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Molecular Epidemiological Analysis of the Spreading Conditions of Trichophyton in Long-Term Care Facilities in Japan.

Tinea pedis and tinea unguium are common infectious diseases, and many elderly people are reported to contract these infections. In this study, to investigate whether strains of the same origin are spreading inside a long-term care facility, we analyzed Trichophyton rubrum and Trichophyton mentagrophytes, isolated from the residents and staff at the facilities located in the Kanto area, using a genomic analytical method targeting tandem repeat regions in the nontranscribed spacer (NTS) region of ribosomal DNA. Five NTS types were confirmed in T. rubrum. T. rubrum of various types (types 1 to 5) was detected at each facility, but there was no isolate specific to one facility only. Eight NTS types of T. mentagrophytes were detected, and T. mentagrophytes that carried an NTS type that was confirmed at one facility only (types C4II, F4II, and D4II) was isolated. These T. mentagrophytes sequence types were isolated from several subjects residing at the same facility. This study proved that a T. mentagrophytes strain of the same type had spread in long-term care facilities. We believe in the importance of cleaning at a long-term care facility as a countermeasure to the spread of Trichophyton species.


INTRODUCTION
Trichophytosis is one of the major infectious diseases that invades human skin by the activation of an exotoxin (keratinase), and the target skin area is mainly located around the toes, where the causative fungus lodges stably. Trichophytosis of the foot is called tinea pedis. The causative fungi of tinea pedis are dermatophytes such as Trichophyton, Epidermophyton, and Microsporum. Especially, Trichophyton rubrum and Trichophyton mentagrophytes var. interdigitale are frequently isolated as pathogenic fungi (1).
Numerous epidemiological studies of skin infections have been conducted: The most prevalent disease in the past 3 surveys in Japan was tinea pedis, followed by tinea unguium (2)(3)(4). In addition, age-specific prevalence rates of tinea pedis and tinea unguium are constantly being investigated, and they are high in elderly people. An epidemiological study on Trichophyton collected from elderly people at long-term care facilities in 2016 revealed that approximately half of the elderly people shed Trichophyton from their feet, and we believe there is a risk for rapid spread of Trichophyton (5). Besides, it has been reported that elderly people with tinea pedis have experienced falling down significantly more frequently than the noninfected people have (6). Infec-tion with a dermatophyte such as T. rubrum or T. mentagrophytes leads to the deterioration of the quality of life.
The details of the pathogenesis of Trichophyton infection have not been clarified yet. Direct infection transmission from patients with tinea pedis to a noninfected person is very rare, and it is believed that the main route of infection is the direct contact of an uninfected person with Trichophyton existing in the environment (7). There are few reports that have examined the infection source at a long-term care facility. In our previous epidemiological study, it was not clear whether Trichophyton isolates collected from the elderly people who live at long-term care facilities are derived from the fungus remaining at the same long-term care facility or have come from other environments (5). Therefore, we assumed that it is necessary to prove the presence or absence of spreading of one original strain at a facility by conducting genomic analysis of Trichophyton.
In this study, to confirm whether strains of the same origin are spreading inside a long-term care facility, we analyzed Trichophyton (T. rubrum and T. mentagrophytes) isolates, collected from the residents and staff working at the facilities, using a genomic analytical method targeting tandem repeat regions in the nontranscribed spacer (NTS) region of ribosomal DNA, which can confirm relatedness and individual identification among the species.

