte infections are rarely invasive but when they involve the scalp or nails, systemic therapy for weeks to months is required [
      • Mayser P.
      • Nenoff P.
      • Reinel D.
      • Abeck D.
      • Brasch J.
      • Daeschlein G.
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      • Gupta A.K.
      • Versteeg S.G.
      • Shear N.H.
      • Piguet V.
      • Tosti A.
      • Piraccini B.M.
      A practical guide to curing onychomycosis: how to maximize cure at the patient, organism, treatment, and environmental level.
      ]. Systemic antifungal therapy is indicated also in cutaneous dermatophytosis presenting with extensive lesions or in patients with lack of response to topical therapy [
      • Hay R.
      Therapy of skin, hair and nail fungal infections.
      ,
      • Ely J.W.
      • Rosenfeld S.
      • Seabury Stone M.
      Diagnosis and management of tinea infections.
      ]. Presumptive diagnosis often relies on clinical findings and direct microscopic examination only, but differential diagnoses are many, systemic therapy is associated with risk of side effects, and resistance in Trichophyton species is rapidly rising [
      • Osborne C.S.
      • Leitner I.
      • Favre B.
      • Ryder N.S.
      Amino acid substitution in Trichophyton rubrum squalene epoxidase associated with resistance to terbinafine.
      ,
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      • Masih A.
      • Chowdhary A.
      • Sardana K.
      • Borker S.
      • Gupta A.
      • et al.
      Correlation of in vitro susceptibility based on mics and squalene epoxidase mutations with clinical response to terbinafine in patients with tinea corporis/cruris.
      ,
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      • Jørgensen K.M.
      • Jørgensen R.
      • Deleuran M.
      • Zachariae C.O.
      • et al.
      Emerging terbinafine resistance in Trichophyton: clinical characteristics, squalene epoxidase gene mutations and a reliable EUCAST method for detection.
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      • Hsieh A.
      • Quenan S.
      • Riat A.
      • Toutous-Trellu L.
      • Fontao L.
      A new mutation in the SQLE gene of Trichophyton mentagrophytes associated to terbinafine resistance in a couple with disseminated tinea corporis.
      ,
      • Monod M.
      • Feuermann M.
      • Salamin K.
      • Fratti M.
      • Makino M.
      • Alshahni M.M.
      • et al.
      Trichophyton rubrum azole resistance mediated by a new ABC transporter, TruMDR3.
      ,
      • Yamada T.
      • Maeda M.
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      • Tanaka R.
      • Yaguchi T.
      • Bontems O.
      • et al.
      Terbinafine resistance of Trichophyton clinical isolates caused by specific point mutations in the squalene epoxidase gene.
      ,
      • Baudraz-Rosselet F.
      • Ruffieux C.
      • Lurati M.
      • Bontems O.
      • Monod M.
      Onychomycosis insensitive to systemic terbinafine and azole treatments reveals non-dermatophyte moulds as infectious agents.
      ,
      • Monod M.
      Antifungal resistance in dermatophytes: emerging problem and challenge for the medical community.
      ,
      • Hsiao Y.-H.
      • Chen C.
      • Han H.S.
      • Kano R.
      The first report of terbinafine resistance Microsporum canis from a cat.
      ,
      • Singh A.
      • Masih A.
      • Monroy-Nieto J.
      • Singh P.K.
      • Bowers J.
      • Travis J.
      • et al.
      A unique multidrug-resistant clonal Trichophyton population distinct from Trichophyton mentagrophytes/Trichophyton interdigitale complex causing an ongoing alarming dermatophytosis outbreak in India: genomic insights and resistance profile.
      ,
      • Singh A.
      • Masih A.
      • Khurana A.
      • Singh P.K.
      • Gupta M.
      • Hagen F.
      • et al.
      High terbinafine resistance in Trichophyton interdigitale isolates in Delhi, India harbouring mutations in the squalene epoxidase gene.
      ,
      • Taghipour S.
      • Shamsizadeh F.
      • Pchelin I.M.
      • Rezaei-Matehhkolaei A.
      • Mahmoudabadi A.Z.
      • Valadan R.
      • et al.
      Emergence of terbinafine resistant Trichophyton mentagrophytes in Iran, harboring mutations in the squalene epoxidase (Sqle) gene.
      ]. Hence, mycological diagnosis and standardized AFST methods for determining the in vitro susceptibilities of clinical isolates of dermatophytes are needed to limit inappropriate therapy, unnecessary toxicity and selection of resistance [
      • Danielsen A.G.
      • Thomsen J.S.
      • Svejgaard E.L.
      Severe skin rash in patients treated with terbinafine.
      ].
      Dermatophytes grow slowly and often require 4–7 days of culture or more. Skin scrapings and nail samples are not sterile and although cultured on selective agars, MIC determination in non-selective broth medium is often challenged by contamination (Fig. 1). Such contamination interferes with end-point reading and often necessitates repetition of the test after isolation, which adds days or even weeks to the turnover time before a result can be provided. For these reasons, European Committee on Antimicrobial Susceptibility Testing (EUCAST) initially investigated if addition of cycloheximide and chloramphenicol to the standard EUCAST AFST growth medium, to render it selective for dermatophytes, interfered with microdilution MIC determination of selected wild-type and mutant Trichophyton isolates. Subsequently, EUCAST investigated the optimal test conditions for a dermatophyte EUCAST microdilution method, including various end-point definitions for the correct separation of susceptible wild-type and resistant non-wild-type clinical isolates of Trichophyton rubrum and Trichophyton interdigitale in a multicentre study [
      • Arendrup M.C.
      • Jørgensen K.M.
      • Guinea J.
      • Lagrou K.
      • Chryssanthou E.
      • Hayette M.
      • et al.
      Multicentre validation of a EUCAST method for the antifungal susceptibility testing of microconidia-forming dermatophytes.
      ].
      Fig. 1
      Fig. 1Terbinafine growth inhibition curves for (a) a resistant Trichophyton rubrum isolate (SSI-7885) harbouring a F397L target gene alteration and (b) a susceptible wild-type T. rubrum isolate (SSI-6714) tested following the E.Def 9.3.2 mould testing method (black curve) and the new E.Def 11.0 dermatophyte testing method with cycloheximide and chloramphenicol-supplemented medium (green curve). Solid lines indicate the inhibition curves, dashed lines the mean of four positive growth controls. Contaminated wells interfering with the susceptibility testing of the resistant isolate with the E.Def 9.3.2 method are indicated with grey arrows. The background is not subtracted (~OD 0.100).
      This first version of the standard is based on the general EUCAST principles for microtitre plate production described in the yeast and mould microdilution method documents (E.Def 7.3.2 and E.Def 9.3.2) with subsequent addition of cycloheximide and chloramphenicol during the inoculation step. This will allow the use of plates already prepared for mould testing (and thus prepared without cycloheximide and chloramphenicol in the plates) rather than requiring production of special plates for dermatophyte testing. Details on plate production are given in the Supplementary material (Appendix S1).

