The clinical samples were collected from patients who visited the dermatology clinic of the Affiliated Hospital of Guizhou Medical University, Guiyang, China, between May 2017 and April 2022. Identification of all dermatophytes except T. rubrum was confirmed by internal transcribed spacer (ITS) rDNA sequencing. A total of 31 isolates of the T. mentagrophytes complex were isolated and examined. The information on source of isolation and other metadata can be found in Table S1. Nail fragments, hair and skin scales of the patients were collected and divided for cultivation and direct microscopy (with/without Calcofluor in 10% potassium hydroxide). The untreated samples were inoculated on the slope of Sabouraud’s Glucose Agar (SGA; homemade) containing 100 mg/L chloramphenicol and cultured at 28°C for two weeks. The strains with good growth, successful isolation and purification, and morphological identification as dermatophytes were included in the study.

Isolates of T. indotineae (JYP18010 and JYP22048) were grown on various agar media, including SGA and potato dextrose agar (PDA; HiMedia) at 30°C. Spore suspension of strains JYP22048 and JYP18010 (10⁶ CFU/mL) were obtained, and 1μL (10³ CFU) suspension was inoculated in the center of a PDA plate (9 cm). Morphology and growth rates of colonies were determined during two weeks in the dark at temperatures of 4, 18, 25, 30, 37 and 40°C. The colonies were documented using a Canon EOS 500D camera. Micromorphology was observed and documented. Production of urease was determined in Christensen’s urea (HuanKai Microbial, China) broth after incubation at 30°C for 3 and 7 days, with a final check after 10 days. Orange and pink tubes were scored as negative and positive, respectively. Strain T. mentagrophytes JYP18049 (=JYP18-3) was used as positive control (30). All strains were sequenced and stored in 15% glycerol in a refrigerator at -80°C.

Identification of isolates was done by phenotype (31) and confirmed by rDNA ITS sequencing. Briefly, isolates were subcultured on SGA (homemade) and incubated at 28°C for one week. DNA extraction was by the cetyltrimethylammonium bromide (CTAB) method (32). ITS of the rDNA operon was amplified with primers ITS1 and ITS4 according to Jiang et al. (32, 33). PCR products were sequenced by TSINGKE Biological Technology (Beijing, China). Sequencing results were assembled by SeqMan (DNASTAR), and were compared by BLAST in GenBank. A total of 31 isolates of the T. mentagrophytes complex were isolated and examined in this study. GenBank accession numbers for new sequences are given in Supplementary Table S1. For global comparison, 115 reference sequences were retrieved from GenBank, including Trichophyton mentagrophytes complex (n=108), T. schoenleinii (n=3), T. tonsurans (n=1), T. equinum (n=1), and with T. simii (n=2) as outgroup. These include the neotype and type strains of Trichophyton species (T. interdigitale, CBS 428.63^NT; T. mentagrophytes, IHEM 4268^NT; T. indotineae, CBS 146623^T; T. equinum CBS 100080^T; T. tonsurans, CBS 338.37^NT; T. simi, CBS 449.65^T). Reference strains for genotypes of T. mentagrophytes (ITS genotypes III, III^*, IV, VII, VIII, XI) and T. interdigitale (ITS genotypes I, II) were included in order to precisely reproduce the population structure (9, 10). Alignment was done with MAFFT v7 (https://mafft.cbrc.jp/), then analyzed using maximum-likelihood (ML) and Bayesian inference (BI) methods. Suitable substitution models of ML trees were determined using MEGA v6.0. ML in the CIPRES web server (https://www.phylo.org). Bayesian posterior probabilities were calculated using MRBAYES v3.2.7 (34). Phylograms are shown using FIGTREE v1.3.1 (35). Bootstrap values ≥ 80% and posterior probabilities ≥ 0.97 were considered as statistically supported and were indicated above thickened branches.

