Trichophyton species of Arthroderma benhamiae - a new infectious agent in dermatology

571© 2014 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd. | JDDG | 1610-0379/2014/1207

Review Article

Submitted: 12.3.2014Accepted: 4.5.2014

DOI: 10.1111/ddg.12390

Trichophyton species of Arthroderma benhamiae – a new infectious agent in dermatology

Summary

In Germany, infections due to the zoophilic dermatophyte Trichophyton (T.) spe-cies of Arthroderma benhamiae are being more frequently diagnosed. The source of infection of this emerging pathogen overlaps with that of the zoophilic species T. interdigitale. The most common source are guinea pigs. T. species of Arthroderma benhamiae causes inflammatory dermatophytosis in children and adolescents. In ad-dition to tinea capitis, it may cause both tinea corporis, tinea manus and frequently tinea faciei. In Germany, T. species of Arthroderma benhamiae is a frequent zoophilic dermatophyte, which in regions is probably more frequent than Microsporum canis. The mycological identification of the isolates with their yellow stained colonies is ba-sed on their macroscopic and microscopic features. However, some exhibit colony features consistent with those of T. interdigitale. These strains only can be identified unambiguously by means of molecular techniques. Using detection methods such as PCR-ELISA or real-time PCR, the dermatophyte can be identified directly from clinical material. Sequencing of the internal transcribed spacer region (ITS) of the ribosomal DNA has been approved as culture confirmation test for T. species of Arthroderma benhamiae. In addition, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is useful. Widespread dermatophytosis due to T. species of Arthroderma benhamiae, in particular of tinea capitis, requires oral an-tifungal agents. Terbinafine is most effective, alternatives are fluconazole and itraco-nazole.

Pietro Nenoff1, Silke Uhrlaß1, Constanze Krüger1, Marcel Erhard2, Uta-Christina Hipler3, Florian Seyfarth4, Jürgen Herrmann1, Tino Wetzig5, Wieland Schroedl6, Yvonne Gräser7

(1) Laboratory for Medical Microbiolo-gy, Mölbis, Germany(2) RIPAC-LABOR LLC, Potsdam-Golm, Germany(3) Department for Dermatology, Uni-versity Hospital Jena, Germany(4) Dermatologists in Private Practice: Priv.-Doz. Dr. Kirsten Jung, Uta Zell & Dr. Florian Seyfarth, Erfurt, Germany(5) Department of Dermatology, Dermatosurgery, and Allergolo-gy, Asklepios Hospital Weißenfels, Germany(6) Institute for Bacteriology and My-cology, Veterinary Faculty, University of Leipzig, Germany(7) Consultant Laboratory for Dermat-ophytes, Institute for Microbiology and Hygiene, Medical Faculty Berlin – Charité, Berlin, Germany

Conflict of interestNone.

IntroductionThus far, dermatophytoses resulting from contact with small rodents, especially guinea pigs, have almost exclu-sively been caused by zoophilic Trichophyton (T.) interdi-gitale strains (formerly T. mentagrophytes) [1]. Over the past five years, however, isolates exhibiting distinctly yel-low colonies have increasingly been detected (Figure 1a–c). Although macroscopically resembling Microsporum (M.) canis, the microscopic features of these yellow-stained der-

matophyte strains, if expressed, are consistent with those of T. interdigitale. Only molecular biology methods, such as sequencing of the internal transcribed spacer (ITS) regi-on of ribosomal DNA, allow for the exact classification of these strains as T. species of Arthroderma (A.) benhamiae [2, 3]. Though known for a long time, this zoophilic der-matophyte species has domestically not been described in recent decades. Nowadays, however, one has to assume that T. species of A. benhamiae is the most common zoophilic

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Review Article Trichophyton species of Arthroderma benhamiae

572 © 2014 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd. | JDDG | 1610-0379/2014/1207

pathogen of dermatophytoses, par t icu larly among ch i l-dren and adolescents , in Germany.

T. species of A. benhamiae may cause various kinds of dermatophytoses, predominantly tinea corporis and tinea fa-ciei, but frequently also tinea capitis and Kerion Celsi. Rare cases of onychomycosis have also been reported. When using conventional cultural detection methods, the diagnostic challenge lies in the differentiation from dermatophytes with similar morphology. Direct detection of pathogen DNA from skin scrapings and hair roots through polymerase chain reac-tion (PCR) has therefore proven diagnostically useful. The-rapeutic agents include topical antimycotics effective against dermatophytes. Extensive infections as well as tinea capitis require the administration of systemic antimycotics. Here, terbinafine is the treatment of choice, with fluconazole and itraconazole representing valid alternatives.

