Antifungal drug dermatophytes

Abstract

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 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

Introduction

Dermatophytes are slow-growing moulds with the ability to invade keratinised tissue such as skin, hair and nails [1]. They are classified into three groups depending on their natural habitat: geophilic [soil], zoophilic [animals] and anthropophilic [humans].

De Hoog et al. [2] revised the taxonomy of dermatophytes in 2017 and emphasised the importance of distinguishing between Trichophyton [T.] mentagrophytes [zoophilic strains] and T. interdigitale [anthropophilic strains] within the T. mentagrophytes complex. Examples of other species in this complex are for example, T. quinckeanum [typical source of infection: cats, dogs and mice] [3], T. equinum [horse] [2] and T. erinacei [hedgehogs] [4]. However, the revised taxonomy is not yet fully implemented. Hence, the term T. mentagrophytes complex is commonly used, and the specific genotype of T. mentagrophytes which shows alarming resistance to terbinafine [TRB] is often referred to as T. interdigitale [5-8]. In this context, it is important to appreciate that the names used in this paper are the original published names used in the articles included in the review which may refer to species names used before the new classification was proposed in 2017. Therefore, some published T. interdigitale may not be T. interdigitale sensu stricto but rather a specific genotype of T. mentagrophytes, that is, T. mentagrophytes ITS genotype VIII, which has recently been identified to cause a morphologically atypical, often extensive, recalcitrant form of tinea corporis [9, 10].

The dermatophytes T. rubrum, T. interdigitale and T. mentagrophytes, are the main aetiological agents of dermatophytosis of skin and nails in humans [11-15]. The prevalence of skin mycosis is 1425% worldwide [16, 17], which makes it one of the most common skin diseases [12, 18]. Medical treatment of dermatophytosis consists of oral [e.g., TRB, itraconazole and griseofulvin] and/or topical antifungal agents [e.g., TRB, ketoconazole, ciclopirox, clotrimazole]. TRB has been preferred as first-line agent against Trichophyton species because of its clinical efficacy and because TRB, in contrast to itraconazole, does not select for azole resistance in the colonising Candida microflora [19]. TRB is a fungicidal allylamine compound that interferes with ergosterol synthesis and thus the fungal cell membrane synthesis via its inhibition of the squalene epoxidase [SQLE] enzyme [20, 21].

TRB therapy of dermatophytosis is usually effective, yet increasing numbers of non-responding cases have been documented [22-26]. Treatment failures and recurrences can be due to host factors such as poor compliance, drug interactions and in cases of onychomycosis local nail factors [27]. TRB resistance in dermatophytes is typically rare [28], but reports have been increasing since 2017. It is associated with various point mutations in the SQLE target gene of T. rubrum and T. interdigitale [22]. In India, TRB resistant dermatophytosis has become epidemic with common resistant or relapsing atypical and widespread ringworm infections [10, 29, 30]. Widespread use of over-the-counter topical creams containing a triple combination of antibacterial and antifungal agents, as well as a steroid may have contributed to the epidemic, as repeated exposures to sub-inhibitory concentrations of antifungals in an immunosuppressed environment caused by the steroid increases the risk of resistance [31-33]. However, cases of TRB-resistant Trichophyton infections are also seen in other Asian and Middle Eastern countries such as Japan and Iran [21, 34, 35]. In Europe, resistant strains of T. rubrum and T. interdigitale with SQLE mutations have been identified in Switzerland [25], Germany [36, 37], Poland [38], Belgium [39] and Denmark [22-24].

Clinical Signs of Antifungal Drug Resistance

It would be ideal if the term Treatment resistant dermatophytosis was used exclusively to describe clinically resistant cases in which antifungal resistance had been confirmed both in vivo and in vitro either through susceptibility testing [demonstrating a high MIC] or molecularly [demonstrating a recognised resistance mutation]. It is not possible in all countries to follow such a stringent definition. What is seen instead is difficult to treat infections with persistent superficial dermatophytosis including chronic [more than 6 months] and recurrent [reappearance of lesions 4 weeks after cessation of standard recommended treatment after clinical cure] forms with poor or no response to standard recommended treatment [31].