       Scope

      This EUCAST standard describes a suitable method for testing the susceptibility of microconidia-producing dermatophytes to antifungal agents by determination of the MIC. MICs show the in vitro activity of a given antifungal drug under the test conditions described, and can be used for patient management in conjunction with other factors, such as pharmacokinetics, pharmacodynamics and resistance mechanisms. The MIC permits microorganisms to be categorized as Susceptible (S), susceptible, Increased exposure (I), or Resistant (R) to an antifungal drug when appropriate breakpoints are applied [
      • Arendrup M.C.
      • Friberg N.
      • Mares M.
      • Kahlmeter G.
      • Meletiadis J.
      • Guinea J.
      Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST)
      How to: Interpret MICs of antifungal compounds according to the revised clinical breakpoints v. 10.0 European Committee on Antimicrobial Susceptibility Testing (EUCAST).
      ]. In addition, MIC distributions can be used to define wild-type or non-wild-type fungal populations when species-specific epidemiological cut-off values (ECOFFs) are applied.
      The method described herein is intended to provide a suitable, easy and economic method for testing the susceptibility to antifungal agents against Trichophyton spp. and to facilitate an acceptable degree of conformity, e.g. agreement within specified ranges, between laboratories. Many factors influence the MIC of filamentous fungi against antifungal agents as shown by Rambali et al. [
      • Rambali B.
      • Fernandez J.A.
      • Van Nuffel L.
      • Woestenborghs F.
      • Baert L.
      • Massart D.L.
      • et al.
      Susceptibility testing of pathogenic fungi with itraconazole: a process analysis of test variables.
      ]. For example the MIC of itraconazole against Aspergillus was profoundly influenced by shape of the microdilution well, inoculum concentration, temperature and length of incubation time. As technical laboratory factors are of utmost importance, this standard focuses on testing conditions including inoculum preparation and inoculum size, incubation time and temperature, and end-point definition. The terms and definitions used as basis for this procedure are found in the Supplementary material (Appendix S1).