Prevalence of Trichophyton species of Arthroderma benhamiae

Japan

The earliest and also most reports on infections by T. species of A. benhamiae come from Japan [4–7]. T. species of A. benhamiae as pathogenic agent in human dermatophytoses was first isolated and described in Japan in the year 2002. The isolates came from two patients with tinea corporis as well as from a rabbit representing the source of infection [5]. At the time, differentiation was based on sequencing of the gene coding for chitin synthase 1 (CHS1) as well as on cros-sing experiments. In 1998, A. benhamiae had already once been isolated from a rabbit in Japan.

Shiraki et al. [6] reported on tinea corporis by T. species of A. benhamiae with an atypical clinical presentation in a Japanese patient working at a pet shop. The authors presu-med that T. species of A. benhamiae had most likely alrea-dy spread throughout Japan, however, to date, M. canis still remains the most common zoophilic dermatophytic patho-gen in that country [8]. It is closely followed in second place by A. benhamiae (T. species of A. benhamiae) transmitted by rabbits, rodents, and white-bellied hedgehogs, all of them being popular pets in Japan, too.

Japan is expecting a future increase in infections by this zoophilic dermatophyte [8]. In a recent study on molecular identification and epidemiology of T. species of A. benhami-ae in Japan, 46 of 61 strains showed high sequence homology within the internal transcribed spacer (ITS) regions of ribo-somal RNA (rRNA) genes. A total of eleven genotypes (NTS types) were differentiated within these 46 strains by means of strain typing through sequencing of the non-transcribed spacer (NTS) gene region of rRNA [9], among them 22 Ja-panese isolates. Of those, ten belonged to the NTS8 type, six

to NTS1, and three to NTS2. Five epidemiologically related strains, i.e. they were gathered from various body sites of the same patient or his pet, revealed identical genotypes.

Figure 1 Trichophyton species of Arthroderma benhamiae: Fungal cultures. Primary culture from skin scrapings in an 8-year-old girl with tinea faciei. Sabouraud 4 % dextrose slant agar shows characteristic beige to yellow, flat, radiating colo-nies (a). Subculture of Trichophyton species of Arthroderma benhamiae on Sabouraud 4 % dextrose agar in a Petri dish: flat, yellow, peripherally radiating, centrally raised colonies (b). Individual colonies of Trichophyton species of Arthroder-ma benhamiae displaying a yellow, radiating thallus (c).

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Prevalence in Germany

So far, T. species of A. benhamiae has only rarely been detec-ted as cause of dermatomycoses in Germany, yet not due to the fact that this fungus does not occur, but rather because T. species of A. benhamiae has been incorrectly identified. This comes as no surprise, for, on the basis of its thallus color and the large number of microconidia, this pathogen cannot be unequivocally differentiated from other zoophilic species, such as T. interdigitale and other morphologically similar dermatophytes (e.g. M. canis). This is in contrast to the actu-al prevalence of T. species of A. benhamiae as dermatophytic pathogen in Germany.

Predilection sites of T. species of A. benhamiae infec-tions are the trunk and arms (tinea corporis) as well as the face (tinea faciei) and scalp (tinea capitis and Kerion Celsi) (Figure 2a, b).

There has been one report of a German patient under im-munosuppressive therapy following a kidney transplant who de-veloped extensive tinea corporis by A. benhamiae[10]. The zo-ophilic pathogen was found on the patient, her husband as well as on several pets (three guinea pigs, three rabbits, and a dog). Here, identification was again based on sequencing of the ITS region of rRNA. The immunosuppressed patient was success-fully treated with oral terbinafine as well as topical ciclopirox.

One of our own patients was a 5-year-old girl with tinea faciei et corporis by T. species of A. benhamiae. Source of infection were two infected guinea pigs. The patient’s 10-ye-ar-old sister as well as her mother also showed tinea corporis. As topical therapy with ciclopirox cream was unsuccessful, oral administration of terbinafine (62.5 mg QD for 2 weeks) was initiated and led to a swift resolution of lesions [11].

