Clonal Outbreak of Trichophyton tonsurans Causing Tinea Capitis Among a Wrestling Team in Beijing, China

Trichophyton tonsurans mostly causes tinea capitis and tinea corporis and often associates with outbreaks among combat sports athletes. Here, we report an outbreak of tinea capitis caused by T. tonsurans among five juvenile athletes in a wrestling team in Beijing, China. Scrapings from the lesions of the five patients were performed by direct microscopic examination and fungal culture. The fungal pathogens were all identified as T. tonsurans by morphology and sequencing of the internal transcribed spacer (ITS) regions. Multilocus genotyping analysis was performed by sequencing of 13 gene loci. The sequences of these markers were identical among the five isolates, revealing a single genotype. Antifungal susceptibilities of terbinafine, itraconazole, fluconazole, ketoconazole, and amphotericin B against T. tonsurans was determined by broth microdilution method according to the Clinical and Laboratory Standards Institute M38-A3 document and these isolates were all susceptible to the common antifungal drugs treating tinea capitis.


Introduction
Trichophyton tonsurans, an anthropophilic dermatophyte mostly associated with tinea capitis and tinea corporis, was described as a predominant pathogen in Mexico as early as the 1940s [1] and now is highly prevalent in Africa, South America and endemic in North America [2]. Outbreaks of T. tonsurans infection among combat sports club members, such as wrestlers, judo athletes, and sumo wrestlers, have been reported frequently in Japan [3]. Tinea capitis is a contagious fungal dermatosis caused by anthropophilic, zoophilic, and geophilic dermatophytes, among which Microsporum canis is the most frequently reported zoophilic agent worldwide, while T. violaceum and T. tonsurans are the predominant anthropophilic agents and have been increasing over time globally [4]. In China, the common pathogens of tinea capitis were M. canis (65.2%), T. violaceum (13.6%) and T. mentagrophytes (6.6%), while T. tonsurans was less common (4.6%) [5].
Classically, dermatophytoses are easily cured by topical and oral antifungal drugs such as triazoles (fluconazole and itraconazole), imidazole (ketoconazole), and allylamine (terbinafine) have been commonly administrated for the treatment of tinea capitis [10]. However, several reports have shown antifungal resistance in dermatophytes isolates [11,12]. The frequent and irregular use of the antifungal drugs in athletes, often for long periods, may drive acquired antifungal resistance [10]. Overall, the resistance rate of dermatophytes isolates was relatively low and has not yet been reported in China until now.
Here, we report an outbreak of tinea capitis caused by T. tonsurans among juvenile athletes in the same collective from a wrestling team in Beijing, China. Multilocus genotyping analysis and antifungal susceptibility were performed on these causative isolates of T. tonsurans.

Case Presentation and Fungal Culture
Five wrestlers aged ten to fourteen presented at our clinic with a complaint of alopecia and erythema on the scalp. The earliest onset of symptoms in one of the patients occurred about 1 month before, and the other four patients joined in the same sporting collective and trained together with him. 1 month later, they all presented with similar symptom and the first patient was progressively aggravated. Scrapings from the lesions of the scalps were performed by direct microscopic examination using 10% potassium hydroxide (KOH) with calcofluor white and fungal culture. The specimens were inoculated onto Sabouraud dextrose agar (SDA) with chloramphenicol. Cultures were incubated at 28°C for 3 weeks for morphological observation.

Genomic DNA Extraction and Species Identification
For molecular identification, genomic DNA of the fungal isolates was extracted using the Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Ltd, Hubei, China) according to the manufactures' instructions. The ITS region was amplified by polymerase chain reaction (PCR) using the primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') in a reaction volume of 25 lL containing 20 ng of genomic DNA, 0.08 lM each of the primers, 12.5 lL 2 9 Taq PCR MasterMix (Tiangen Biotech Ltd; Beijing, China). The reaction condition was 95°C for 5 min for denaturation, 95°C for 30 s for denaturation, 58°C for 30 s for annealing, and 72°C for 1 min for extension for 30 cycles, with 5 min of extension at 72°C used for the final cycle. Amplification products were sequenced by BGI Company (Beijing, China). The sequences were analyzed against the CBS database (https://wi.knaw.nl/page/Pairwise_ alignment).

