First terbinafine-resistant Trichophyton indotineae isolates with Phe397Leu and/or Thr414His mutations in Turkey: Differences in response to resveratrol treatment

doi:10.21203/rs.3.rs-2132702/v1

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First terbinafine-resistant Trichophyton indotineae isolates with Phe397Leu and/or Thr414His mutations in Turkey: Differences in response to resveratrol treatment

https://doi.org/10.21203/rs.3.rs-2132702/v1

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published 19 Jan, 2023

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The increase in terbinafine-resistant superficial dermatophytosis has become a major problem over the last decade. In this report, we present two cases of infection with terbinafine-resistant Trichophyton indotineae, a species diagnosed for the first time in Turkey. One of the patients harbored a CD36 mutation. Both strains harbored the same amino acid substitution in the squalene epoxidase (SQLE) gene, whereas one of the isolates had another as yet unknown mutation. Clinical improvement was observed with resveratrol treatment in the patient with the CD36 mutation, but this compound was not effective in the other patient.

Resveratrol

Fluconazole

Itraconazole

Terbinafine

Trichophyton indotineae

Dermatophyte infections have gained renewed interest since the sudden emergence of a novel dermatophyte now known as Trichophyton indotineae, a species with high virulence and significant terbinafine resistance [1]. This species is a member of the Trichophyton mentagrophytes complex, which contains genotypes that are generally thought to have an animal origin [2] but are commonly transmitted by human hosts [3]. The fungus was first detected in India [4, 5] and has since spread rapidly over Southern Asia, Australia, and Europe [6]. The cases presented in this report are the first from Turkey. One of the reasons for its emergence is thought to be the inappropriate use of antifungal creams with corticosteroids by the general public [7], which has promoted antifungal resistance in the species. Patients generally show extended skin and crural lesions, suggesting significant virulence, which indicates a recent animal host [8]. No specific animal host has been identified for T. indotineae, although Tartor et al. [9] reported an isolate from a cow in Egypt.

In general, affected patients are immunocompetent and do not have underlying immune disorders. One of the two patients described in this study had a mutation in the CD36 gene, which encodes a membrane protein involved in fatty acid transport and scavenging receptors. As both strains of T. indotineae proved to be terbinafine resistant, we applied an alternative therapy with resveratrol. The course of events in these two cases is outlined below.

Skin scraping samples were inoculated onto Sabouraud glucose agar plates (SGA; Merck, Darmstadt, Germany) containing 100 µg/ml chloramphenicol (Sigma, Steinheim, Germany) and 50 µg/ml gentamicin (Sigma). The plates were incubated at 28°C for 14 days. DNA was extracted from fresh colonies using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The rDNA operon with the internal transcribed spacer (ITS) and D1-D2 region of the large subunit (LSU) were amplified using primers ITS4-ITS5 and LR0R-LR5, respectively [10]. PCR amplicons were visualized on a 1.5% agarose gel, and sequencing was performed with the same primer pairs used for PCR amplification using Applied Biosystems BigDye Terminator version 3.1 (Thermo Fisher Scientific). The sequences were edited and assembled using Geneious R11 [11]. Phylogenetic analysis of ITS data was performed using maximum likelihood (ML) methods implemented in IQ-TREE software [12, 13]. The squalene epoxidase (SQLE) gene was amplified and sequenced using primers described by Kong et al. [14]. The detected mutations and GenBank accession numbers of the sequences are listed in Table 2. Antifungal susceptibility testing was performed according to the EUCAST E. Def 9.3.1 protocol [15]. Isolates were deposited in the CBS reference collection with accession numbers CBS 149165 and CBS 149166.