Certain clinical characteristics are useful indicators of drug resistance. The most important is a widespread dermatophytosis that has been previously treated, often with multiple regimens. These patients often present with large, sometimes coalescing lesions of superficial dermatophytosis with severe itching. In these cases, the lesions can range from highly inflammatory ones that are raised with erythema and variable scaling, to minimally inflammatory ones with uniform small whitish appearing scales. Most of the patients have concomitant tinea corporis and tinea cruris which often extends posteriorly to the perineum, posterior thighs and buttocks [Fig.1]. Tinea faciei often develops secondarily in these patients, at times extending from the neck to the face, including ears, and in some cases to the scalp, but usually not very far inside the hair margins. Occasionally, TRB resistance is seen in patients with a localised infection, which do not respond to treatment regimens that would normally cure the infection [Fig.2].

Resistance to antifungal compounds is determined by antifungal susceptibility testing [AFST]. A micro-organism is defined as clinically susceptible [S] by a level of antimicrobial activity associated with a high likelihood of therapeutic success using a standard dosing regimen of the agent, whereas it is considered clinically resistant [R] when therapeutic failure is expected [40]. Various methods have been adopted for the testing of yeast or moulds including disk diffusion, agar dilution, E-test, flow cytometry and broth micro- and macrodilution techniques with or without a colour indicator [31, 41]. AFST of dermatophytes is not routinely performed because it is complicated by the slow growth of the fungi and the associated risk of contamination, because no commercial susceptibility tests are validated for dermatophyte testing, because interpretative clinical breakpoints that allow interpretation of the specific MIC as susceptible or resistant have not been established, and because the false perception of low resistance rates creates an assumption that testing is not needed. Numerous critical parameters such as inoculum size, incubation time, type of microdilution plate, choice of medium and endpoint determination affect susceptibility test results. Currently, there are only two standardised techniques for in vitro AFST of dermatophytes; one from the Clinical Laboratory Standards Institute [CLSI] and the other from the European Committee on Antimicrobial Susceptibility Testing [EUCAST] [42, 43]. Both of these reference methods are performed in microtitre plates containing broth culture media with serial dilutions of antifungal drugs, wherein a standardised amount of dermatophyte cells are added and the fungus growth is compared with positive and negative controls. The main differences of the two methods are that the CLSI method recommends a visual no-growth endpoint in round bottom plates, whereas the EUCAST adopted flat-bottom plates and an automated spectrophotometer reading to avoid subjectivity in endpoint determination. Moreover, the EUCAST has recently established tentative epidemiological cut off values [ECOFFs, defined as the upper limit of the wild-type population [44]] that allows classification of the tested organism as either wild type [normal, and thus you can trust your clinical experience with respect to efficacy of medical treatment] or non-wild type [less susceptible than normal, and thus the dermatophyte may not respond as well to the typical therapy].

Despite the lack of routine susceptibility testing, increasing numbers of clinically resistant dermatophytes due to isolates with high TRB MICs and with target gene mutations have been reported in the past few years. This review evaluates the reported MICs for T. rubrum, T. mentagrophytes and T. interdigitale with the broth microdilution method in the literature and addresses the problem of extrapolation from the laboratory bench to patients.

Method

This systematic review was conducted in accordance with the PRISMA guidelines [45] [Fig.3]

PubMed was searched for TRB susceptibility testing of T. rubrum, T. mentagrophytes and T. interdigitale with the following search string: ["disease susceptibility"[MeSH Terms] OR ["disease"[All Fields] AND "susceptibility"[All Fields]] OR "disease susceptibility"[All Fields] OR "susceptibility"[All Fields]] AND ["terbinafine"[MeSH Terms] OR "terbinafine"[All Fields]] AND ["trichophyton"[MeSH Terms] OR "trichophyton"[All Fields]]. The literature search and screening were conducted on October 20, 2020, by two individual researchers [J.J.S. and D.M.L.S.]. The inclusion criteria were in vitro terbinafine susceptibility testing of human isolates of T. rubrum, T. mentagrophytes and T. interdigitale with the broth microdilution technique. The exclusion criteria were papers in languages other than English. See Figure 1 for additional details of the literature search. Data from the included papers were extracted in a predefined Excel chart, and outcomes were reported as MIC range, MIC in which 50% of isolates were inhibited [MIC50], MIC in which 90% of isolates were inhibited [MIC90], geometric mean [GM-MIC], most frequent MIC [Modal MIC], method, number of isolates and test material. Whenever data was not available this was stated as NA.

