Clonal outbreak of Trichophyton tonsurans causing tinea capitis among a wrestling team in Beijing, China

doi:10.21203/rs.3.rs-2112370/v1

Download PDF

Research Article

Clonal outbreak of Trichophyton tonsurans causing tinea capitis among a wrestling team in Beijing, China

https://doi.org/10.21203/rs.3.rs-2112370/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Trichophyton tonsurans, an anthropophilic dermatophyte, mostly causes tinea capitis and tinea corporis and often associates with outbreaks among athletes involving in combat sports. Here, we report an outbreak of tinea capitis caused by T. tonsurans among five juvenile athletes aged ten to fourteen 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 the five gene loci including the ITS and non-transcribed spacer (NTS) of the ribosomal RNA (rRNA) locus, alkalineprotease-1 (ALP1), metalloprotease-5 (MEP5), carboxypeptidases Y (CarbY). The sequences of these markers were identical among the five isolates, revealing a single genotype. Antifungal susceptibilities of terbinafine (TBF), itraconazole (ITC), fluconazole (FLC), ketoconazole (KTC), and amphotericin B (AMB) against T. tonsurans was determined by broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) M38-A3 document and the minimum inhibitory concentrations (MICs) were 0.015 µg/mL, 0.015 µg/mL, 8 µg/mL, 0.06 µg/mL, and 0.5 µg/mL, respectively. In conclusion, we report an outbreak of tinea capitis caused by T. tonsurans among wrestlers 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.

Trichophyton tonsurans

tinea capitis

outbreak

multilocus genotyping

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].

Molecular epidemiology of T. tonsurans enhances understanding of the transmission and outbreaks of this contagious pathogen. The genetic variability of T. tonsurans at the rRNA locus was firstly carried out by Abdel-Rahman, et al. [6], and then they discriminated multi-strain T. tonsurans infections by use of a multilocus approach in combination of rDNA nontranscribed spacer (NTS), alkalineprotease-1 (ALP1), metalloprotease-5 (MEP5), carboxypeptidases Y (CarbY) [7]. Intriguingly, they reported that recently diverge anthropophilies are associated with diminished inflammatory involvement based on phylogenetic analyses of NTS and ALP1 sequences [8]. Additionally, they identified 47 distinct strain types in 198 isolates from 14 countries through a mixed-marker strategy utilizing 13 gene loci [9].

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 a wrestling team in Beijing, China. Multilocus genotyping analysis and antifungal susceptibility were performed on these causative isolates of T. tonsurans.

Sample collection and fungal culture

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 ℃ 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 internal transcribed spacers (ITS) gene 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 µL containing 20 ng of genomic DNA, 0.08 µM each of the primers, 12.5 µL 2 × 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).

Multilocus Genotyping

Multilocus genotyping analysis was performed by sequencing of the five gene loci including the ITS and non-transcribed spacer (NTS) [6] of the ribosomal RNA (rRNA) locus, alkalineprotease-1 (ALP1), metalloprotease-5 (MEP5), carboxypeptidases Y (CarbY) [7], which show intraspecies diversity and can be applied to epidemiological investigations and determining the route of infection transmission.

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℃ 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.

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).