Case 1

A 27-year-old man presented with a fungal infection in the inguinal and gluteal regions that did not improve despite treatment with systemic terbinafine and itraconazole for the last 5 years. The patient was a doctor in the emergency department and had no history of animal contact. His symptoms began in the inguinal region and spread to the pubic and gluteal areas. The patient had been diagnosed with tinea cruris and used topical antifungal agents, i.e., terbinafine (250 mg/day) for 6 months and itraconazole (2 ⋅ 100 mg/day) for 2 months. However, his symptoms continued to progress. Dermatological examination revealed scaly erythematous pruritic patches and plaques on the penis, scrotum, groin, and pubic and gluteal regions (Figs. 1a and 1b). KOH examination revealed dense hyphae and spores, consistent with dermatophytosis. Histopathologically, fungal elements were observed only in the stratum corneum without dermal invasion, which is compatible with superficial dermatophytosis. All lesions healed with high-dose fluconazole treatment (2 ⋅ 100 mg/day); however, when the drug was discontinued, the lesions recurred in the same area. Additional skin scraped samples were collected for fungal culture. Although there was no history of other infections, complete blood count and immunoglobulin levels were checked, and flow cytometry analysis was performed to detect possible immunosuppressive diseases in the patient; however, no pathology was found. The patient tested negative for Anti-HIV antibodies. Genetic testing revealed a heterozygous p. N220fs*4 variant in the CD36 gene. Resveratrol tablets were recommended to the patient twice daily, and the clinical lesions resolved without further antifungal treatment (Figs. 1c and 1d). The fungus isolated from clinical samples was cultured on SGA and was found to be consistent with T. indotineae based on ITS and LSU sequencing (Table 1 and Fig. 2). The isolate was resistant to fluconazole and terbinafine in vitro; additional antifungal susceptibility testing (AFST) data are listed in Table 2. Two nonsynonymous mutations were found in SQLE (1189 T > C and 1240 T > C). A subculture was deposited in the CBS collection under accession number CBS 149166.

Table 1

GenBank accession numbers for the sequences and detected *SQLE* mutations.
Case (CBS) numbers	GenBank accession numbers			Mutation(s)
Case (CBS) numbers	ITS	LSU	SQLE
1 (149166)	ON528187	ON528577	ON863899	1189 T > C, 1240 T > C**
2 (149165)	ON528186	ON528576	ON863900	1189 T > C*
CBS, Culture collection of the Westerdijk Biodiversity Institute; ITS, internal transcribed spacer rDNA; LSU, large subunit rRNA; , Phe³⁹⁷Leu; *, The⁴¹⁴His.

Table 2

Antifungal susceptibility results of the *Trichophyton indotineae* isolates.
Case (CBS) numbers	MIC values (mg/L)
Case (CBS) numbers	CPX	GSF	FLC	ITR	VRC	MCZ	TRB	KET
1 (149166)	0.25	2	16	0.031	0.125	1	> 16	0.125
2 (149165)	0.50	1	32	0.031	0.250	1	> 16	0.250
CBS, Culture collection of the Westerdijk Biodiversity Institute; CPX, ciclopirox; GSF, griseofulvin; FLC, fluconazole; ITR, itraconazole; VRC, voriconazole; MCZ, miconazole; TRB, terbinafine; KET, ketoconazole.

Case 2

A 25-year-old woman presented with a fungal infection in the bilateral posterior thigh and gluteal region that did not respond to systemic terbinafine treatment for 2-years (Figs. 3a and 3b). She was diagnosed with tinea corporis, and her symptoms did not improve despite topical and systemic terbinafine (250 mg/day, 6 months) treatment. Her blood immunoglobulin levels and complete blood counts were measured. Flow cytometric analysis was also performed, and no immunological defects were detected. The patient tested negative for Anti-HIV antibodies. A 4 mm punch biopsy sample was obtained for histopathological examination and was found to be compatible with superficial dermatophytosis. Genetic analysis could not be performed because of the lack of patient consent. The patient was treated with oral itraconazole (2 ⋅ 100 mg/day, 4 weeks), and the lesions resolved with post-inflammatory pigmentation (Figs. 3c and 3d). One month later, the patient returned with relapsed lesions in the same body parts. Since patient 1 responsed well to resveratrol, the same treatment was recommended for this patient; however, no clinical improvement was observed. Therefore, systemic itraconazole treatment was initiated. No recurrence was observed after 2-months of systemic itraconazole treatment. The etiological agent isolated from clinical samples was cultured on SGA and was found to be consistent with T. indotineae based on ITS and LSU sequencing (Table 1 and Fig. 2). The isolate was resistant to fluconazole and terbinafine in vitro; additional AFST data are listed in Table 1. A mutation was found in the SQLE gene (1189 T > C). A subculture was deposited in the CBS collection under accession number CBS 149165.