A 9-year-old boy presented with a painful, oozing, pu-rulent swelling with abscess formation on the scalp that had developed a few weeks earlier. He initially received antibiotic treatment with cefuroxime axetil. The family had three pets: one cat and two guinea pigs, one of which showed fur lesi-ons. Conventional and molecular biology workup from swab material and epilated hairs (from the boy) revealed T. species of A. benhamiae. Administration of oral terbinafine over the course of eight weeks resulted in complete resolution of tinea capitis profunda (Kerion Celsi) lesions [12].

After an 11-year-old girl had suffered from tinea corporis for several weeks, she additionally developed tinea capitis with round alopecia and a hyperkeratotic scab (Figure 3a, b). Initially, she was topically treated with an antimycotic and antiinflammatory agent (fluprednidene 21-acetate and mico-nazole nitrate). She had contact with a cat (at her grandmo-ther's place), guinea pigs at home, and mice (in biology class at school). Fungal cultures as well as PCR from scalp scra-pings showed T. species of A. benhamiae. In addition to to-pical terbinafine and ciclopirox, oral terbinafine 125 mg QD

was administered in the context of a so-called “individual therapeutic attempt” over the course of 14 days. The father’s written informed consent had to be obtained, as terbinafine is not approved for the treatment of children. The intensive therapy resulted in complete resolution of tinea capitis lesions within two weeks [13].

Two of our own patients suffered from onychomycosis caused by T. species of A. benhamiae: a teenager showed in-volvement of the fingernails, a woman of the toenails (own observation).

Due to the clinical suspicion of pyoderma, a 24-year-old patient with pustular nodular lesions on the chin and cheeks was initially treated with oral amoxicillin and subsequent-ly as inpatient with piperacillin/tazobactam IV. The correct diagnosis of tinea barbae profunda by T. species of A. ben-hamiae was only made after identification of the isolate from epilated hairs by means of PCR [14]. The pathogen was also found on a guinea pig kept as pet. Treatment with oral flu-conazole 100 mg QD and topical ciclopirox cream led to the resolution of tinea barbae lesions.

Figure 2 Tinea corporis by Trichophyton species of Arthro-derma benhamiae in a 43-year-old man. Source of infection for him and his 21-year-old daughter was a guinea pig (a). Tinea manus caused by the same pathogen (and strain) in the patient’s 21-year-old daughter (b).

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The laboratory in Mölbis has analyzed a total of 8,464 samples from 7,680 patients collected over a 3-year peri-od (March 2010 until March 2013), in order to ascertain the prevalence of this particular dermatophyte species in Germany. Samples came from skin scrapings, hairs or hair roots as well as skin swabs. All samples were analyzed by culture and a large percentage (> 90 %) was also subjected to in-house PCR for dermatophyte detection, among them

T. species of A. benhamiae. In 231 (2.9 %) of 7,680 pati-ents, T. species of A. benhamiae was identified by means of culture (yellow strains) and/or PCR [15]. Surprisingly, M. canis was only the second most common zoophilic dermat-ophyte with a detection rate roughly 50 % lower. Among patients infected by T. species of A. benhamiae, children and adolescents up to 19 years of age were most frequently affected (61.3%). In the Mölbis lab (serving Leipzig and surrounding counties as well as parts of Middle Germany), T. species of A. benhamiae is currently the most common zoophilic dermatophytic pathogen. This, however, does not necessarily reflect its overall prevalence within Germany. Further studies are required, in order to ascertain whether this pathogen shift will persist.

Trichophyton species of Arthroderma benhamiae in Switzerland and Belgium

Nine rapidly growing dermatophytes isolated from eight chil-dren and one adult in Switzerland have been retrospectively classified as T. species of A. benhamiae by sequencing of the ITS region. Eight of the nine patients had contact with ro-dents, mostly guinea pigs [16].

A dermatophyte recently isolated from a Belgian child with vesicular and markedly inflammatory tinea corporis [17] morphologically resembled T. erinacei (formerly T. men-tagrophytes var. erinacei). Because of the unusual infection source “guinea pig” (T. erinacei is almost always transmit-ted by hedge hogs), the authors suggested the name T. men-tagrophytes var. porcellae for this “new” subspecies. The authors of the present review article, however, believe that their colleagues were rather dealing with a “yellow isolate” of T. species of A. benhamiae, for differentiation was merely predicated on morphologic criteria, as no molecular biology methods were implemented.

