Jpn. J. Infect. Dis.

, 64, 458-462, 2011

Original Article

Molecular Epidemiology of Trichophyton tonsurans Strains Isolated in Japan between 2006 and 2010 and Their Susceptibility to Oral Antimycotics
Kazushi Anzawa1,2, Takashi Mochizuki1,2*, Akiko Nishibu1, Hiroshi Ishizaki2, Katsuhiko Kamei3, Yoko Takahashi3, Machiko Fujihiro4, and Hidekazu Shinoda5

of Dermatology and 2Division of Dermatomycology (Novartis Pharma), Research Institute of Medical Science, Kanazawa Medical University, Ishikawa 920-0293; 3Division of Fungal Infection, Medical Mycology Research Center, Chiba University, Chiba 260-8673; 4Department of Dermatology, Ibi Welfare Hospital, Gifu 501-0619; and 5Shinoda Dermatological Clinic, Saga 843-0023, Japan
(Received July 11, 2011. Accepted August 31, 2011) SUMMARY: Trichophyton tonsurans has been isolated among judo practitioners, wrestlers, and sumo wrestlers during an epidemic of tinea corporis and tinea capitis in Japan. A previous study using restriction fragment length polymorphism (RFLP) analysis of the non-transcribed spacer (NTS) region of the ribosomal RNA gene revealed different sources for the causative fungus in epidemics among judo practitioners and among wrestlers. Many different fungal strains have since been isolated from practitioners of these sports. The present study evaluated fungal characteristics of strains newly isolated between July 2006 and December 2010 using this molecular method. PCR-RFLP analysis using MvaI and AvaI was performed on 263 strains, composed of 186 isolates from judo practitioners, 32 from wrestlers, 30 from sumo wrestlers, 5 from other sports, 7 from family members or friends of the sports practitioner patients, and 3 from sporadic (non-epidemic) cases. Four molecular types, NTS I, II, III, and VII were detected. Of these, NTS I was the most predominant, occurring in 243 of 263 strains (92.4z). All of the 30 strains isolated from sumo wrestlers were classified as NTS I, suggesting that the epidemic among sumo wrestlers originated from an earlier epidemic among judo practitioners. Thirteen strains were classified as NTS II; all were related to wrestling and were isolated mainly from the Chubu and Kansai areas in the central part of Honshu island. NTS III was detected in 6 strains, and one strain classified as NTS VII was isolated from a sporadic case of tinea capitis in a Peruvian immigrant. The minimum inhibitory concentrations (MICs) of terbinafine, itraconazole, fluconazole, and griseofulvin on 10 strains of NTS I and NTS II and 4 strains of NTS III were examined; there were no differences in MIC between these molecular types. targeting the VIR region in the NTS region; among these, they identified 7 sub-variants with different single nucleotide polymorphisms (SNPs) identified by restriction fragment length polymorphism (RFLP) analyses using 7 restriction enzymes. In 2006, Sugita et al. (5) sequenced the VIR region in the NTS region from 101 strains isolated from Japanese judo practitioners; based on the genetic homogeneity of the isolates, they concluded that the causative agent was clonal. Our previous study used RFLP analysis of PCR amplicons of the VIR region in the NTS region to examine 232 strains isolated between 2001 and 2006; our results suggested that the epidemics among judo practitioners and wrestlers had different origins (6). The fungal source of the epidemic among sumo wrestlers was probably derived from T. tonsurans spread by infected judo practitioners (3). In order to understand the contemporary epidemic in Japan, we evaluated the molecular characteristics of recently isolated strains, including strains from sumo wrestlers. In vitro susceptibility against oral antifungal agents was also measured in these newly isolated strains to provide insight into clinical treatment.