The identification of species in the T. mentagrophytes complex is highly controversial. Tang et al. [19] noted the emergence of a large number of ITS-based genotypes representing the species’ host shift to humans after its probable origin from domesticated animals. The authors suggested restricting the naming of entities to those that are phenotypically different and clinically relevant. In accordance with the epidemiological data presented by Taghipour et al. [18], European and Asian clusters of genotypes could be recognized, with different clinical predilections in some but not in all genotypes. The most deviant genotype was VIII, which was described as T. indotineae. A bootstrap-supported cluster of this species, also comprising genotypes XIII and XIV, was formed in the ITS tree (Fig. 2). Genotypes XIII and XIV were listed by Taghipour et al. [18]; however, no information on these strains was provided. The two isolates reported in the present study were identical to the type strain of T. indotineae (ITS genotype VIII).

The first two cases of superficial dermatophytosis caused by T. indotineae in Turkey are presented here. In both cases, pronounced resistance to terbinafine was found, a feature that has been noted since the first emergence of this species in India [4]. The most common causes of acquired resistance in dermatophytes are the combined use of antifungal creams containing steroids, non-adherence to the prescribed clinical course, treatment incompatibility, humid and warm living conditions, enhanced proliferation of dermatophytes, poor skin hygiene, repetitive contact with possible sources of infection, decreased hydration of the stratum corneum due to barrier function defects, and defective immune responses [20]. Although treatment compliance was good in both our patients, the T. indotineae infections were highly recalcitrant and relapsed when treatment was discontinued.

During the last decade, several outbreaks of multidrug-resistant dermatophytes have been reported in India [16]. Mycological studies during these outbreaks revealed that the most common dermatophytes causing resistant infections in India were members of the T. mentagrophytes complex [1, 16, 21], followed by the T. rubrum complex [22]. Trichophyton mentagrophytes genotype VIII, now known as T. indotineae, was first detected between 2004 and 2013 in India, later emerging in Australia, Iran, and Oman, and recently even reaching the USA and several European countries [6]. Cases outside India are sporadic. The crural prevalence of T. indotineae may enhance its distribution through sexual contact [23]. Neither of our patients had a history of travel to a hyperendemic region for T. indotineae.

Terbinafine resistance is often associated with resistance to voriconazole and itraconazole [14, 21]. To investigate the resistance in our isolates, mutations were screened in the SQLE gene, which encodes the target enzyme of the drug. Resistant Trichophyton isolates have been reported to have amino acid substitutions at positions Lys276, Leu393, Phe397, Glu408, Phe415, His440, and Ala448 [14, 24–28]. Both isolates obtained in the present study revealed a Phe³⁹⁷Leu substitution (T ◊ C transition at position 1189), whereas the isolate from patient 1 had an additional Thr⁴¹⁴His mutation (T ◊ C transition at position 1240) (Fig. 4). To the best of our knowledge, this mutation has not been reported in any terbinafine-resistant T. indotineae strain before. Since the isolate already harbors another substitution and the patient has a CD36 defect, it is not clear whether this substitution enhances antifungal resistance.

There are no guidelines for the management of terbinafine-resistant superficial dermatophyte infections. Posso-De Los Rios et al. [29] reported eight cases of terbinafine-resistant dermatophyte infections from Canada and outlined a clinical approach for such cases, with administration of fluconazole (400 mg/week for 12 weeks) or itraconazole (200 mg/day for 4 weeks) as the most important component. Although patient 2 responded well to systemic itraconazole treatment, patient 1 did not respond to itraconazole or fluconazole therapy. The lesions in patient 1 resolved with high-dose fluconazole (200 mg/day) treatment but relapsed when the treatment was discontinued.

Genetic tests were performed in patient 1, which revealed a mutation in the CD36 gene. This gene encodes a glycoprotein ligand that can be found in different cell membrane structures and is involved in the membrane transport of lipids and fatty acids. The functions of ligands vary according to the cell in which they are located. Ligands in platelets are involved in angiogenesis, while they act as pattern recognition receptors in phagocytic cells [30]. CD36 recognizes fungal β-glucans [31] and binds to lipoprotein and lipid components [32]. The activation of CD36 receptors after contact with Cryptococcus neoformans and Candida albicans leads to the release of pro-inflammatory cytokines and chemokines from phagocytic cells [31]. In a study conducted in China, the frequency of CD36 mutations varied according to the ethnic population [33]. CD36 mutation also predisposes individuals to malaria and attenuate experimental mycobacterial infections [34, 35]. Given these data, enhancement of susceptibility to fungal infections is plausible.