       Test procedures

      The general test procedures concerning type of microtitre plates, choice of medium, preparation of stock and working solutions of antimicrobial agents, and preparation and storage of prepared plates with antifungals are identical to the recommendations in the mould (E.Def 9.3.2) reference method document, available at https://www.eucast.org/astoffungi/. The details are found in the Supplementary material (Appendix S1) and Table 1, Table 2.
      Table 1The amount of powder or diluent required to prepare a standard solution of antifungal powder, cycloheximide or chloramphenicol may be calculated as follows
      Weight(g)=Volume(L)×Concentration(mg/L)Potency(mg/g)
      Volume(L)=Weight(g)×Potency(mg/g)Concentration(mg/L)
      Table 2Solvents for preparation of stock solutions, characteristics and appropriate test concentration ranges for antifungal agents
      Antifungal agentSolventCharacteristicsTest range (mg/L)
      AmorolfineDMSOHydrophobic0.008–4
      ItraconazoleDMSOHydrophobic0.008–4
      PosaconazoleDMSOHydrophobic0.008–4
      TerbinafineDMSOHydrophobic0.004–2
      VoriconazoleDMSOHydrophobic0.008–4
      DMSO, dimethyl sulfoxide.

       General

      The test is performed in flat-bottom well microdilution plates. Different plastics are likely to impact on drug binding, which may affect MIC values. The validation of this method has been performed using tissue-treated microdilution plates, and the use of such plates is therefore more likely to yield similar MIC values. The method is based on the preparation of antifungal agent working solutions in 100-μL volumes per well to which 100 μL of inoculum supplemented with cycloheximide and chloramphenicol is added.
      Cycloheximide and chloramphenicol working solutions are prepared in the following concentrations: cycloheximide: 100 mg/mL (= 100 000 mg/L) in dimethylsulphoxide, followed by filtration (0.2 mm) (available ready to use from Sigma-Aldrich, St Louis, MO, USA; Cat. No. C4859). Chloramphenicol: 50 mg/mL (= 50 000 mg/L) in ethanol.
      Solutions must be prepared taking into account the potency of the lot of antifungal drug powder that is being used. The amount of powder or diluent required to prepare a standard solution may be calculated as described in Table 1.

       Preparation of inoculum supplemented with cycloheximide and chloramphenicol

      Standardization of the inoculum is essential for accurate and reproducible antifungal susceptibility tests. The final inoculum must be between 1 × 105 CFU/mL and 2.5 × 105 CFU/mL.