Table 1

Clinical information, antifungal susceptibility, and GenBank accession numbers of *Trichophyton tonsurans* isolates.
Patient No.	1	2	3	4	5
Age/gender	12/M	10/M	13/M	14/M	12/M
Type	kerion	tinea alba	kerion	tinea alba	tinea alba
Pathogenic isolate No.	BMU11797	BMU11798	BMU11799	BMU11800	BMU11801
Treatment	Oral TBF 125 mg BID + AZM 250 mg QD + topical 2% KTC cream, for 4 wks	Oral TBF 125 mg BID + topical 2% KTC cream, for 4 wks	Oral TBF 125 mg BID + AZM 250 mg QD + topical 2% KTC cream, for 4 wks	Oral TBF 125 mg BID + topical 2% KTC cream, for 4 wks	Oral TBF 125 mg BID + topical 2% KTC cream, for 4 wks
Outcome	Cured	Cured	Cured	Cured	Cured
Antifungal susceptibility
TBF	0.015	0.015	0.015	0.015	0.015
ITC	0.015	0.015	0.015	0.015	0.015
FLC	8	8	8	8	8
KTC	0.06	0.06	0.06	0.06	0.06
AMB	0.5	0.5	0.5	0.5	0.5
GenBank accession No.
ITS	ON125554	ON125555	ON125556	ON125557	ON125558
NTS	ON060591	ON060592	ON060593	ON060594	ON060595
MEP5	ON060596	ON060597	ON060598	ON060599	ON060600
CarbY	ON060601	ON060602	ON060603	ON060604	ON060605
ALP1	ON060606	ON060607	ON060608	ON060609	ON060610
TBF: terbinafine, AZM: azithromycin

The patients were treated with oral TBF (125 mg BID) combined with topical 2% KTC cream for four weeks. In parallel, the two patients with kerion were also applied with oral azithromycin (AZM, 250 mg QD) for four weeks. The lesions relieved significantly, and no relapse was noted at a four-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, and CarbY (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.

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 µg/mL, 0.015 µg/mL, 8 µg/mL, 0.06 µg/mL, and 0.5 µg/mL, respectively (Table 1), and showed no difference between the five causative T. tonsurans isolates.

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 appropriate clinical management are important for treating the infected athlete, minimizing the risk of transmission and preventing large outbreaks.

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 ITS, NTS, ALP1, MEP5, and CarbY 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. Recent research about antifungal susceptibilities against a large collection of T. tonsurans isolated from wrestlers revealed the MIC was the lowest for tolnaftate (0.022 µg/mL), followed by itraconazole (0.026 µg/mL) and terbinafine (0.033 µg/mL) [10]. 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 µg/mL), followed by itraconazole (0.004-0.5 µg/mL) [27]. Our results also revealed that terbinafine and itraconazole showed lowest MICs (0.015 µg/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 [28]. 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.

We report a small outbreak of tinea capitis caused by T. tonsurans among wrestlers 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.

Funding

This work was supported by the National Key Research and Development Program of China (Grant No. 2021YFC2302005) and National Natural Science Foundation of China (Grant No. 81971912).

Conflict of interest

The authors report no conflicts of interest in this work.