Sources of infection

Guinea pigs are the main source of infection for T. species of A. benhamiae (Figure 4). Other small rodents, however, may also be potential carriers of this zoophilic dermatophyte, e.g. hamsters and rats. Over a 14-month period, a Swiss vete-rinary clinic, apart from M. canis, also found T. species of A. benhamiae and zoophilic T. interdigitale strains in pets. While T. species of A. benhamiae was frequently isolated from guinea pigs, zoophilic T. interdigitale strains mostly came from European shorthair cats (predominantly “strays”) and occasionally from dogs [18]. We have also been able to identify T. species of A. benhamiae in a cat and a group from Illinois, USA, recently isolated this strain from several dogs affected by dermatophytosis [19]. A human infection by A. benhamiae following contact with a Canadian porcupine in

Figure 3 Tinea capitis by Trichophyton species of Arthroder-ma benhamiae in an 11-year-old girl. Right parietally, there is a circular, centrifugally growing, erythemato-squamous, centrally hyperkeratotic, scabbed area, 4 × 5 cm in diameter, showing centrifugal growth and alopecia as well as causing pruritus. Once more, guinea pigs were the source of infection (from: P. Nenoff, C. Krüger. Dermatophyten-Infektionen der Haut, Haare, Nägel – ein Update. Teil 1: Klinische Aspekte. Akt Dermatol 2012; 38: 347–59) (a). Following treatment with oral terbinafine 125 mg QD for 14 days. The tinea capitis lesions have completely healed. The remaining intact hair follicles suggest a resolution without scar formation in terms of pseu-dopelade of Brocq (b).

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a Japanese zoo has also been described [20].

Pathogenesis of dermatomycoses by Trichophy-ton species of Arthroderma benhamiae

Grumbt et al. [21] studied virulence genes of the human pathogen A. benhamiae (better T. species of A. benha-miae). While examining infections by A. benhamiae in guinea pigs, the same work group had previously already identified the gene coding for malate synthase AcuE, a key enzyme in the glyoxylate cycle. Malate synthase facilita-tes growth of A. benhamiae on lipids, which constitute an essential structural component of skin. It remained unc-lear, however, in what way the enzyme mutants devised by this work group actually played a pathogenetic role as virulence factor in this guinea pig model of A. benhamiae infections.

Burmester et al. [22] analyzed the genome of A. ben-hamiae and T. verrucosum, in order to gain insight into the pathogens’ virulence and pathogenicity. With respect to A. benhamiae, they found genomic evidence for secretory pro-

teases and other hydrolytic enzymes responsible for keratin degradation.

Cambier et al. [23] recently examined cutaneous im-mune responses to infections by T. species of A. benha-miae and zoophilic T. interdigitale strains in a mouse mo-del. They were able to show that cutaneous symptoms and microscopic changes caused by both dermatophytes in a guinea pig model as well as in humans, particularly the co-lonization of epidermal and follicular structures, were very similar. The cutaneous inflammatory infiltrate consisted of macrophages, dendritic cells, and especially polymorpho-nuclear neutrophils. According to the authors of that study, the latter are known to represent the “histologic key” to the diagnosis of dermatophytosis. The cytokine profile of the infection in situ was characterized by overexpression of TGF β, interleukin (IL) 1β, and IL6 mRNA, indicating the significance of the Th17 pathway in the immune response [23].

Unlike anthropophilic dermatophytes, e.g. T. ton-surans, T. species of Arthroderma benhamiae frequently causes highly inflammatory infections (Figure 5), which is consistent with the, by now known, keratinocytic cy-tokine pattern. Infections by T. species of A. benhamiae result in the secretion of numerous, particularly proin-flammatory cytokines, chemokines and immunomodula-ting cytokines. Specifically, there is an upregulation in the expression of genes coding for IL1ß, IL2, IL4, IL6, IL10, IL13, IL15, IL16, IL17, and interferon gamma [24]. On the other hand, T. tonsurans causes an increase in gene activity of only few cytokines (IL1ß and IL16), lea-ding to a distinctly more discreet inflammatory response. Both dermatophytes increase IL8 mRNA expression in keratinocytes.

Diagnostic workup

Microscopic workup and Wood light

In case of tinea capitis, spores invade the hair shaft in an en-dothrix pattern typical for Trichophyton species. T. species of A. benhamiae may not be detected by Wood light.