       Microconidia suspension method

      The isolates are subcultured on Sabouraud dextrose, potato dextrose agar or malt agar supplemented with cycloheximide (300 mg/L) and chloramphenicol (50 mg/L) and incubated at 25°C–28°C for 4–7 days. It may be advisable to inoculate two or three agar plates per isolate to ensure that a sufficient amount of microconidia can be harvested. Other culture media selective for dermatophyte growth, and where the fungus is able to sporulate sufficiently, can be used. Inoculum suspensions are prepared from fresh, mature cultures. In some cases, an extended incubation is required for proper sporulation of the isolate.
      Colonies are covered with approximately 5 mL of sterile water supplemented with 0.1% Tween-20. Then, the microconidia are carefully rubbed with a sterile cotton swab and transferred with a pipette to a sterile tube. Alternatively, a damp sterile cotton swab could be used to gently touch the culture, and the microconidia transferred to a sterile tube containing 5 mL water supplemented with 0.1% Tween-20. The suspension is vortexed for 15 seconds with a gyratory vortex mixer at approximately 2000 rpm and transferred to a sterile syringe attached to a sterile filter with a pore diameter of 11 μm, filtered and collected in a sterile tube. This step removes hyphae and yields a suspension composed of microconidia.
      The suspension is adjusted with sterile distilled water to 2 × 106 to 5 × 106 microconidia/mL by counting the microconidia in a haemocytometer chamber. Alternatively, a spectrophotometer can be used to adjust the filtered suspension to a concentration equivalent to McFarland 0.5 [
      • Rodriguez-Tudela J.L.
      • Chryssanthou E.
      • Petrikkou E.
      • Mosquera J.
      • Denning D.W.
      • Cuenca-Estrella M.
      Interlaboratory evaluation of hematocytometer method of inoculum preparation for testing antifungal susceptibilities of filamentous fungi.
      ,
      • Arendrup M.C.
      • Howard S.
      • Lass-Flörl C.
      • Mouton J.W.
      • Meletiadis J.
      • Cuenca-Estrella M.
      EUCAST Testing of isavuconazole susceptibility in Aspergillus: comparison of results for inoculum standardization using conidium counting versus optical density.
      ]. The suspension is then diluted 1:10 with sterile distilled water to obtain a final working inoculum of 2 × 105 to 5 × 105 CFU/mL [
      • Rodriguez-Tudela J.L.
      • Chryssanthou E.
      • Petrikkou E.
      • Mosquera J.
      • Denning D.W.
      • Cuenca-Estrella M.
      Interlaboratory evaluation of hematocytometer method of inoculum preparation for testing antifungal susceptibilities of filamentous fungi.
      ,
      • Arendrup M.C.
      • Howard S.
      • Lass-Flörl C.
      • Mouton J.W.
      • Meletiadis J.
      • Cuenca-Estrella M.
      EUCAST Testing of isavuconazole susceptibility in Aspergillus: comparison of results for inoculum standardization using conidium counting versus optical density.
      ,
      • Aberkane A.
      • Cuenca-Estrella M.
      • Gomez-Lopez A.
      • Petrikkou E.
      • Mellado E.
      • Monzón A.
      • et al.
      Comparative evaluation of two different methods of inoculum preparation for antifungal susceptibility testing of filamentous fungi.
      ,
      • Petrikkou E.
      • Rodríguez-Tudela J.L.
      • Cuenca-Estrella M.
      • Gómez a.
      • Molleja a.
      • Mellado E.
      Inoculum standardization for antifungal susceptibility testing of filamentous fungi pathogenic for humans.
      ].

       Inoculum supplementation with cycloheximide and chloramphenicol

      Each inoculum suspension is supplemented with cycloheximide and chloramphenicol in an amount that results in a double-strength final concentration (100 mg/L chloramphenicol and 600 mg/L cycloheximide, respectively). This allows further dilution when the inoculum is added to the test plate, resulting in a final concentration of 50 mg/L chloramphenicol and 300 mg/L cycloheximide in the inoculated plate. In Table 3, examples of a final inoculum of 8 mL are prepared for microdilution plates with four antifungals in a horizontal format, and of 12 mL for a full plate with eight compounds. The volumes can be adjusted to the preferred use in the individual laboratory. The total volume of chloramphenicol and cycloheximide corresponds to 0.8% of the total volume of the inoculum. This will result in a small dilution of the inoculum suspension; but this is below the limit of precision for AFST.
      Table 3Instructions on calculating the volume needed of chloramphenicol and cycloheximide for addition to the inoculum preparation depending on the final inoculum volume prepared
      Inoculum volumeVolume needed of chloramphenicol stock solution (50 000 mg/L)Volume needed of cycloheximide stock solution (100 000 mg/L)Total volume added to the inoculum
      8 mL100mg/L×8000μL50000mg/L=16μL600mg/L×8000μL100000mg/L=48μL64 μL
      12 mL100mg/L×12000μL50000mg/L=24μL600mg/L×12000μL100000mg/L=72μL96 μL

       Inoculation of microdilution plates

      The microdilution plates should be inoculated within 30 min of the preparation of the inoculum suspension to maintain viable microconidia concentration.
      The cycloheximide and chloramphenicol supplemented inoculum suspension is vortexed and each well of the microdilution plate is inoculated with 100 μL of the 2 × 105 to 5 × 105 CFU/mL microconidial suspension, without touching the contents of the well. This will give the required final drug concentration and inoculum density (final inoculum 1 × 105 to 2.5 × 105 CFU/mL). The growth control wells (column 11), which contained 100 μL of sterile drug-free medium, are also inoculated with 100 μL of the same inoculum suspension. Column 12 of the microdilution plate is filled with 100 μL of sterile distilled water from the lot used to prepare the inoculum as a sterility control for medium and distilled water (drug-free medium only). Quality control organisms are tested using the same method each time an isolate is tested.
      Viability counts should be performed for quality control purposes to ensure that test wells contain between 1 × 105 and 2.5 × 105 CFU/mL, as follows. Ten microlitres of the inoculum suspension should be diluted in 2 mL of sterile distilled water supplemented with 0.1% Tween-20. The suspension is then vortexed with a gyratory vortex mixer at 2000 rpm. Then 100 μL of this suspension is spread over the surface of a suitable agar plate (such as Sabouraud dextrose agar with cycloheximide and chloramphenicol), which is then incubated at 25°C–28°C until colonies can be counted. One hundred to 250 colonies are expected from an acceptable test suspension. A further dilution of 100 μL suspension in 900 μL sterile distilled water supplemented with 0.1% Tween-20, vortexing, and 100 μL plated out would provide an optional/additional count – 10 to 50 colonies would be expected. It is recommended that this is completed for every isolate when the laboratory is setting up this test or conducts the test rarely, when unexplained results are suspected, or periodically (to be locally defined dependent on need).