Gonzalez-Ochoa A, V.B. Dermatofitosis causantes de tina lapiel cabellula en la ciudad de Mexico. Rev d Inst salub y enferm trop 1945, 6, 145-148.
Cogliati, M.; Cruz Choappa, R.; Vieille Oyarzo, P.; Noguera, M. Trichophyton tonsurans in Chile: Genotyping in search of an origin. Med Mycol 2022, 60, doi:10.1093/mmy/myab077.
Hiruma, J.; Ogawa, Y.; Hiruma, M. Trichophyton tonsurans infection in Japan: epidemiology, clinical features, diagnosis and infection control. J Dermatol 2015, 42, 245-249, doi:10.1111/1346-8138.12678.
Rodríguez-Cerdeira, C.; Martínez-Herrera, E.; Szepietowski, J.C.; Pinto-Almazán, R.; Frías-De-León, M.G.; Espinosa-Hernández, V.M.; Chávez-Gutiérrez, E.; García-Salazar, E.; Vega-Sánchez, D.C.; Arenas, R.; et al. A systematic review of worldwide data on tinea capitis: analysis of the last 20 years. J Eur Acad Dermatol Venereol 2021, 35, 844-883, doi:10.1111/jdv.16951.
Chen, X.Q.; Zheng, D.Y.; Xiao, Y.Y.; Dong, B.L.; Cao, C.W.; Ma, L.; Tong, Z.S.; Zhu, M.; Liu, Z.H.; Xi, L.Y.; et al. Aetiology of tinea capitis in China: a multicentre prospective study. Br J Dermatol 2021, doi:10.1111/bjd.20875.
Gaedigk, A.; Gaedigk, R.; Abdel-Rahman, S.M. Genetic heterogeneity in the rRNA gene locus of Trichophyton tonsurans. J Clin Microbiol 2003, 41, 5478-5487, doi:10.1128/JCM.41.12.5478-5487.2003.
Abdel-Rahman, S.M.; Preuett, B.; Gaedigk, A. Multilocus genotyping identifies infections by multiple strains of Trichophyton tonsurans. J Clin Microbiol 2007, 45, 1949-1953, doi:10.1128/JCM.02610-06.
Abdel-Rahman, S.M.; Talib, N.; Solidar, A.; Nopper, A.J.; Wyckoff, G.J. Examining Trichophyton tonsurans genotype and biochemical phenotype as determinants of disease severity in tinea capitis. Med Mycol 2008, 46, 217-223, doi:10.1080/13693780701787840.
Abdel-Rahman, S.M.; Sugita, T.; Gonzalez, G.M.; Ellis, D.; Arabatzis, M.; Vella-Zahra, L.; Viguie-Vallanet, C.; Hiruma, M.; Leeder, J.S.; Preuett, B. Divergence among an international population of Trichophyton tonsurans isolates. Mycopathologia 2010, 169, 1-13, doi:10.1007/s11046-009-9223-7.
Kermani, F.; Javidnia, J.; Hedayati, M.T.; Abastabar, M.; Haghani, I.; Didehdar, M.; Fami Zaghrami, M.; Shokohi, T. In vitro activities of antifungal drugs against a large collection of Trichophyton tonsurans isolated from wrestlers. Mycoses 2020, 63, 1321-1330, doi:10.1111/myc.13172.
Thatai, P.; Sapra, B. Critical review on retrospective and prospective changes in antifungal susceptibility testing for dermatophytes. Mycoses 2016, 59, 615-627, doi:10.1111/myc.12514.
Dogra, S.; Shaw, D.; Rudramurthy, S.M. Antifungal drug susceptibility testing of dermatophytes: Laboratory findings to clinical implications. Indian Dermatol Online J 2019, 10, 225-233, doi:10.4103/idoj.IDOJ_146_19.
CLSI. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi, 3rd ed. CLSI standard M38. Wayne, PA: Clinical and Laboratory Standards Institute. 2017.
Leung, A.K.C.; Hon, K.L.; Leong, K.F.; Barankin, B.; Lam, J.M. Tinea capitis: An updated review. Recent Pat Inflamm Allergy Drug Discov 2020, 14, 58-68, doi:10.2174/1872213X14666200106145624.
Beller, M.; Gessner, B.D. An outbreak of tinea corporis gladiatorum on a high school wrestling team. J Am Acad Dermatol 1994, 31, 197-201, doi:10.1016/s0190-9622(94)70145-8.
Williams, C.; Wells, J.; Klein, R.; Sylvester, T.; Sunenshine, R. Notes from the field: outbreak of skin lesions among high school wrestlers--Arizona, 2014. MMWR Morb Mortal Wkly Rep 2015, 64, 559-560.