Antifungal Susceptibility Testing
Antifungal susceptibility of terbinafine (TBF), itraconazole (ITC), fluconazole (FLC), ketoconazole (KTC), and amphotericin B (AMB) against the causative fungal isolates was determined by broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) M38-A3 document [13]. The reference strain, T. mentagrophytes ATCC-MYA4439, was included as quality-control. The isolates were inoculated onto potato dextrose agar (PDA) at 28°C for 5-7 days until good conidial growth is present. MICs were determined after incubating for 4 days. The MIC was estimated as the lowest concentration of the antifungal, inducing at least an 80% reduction (for TBF, ITC, FLC, KTC) or a 100% reduction (for AMB) in fungal growth from that of the control. All tests were performed in duplicate on different days.

Results
Five wrestlers aged ten to fourteen presented with alopecia, erythema, and scales on the scalp, and two patients also accompanied with pustule (Fig. 1A). The specimens from scalp were performed by direct microscopic examination and spores and hyphae were observed in all five patients (Fig. 1B). Fungal culture on SDA was positive for suede-like, radially furrowed, white to yellowish colonies ( Fig. 2A). Therefore, the five patients were diagnosed with tinea capitis because of the scalp lesions, positive microscopic examination, and positive fungal culture. The causative isolates were all identified as T. tonsurans by macroscopic and microscopic characteristics (Fig. 2) and sequencing of ITS regions (GenBank accession numbers were listed in Table 1).
The patients were treated with oral TBF (125 mg BID) combined with topical 2% KTC cream for 4 weeks. In parallel, the two patients with kerion were also applied with oral azithromycin (AZM, 250 mg QD) for 4 weeks. The lesions relieved significantly, and no relapse was noted at a 4-month follow-up visit.
In order to determine the genotype of the causative T. tonsurans isolates among the five patients, multilocus genotyping analysis was performed by sequencing of ITS, NTS, ALP1, MEP5, CarbY, CarbM14, LAP1, LAP2, DPP4, SUB2, SUB3, SUB5 (GenBank accession numbers were listed in Table 1). The result showed that the sequences of these gene loci were identical among the five isolates, revealing a single genotype. However, the T. tonsurans isolated in this report harbored different genotype with the strain isolated in Asia reported by Abdel-Rahman, S.M. [9], with NTS VIR-B, ALPr5, SUB2-GTT10.
Finally, antifungal susceptibility of the common antifungal drugs treating tinea capitis, including TBF, ITC, FLC, KTC, and AMB, against the T. tonsurans isolates was performed and compared between the isolates. The minimum inhibitory concentrations (MICs) of TBF, ITC, FLC, KTC, and AMB against T. tonsurans isolates were 0.015 lg/mL, 0.015 lg/ mL, 8 lg/mL, 0.06 lg/mL, and 0.5 lg/mL,  (Table 1), and showed no difference between the five causative T. tonsurans isolates.