Cultural features of Trichophyton species of Arthroderma benhamiae

On Sabouraud agar, T. species of A. benhamiae forms flat radiating colonies with beige to yellow mycelium and a dense velvety surface (Figure 6). The reverse side of the colonies shows a strong, sometimes even bright, yellow (Figure 7), but may at times also appear ocher to brown or auburn. A smaller percentage of isolates exhibits a different colony mor-

Figure 4 Dermatophytosis by Trichophyton species of Ar-throderma benhamiae in a guinea pig. The pathogen was identified by culture as well as molecular biology methods. Caudally, there is a bald, sharply demarcated lesion with sil-very shiny hyperkeratoses. The animal was the infection sour-ce for tinea corporis in two siblings.

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phology, resembling that of T. interdigitale (formerly T. men-tagrophytes), with white granular, sometimes powdery, but also radiating and flat colonies, at times slightly yellowish at the margins (Figure 8). These roughly 20 % of T. species of A. benhamiae strains (percentage estimate based on figures from our own mycology lab) may morphologically not be dis-tinguished from T. interdigitale.

Using lactophenol cotton blue stains, microscopic fea-tures include predominantly round and occasionally oval to clavate microconidia laterally and terminally inserting at the hyphae (Figure 9a, d). The botrytis-like (grape-like) arrange-ment of microconidia corresponds to the micromorphology of T. interdigitale (Figure 9 b, c). Apart from that, microcon-idia may also laterally insert at the hyphae in an “ear of corn” fashion. Spiral hyphae may also occur, indicating they are not species-specific for T. interdigitale. The cigarette-shaped or sometimes clavate macroconidia are transversely septated (three to eight septa) [25].

Yellow T. species of A. benhamiae colonies may be confused with M. canis, T. erinacei, and even T. soudanen-se. With regard to the latter anthropophilic dermatophyte, however, the history – the patient is either of African descent or the infection was contracted on a trip to Africa – should lead the mycologic workup in the right direction. It should also be mentioned here that T. soudanense is generally no longer regarded a separate species, as it genotypically corres-ponds to the African population of T. rubrum. The morpho-logic differentiation between T. species of A. benhamiae and T. erinacei is feasible, though, if the phenotypic characteri-stics are fully developed. The urea hydrolysis test is negative

in T. erinacei, whereas T. species of A. benhamiae shows a positive reaction. M. canis may easily be differentiated on the basis of its characteristic macroconidia.

Furthermore, there is a genetic relationship between T. species of A. benhamiae and T. verrucosum, the pathogen cau-sing ringworm in cattle, which, according to an initial study [26], seems to also be corroborated by crossbreeding patterns. This genetic relationship is diagnostically significant, because

Figure 7 Trichophyton species of Arthroderma benhamiae: Subculture from skin scrapings in tinea capitis. On Sabouraud 4 % dextrose agar, the colonies’ reverse side shows a bright yellow.

Figure 6 Trichophyton species of Arthroderma benhamiae: Swab isolate from the arm of a 9-year-old girl with tinea cor-poris. The colonies with their yellow reverse side on Sabour-aud 4 % dextrose agar are similar to those of Microsporum canis, Trichophyton interdigitale, and Trichophyton erinacei.

Figure 5 Tinea corporis by Trichophyton species of Arthro-derma benhamiae in a 43-year-old woman. The mammary lesion shows an inflammatory erythema, erosion, scaling, and accentuation at the periphery.

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some molecular detection assays for T. species of A. benhamiae may show a certain degree of cross reactivity. In routine clinical practice, it is therefore advisable to validate the species-speci-ficity of the respective detection test. In case of clinical suspi-cion, particularly if the culture does not unequivocally show a rapidly growing dermatophyte, species-specific PCR for T. verrucosum or sequencing of the ITS region should additio-nally be performed. The latter pathogen is, however, much less common than T. species of A. benhamiae and, as already men-tioned, is transmitted by a different animal species.

Urea hydrolysis for the differentiation of Tricho-phyton species of Arthroderma benhamiae

Urea hydrolysis on Christensen's urea agar (Heipha Dia-gnostika Dr. Müller, Heidelberg) is positive (indicator: phenolphthalein, reactive = red agar staining). As M. canis and T. interdigitale are also urease positive, differentiation from these two species is therefore not feasible using this method. On the other hand, T. erinacei, which displays no or only delayed urea hydrolysis, may be ruled out by this technique.