Since the lesions recurred when antifungal therapy was discontinued in patient 1, who had the CD36 mutation, we searched for an alternative treatment that might counteract this defect. Resveratrol is a stilbene phytoalexin found in certain plants such as red grapes, peanuts, cranberries, and blueberries [36]. This molecule shows antifungal activity by inducing apoptosis in C. albicans [37]. In addition, resveratrol has been observed to inhibit different dermatophytes, such as T. mentagrophytes, T. tonsurans, T. rubrum, Epidermophyton floccosum, and Nannizzia gypsea [38]. In our patients, the response to resveratrol differed from proven CD36 deficiency. It remains unclear whether resveratrol acted because of its antifungal effect or because of the defective CD36 pathway. Nevertheless, the response to resveratrol treatment in the first patient but not in the second, suggests that CD36 played a role.

In conclusion, owing to the growing number of terbinafine-resistant dermatophytes in case reports, guidelines are required to improve its clinical management. Further studies are needed to investigate the relationship between CD36 pathway defects and dermatophyte infection.

Acknowledgments

We acknowledge Marlou Tehupeiory-Kooreman and Xue Kong for their advice and technical assistance. We also appreciate both of patients for their kindness of cooperation and permission.

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Funding

No specific funding was provided for this study.

Ethical approval

Ethical approval for this study was not required.

Consent of publish

The authors affirm that the human research participants provided informed consent for publication of the images in Figs. 1 and 3.