Antifungal susceptibility testing was performed for isolates JYP18010 and JYP 22048 using the Clinical and Laboratory Standards Institute (CLSI) M38-A2 broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi (36). MICs were determined after 24 h of incubation at 35°C. Candida parapsilosis ATCC 22019 and Trichophyton mentagrophytes ATCC MYA-4439 were used as quality controls. Terbinafine (TBF; CFDA Co., Beijing, China), luliconazole (LLCZ; Higher Biotech Co, Shanghai, China), itraconazole (ITZ; CFDA), voriconazole (VCZ), fluconazole (FCZ; Sigma Aldrich), ketoconazole (KTZ; CFDA) were applied. Stock solutions of all drugs were prepared in dimethyl sulfoxide (DMSO) at a concentration of 1600 µg/ml (except FCZ, which was dissolved in distilled water at final concentration 6400 µg/ml).

The isolates of T. indotineae (JYP18018 and JYP22048) were screened for mutations in the squalene epoxidase (SQLE) gene. DNA was amplified using primers SQLE3S (5’-GTGTAAAGGGTCACATGCGG) and SQLE4R-2 (5’-AAGTTCGGCAAATACGAAAG) (7). PCR amplification (T100 Thermal Cycler; BIO-RAD, America) was carried out for 30 cycles consisting of denaturation for 30 sec at 95°C (Preheating for 5 min at 95°C), annealing for 30 sec at 55°C and extension for 30 sec at 72°C. A final extension was performed at 72°C for 5 min. The amino acid sequences of SQLE of the two T. indotineae isolates in the present study were compared with the reference sequence of T. mentagrophytes SQLE (TIMM 2789, GenBank accession no. KU242352).

We performed a search of published cases/GenBank accession no. of T. mentagrophytes VIII or T. indotineae in PubMed and Embase using the keywords “dermatophytoses”, “terbinafine resistance”, “Trichophyton. indotineae”, “T. mentagrophytes Genotype VIII” and “ITS region”. We only reviewed cases with clinical information and laboratory/sequence data to identify the fungus as T. mentagrophytes genotype VIII or T. indotineae, for cases and GenBank accession numbers of strains reported from India and Iran is referred to literature. Data recorded included case year, race where known, and country of origin.

A 32-year-old Indian male patient presented at the Dermatology clinic of our hospital in March 2022 with a 6-month history of pruritic eruptions. The rash started on the buttocks and then spread to the abdomen and left eyebrow, presenting as irregular dark erythema ranging from coin to palm size, with a small amount of fine furfur-like desquamation on the surface (Figures 1A–C). He has lived in Guiyang for four years and had not traveled back to India or elsewhere during past four years. There was no history of animal contact or of contact with similar patients, but his father in India also developed rashes and itching for nearly one year. The patient was initially diagnosed with tinea corporis and tinea cruris in another hospital four months earlier. Fluconazole 50 mg once a day was taken orally for two weeks, and miconazole nitrate cream was applied locally for 4 months, but there was no visible improvement. Direct examination (KOH 10% and Blankophor) showed septate, bright hyphal elements (Figures 2A, B) in samples from scales of the buttocks, abdomen and left eyebrow. Cultures from lesions of the patient were performed on SGA incubated for two weeks at 28°C. Only one velvety isolate (JYP22048) (Figure 2C) was obtained from the eyebrow sample and was preliminarily identified based on morphological features as a zoophilic strain of Trichophyton interdigitale according to the concept of Heidemann et al. (37). The identification was subsequently corrected to T. mentagrophytes type VIII after ITS rDNA sequencing and phylogenetic analysis. Oral treatment with itraconazole (200 mg daily) in combination with bifonazole (1% cream, two times a day) and ketoconazole (2% lotion, once a day) was initiated based on the mycological result. To prevent anaphylaxis, oral glycyrrhizin tablets (100 mg/d p.o. every 12 h) was given for 2 weeks. Following this approach, remission was observed, and the lesions became smaller and without scaling. A cure was achieved 4 weeks after starting the treatment, with only pigmentation remaining (Figures 1D–F). No recurrence has been observed to date.