1Department

INTRODUCTION

Trichophyton tonsurans is known to be the causative agent of a nationwide epidemic of tinea capitis and tinea corporis among judo practitioners and wrestlers in Japan. The epidemic was first identified in a high school wrestling team (1), but an explosive epidemic then occurred among high school and university judo teams (2). An epidemic among young trainees of sumo wrestling, a form of traditional Japanese wrestling, was reported in 2005 (3). Several recent studies have examined the variable internal repeat (VIR) region of the non-transcribed spacer (NTS) region of ribosomal RNA (rRNA) genes, thus detecting different molecular types among T. tonsurans isolates (48). Gaedigk et al. (4) detected 5 variants differing in size by polymerase chain reaction (PCR)
*Corresponding author: Mailing address: Department of Dermatology, Kanazawa Medical University, Daigaku 1-1, Uchinada, Ishikawa 920-0293, Japan. Tel: 81 76 218 8141, Fax: 81 76 286 6369, E-mail: mocizuki kanazawa-med.ac.jp
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Table 1. Origins of strains and their molecular types (20062010) Origin of the strains NTS type NTS NTS NTS NTS Total I II III VII Judo 181 0 5 0 186 Wrestling 19 12 1 0 32 Sumo 30 0 0 0 30 Other sports 5 0 0 0 5 Family/Friends 6 1 0 0 7 Sporadic 2 0 0 1 3 Total 243 13 6 1 263

MATERIALS AND METHODS Fungal strains (Table 1, Fig. 1): We examined 263 isolates of T. tonsurans from 34 of all 47 prefectures in Japan between July 2006 and December 2010. As previously described (6), some of these were cultured from skin lesions of the heads of active martial arts participants, their family members, and close friends using the hairbrush sampling method. This study also included 64 strains isolated from a screening examination among high school judo practitioners at the 2007 All Japan Championships in Saga (9). Individuals' activities were recorded and were classified as follows: judo (n 186), wrestling (n 32), sumo (n 30), other sports (n 5). Other sports comprised 2 martial arts practitioners, 1 kendo player, 1 sports instructor, and 1 gymnast. The analysis also included another 7 strains isolated from friends or family members of tinea patients involved in these activities and 3 strains isolated from patients unrelated to sports, including a Peruvian immigrant (7,10). The geographical origins of the strains were: Tohoku (northern part of Honshu island: Aomori, Akita, Iwate, Yamagata, Miyagi, and Fukushima; n 26); KantoKoshinetsu (central-east Honshu island: Tokyo, Kanagawa, Chiba, Niigata, Ibaraki, Saitama, and Gunma; n 47); Chubu (central Honshu island: Aichi, Shizuoka, Gifu, Fukui, Ishikawa, and Toyama; n 121); Kansai (central-western Honshu island: Kyoto, Osaka, Nara, Shiga, Hyogo, and Wakayama; n 28); and Chugoku, Shikoku, and Kyushu (western Honshu island, Shikoku and Kyushu islands: Hiroshima, Yamaguchi, Ehime, Nagasaki, Fukuoka, Oita, Saga, Kagoshima, and Miyazaki; n 41). For species-level identification of the strains, we first examined the gross colony morphology and then confirmed that the strains were T. tonsurans by PCR-RFLP analysis of the ITS regions of rRNA genes (11). Preparation of template DNA, PCR conditions, and RFLP analysis: The NTS region was analyzed as previously described (6,7). Briefly, total cellular DNA was extracted from colonies cultured on Sabouraud's dextrose agar slants or plates with or without antibiotics by a rapid preparation method (12) and used as the template for PCR. PCR amplicons were generated using the templates and the primer pair L663 (5?-TTGTAGAAT GCTCCCAACCAC-3?) and R1145 (5?-ACAAGGGC GGAACTATCAGAC-3?), which targeted the middle part of the NTS region. The thermal cycling conditions C, followed by 28 cycles of were as follows: 4 min at 949 C, 1 min at 589 C, and 1 min at 729 C. The 1 min at 949 amplicons were digested with MvaI and AvaI (Toyobo
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Fig. 1. Geographical origins of examined strains and distribution of NTS I strains (No. of NTS I strains/examined strains) found in Japan (20062010).