Poisson, D.M.; Rousseau, D.; Defo, D.; Estève, E. Outbreak of tinea corporis gladiatorum, a fungal skin infection due to Trichophyton tonsurans, in a French high level judo team. Euro Surveill 2005, 10, 187-190.
Bonifaz, A.; Araiza, J.; Tirado Sanchez, A.; Barbosa Zamora, A.; Gomez Saenz, A.; Mendez Juarez, A. Tinea gladiatorum due to Trichophyton tonsurans in a school wrestling team in Mexico: A case series. Curr Med Mycol 2020, 6, 62-65, doi:10.18502/cmm.6.4.5439.
Hedayati, M.T.; Afshar, P.; Shokohi, T.; Aghili, R. A study on tinea gladiatorum in young wrestlers and dermatophyte contamination of wrestling mats from Sari, Iran. Br J Sports Med 2007, 41, 332-334, doi:10.1136/bjsm.2006.030718.
Schießl, J.; Uhrlaß, S.; Wichmann, K.; Wilde, D.; Krüger, C.; Nenoff, P. Trichophyton tonsurans-an emerging pathogen in wrestling in Germany. Hautarzt 2021, 72, 878-891, doi:10.1007/s00105-021-04803-7.
Ilkit, M.; Ali Saracli, M.; Kurdak, H.; Turac-Bicer, A.; Yuksel, T.; Karakas, M.; Schuenemann, E.; Abdel-Rahman, S.M. Clonal outbreak of Trichophyton tonsurans tinea capitis gladiatorum among wrestlers in Adana, Turkey. Med Mycol 2010, 48, 480-485, doi:10.3109/13693780903278051.
Kermani, F.; Moosazadeh, M.; Hosseini, S.A.; Bandalizadeh, Z.; Barzegari, S.; Shokohi, T. Tinea gladiatorum and dermatophyte contamination among wrestlers and in wrestling halls: A systematic review and meta-analysis. Curr Microbiol 2020, 77, 602-611, doi:10.1007/s00284-019-01816-3.
Kermani, F.; Moosazadeh, M.; Hedayati, M.T.; Abastabar, M.; Haghani, I.; Aghili, S.R.; Shokohi, T. Molecular epidemiology of Tinea gladiatorum in contact sports in northern Iran. Mycoses 2020, 63, 509-516, doi:10.1111/myc.13069.
Abdel-Rahman, S.M. Strain differentiation of dermatophytes. Mycopathologia 2008, 166, 319-333, doi:10.1007/s11046-008-9108-1.
Sugita, T.; Shiraki, Y.; Hiruma, M. Genotype analysis of the variable internal repeat region in the rRNA gene of Trichophyton tonsurans isolated from Japanese Judo practitioners. Microbiol Immunol 2006, 50, 57-60, doi:10.1111/j.1348-0421.2006.tb03771.x.
Gupta, A.K.; Mays, R.R.; Versteeg, S.G.; Piraccini, B.M.; Shear, N.H.; Piguet, V.; Tosti, A.; Friedlander, S.F. Tinea capitis in children: a systematic review of management. J Eur Acad Dermatol Venereol 2018, 32, 2264-2274, doi:10.1111/jdv.15088.
Ansari, S.; Hedayati, M.T.; Zomorodian, K.; Pakshir, K.; Badali, H.; Rafiei, A.; Ravandeh, M.; Seyedmousavi, S. Molecular characterization and in vitro antifungal susceptibility of 316 clinical isolates of dermatophytes in Iran. Mycopathologia 2016, 181, 89-95, doi:10.1007/s11046-015-9941-y.
Arendrup, M.C.; Kahlmeter, G.; Guinea, J.; Meletiadis, J. How to: perform antifungal susceptibility testing of microconidia-forming dermatophytes following the new reference EUCAST method E.Def 11.0, exemplified by Trichophyton. Clin Microbiol Infect 2021, 27, 55-60, doi:10.1016/j.cmi.2020.08.042.

Download PDF

Reviewers agreed at journal
04 Oct, 2022
Reviewers invited by journal
04 Oct, 2022
Editor invited by journal
04 Oct, 2022
Editor assigned by journal
30 Sep, 2022
First submitted to journal
28 Sep, 2022

You are reading this latest preprint version

Clonal outbreak of Trichophyton tonsurans causing tinea capitis among a wrestling team in Beijing, China

Status:

Version 1

Abstract

Figures

Introduction

Material And Methods

Sample collection and fungal culture

Genomic Dna Extraction And Species Identification

Multilocus Genotyping

Antifungal Susceptibility Testing

Results

Discussion

Conclusion

Declarations

References

Status:

Version 1