The second case concerned a 20-year-old Indian girl who presented to our outpatient clinic in July 2018 with rash and pruritus at the root of both thighs. The patient, an international student of Guizhou Medical University, had been in Guiyang for 3 years before onset of illness, during which she had not returned to India and had no contact history. Tinea cruris was diagnosed after positive microscopic examination of skin scales. Itraconazole (200 mg orally daily) was given for 1 week and the disease was relieved. There was no recurrence in the telephone follow-up. Clinical and microscopic images were not collected, but positive strain isolation was achieved from a skin scale of the patient and referred to as JYP18010; this strain was confirmed to be T. mentagrophytes genotype VIII by ITS sequencing. The source of the infection was not found.

In total, 30 cases of dermatophytosis were caused by members of the T. mentagrophytes complex. Strains were identified during retrospective analysis of the rDNA internal transcribed spacer generated during epidemiological surveys in the Affiliated Hospital of Guizhou Medical University between 2017 and 2022. The cases are summarized in Table 1. Combined with molecular sequencing and phylogenetic analysis in this study, 30 patients (31 strains) belonged to four ITS genotypes of the T. mentagrophytes complex, including T. interdigitale types I and II (16 cases), T. mentagrophytes type VII (3 cases), T. mentagrophytes type IX (9 cases) and T. mentagrophytes type VIII (2 cases). Except for the two hosts of T. mentagrophytes type VIII, which were Indian, all other patients were Chinese nationals from Guizhou.

Trichophyton interdigitale types I and II patients were mostly adults, with an average age of 34 years (range 4–66 years, with 5 children under 8 years old). The infection sites of 5 pediatric patients were all scalp (1 case) and face (4 cases). Patient with isolate JYP21091 was an 8-year-old child with a history of contact with cats and rabbits, which resulted in severe inflammation of tinea capitis, and a scalp scar and permanent hair loss were left after 6 months of treatment. The main site of infection in 11 adult cases was nail (9/11, 82%), in addition to one causing tinea corporis and tinea cruris with severe inflammation (strain JYP21100), and one causing tinea faciei in a 44-year-old woman (strain JYP21071). Nine cases of T. mentagrophytes type IX were mainly children under the age of eight (5/9, 55.6%), presenting with tinea capitis and tinea faciei. In one case, a 73-year-old male (strain JYP18108) presented with an infected toenail. Trichophyton mentagrophytes type VII (3 cases) concerned a family of three previously reported (30), with a history of close contact with rabbits.

For phylogenetic analysis, we used 115 previously published ITS sequences from members of the T. mentagrophytes complex, as well as the type strains of T. tonsurans and T. equinum that were previously confirmed to branch within the clade (38). The phylogenetic analysis indicated that the group of T. mentagrophytes complex, T. tonsurans, and T. equinum formed a single terminal cluster without prominent branches with strong bootstrap support (ML/BI 100/1.00). Trichophyton schoenleinii was placed in a basal position to this cluster with a Bayesian posterior probability of 1.00 (Figure 3). The 31 strains in this study were scattered in four subclades with strong bootstrap support, 16 strains clustered in ITS type I and II subclade (ML/BI 97/0.98), 10 strains clustered in ITS type IX subclade (ML/BI 80/1.00), 3 strains in ITS type VII subclade (ML/BI 81/0.98) and 2 strains in ITS type VIII subclade (ML/BI 93/1.00). So far, no strains collected from Chinese Guizhou patients were found to cluster in the ITS type VIII subclade, which has been described as T. indotineae. Compared to CBS 428.63 (neotype of T. interdigitale), our two isolates (JYP18010 and JYP22048) harbored three single polymorphisms (SNPs) at position 94 (C), 125 (T) and 462 (T), identical to the remaining strains located in the subclade (Figure 4), whereas they differed from IHEM 4268 (neotype of T. mentagrophytes) in 2 positions in ITS.