       Incubation of microdilution plates

      Microdilution plates are incubated without agitation at 25°C–28°C in ambient air [
      • Arendrup M.C.
      • Jørgensen K.M.
      • Guinea J.
      • Lagrou K.
      • Chryssanthou E.
      • Hayette M.
      • et al.
      Multicentre validation of a EUCAST method for the antifungal susceptibility testing of microconidia-forming dermatophytes.
      ]. Most Trichophyton isolates should be read at day 5, whichwas previously found appropriate for sufficient growth and validated in a multicentre study [
      • Arendrup M.C.
      • Jørgensen K.M.
      • Guinea J.
      • Lagrou K.
      • Chryssanthou E.
      • Hayette M.
      • et al.
      Multicentre validation of a EUCAST method for the antifungal susceptibility testing of microconidia-forming dermatophytes.
      ]. A, incubation longer than 7 days is not recommended.

       Reading results

      During the validation process, spectrophotometric readings (most experience with 490 nm but 405–540 nm applied) using 50% and 90% reduction of the optical density of the growth control when the background was subtracted were compared with visual reading of the plates [
      • Arendrup M.C.
      • Jørgensen K.M.
      • Guinea J.
      • Lagrou K.
      • Chryssanthou E.
      • Hayette M.
      • et al.
      Multicentre validation of a EUCAST method for the antifungal susceptibility testing of microconidia-forming dermatophytes.
      ]. However, for itraconazole, trailing growth complicated visual and 90% spectrophotometric inhibition end-point readings. The performance for correct separation of terbinafine wild-type and non-wild-type isolates harbouring target gene mutations was comparable across these end-point methods provided the end-point specific wild-type upper limits were adopted. Hence, the spec-50% end-point was regarded as preferable because it allows an objective end-point determination applicable to the four drugs evaluated. This will avoid subjectivity and lower interlaboratory variation and hopefully facilitate a broader implementation of susceptibility testing of dermatophytes also in laboratories that are less experienced with visual end-point reading.

       Interpretation of results

      Clinical breakpoints for antifungal agents and dermatophyte species have not yet been established because sufficient MIC and clinical outcome data are not yet available. Interpretation of MICs in the absence of breakpoints is challenging and should be done very carefully taking into account any available data including clinical experience and drug exposure during therapy. However, the MIC may still provide some information regarding susceptibility and, importantly, generation of MICs for dermatophytes is a vital prerequisite for future ECOFF and breakpoint selection. EUCAST established tentative ECOFFs for terbinafine, itraconazole, voriconazole and amorolfine against T. interdigitale and T. rubrum at the time of establishing this method and they are available at https://www.eucast.org/astoffungi/. These may serve to categorize the organism as presumably wild-type or non-wild-type until final ECOFFs and breakpoints are set. Non-wild-type isolates harbour resistance mechanisms and may respond less well to standard therapy.

       Quality control

      Control procedures are the means by which the quality of results is assured and are described in detail by the CLSI. The routine quality of test results is monitored by the use of control strains.