Differentiation of Trichophyton species of Arthroderma benhamiae by chromogenic agar

Identification of T. species of A. benhamiae by means of CandiSelect™ 4 medium (Bio-Rad Laboratories, Munich)

has recently been described [27]. The CandiSelect medium is a chromogenic agar for the differentiation of yeasts, par-ticularly Candida species. This procedure is easy to per-form, as the colonies’ change in color denotes the respective Candida species. All 21 M. canis strains developed a pink to violet colony color on chromogenic agar. On the other hand, Arthroderma benhamiae colonies were predominant-ly (25 of 30 strains) turquoise. Five of the 30 A. benhamiae strains, however, displayed a violet color like M. canis. It ought to be stressed that the chromogenic agar method has been designed as quick test, since conidia formation takes a long time.

Molecular methods for the direct detec-tion of Trichophyton species of Arthro-derma benhamiae

Presently, unambiguous identification of T. species of A. ben-hamiae may only be achieved by means of molecular biolo-gy techniques. Specific ITS-based PCR constitutes a reliable method for direct pathogen detection from clinical sample material [28–31].

PCR ELISA for the detection of Trichophyton spe-cies of Arthroderma benhamiae

Using a uniplex PCR ELISA (enzyme-linked immunosorbent assay), whose primer pair amplifies specific sections of the to-poisomerase 2 gene, allows for the detection of T. rubrum, T. interdigitale, Epidermophyton floccosum, M. canis, T. ton-surans, T. verrucosum, and T. violaceum. The target gene for the detection of T. species of A. benhamiae, however, is the ITS 1 gene region (internal transcribed spacer). In general, amplified gene segments are labeled with digoxigenin by one of the primers and, using specific ELISA probes, subsequent-ly optically or photometrically detected [28, 32, 33].

Molecular biology differentiation of yellow and white colonies of Trichophyton species of Arthro-derma benhamiae

With respect to the A. benhamiae (T. species of A. benha-miae) strains isolated in Switzerland, sequencing of the ITS region as well as parts of the 28S rRNA gene revealed the presence of two infraspecific groups corresponding to the two markedly different colony phenotypes. Thus, group I with white colonies may be differentiated from group II with yellow colonies [34]. This classification ultimately also ser-ves an important practical purpose, for it is impossible to identify white colonies as T. species of A. benhamiae solely based on morphologic colony features. The sophisticated

Figure 8 Trichophyton species of Arthroderma benhamiae (white colonies). Isolate from a 14-year-old girl with tinea faciei. Sabouraud 4 % dextrose slant agar shows white, gra-nular, powdery, and radiating colonies whose morphology is similar to that of Trichophyton interdigitale (formerly T. mentagrophytes). Identification as Trichophyton species of Arthroderma benhamiae is based on molecular biology me-thods, e. g. PCR ELISA.

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molecular biology method of sequencing has, however, not yet become generally available for routine lab diagnostics.

MALDI-TOF mass spectrometry for the identifi-cation of Trichophyton species of Arthroderma benhamiae

In order to confirm results obtained from cultures, MAL-DI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (MS) may be used. The combination of MALDI-TOF MS and e.g. AnagnosTec “SARAMIS” (for-merly: spectral archiving and microbial identification system; nowadays: VITEK MS Plus, bioMèrieux, Nürtingen, Germa-ny), a software and database, constitutes a quick and specific procedure for the identification of bacteria and fungi. Here, samples are prepared and analyzed without prior purification, eventually yielding an unequivocal so-called fingerprint mass spectrum of the microorganism in question. This fingerprint is individual and may therefore, depending on the respecti-

ve group of microorganisms, be used in the identification of species, subspecies, and ultimately even strains. Protein mass spectra allow for the detection of genus-, species-, type-, and strain-specific signals (Figure 10a–d). The sample material – fungal colonies of dermatophyte cultures – thus ultimately biomolecules – is embedded into a matrix and subsequently desorbed and ionized by means of a laser. Acceleration of ions thus generated in the gaseous phase by an electromagnetic field is then followed by time-dependent detection after a flight di-stance of 1.2 meters. Flight times may then be matched with molecular masses according to prior calibration. This method offers an easy and outstandingly accurate way to differentia-te all clinically relevant dermatophytes and even rare species, provided they have been entered into the database. A compre-hensive study on the establishment of MALDI-TOF MS in 285 dermatophytic isolates belonging to 21 different species also included 17 T. species of A. benhamiae strains isolated from actual patients [35]. While conventional methods had failed to unambiguously identify these strains, MALDI-TOF

Figure 9 Microarchitecture of Trichophyton species of Arthroderma benhamiae, lactophenol cotton blue stain: microsco-py shows a vast number of small round microconidia (a). Clusters of round, partially relatively large microconidia (incipient chlamydospore formation) (b). Botrytis-like (grape-like) microconidia (c). Microconidia, thickened septate mycelium, and chlamydospores (d).