Co. Ltd., Osaka, Japan), separated by 5z polyacrylamide gel electrophoresis, stained with ethidium bromide, and visualized under a UV lamp. The strains were sub-typed based on their banding profiles, as previously reported (6,7). Susceptibility of the fungal strains against antimycotics in vitro: Ten of 243 NTS I strains, 10 of 13 NTS II strains, and 4 of 6 NTS III strains were selected for further examination based on their geographical origin and the type of sports activity. Susceptibility tests were performed using a slightly modified broth microdilution method proposed by the Japanese Society of Medical Mycology (13). The strains were inoculated onto potato dextrose slants and incubated at room temperature for 2 weeks. Conidia were suspended in saline by vigorously scraping the surface of the colony with a loop; the suspensions were filtered with gauze folded 4 times and adjusted to a final concentration of 2.5 104 conidia/mL using a hemocytometer. Serial dilutions of antifungal agents were prepared in 96-well plates using stock solutions

diluted in culture medium composed of RPMI-1640 (Sigma-Aldrich, St. Louis, Mo., USA), buffered with 0.165 M morpholinopropane sulfonic acid (MOPS; Wako, Tokyo, Japan), and supplemented with glutamine and Alamar Blue 10z (v/v). The stock solutions of antimycotics were prepared as follows: terbinafine (TBF) and itraconazole (ITCZ), 0.5 mg/mL in dimethyl sulfoxide (DMSO); griseofulvin (GRF), 64 mg/mL in DMSO; and fluconazole (FCZ), 3.2 mg/mL in distilled C until water. These stock solutions were stored at 809 use. The working concentrations were 0.0010.5 mg/mL for TBF and ITCZ and 0.12564 mg/mL for GRF and FCZ. The final concentration of DMSO was adjusted to 0.5z (v/v). The prepared conidia were inoculated into each well (0.08 mL) to a concentration of 2 103/well; they were incubated at 309 C and observed every 24 h for up to 7 days. Growth of the strains was confirmed by a color change in the control well from blue to red. Wells that changed color were classified as being positive for growth, while those whose color remained unchanged were classified as negative for growth. The panel of antimycotics used in the present study was selected on the basis of a previous report (14), and all of them may be used to treat T. tonsurans infections. RESULTS On the basis of their banding profiles, the 263 strains were classified into 4 molecular types: NTS I, II, III or

VII (Table 1). Of the 263 strains, NTS I was the predominant type (243 strains, 92.4z) and was ubiquitously distributed throughouot Japan (Fig. 1). The next most common molecular type was NTS II, which accounted for only 13 strains (4.9z). The geographical distribution of NTS II was not uniform, with incidences of 6.6z (8/121) in the Chubu region and 14.3z (4/28) in the Kansai region, but only 0.9z (1/114) in other regions (Fig. 1). Six of the strains were identified as NTS III. Four of the 6 NTS III strains were isolated from judo practitioners in Gifu; 1 was isolated from a wrestler in Tokyo, and 1 was isolated from a judo practitioner in Chiba (Kanto). The 1 remaining strain was classified as NTS VII and was isolated from a Peruvian immigrant (10). Among sports practitioners, 181 of 186 strains (97.3z) isolated from the judo practitioners were classified as NTS I, and the rest were classified as NTS III (5/186, 2.7z); none were classified as NTS II. Of the 32 strains isolated from the wrestlers, 19 (59.4z) were classified as NTS I; 12 (37.5z) were classified as NTS II, and 1 (3.1z) was classified as NTS III. All of the 30 strains isolated from sumo wrestlers were classified as NTS I. All the 5 strains derived from participants of other sports activities were also classified as NTS I. The strains examined in this study and their minimum inhibitory concentrations (MICs) are shown in Table 2. The MIC range measured for TBF, including a reference strain for the MIC test of Trichophyton men-

Table 2. MICs of antifungal agents against strains of Trichophyton tonsurans determined by the microdilution method NTS type I KMU strain no. /Origin 6190/Judo 6218/Judo 6220/Judo 6241/Wrestling 6313/Judo 6324/Sumo 6359/Judo 6364/Wrestling 6453/Sumo 6462/Sumo 5159/Wrestling 5160/Wrestling 5205/Wrestling 6063/Wrestling 6087/Wrestling 6124/Wrestling 6145/Wrestling 6146/Wrestling 6147/Wrestling 6400/Wrestling 6162/Judo 6164/Judo 6246/Wrestling 6479/Judo MIC QC1) TBF (EP)
1 (6) 1 (5) 1 (6) 1 (7) 1 (6) 1 (6) 1 (6) 1 (5) 1/2 (7) 1/8 (7) 1 (5) 4 (7) 1 (6) 1 (6) 2 (7) 1 (7) 4 (6) 2 (6) 1 (5) 1/2 (6) 2 (6) 1/4 (6) 1/4 (6) 1 (6)