Singh A, Masih A, Monroy-Nieto 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. Fungal Genet Biol. 2019;133:103266.
van Rooij P, Detandt M, Nolard N. Trichophyton mentagrophytes of rabbit origin causing family incidence of kerion: An environmental study. Mycoses. 2006;49(5):426–30.
Tang C, Kong X, Ahmed SA, et al. Taxonomy of the Trichophyton mentagrophytes/T. interdigitale species complex harboring the highly virulent, multiresistant genotype T. indotineae Mycopathologia. 2021;186(3):315–26.
Verma S, Madhu R. The great Indian epidemic of superficial dermatophytosis: An appraisal. Indian J Dermatol. 2017;62(3):227–36.
Singh A, Masih A, Khurana A, et al. High terbinafine resistance in Trichophyton interdigitale isolates in Delhi, India harbouring mutations in the squalene epoxidase gene. Mycoses. 2018;61(7):477–84.
Jabet A, Brun S, Normand AC, et al. Extensive dermatophytosis caused by terbinafine-resistant Trichophyton indotineae, France. Emerg Infect Dis. 2022;28(1):229–33.
Rana P, Ghadlinge M, Roy V. Topical antifungal-corticosteroid fixed-drug combinations: Need for urgent action. Indian J Pharmacol. 2021;53(1):82–4.
Chollet A, Cattin V, Fratti M, Mignon B, Monod M. Which fungus originally was Trichophyton mentagrophytes? Historical review and illustration by a clinical case. Mycopathologia. 2015;180(1–2):1–5.
Tartor YH, El-Neshwy WM, Merwad AMA, et al. Ringworm in calves: risk factors, improved molecular diagnosis, and therapeutic efficacy of an Aloe vera gel extract. BMC Vet Res. 2020;16(1):421.
de Hoog GS, Dukik K, Monod M, et al. Toward a novel multilocus phylogenetic taxonomy for the dermatophytes. Mycopathologia. 2017;182(1–2):5–31.
Kearse M, Moir R, Wilson A, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28(12):1647–9.
Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32(1):268–74.
Minh BQ, Schmidt HA, Chernomor O, et al. IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 2020;37(5):1530–4.
Kong X, Tang C, Singh A, et al. Antifungal susceptibility and mutations in the squalene epoxidase gene in dermatophytes of the Trichophyton mentagrophytes species complex. Antimicrob Agents Chemother. 2021;65(8):e0005621.
Arendrup MC, Guinea J, Cuenca-Estrella M, et al.; and the Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST)*. Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia forming moulds. EUCAST E.DEF 9.3 December, 2015.
Nenoff P, Verma SB, Vasani R, et al. The current Indian epidemic of superficial dermatophytosis due to Trichophyton mentagrophytes – A molecular study. Mycoses. 2019;62(4):336–56.
Kano R, Kimura U, Kakurai M, et al. Trichophyton indotineae sp. nov.: A new highly terbinafine-resistant anthropophilic dermatophyte species. Mycopathologia. 2020;185(6):947–58.
Taghipour S, Pchelin IM, Zarei Mahmoudabadi A, et al. Trichophyton mentagrophytes and T. interdigitale genotypes are associated with particular geographic areas and clinical manifestations. Mycoses. 2019;62(11):1084–91.
Tang C, Ahmed SA, Deng S, et al. Detection of emerging genotypes in Trichophyton mentagrophytes species complex: A proposal for handling biodiversity in dermatophytes. Front Microbiol. 2022;13:960190.
Bhargava P, Nijhawan S, Singdia H, Mehta T. Skin barrier function defect – A marker of recalcitrant tinea infections. Indian Dermatol Online J. 2020;11(4):566–9.
Ebert A, Monod M, Salamin K, et al. Alarming India-wide phenomenon of antifungal resistance in dermatophytes: A multicenter study. Mycoses. 2020;63(7):717–28.
Thakur R, Kalsi AS. Outbreaks and epidemics of superficial dermatophytosis due to Trichophyton mentagrophytes complex and Microsporum canis: Global and Indian scenario. Clin Cosmet Investig Dermatol. 2019;12:887–93.
Luchsinger I, Bosshard PP, Kasper RS, Reinhardt D, Lautenschlager S. Tinea genitalis: a new entity of sexually transmitted infection? Case series and review of the literature. Sex Transm Infect. 2015;91(7):493–6.
Yamada T, Maeda M, Alshahni MM, et al. Terbinafine resistance of Trichophyton clinical isolates caused by specific point mutations in the squalene epoxidase gene. Antimicrob Agents Chemother. 2017;61(7):e00115–117.
Khurana A, Masih A, Chowdhary 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. Antimicrob Agents Chemother. 2018;62(12):e01038-18.
Hsieh A, Quenan S, Riat A, Toutous-Trellu L, Fontao L. A new mutation in the SQLE gene of Trichophyton mentagrophytes associated with terbinafine resistance in a couple with disseminated tinea corporis. J Mycol Med. 2019;29(4):352–5.
Siopi M, Efstathiou I, Theodoropoulos K, Pournaras S, Meletiadis J. Molecular epidemiology and antifungal susceptibility of Trichophyton isolates in Greece: Emergence of terbinafine-resistant Trichophyton mentagrophytes type VIII locally and globally. J Fungi. 2021;7(6):419.
Astvad KMT, Hare RK, Jørgensen KM, Saunte DML, Thomsen PK, Arendrup MC. Increasing terbinafine resistance in Danish Trichophyton isolates 2019–2020. J Fungi. 2022;8(2):150.
Posso-De Los Rios CJ, Tadros E, Summerbell RC, Scott JA. Terbinafine resistant Trichophyton indotineae isolated in patients with superficial dermatophyte infection in Canadian patients. J Cutan Med Surg. 2022;26(4):371–6.
Silverstein RL, Febbraio M. CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior. Sci Signal. 2009;2(72):re3.
Means TK, Mylonakis E, Tampakakis E, et al. Evolutionarily conserved recognition and innate immunity to fungal pathogens by the scavenger receptors SCARF1 and CD36. J Exp Med. 2009;206(3):637–53.
Hoebe K, Georgel P, Rutschmann S, et al. CD36 is a sensor of diacylglycerides. Nature. 2005;433(7025):523–7.
Liu J, Shao Y, Ding H, et al. Distribution of CD36 deficiency in different Chinese ethnic groups. Hum Immunol. 2020;81(7):366–71.
Kajeguka D, Mwanziva C, Daou M, et al. CD36 c.1264 T > G null mutation impairs acquisition of IgG antibodies to Plasmodium falciparum MSP1₁₉ antigen and is associated with higher malaria incidences in Tanzanian children. Scand J Immunol. 2012;75(3):355–60.
Hawkes M, Li X, Crockett M, et al. CD36 deficiency attenuates experimental mycobacterial infection. BMC Infect Dis. 2010;10:299.
Redondo-Blanco S, Fernández J, López-Ibáñez S, Miguélez EM, Villar CJ, Lombó F. Plant phytochemicals in food preservation: Antifungal bioactivity: A review. J Food Prot. 2020;83(1):163–71.
Lee J, Lee DG. Novel antifungal mechanism of resveratrol: apoptosis inducer in Candida albicans. Curr Microbiol. 2015;70(3):383–9.
Chan MM. Antimicrobial effect of resveratrol on dermatophytes and bacterial pathogens of the skin. Biochem Pharmacol. 2002;63(2):99–104.

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First terbinafine-resistant Trichophyton indotineae isolates with Phe397Leu and/or Thr414His mutations in Turkey: Differences in response to resveratrol treatment

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