Evaluation of terbinafine resistance of Trichophyton rubrum and T. interdigitale in Portugal and characterization of the associated resistance mechanisms

Henriques, Camila; Simões, Helena; Veríssimo, Cristina; Sabino, Raquel

http://hdl.handle.net/10400.18/8093

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Objectives: Superficial mycoses caused by Trichophyton rubrum are among the most common infections worldwide. In Portugal, it is estimated that 1,510,391 of Portuguese suffer from dermatophytosis, corresponding to an incidence of 14,300 per 1,000,000 inhabitants. Superficial mycoses caused by Trichophyton rubrum are among the most common infections worldwide, being difficult to treat and often associated with recurrences after interruption of the antifungal therapy. Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophytes’ infections. The emergence of resistance to terbinafine in Trichophyton species has been recently described and is associated with point mutations in the SQLE gene. The increasing number of patients with Tinea pedis or Tinea unguium resistant to terbinafine treatment prompted us to screen the terbinafine resistance in Trichophyton isolates obtained by culture at the Mycology Reference Laboratory of the National Institute of Health Doctor Ricardo Jorge. We aimed to determine the frequency of terbinafine resistance and associated mechanisms of resistance. Materials & Methods: Dermatophytes collected during 2017–2020 were identified by culture and mass spectrometry (MALDI-TOF-MS) and/or by sequencing the ITS (Internal Transcribed Spacers) region of the rDNA. All isolates were grown onto agar supplemented with terbinafine (0.06 and 0.125 mg/L) in order to detect potential resistant isolates. Antifungal susceptibility testing was performed following CLSI M38A2 broth microdilution method. The squalene epoxidase gene (SQLE) was sequenced and mutations described as conferring resistance to terbinafine were screened. Results: Among 102 T. rubrum and 17 of T. interdigitale isolates that identified and further tested in what concerns to their antifungal susceptibility, 1 T. rubrum isolate (≈1%) and 3 T. interdigitale isolates (≈18%) showed to be resistant to terbinafine by screening agar. According to the microdilution method, 2.44% of the isolates were less susceptible to terbinafine. Overall, three isolates (1 T. rubrum and 2 T. interdigitale) showed high terbinafine resistance (MICs, 4 to ≥ 8 mg/L) and one isolate (T. interdigitale) displayed moderate terbinafine resistance (1 to 2 mg/L). The sequencing of the SQLE gene allowed the detection of the following single point mutations: in one T. rubrum and in one T. interdigitale, at the position 1189 in the ORF of the SQLE gene (corresponding to a substitution of a phenylalanine at the position 397 by an isoleucine or by a leucine, respectively). In the remaining resistant T. interdigitale isolates, no mutations were found in the gene encoding SQLE. Although the most common substitutions that lead to higher resistance to terbinafine are L393F and F397L, the F397I substitution detected in our study suggests to be associated with high terbinafine resistance. Conclusions: This study allowed us to detect, for the first time in Portugal, Trichophyton isolates resistant to terbinafine. It was also possible to estimate, within the studied isolates, a resistance frequency of 2.4%. Taken together, our results prompt the current knowledge about the necessity of antifungal susceptibility testing to select effective strategies for management of clinical cases of dermatophytosis not only in Portugal but worldwide.