Fungal isolates:
In 2016, 23 isolates of T. rubrum (Table 1) and 20 isolates of T. mentagrophytes (Table 2) were collected from the feet of elderly subjects, who were aged 65 or more and the staff working at long-term care facilities (facilities A, B, C, D, and E) located in the Kanto area, Japan. Sample collection and analysis in the study were approved by the ethics review committee of Bunkyo Gakuin University (Approval number: 2016-0010). These collected isolates were identified by the macroscopic examination of their colony morphology (pigmentation, the growth rate, and texture) on Sabouraud dextrose agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), microscopic examination of conidia, and PCR restriction fragment length polymorphism analysis of the amplified ITS region fragments by means of the MvaI restriction enzyme (8). The identified isolates were cryopreserved at −80°C until experiments were performed.
DNA extraction method: Conserved Trichophyton was applied to Sabouraud dextrose agar and cultured until colonies were confirmed. DNA extraction was performed with the modified Rapid Mini-Preparation method (9). A small amount of a fungus was added to the lysis solution (400 mM Tris-HCl Buffer [pH 8.0], 60 mM EDTA [pH 8.0], 150 mM NaCl, 1% of sodium dodecyl sulfate) and heated at 100°C for 10 min. Potassium acetate was added to the fungus solution, and the mixture was incubated at −20°C for 5 min. After centrifugation (10,000 × g, 1 min), the supernatant was recovered. An equal volume of isopropyl alcohol was added to the supernatant and mixed, the mixture was centrifuged at 10,000 × g for 2 min, and the supernatant was removed. The precipitated DNA pellet was washed with 70% ethanol, centrifuged at 10,000 × g for 1 min, and the supernatant was again removed. The DNA pellet was air dried and dissolved in Tris-EDTA (TE) buffer.
Analysis of molecular polymorphism: The NTS region of ribosomal DNA was amplified by PCR, and molecular polymorphism was analyzed via electrophoretic patterns. Because the primers used were different among the fungal species, primers that amplify tandemly repetitive subelement 1 (TRS-1) and tandemly repetitive subelement 2 (TRS-2) in the NTS region were used with T. rubrum (Table 3) (10). Primers targeting the S0 tandem repeat region (TmiS0), the S1 tandem repeat region (TmiS1), and the S2 tandem repeat region (TmiS2) in the NTS region were used for T. mentagrophytes  D  1  II  D1II  BGUTM21  A the elderly TM  D  6  I  D6I  BGUTM7  B the elderly TM  D  2  II  D2II  BGUTM8  B the elderly TM  D  2  II  D2II  BGUTM9  B the elderly TM  D  2  II  D2II  BGUTM10  B the elderly TM Z (new) 2  II  Z2II  BGUTM11  C the elderly TM  D  4  II  D4II  BGUTM14  C the elderly TM  D  4  II  D4II  BGUTM12  C the elderly TM  F  4  II  F4II  BGUTM13  C the elderly TM  F  4  II  F4II  BGUTM16  D the elderly TM  C  4  II  C4II  BGUTM18  D the elderly TM  C  4  II  C4II  BGUTM19  D  staff  TM  C  4  II  C4II  BGUTM17  D the elderly TM  D  2  II  D2II  BGUTM20  D  staff  TM  D  2  II  D2II  BGUTM24  E the elderly TM  C  8  II  C8II  BGUTM22  E the elderly TM  D  2  II  D2II  BGUTM23  E the elderly TM  D  2  II  D2II  BGUTM25  E the elderly TM  D  2  II  D2II  BGUTM26  E the elderly TM  D  2  II  D2II 1) : The combinations of TmiS0, TmiS1 and TmiS2 fingerprint patterns classified the isolates into eight types. TM, Trichophyton mentagrophytes.  Each amplification product was applied to a 2% agarose gel, and electrophoresis was carried out; bands were visualized by staining with ethidium bromide. Polymorphisms of PCR products from each locus were identified by comparative gel electrophoresis, and the NTS type was determined (10,11).

Molecular polymorphism analysis of T. rubrum:
By amplifying TRS-1 and TRS-2 obtained from the NTS region of T. rubrum and by analyzing each electrophoretic pattern, five types (1, 2, 3, 4, and 5) based on TRS-1 were confirmed (Fig. 1). The type of TRS-1 for one isolate of BGUTR22 could not be determined. In terms of TRS-2, only one type (type I) was observed (Fig. 1). Analysis was performed by combining the results of TRS-1 and TRS-2, and a total of five NTS types (1, 2, 3, 4, and 5) were identified. Table 1 shows the analysis results on the NTS type from 23 isolates of T. rubrum. BGUTR2, BGUTR3, and BGUTR4 isolated from subjects at facility A were type 1 of the same NTS type. At other facilities, partial concordance of the NTS type was confirmed. Nonetheless, T. rubrum of various types (types 1 to 5) was detected at each facility, and there were no isolates specific to one facility only. The isolation frequency of the NTS types of T. rubrum is shown in Table 4. The most prevalent confirmed NTS type was type 1 (six isolates), and type 5 was the least prevalent (two isolates).

Molecular polymorphism analysis of T. mentagrophytes:
The NTS region of T. mentagrophytes was analyzed, and four PCR types (C, D, F, Z) were identified on the basis of TmiS0 ( Fig. 2A). We confirmed five types (1, 2, 4, 6, and 8) on the basis of TmiS1 ( Fig. 2A) and two types (I and II) based on TmiS2 (Fig. 2B). By analyzing the above PCR types, we identified a total of eight NTS types (C4II, C8II, D1II, D2II, D4II, D6I, F4II, and Z2II). The NTS types of T. mentagrophytes (20 isolates) collected from subjects residing at each facility are shown in Table 2. An isolate that had an NTS type confirmed at one facility only was detected at some    facilities (facility C, types D4II and F4II; facility D, type C4II), and these isolates were isolated from several subjects residing at the same facility. At facilities B and E, almost all the NTS types of T. mentagrophytes were type D2II (3 out of 4 and 4 out of 5, respectively). The isolation frequencies of the NTS types are shown in Table 4. The most frequently isolated NTS type of T. mentagrophytes was type D2II: this type accounted for approximately a half of the total.