MIC ( mg/mL) ITCZ (EP) 0.008 (7) 0.008 (5) 0.004 (5) 0.13 (5) 0.002 (6) 0.002 (6) 0.001 (6) 0.002 (6) 0.004 (7) 0.008 (5) 0.002 (6) 0.004 (7) 0.002 (6) 0.008 (6) 0.002 (6) 0.004 (6) 0.002 (6) 0.015 (6) 0.002 (6) 0.03 (6) 0.004 0.004 0.004 0.002 (6) (7) (6) (6) FCZ (EP) 16 (7) 1 (5) 2 (5) 16 (6) 2 (6) 0.5 (6) 2 (6) 2 (6) 2 (7) 2 (5) 0.5 (6) 16 (7) 4 (6) 8 (6) 4 (6) 4 (6) 2 (6) 32 (6) 4 (6) 32 (6) 2 (5) 8 (7) 4 (6) 0.5 (5) GRF (EP) 1 (6) 1 (5) 1 (7) 2 (7) 2 (6) 1 (6) 1 (6) 2 (5) 2 (7) 0.25 (7) 1 (5) 1 (7) 1 (6) 1 (6) 2 (7) 2 (7) 1 (6) 1 (6) 0.5 (5) 1 (6) 2 2 1 4 (6) (6) (7) (6)

II

III

1):

QC, comparison with the quality control strain of Trichophyton mentagrophytes IFM 5218 (ATCC 18748), for which the MIC of TBF is known to be 0.016 mg/mL. EP, end point (days).

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tagrophytes IFM 5218 (ATCC18748) (13), was considerably higher than in previous studies (1416). Therefore, we reported the MIC in comparison with that of the tester strain, which was reported to be 0.016 mg/mL (13). The ranges of MIC for the NTS I strains were as follows: TBF, 1/81; ITCZ, 0.0010.13 mg/mL; FCZ, 0.516 mg/mL; and GRF, 0.252 mg/mL. The ranges of MIC for NTS II strains were similar to those for NTS I strains: TBF, 1/24; ITCZ, 0.0020.03 mg/mL; FCZ, 0.532 mg/mL; and GRF, 0.52 mg/mL. The ranges of MIC for NTS III strains were similar to those for NTS I and II strains: TBF, 1/42; ITCZ, 0.0020.004 mg/mL; FCZ, 0.58 mg/mL; and GRF, 14 mg/mL. Therefore, there were no apparent differences in the MICs between the molecular types. Although the MIC of TBF in NTS II tended to be higher than that in NTS I, the difference was not significant ( P 0.05, Mann-Whitney test). Of the 24 total strains, the MIC90 values for TBF, ITCZ, FCZ, and GRF were 2 (n 24), 0.015 mg/mL (n 24), 16 mg/mL (n 24), and 2 mg/mL (n 24), respectively. GRF had a narrow MIC range, whereas ITCZ and FCZ had wider MIC ranges. The 3 strains with higher MIC of ITCZ (KMU 6241, NTS I: 0.13 mg/mL; KMU 6146, NTS II: 0.015 mg/mL; KMU 6400, NTS II: 0.03 mg/mL) also showed higher MIC of FCZ (KMU 6241: 16 mg/mL; KMU 6146: 32 mg/mL; KMU 6400: 32 mg/mL).
DISCUSSION The results of the present study examining strains isolated between 2006 and 2010 were similar to those of our previous study on strains isolated between 2001 and 2006 (6). The predominant type was NTS I. One hundred eighty-one of the 186 strains isolated from judo practitioners (97.3z) were classified as NTS I; this ratio is almost the same as that in our previous study (160/164 strains, 97.6z) (6). Sugita et al. (5) examined 101 strains isolated from Japanese judo practitioners before 2005 and found only 1 molecular type, ``type II,'' which corresponds to NTS I in our studies. Thirtyone of these Japanese strains were later found to be identical by a mixed-marker method using 27 sequence variations in 13 gene loci, which discriminated 198 strains into 47 distinct types (8). The molecular profile of their ``EvS03'' was also detected among strains isolated from USA and Canada (8). Aside from NTS I, the present study identified only a small number of NTS III strains and no NTS II strains among isolated from judo practitioners. Of the 32 strains isolated from wrestlers, 19 (59.4z) were classified as NTS I, and 12 (37.5z) were classified as NTS II in the present study. The incidence of NTS II among wrestlers was 35.4z (17/48 strains) in our previous study (6); the present results indicate that the clone may be preserved among Japanese wrestlers. In addition, all of the NTS II strains were isolated from the Chubu and Kansai regions with the exception of 1 strain isolated from Kanagawa (KantoKoshinetsu, Fig. 1). The heterogeneous geographic distribution observed in the present study is consistent with previous series, in which 17 of 21 NTS II strains were isolated from these regions (6). The NTS II classification in our study is related to early designations from previous reports; it may be ``type II'' in Gaedigk's clas461