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MS analysis enabled the distinct classification as T. species of A. benhamiae. Sequencing of the ITS gene region eventually confirmed the identification. Similar results have recently been reported using an AXIMA confidence mass spectrometer (Shi-madzu Biotech, Kyoto, Japan) [36].

In this context, it ought to be emphasized that although MALDI-TOF MS is extremely useful in unequivocally identifying mature T. species of A. benhamiae colonies, the differentiation between yellow and white T. species of A. benhamiae colonies, is not possible. Unlike PCR, direct dermatophyte detection from skin scrapings or hair roots by means of MALDI-TOF MS is currently not feasible, either.

Treatment of infections by Trichophyton species of Arthroderma benhamiae

With regard to topical treatment of T. species of A. benhamiae infections, any antimycotic agent effective against dermatophy-

tes may be used, e.g. imidazoles (clotrimazole, bifonazole), ciclo-pirox, or terbinafine. Extensive dermatophytoses by T. species of A. benhamiae, especially tinea capitis, are generally treated with oral antimycotics. Here, terbinafine is the drug of first choice, with fluconazole and itraconazole representing valid alternatives.

The excellent efficacy of terbinafine in T. species of A. benhamiae infections is mirrored by its low minimal inhi-bitory concentration (MIC) value of 0.0156 μg/ml shown in an in vitro study [37]. All other antimycotics revealed higher MIC values with respect to T. species of A. benhamiae, to wit, griseofulvin 1 μg/ml, itraconazole 0.25 μg/ml, ketoco-nazole 16 μg/ml, fluconazole 32 μg/ml, voriconazole 1 μg/ml, clotrimazole 0.0625 μg/ml, ciclopirox 16 μg/ml, and amoro-lfine 0.25 μg/ml. It is unclear, however, to what extent these MIC values actually reflect the in vivo situation in patients. In our own experience, topical terbinafine and ciclopirox both work well. Systemically, terbinafine has been very effec-tive and well tolerated, yet the administration of fluconazole has also yielded successful results.

Conclusion

In Germany, infections by T. species of A. benhamiae initially went largely unnoticed. For the past five years, however, der-matophytoses caused by this new (emerging) pathogen have been described all over the country. Dermatologists, but also pediatricians, and ultimately even veterinarians, local health authorities, and also pet shop employees should take note of the rise in infections by this zoophilic dermatophyte in children and adolescents and take appropriate action. Avoiding contact with infected pets (guinea pigs) is the best form of prevention. Infec-ted pets should therefore not be admitted to any household. T. species of A. benhamiae causes markedly inflammatory and even purulent, abscess-forming dermatophytoses, frequently on the face and scalp. Although simple cultures easily provide evidence for a fungus, the exact classification of a fungus as T. species of A. benhamiae still poses difficulties. Newer, swiftly practicable molecular methods such as PCR or MALDI TOF MS allow for a specific mycologic workup.

Extensive tinea cases as well as tinea capitis should always be treated with systemic antimycotic agents. Terbinafine has proven to be effective and safe, with fluconazole and itracona-zole representing valid alternatives. Oral antimycotic treatment in children, however, requires the parents’ written informed consent and should only be administered as so-called “indi-vidual therapeutic attempt” according to German drug laws.

Acknowledgements

The excellent macroscopic pictures of fungal cultures were taken by the photographer Uwe Schoßig (Leipzig).

Figure 10 MALDI-TOF mass spectra: Trichophyton species of Arthroderma benhamiae, yellow strain (a). Trichophy-ton species of Arthroderma benhamiae (b). Trichophyton interdigitale (formerly Trichophyton mentagrophytes), zoophilic strain, isolated from a child with onychomycosis following contact with a pet (c). Trichophyton rubrum (d).

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Correspondence to

Prof. Dr. med. Pietro NenoffHaut- und Laborarzt/Allergologie, AndrologieLabor für medizinische MikrobiologiePartnerschaft Prof. Dr. med. Pietro Nenoff & Dr. med. Constanze Krüger

Straße des Friedens 804579 MölbisGermany

E-mail: [email protected]

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Trichophyton species of Arthroderma benhamiae - a new infectious agent in dermatology