Results

T. interdigitale

TRB susceptibility of a total of 829 T. interdigitale isolates were reported between 2001 and 2020 in 24 studies by the following reference standards: CLSI-M38-A2 [19 studies], EUCAST E.Def 11.0 [2 studies], EUCAST E.Def 9.3.1 [1 study], EUCAST E.Def 9.2 [1 study], CLSI-M38-A [1 study] and CLSI-M38-P/NCCLS-M38-P [1 study] [Table1]. For all T. interdigitale isolates, the MIC ranged from 0.001 to >32 mg/L [21 studies] using the CLSI/NCCLS method and from 8 mg/L [4 studies] using the EUCAST method. Details of MIC50, MIC90, GM-MIC and modal MIC are available in Table1. Overall, 85 isolates were from patients with clinical failures, and two isolates were deemed resistant because of high MIC and SQLE mutations. For T. interdigitale isolates from clinically non-responding patients, the MIC ranged from 32 mg/L and 8 using the CLSI/NCCLS method [6 studies] and EUCAST method [2 studies], respectively. Six studies performed SQLE gene sequencing detecting the following SQLE mutations: F397L, L393F and F397L/A448T. Three studies proposed the following breakpoints [susceptible [S] and resistant [R]] for TRB against T. interdigitale strains using CLSI reference standard: R 1 mg/L [46], R 4 mg/L [12, 47], and one study also using CLSI as reference proposed high TRB MIC at 2 mg/L [11]. One study [48] proposed the following wild-type upper limits [WT-UL] using EUCAST as reference standard: WT-UL 0.25 mg/L [by visual endpoint reading and spectrophotometric endpoint reading with a 90% growth inhibition [spec-90%], WT-UL 0.125 mg/L [by spectrophotometric endpoint reading with a 50% growth inhibition [spec-50%]]. The spec-50% WT-UL values have subsequently been accepted as EUCAST tentative ECOFF for the EUCAST 11.0 method [43].

T. mentagrophytes

TRB susceptibility of a total of 1,399 T. mentagrophytes isolates were reported between 1995 and 2020 in 41 studies by the following reference standards: CLSI-M38-A2 [18 studies], CLSI-M38-A/NCCLS-M38-A [10 studies], CLSI-M38-P/NCCLS-M38-P [5 study], CLSI-M38 [1 study], CLSI-M27-A/NCCLS-M27-A [3 studies], EUCAST E.Def 9.3.1 [1 study], EUCAST E.Dis 7.1 [1 study] and unspecified broth microdilution method [2 studies] [Table1]. For all T. mentagrophytes isolates, the MIC ranged from 0.001 to >32 mg/L using the CLSI/NCCLS method [37 studies] and 32 mg/L using the CLSI/NCCLS method [5 studies], and one EUCAST study reported TRB MIC 4 mg/L [39]. Overall, 36 isolates were from patients with clinical failures, and 210 isolates were suspected as clinically resistant because of high MIC and SQLE mutations. Five studies performed SQLE gene sequencing detecting the following SQLE mutations: F397L, L393F, L393S, H440Y, S443P, A448T, L335F/A448T, S395P/A448T, L393S/A448T, Q408L/A448T and F397L/A448T. Two studies using the CLSI reference method defined TRB resistance as R 1 mg/L [9] and R 4 mg/L [47].

T. mentagrophytes-T. interdigitale complex

TRB susceptibility of a total of 548 isolates reported as belonging to the T. mentagrophytes-T. interdigitale complex were reported between 2019 and 2020 in 4 studies by the CLSI-M38-A2 reference standard [Table1]. For all T. mentagrophytes-T. interdigitale complex isolates, the MIC ranged from 0.015 to 32 mg/L [4 studies]. Overall, 16 isolates were from patients with clinical failures, and 74 isolates were suspected as clinically resistant because of high MIC and SQLE mutations. The MIC ranged from 1 to 32 mg/L [3 studies]. Three studies that performed SQLE gene sequencing detected the following SQLE mutations: F397L and Q408L. One study proposed WT-UL 8 mg/L [5], and one study proposed high TRB MIC at >2 mg/L [6] both using CLSI as reference standard.

