Molecular Epidemiology and Antifungal Susceptibility of Trichophyton Isolates in Greece: Emergence of Terbinafine-Resistant Trichophytonmentagrophytes Type VIII Locally and Globally

Abstract

Trichophyton isolates with reduced susceptibility to antifungals are now increasingly reported worldwide. We therefore studied the molecular epidemiology and the in vitro antifungal susceptibility patterns of Greek Trichophyton isolates over the last 10 years with the newly released EUCAST reference method for dermatophytes. Literature was reviewed to assess the global burden of antifungal resistance in Trichophyton spp. The in vitro susceptibility of 112 Trichophyton spp. molecularly identified clinical isolates (70 T. rubrum, 24 T. mentagrophytes, 12 T. interdigitale and 6 T. tonsurans) was tested against terbinafine, itraconazole, voriconazole and amorolfine (EUCAST E.DEF 11.0). Isolates were genotyped based on the internal transcribed spacer (ITS) sequences and the target gene squalene epoxidase (SQLE) was sequenced for isolates with reduced susceptibility to terbinafine. All T. rubrum, T. interdigitale and T. tonsurans isolates were classified as wild-type (WT) to all antifungals, whereas 9/24 (37.5%) T. mentagrophytes strains displayed elevated terbinafine MICs (0.25-8 mg/L) but not to azoles and amorolfine. All T. interdigitale isolates belonged to ITS Type II, while T. mentagrophytes isolates belonged to ITS Type III* (n = 11), VIII (n = 9) and VII (n = 4). All non-WT T. mentagrophytes isolates belonged to Indian Genotype VIII and harbored Leu393Ser (n = 5) and Phe397Leu (n = 4) SQLE mutations. Terbinafine resistance rates ranged globally from 0-44% for T. rubrum and 0-76% for T. interdigitale/T. mentagrophytes with strong endemicity. High incidence (37.5%) of terbinafine non-WT T. mentagrophytes isolates (all belonging to ITS Type VIII) without cross-resistance to other antifungals was found for the first time in Greece. This finding must alarm for susceptibility testing of dermatophytes at a local scale particularly in non-responding dermatophytoses.

Keywords: Greece; Trichophyton spp.; antifungal resistance; dermatophytes; terbinafine.

Figure 1

Maximum likelihood phylogenetic tree of T. interdigitale (Ti) and T. mentagrophytes (Tm) genotypes based on ITS sequencing. Values at the nodes indicate bootstrap percentages based on 1000 replicates and only branches with bootstrap values above 50% are shown (different clades are highlighted using different colors). ITS sequences of several reference isolates and clinical strains retrieved from the GenBank [21,22] were used for comparative analysis. The isolates of the present study are marked with the prefix AUH.

Figure 2

(A) Colonies of all but one (Nr.1687) T. mentagrophytes ITS Type VIII (Tm VIII) isolates had yellowish reverse pigment (upper line), as opposed to T. interdigitale (Ti) as well as T. mentagrophytes Type III* (Tm III*) and Type VII (Tm VII) isolates (lower line). (B) Urease test on Christensen urease agar was positive for T. interdigitale and T. mentagrophytes ITS Type III* and Type VII, but negative for T. rubrum (Tr) and T. mentagrophytes Type VIII after 7 days of incubation.

Figure 3

Global distribution of T. rubrum isolates exhibiting reduced susceptibility to terbinafine (detailed in Table 3). The country of origin is colored in red, while the type of study (case report: CR, case series: CS, single-center study: SS, multicenter study: MS) along with the recovery rate of isolates displaying non-wild-type (WT) phenotype, terbinafine minimum inhibitory concentrations (mg/L) and amino acid positions with hot-spot mutations in the squalene epoxidase target gene (where available) are presented.

Figure 4

Global distribution of T. interdigitale/T. mentagrophytes isolates exhibiting reduced susceptibility to terbinafine (detailed in Table 4). The country of origin is colored in red, while the type of study (case report: CR, case series: CS, single-center study: SS, multicenter study: MS) along with the recovery rate of isolates displaying non-wild-type (WT) phenotype, terbinafine minimum inhibitory concentrations (mg/L) and amino acid positions with hot-spot mutations in the squalene epoxidase target gene (where available) are presented.