Discussion
To our knowledge, the most frequent agents causing tinea capitis were M. canis and T. violaceum either worldwide [4] or throughout China [5], despite heterogeneous distribution depending on the region. However, it should be mentioned that T. tonsurans is an emerging pathogen, and has become the predominant pathogen in the USA, UK, and Brazil [14], probably be explained by increasing immigration and the anthropophilic characteristics of the fungus, favoring its transmission due to changes in the lifestyle [4]. In this study, all the patients with tinea capitis were caused by T. tonsurans and two of them presented with kerion, an inflammatory variant of tinea capitis caused by a dramatic immune response to dermatophytes, most commonly zoophilic M. canis and, less commonly, by anthropophilic T. tonsurans and T. violaceum [14]. Thus, the atypical manifestation of the common infection is one of the characteristics of our report.
Tinea gladiatorum is a type of dermatophytosis that is transmitted by human-to-human contact; accordingly, it is common in combat athletes, such as wrestlers and judo fighters. It has been commonly reported worldwide, include the USA [15,16], France   17], Mexico [18], Iran [19], Japan [3], Germany [20], and Turkey [21]. The most common clinical manifestation are tinea corporis, followed by tinea capitis. The most prevalent causative dermatophyte is T. tonsurans [22]. Previous reports revealed that a high proportion of wrestlers are asymptomatic carriers [3,17], which probably explains why wrestlers tend to be infected with T. tonsurans. One report isolated T. tonsurans from all the wrestling mats indicating the burden of contamination in our study was high [19] and the contamination of wrestling mats with T. tonsurans has a crucial role in transmission. While another study demonstrated that no positive culture of dermatophyte was obtained from mats, it seems that body-to-body contact would be the most probable mode of transmission among wrestlers [23]. Early diagnosis and TBF: terbinafine, AZM: azithromycin, ITS: internal transcribed spacer, NTS: the nontranscribed spacer, MEP5: metalloprotease-5, CarbY: carboxypeptidases Y, ALP1: alkaline protease-1, DPP4: dipeptidylpeptidase IV, SUB3: subtilisinlike proteases 3, CarbM14: carboxypeptidases M14, LAP1: leucine aminopeptidases 1, LAP2: leucine aminopeptidases 2, SUB2: subtilisinlike proteases 2, SUB5: subtilisinlike proteases 5 appropriate clinical management are important for treating the infected athlete, minimizing the risk of transmission and preventing large outbreaks. In addition, conducting mass screenings for the sports groups to which the patients belonged and treating asymptomatic carrier patients is important to stop infection and prevent pandemics. The development of molecular methods of detecting intraspecies subtypes within dermatophytes and further strain level differentiation may make it possible to track infections, determine sources of infections and recurrence or reinfection after treatment, and analyze their virulence and drug resistance [24]. As for T. tonsurans, molecular polymorphisms were first identified by length variations of amplicons targeting the VIR in the NTS gene [6]. Subsequently, based on phylogenetic studies of the rDNA and ALP1 locus, they reported a relationship between variations in nucleotide sequences and the severity of infections [7]. Genotype analysis of the VIR region in the rRNA gene of T. tonsurans isolated from Japanese Judo practitioners revealed that all 101 isolates had identical genotype, suggesting that a specific genotype strain occurs throughout Japan [25]. Similarly, we performed multilocus genotyping analysis using 13 gene loci and the result showed that the sequences of these gene loci were identical among the five isolates, revealing a single genotype. It highly indicated that these causative isolates originated from the same strain and existed a possible spread in the wrestling team.
Oral antifungal agents such as terbinafine, griseofulvin, itraconazole, and fluconazole are optional drugs of treatment with a usual duration of 4-6 weeks. A recent systematic review which included 21 randomized controlled trials and 17 clinical trials for a total of 4856 children showed that terbinafine is more effective against Trichophyton species whereas griseofulvin is more effective against Microsporum species [26]. Combined therapy with topical and oral antifungals may decrease shedding of fungal spores and increase the cure rate. Antibiotics should be given for inflammatory tinea capitis such as kerion to control secondary bacterial infections [14]. All the patients were successfully cured in our report. In vitro antifungal susceptibility data of dermatophytes are variable. A recent study screened terbinafine susceptibility in 237 T. tonsurans strains isolated between 2000 and 2020 in Japan revealed all the isolates were susceptible to terbinafine [27]. Another study about 316 clinical isolates of dermatophytes in Iran revealed that terbinafine was the most potent antifungal against all isolates (0.002-0.25 lg/mL), followed by itraconazole (0.004-0.5 lg/mL) [28]. Our results also revealed that terbinafine and itraconazole showed lowest MICs (0.015 lg/mL) against T. tonsurans isolates. Although rare, refractory/recurrence after antifungal therapy in patients with tinea has been reported and there are several reports of antifungal resistance, notably in India and involve the spread of a unique clade related to the T. mentagrophytes/T. interdigitale complex [29]. The frequent and arbitrary use of antifungal drugs in athletes, often for long periods, probably drives acquired antifungal resistance [10]. Although no resistant dermatophytes have been reported in China yet, antifungal susceptibility tests would play an important role in selecting the effective antifungal drug, management and accelerating the recovery of tinea gladiatorum.
In conclusion, we report a small outbreak of tinea capitis caused by T. tonsurans among wrestlers in the same sporting collective in Beijing, China. Multilocus genotyping analysis revealed that all isolates consisted of a single genotype, suggesting the outbreak may be caused by a single strain of T. tonsurans. The isolates were all susceptible to the common antifungal drugs treating tinea capitis. In the case of T. tonsurans outbreak, accurate species identification and genotyping is helpful to trace the source and control the spread. More sensitive and rapid typing methods need to be developed in the future. Early detection and treatment of patients, including asymptomatic carriers, is crucial to control transmission.