T. rubrum

In the years between 1995 and 2020, 62 out of the 74 included studies tested TRB susceptibility of T. rubrum with the following standards: CLSI-M38-A2 [31 studies], CLSI-M38-A/NCCLS-M38-A [13 studies], CLSI-M38-P/NCCLS-M38-P [8 studies], CLSI-M27-A/NCCLS-M27-A [4 studies] EUCAST E.Def 11.0 [2 studies], EUCAST E.Def 9.3.1 [1 study], EUCAST E.Def 9.2 [1 study], EUCAST E.Dis 7.1 [1 study] and unspecified broth microdilution method [3 studies] [Table2]. A total of 2,235 T. rubrum isolates were included, of which 113 isolates were from patients with clinical resistance, and 26 isolates were suspected as clinically resistant because of high MICs and SQLE mutations. The MICs for all T. rubrum isolates ranged from 64 mg/L using the CLSI/NCCLS method [56 studies] and 8 mg/L using the EUCAST method [5 studies] [Table2]. For T. rubrum isolates with clinical resistance, the MIC ranged from 0.003 to >32 mg/L using the CLSI/NCCLS method [8 studies] and 0.03 to >8 mg/L using the EUCAST method [2 studies]. Two studies performed SQLE gene sequencing detecting the following SQLE mutations: F397L, L393F, L393S, H440Y, F393I, F393V, F415I, F415S, F415V, I121M/V237I and H440Y/F484Y. Three studies using the CLSI reference standard proposed the following breakpoints for T. rubrum strains: TRB resistance was defined as R 1 mg/L [18, 49], R 4 mg/L [47], and one study proposed high TRB MIC at 2 mg/L [11]. One study using EUCAST protocol reported following WT-UL: WT-UL 0.125 mg/L [by visual and spec-90% endpoint reading] [48], WT-UL 0.03 mg/L [with spec-50% endpoint reading in plates containing chloramphenicol and cycloheximide [CC]] [48], S 0.06 mg/L [with spec-50% endpoint reading in plates without CC] [48], and one study proposed ECOFF of 0.06 or 0.125 [22]. EUCAST has recently established a tentative ECOFF with the spec-50% endpoint reading of 0.03 mg/L for T. rubrum and E.Def 11.0 [43].

Discussion

The number of patients with clinical and laboratory-confirmed TRB-resistant dermatophytosis is rising [5, 22]. Until this autumn [2020], there were no official guidelines on how to interpret the results and furthermore, there is a huge intraspecies variation in reported MIC values for the most prevalent species T. rubrum, T. mentagrophytes and T. interdigitale. This variation can be due to different methods in AFST and technical issues related to testing, or it can reflect true differences in susceptibility due to acquisition of point mutations in the SQLE gene altering the binding site of TRB and conferring loss of susceptibility to TRB therapy [11, 12, 22, 28, 48].

However, clinical resistance does not always correspond to the in vitro resistance and vice versa. This is because several factors influence outcome of an infection including the severity of infection, the host immunity, the timing and dosing of the therapy, compliance and the susceptibility of the infecting organism. A newer study from 2018 investigated risk factors and performed AFST of both T. rubrum and T. interdigitale strains from 39 recurrent and clinically unresponsive tinea corporis and tinea cruris patients [47]. TRB MIC ranging from 0.015 to 16 mg/L were found, including 1 T. interdigitale and 3 T. rubrum isolates with MICs of >2 mg/L, which is only 10% of the total number of clinically resistant cases. The authors concluded that most of the strains were not drug resistant, and the increase in MIC was not the only factor responsible for clinical resistance in dermatophytosis.

The management of TRB-resistant infections is challenging as few alternatives are available. There is compelling evidence that itraconazole is the preferred agent for TRB-resistant Trichophyton infections [5, 29, 33, 50, 51]. The optimal dosing regimen and treatment duration, however, have not been established. Consequently, practises vary, and elevated doses above 200400 mg daily are sometimes adopted for prolonged duration despite lack of scientific rationale or clinical validation [52, 53]. The current prevailing sentiment in India is to reduce unnecessary prescription of itraconazole as an attempt to avoid emergence of itraconazole resistance in Trichophyton as emergence of multidrug resistant infections would have grave consequences. Azole resistant dermatophytes have previously been reported [54], and few cases have been found to be resistant to griseofulvin since the 1960s [55, 56]. Cross-resistance within the same antifungal group has also been noted for example, resistance to other allylamines [e.g., butenafine, naftifine] in TRB-resistant isolates [57]. The antifungal mode of action differs, as azoles and TRB both inhibit the ergosterol biosynthesis [but inhibit different enzymes], and griseofulvin interferes with the microtubule formation [58, 59]. Nevertheless, multidrug resistance to several drug classes has been reported after serial passages on drug containing agars in vitro [60]. Moreover, multidrug resistance has been sporadically reported in the clinical setting of TRB-resistant T. mentagrophytes/interdigitale infection in India [59]. The Indian epidemic is probably somewhat unique and explained by the emergence of a new and more virulent T. mentagrophytes subspecies [genotype VIII] in response to a high use of antifungal agents potentially facilitated/enhanced by an animal reservoir and challenges in infection control due to crowded housing conditions. However, TRB resistance is increasing elsewhere despite the lack of routine testing and the prevalence of multidrug resistance largely unknown [10, 18, 29, 37].