DISCUSSION
Despite the large number of elderly people with tinea pedis and tinea unguium, there are few reports addressing conditions for the spread of Trichophyton at longterm care facilities in Japan. In addition, there is no study on T. rubrum or T. mentagrophytes of the same NTS type that spreads at a long-term care facility. In this study, by identifying TRS-1 and TRS-2 of T. rubrum and TmiS0, TmiS1, and TmiS2 of T. mentagrophytes, the NTS types of Trichophyton collected from the subjects at each facility were examined, and individual identification of each isolate was sought.
The most prevalent NTS type of T. rubrum isolated in the countries such as the United Kingdom and Brazil is type 1 (10,12). Similarly, the most frequently confirmed NTS type in our study was type 1. On the contrary, an epidemiological study conducted by Jackson et al. in 2000 revealed that most of the NTS types of T. rubrum derived from the Japanese is a type other than type 1, and only one out of 11 isolates belong to type 1 (10). There is a difference between our research results and those of the preceding study (Jackson et al.). Despite the rapid globalization in Japan, a molecular epidemiological study on T. rubrum has not been carried out since 2000 (10), and there is a great difference between their study period and ours. Due to the globalization in Japan, we propose that T. rubrum of type 1, which is the most prevalent NTS type worldwide, has invaded Japan from all over the world. Alternatively, due to the small number of T. rubrum strains derived from the Japanese investigated by Jackson et al., it is possible that type 1 was not detected.
At facility A, T. rubrum isolates that had the same NTS type (type 1) were prevalent. On the contrary, at other facilities, various NTS types of T. rubrum were confirmed (types 1 to 5). Considering that many elderly people have tinea pedis (2)(3)(4), we believe that the elderly may have brought T. rubrum from outside of that facility. Besides, type 1, which is the NTS type of the majority of T. rubrum isolates collected from the elderly people residing at facility A, is the most prevalent NTS type globally (10,12). T. rubrum of type 1 confirmed at facility A may be the fungi acquired in other environments outside of that facility. Therefore, we concluded that the spread of T. rubrum only inside a long-term care facility is unlikely.
The T. mentagrophytes NTS type that was most frequently isolated in this study is D2II, and the number of isolates was approximately a half of the total (45%). Type D2II is the most prevalent NTS type (~40%) (11,13), and this finding is consistent with our research results. We confirmed that type D2II is the most prevalent NTS type of T. mentagrophytes.
T. mentagrophytes isolates that carried an NTS type confirmed at one facility only were collected. These T. mentagrophytes isolates (types C4II, F4II, and D4II) collected from several subjects residing at the same facility were not confirmed at other facilities. In addition, the prevalence rates of these types (C4II and D4II) are low worldwide (0% and 2.4%, respectively) and in Japan (1.8% and 5.0%) as well (11,13). Additionally, type F4II is the NTS type confirmed for the first time in this study. Due to the presence of T. mentagrophytes isolates that carried an NTS type confirmed at one facility only and were collected from several subjects at the same facility, we showed that long-term care facilities are responsible for the spread of T. mentagrophytes. Similarly, T. mentagrophytes of type D2II, which represented the majority of isolates collected at facilities B and D, may have originated from only one individual at each facility. However, type D2II is the most prevalent NTS type (11,13). To prove the mode of the spread of T. mentagrophytes inside such facilities, it is necessary to increase the number of facilities and subjects, and re-examination should be performed.
Although the spread of T. mentagrophytes at longterm care facilities is highly likely, the probability of the spread of T. rubrum is remarkably low. Although the reason has not been elucidated, it has been confirmed that T. mentagrophytes can grow in floor dust for 9 months (14). On the contrary, it has been reported that T. rubrum cannot survive in floor dust (14). The lifetime of T. rubrum and T. mentagrophytes in the environment might be different; it may be a factor only T. mentagrophytes spreads inside a facility.
This study proved that long-term care facilities are involved in the spread of T. mentagrophytes. In a recent study, Trichophyton with low susceptibility to an antifungal drug was discovered in Japan (15), and trichophytosis may become more intractable. Keeping the living environment clean is effective as a layer of protection against dermatophyte infections, and our research results strongly indicate the importance of cleaning at a long-term care facility as a countermeasure to the spread of Trichophyton. This study did not elucidate the virulence factor that is involved in the spread of infection. To prevent further spread of infectious diseases such as tinea pedis and tinea unguium, additional studies should be conducted.