       Control strains

      The two Aspergillus flavus EUCAST QC strains ATCC 204304 and CLM-CM1813 can be used as quality control of the plates with a 2-day incubation provided the inoculum is prepared without cycloheximide and chloramphenicol supplementation. Of note, the MICs for the Aspergillus strains should be read adopting the visual no growth end-point criterion used for mould testing, as outlined in the E.Def 9.3.2 standard. This will allow a quick quality control of prepared plates, without the need for 5 days of incubation. For quality control of dermatophyte testing, two new quality control strains may be used: Trichophyton interdigitale SSI-9396 and Trichophyton rubrum SSI-7583. Both strains are wild-type and have tentative MIC targets and ranges established in parallel with the multicentre validation of the method [
      • Arendrup M.C.
      • Jørgensen K.M.
      • Guinea J.
      • Lagrou K.
      • Chryssanthou E.
      • Hayette M.
      • et al.
      Multicentre validation of a EUCAST method for the antifungal susceptibility testing of microconidia-forming dermatophytes.
      ]. The recommended MIC target and ranges as well as information on availability of the recommended control strains are available at https://www.eucast.org/astoffungi/.

       Storage of control strains

      Fungal isolates may be stored lyophilized or frozen at –70°C or below [
      • Pasarell L.
      • McGinnis M.R.
      Viability of fungal cultures maintained at -70 degrees C.
      ]. Cultures can be stored short term (<2 weeks) on Sabouraud dextrose agar or potato dextrose agar slopes (Aspergillus) at 2°C–8°C, or Sabouraud dextrose agar supplemented with cycloheximide and chloramphenicol (Trichophyton spp.), with new cultures prepared from frozen stocks every 2 weeks.

       Routine use of control strains

      For routine use of control strains, fresh cultures must be prepared from agar slopes, frozen or lyophilized cultures by inoculation on nutritive agar medium (e.g. Sabouraud dextrose agar or potato dextrose agar for Aspergillus spp. or Sabouraud dextrose agar supplemented with cycloheximide and chloramphenicol for Trichophyton spp.)
      • 1.
        At least one control strain must be included per test run and the MICs should be within the control ranges (available at https://www.eucast.org/astoffungi/). Two or more strains are needed if the MIC for the quality control strain falls outside the concentration range tested for one or several compounds. If control strain MIC results are out of range, the test should be repeated. If more than one in 20 tests is out of range the source of error must be investigated.
      • 2.
        Each test must include a well of medium without antifungal drug to demonstrate growth of the test organism and to provide a turbidity control for reading end-points.
      • 3.
        Subculture inoculum on a suitable agar medium to ensure purity and to provide fresh colonies if re-testing is required.
      • 4.
        Test each new batch/lot of medium, microdilution plate, and RPMI-1640 2% glucose broth with at least two of the quality control strains (available on the EUCAST website http://www.EUCAST.org) to ensure that MICs fall within the expected range.

      EUCAST-AFST

      EUCAST-AFST: M.C. Arendrup (Chairman, Denmark), J. Meletiadis (Scientific Data Coordinator, Greece), J. Guinea (Scientific Secretary, Spain), G. Kahlmeter (EUCAST steering committee representative), S. Arikan-Akdagli (Steering Committee, Turkey), N. Friberg (Steering Committee, Finland), F. Barchiesi (Italy), M. Castanheira (USA), P. Hamal (Czech Republic), H. Järv (Estonia), I. Hilmarsdottir (Iceland), N. Klimko (Russia), O. Kurzai (Germany), K. Lagrou (Belgium), C. Lass-Flörl (Austria), T. Matos (Slovenia), C.B. Moore (UK), K. Muehlethaler (Switzerland), T.R. Rogers (Ireland), A. Velegraki (Greece).

      Funding

      None.

      Transparency declarations

      The authors have no conflicts with respect to the current study. Outside the current work MCA has, over the past 5 years, received research grants/contract work (paid to the SSI) from Amplyx , Basilea , Cidara , F2G , Gilead , Novabiotics , Scynexis and T2Biosystems and speaker honoraria (personal fee) from Astellas , Gilead , MSD, SEGES and Pfizer . She is the current chairman of the EUCAST-AFST. GK has nothing to declare. JM has, over the past 5 years, received research grants/contract work (paid to the NKUA) from F2G , Gilead , Astellas , MSD and Pfizer . He is the current clinical data coordinator of the EUCAST-AFST. JG has received funds for participating in educational activities organized on behalf of Astellas , Gilead , MSD , Scynexis and Biotoscana -United Medical; he has also received research funds from FIS , Gilead , Scynexis and Cidara , outside the submitted work.

      Author contributions

      MCA contributed to the conceptualization and wrote the original draft. GK, JM and JG contributed to the conceptualization and to review and editing of the article. All other authors contributed to the review and editing of the article.

      Acknowledgements

      None.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:

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