It should be noted that our data did not allow to resolve the delimitation of T. mentagrophytes and T. interdigitale, since genotypes I and II representing T. interdigitale and III, III^*, VII, IX, and VIII representing T. mentagrophytes were all mixed together and did not form monophyletic clades, respectively. Moreover, the neotype strains of T. mentagrophytes (IHEM 4268), T. interdigitale (CBS 428.63) and T. mentagrophytes type VIII (T. indotineae CBS 146623) were located in the same clade with strong Bayesian posterior probability (ML/BI, 81/0.97). The relationships of these three species with other taxa (T. tonsurans and T. equinum) in the T. mentagrophytes clade are poorly resolved due to insufficient statistical support. Consequently, the level of intraspecific variability, along with the precise numbers of species-specific substitutions or indels useful for their differentiation, needs to be confirmed in future studies when more strains will be available.

Macromorphology of T. mentagrophytes type VIII (T. indotineae) is shown in Figure 5. Colonies of the isolates (JYP18010 and JYP22048) were flat, white to cream in color, with a velvety surface, and light orange yellow reverse with no diffusible pigment on PDA at 30°C for 2 weeks (Figures 5A, B). Numerous subspherical to pyriform microconidia were present and spiral hyphae were absent (Figures 5E, F). Macroconidia were few and cigar- to club-shaped, with three to five septa, and were smooth and thin-walled with narrow attachment bases (Figure 5C). Isolate JYP22048 demonstrated beaded chains of chlamydoconidia on PDA at 30°C (Figure 5D). The optimum growth temperature of the two strains was 25–30°C on PDA reaching a colony diameter of 70 mm in 14 days, 7 mm diameter at 37°C, and slow growth at 40°C for JYP22048, without growth at 40°C for JYP18010; minimum growth temperature 4°C (Figure 6). Absence of urea hydrolysis was observed in the two strains of T. indotineae (JYP18010 and JYP 22048) after incubation for 10 d at 30°C.

Figure 7 compares the MIC values of 6 antifungal drugs against the two clinical isolates of T. indotineae. Except for TBF, the remaining drugs showed similar antifungal activities against JYP18010 and JYP22048. Isolate JYP22048 had a high MIC value (4 µg/ml) against TBF, whereas isolate JYP18010 had a low MIC of < 0.03 µg/ml. Among triazoles, FCZ showed reduced susceptibility against T. mentagrophytes type VIII strains and the two isolates proved to be resistant, with MIC values of 16 µg/ml and 32 µg/ml, respectively. LLCZ was the most active antifungal drug against the two strains, with lowest MIC (< 0.03 µg/ml). KTZ showed good antifungal activity, similar to VCZ and ITZ, with MIC values of 0.25 µg/ml (JYP18010) and 0.125 µg/ml (JYP22048), respectively.

The partial SQLE sequence was successfully amplified in our two T. mentagrophytes type VIII strains (JYP18010 and JYP22048). The TBF-resistant strain (JYP22048) encoded Leu at codon 397 instead of Phe (Phe³⁹⁷Leu) in T. mentagrophytes strain TIMM2789 (GenBank accession number KU242352). The susceptible isolate JYP18010 showed no mutation. The SQLE sequence has been deposited in the GenBank database under accession number OQ054983 and OQ054984.

We reviewed 15 articles containing 100 cases or sequences (including the current study) of T. mentagrophytes type VIII/T. indotineae infection published in the period 2008−2022 (Table 2, Figure 8). In addition to India and Iran, 15 countries have reported cases or collected strains of this genotype so far. A total of 80 (80%) cases were reported from Europe, two from Australia, ten (10%) from Asia and eight from U.S.A., respectively, while no case was from Africa. However, the host origin of cases or strains found locally in these countries was still India (35 cases, 35%), followed by Bangladesh (11 cases, 11%); The remaining 29 cases were of no race or nationality. Strain collection time, patient race and strain numbers or GenBank accession numbers are shown in Table 2.