sification (4), ``type IV'' in Sugita's classification (5), and ``C'' in Abdel-Rahman's classification (8). The NTS II equivalent type was the most prevalent classification among US and European isolates and the second most prevalent among Mexican isolates (8). Among Korean isolates, the incidence of the molecular type related to NTS II was 45.5z (5/11) among isolates from wrestlers and 27.3z (3/11) among isolates from judo practitioners (Seung and Choi, personal communication). The present study examined 30 strains isolated from sumo wrestlers who lived in 9 prefectures in 5 regions; Kyushu (n 9), Kanto-Koshinetsu (n 8), Chubu (n 7), Tohoku (n 4), and Kansai (n 2). The first culture-proven case of T. tonsurans infection among sumo wrestlers was observed in March 2004 in Gifu in the Chubu region (3). All of the subjects were members of a local private sumo studio or high school sumo team, and 1 had trained with a high school judo practitioner with tinea corporis caused by T. tonsurans (3). Subsequently, a recalcitrant epidemic was reported in Nagasaki in Kyushu island between 2006 and 2009 (17,18). During this period, we obtained 30 sumo-related strains from institutes located in the above regions. The results of the present study indicate that all sumorelated strains were NTS I, consistent with the clinical observation that the epidemic among sumo wrestlers originated from judo practitioners (3). Another important observation from this study is that there was an increase in cases among the friends and families of the epidemic cases. Among them, 1 strain showing NTS II was isolated from a 3-year-old girl in Wakayama in Kansai, who was the daughter of a wrestling coach infected with an NTS II strain. Not only NTS I strains (6) but also NTS II strains may have contributed to epidemic outbreaks in the community. With the exception of this case, all other strains were classified as NTS I in the present study, reflecting the prevalence of NTS I in the present epidemic in Japan. The biological and pathogenic differences between the molecular types remain an area of interest. The ratio of NTS I to NTS II strains among wrestlers has remained constant over the last 10 years, suggesting that these 2 molecular types do not differ in pathogenesis. In addition, we examined the MICs of oral antifungal agents and found no significant differences in MICs of 4 antifungal agents among the 3 major molecular types, NTS I, NTS II, and NTS III. Further studies are needed to compare the pathogenesis of strains of different molecular types. The MIC values in the present study were comparable with those of previous studies reported in 2000 (14) and 2001 (15). For example, the MIC90 values of TBF were 2 (0.032 mg/mL) in our study, compared with 0.0080.01 mg/mL in the previous studies (14,15). Here, MIC values of ITCZ were 0.015 mg/mL, as compared with 0.030.06 mg/mL in previous studies (14,15); those of FCZ were 16 mg/mL, as compared with 8 mg/mL (14,15), and those of GRF were 2 mg/mL, as compared with 4 mg/mL (14). The present study measured a rather high MIC90 value of FCZ, although FCZ is not used as first-line therapy for dermatophytoses in Japan. The presence of strains showing higher MICs of both ITCZ and FCZ may suggest the existence of azole-resistant

strains, and the efficacy of FCZ for the treatment of ITCZ-resistant T. tonsurans infections may be limited.
Acknowledgments This study was supported in part by Grants-inAid for Emerging and Reemerging Infectious Diseases (H21-ShinkoIppan 008, H22-Shinko-Ippan 008) from the Ministry of Health, Labour and Welfare of Japan, and Cooperative Research Program of Medical Mycology Research Center, Chiba University, Japan (Grant No. 1019).

Conflict of interest None to declare. REFERENCES

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