Interpretative clinical breakpoint values were not established until recently. CLSI has noted that most TRB MICs are 0.25 mg/L for dermatophytes [61]. This value is, however, several fold higher than the recommended CLSI MIC range for the T. mentagrophytes quality control strain [0.0020.008 mg/L] raising a concern that not all isolates with a MIC up towards 0.25 mg/L may be without resistance mechanisms and clinically susceptible. Of note, some studies using the CLSI method suggest even higher breakpoints for resistance for T. interdigitale between 1 and 4 mg/L [11, 12, 46, 47], T. mentagrophytes between 1 and 4 mg/L [18, 47], T. rubrum between 1 and 4 mg/L [18, 47, 49] and T. mentagrophytes-T. interdigitale complex WT-UL 8 mg/L [5]. Thus, further standardisation and establishment of CLSI breakpoints are urgently needed.

Recently, EUCAST developed a reference method that allows an automated endpoint reading and provides quality control ranges for several control strains [43]. This method will facilitate improved inter-laboratory reproducibility. Moreover, tentative ECOFFs were established which allow classification of isolates as wild type or non-wild type. Several aspects are considered and integrated into the process of setting breakpoints for antifungal agents. These include dosing information, MIC distributions, ECOFF setting, pharmacokinetics, pharmacodynamics and clinical outcome data. Until such data is available, adoption of the EUCAST method with the tentative ECOFFs will allow guidance for TRB, itraconazole and voriconazole use against T. rubrum and T. interdigitale. Wildtype isolates are normal and will respond normally to treatment, meaning the clinician can expect the typical outcome for treatment with a specific drug-bug combination. Non-wild-type isolates may or may not respond to therapy depending on the degree of MIC elevation and the drug exposure that can be obtained. A rule of thumb is nevertheless, that the clinician should consider if alternative therapy would be warranted. Most important is to realise that MICs, ECOFFs and breakpoints are method dependent, and just like a distance is a different number if measured in metres or feet, MIC values across methods will vary and can only be interpreted correctly by the method-specific associated ECOFFs and breakpoints. This is also true for susceptibility results obtained by commercial test systems. Such results are only reliable if the method is correctly standardised against the reference method from which the breakpoints are adopted.

Conclusion

Overall, high TRB MICs were reported in the dermatology literature for T. mentagrophytes, T. interdigitale and T. rubrum and more than 15 individual target protein alterations have been identified in isolates from treatment-resistant cases [62-65]. Clinically, the number of patients with resistant dermatophytosis is also rising, supporting the increasing clinical relevance of AFST in non-responding cases and where resistance is prevalent [5, 10, 22]. AFST is unfortunately not considered a routine test but recent progress allows for standardisation and a broader implementation of dermatophyte susceptibility testing. Hopefully, this will help bring AFST a step closer to becoming a routine test and guide, as susceptibility testing is for so many other infections.

Key Message

Clinical suspected terbinafine resistance can be mycologically proven by microdilution methods. Recent progress allows for standardisation.

Acknowledgement

The authors thank the patients for giving permission to use their photos and Dr. Lisa Travis for English proofreading.

Statement of Ethics

Written informed consent was obtained from the patients for publication of the details of their medical case and any accompanying images.

Conflict of Interest Statement

M.C. Arendrup has, outside the current work, 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, and SEGES over the past 5 years. She is the current chairman of the EUCAST-AFST.

S. Verma has delivered one lecture and has attended an Ad Board meeting once, both organised by Janssen Pharmaceuticals, India, for which he received a honorarium.

D.M.L. Saunte was a paid consultant for advisory board meetings by AbbVie, Janssen, Sanofi, Leo Pharma and received speakers honoraria and/or received grants from the following companies: Abbvie, Desitin, Pfizer, Galderma, Novartis and Leo Pharma during the last 5 years.

J.J. Shen has no conflict of interests.

Funding Sources

J.J. Shen received a research grant from the Dept. of Dermatology, Zealand University Hospital, Roskilde, Denmark.

Author Contributions

D.M.L. Saunte initiated the article, J.J. Shen and D.M.L. Saunte performed the systematic review of included articles. All authors wrote and critically reviewed the manuscript. All authors have accepted the final version of the manuscript.

References

Author Contacts

Article / Publication Details

Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor[s]. The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor[s] disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.