Cases of dermatophytosis caused by Trichophyton benhamiae var. luteum and T. europaeum, newly described dermatophytes within the T. benhamiae complex

Abstract Trichophyton benhamiae var. luteum and T. europaeum – recently described dermatophytes within the T. benhamiae complex – were identified in nine cases of dermatophytosis involving guinea pigs, chinchillas and dogs. The diagnosis was obtained through direct hair/scale examination, culture and sequencing of the internal transcribed spacer region of ribosomal DNA.


INTRODUCTION
Dermatophytes are fungal pathogens that invade the keratinised tissues of humans and animals. 1 The taxonomy of these organisms has been revised recently through a multilocus phylogenetic approach 2 and is still rapidly evolving. One fungus mainly involved in these changes is Trichophyton benhamiae, included in the homonymous species complex. 2 As summarised in Čmoková et al., 3 this species was first described in 1967 as Arthroderma benhamiae from human and canine infections in North America. Subsequently, two races were recognised based on biological compatibility experiments: an "Americano-European" and an "African" race. Furthermore, two phenotypic variants (a "yellow" and a "white" phenotype) were progressively identified among the Americano-European race. Human infections caused by yellow phenotype strains, contracted mostly from guinea pigs, have increased since 2010 in various European countries. The fungus is distributed widely in guinea pig breeding populations and pet shops, with animals typically showing scarce or no evidence of disease. 4 The reason for the sudden increase of this variety is unclear, because the popularity of guinea pigs as pets began before the rise of human infections. Therefore,the cause may be the rise of a new, virulent and highly transmissible genotype. 3 White phenotype strains probably have existed worldwide for many years. Indeed, sporadic human and animal infections were described before the diffusion of yellow phenotype strains in Europe. 3 Notably, the white phenotype is very similar to T. mentagrophytes. Thus, the epidemiology of T. benhamiae infection has been elucidated only as a result of the increasing application of molecular techniques for fungal identification. 2,5 Finally, a recent study 3 showed that isolates previously designated as the Americano-European race of A. benhamiae harbour three species and two | 441

INFECTIONS BY TRICHOPHYTON BENHAMIAE-CLADE SPECIES
varieties. The yellow phenotype strains correspond to T. benhamiae var. luteum, which has a wide distribution in guinea pigs in Europe. 3,4 The white phenotype corresponds to T. benhamiae var. benhamiae (found mainly in the USA), T. japonicum (distributed mainly in Asia) and T. europaeum (the second most common species affecting guinea pigs in Europe). 3 The African race is more distantly related phylogenetically and is now classified as T. africanum. Its distribution, although less well-characterised as a consequence of the low number of available isolates, is mainly on the African continent. 3 In order to aid veterinary practitioners in becoming familiar with these newly described dermatophytes, we herein describe some cases of infection in various animal species (guinea pig, chinchilla, dog) due to T. benhamiae var. luteum and T. europaeum.

CASE DESCRIPTIONS AND METHODOLOGY
Nine cases (some involving multiple animals), examined at the Department of Veterinary Sciences (Turin University, Italy) between 2007 and 2021, showed alopecic patches with various degrees of inflammation (Table 1; Figures 1 and 2). The material collected for laboratory diagnosis included hairs plucked using sterile forceps, and hair and scales obtained by pressing a piece of transparent cellophane tape against the lesions (sticky tape technique). 6 The procedure for microscopic examination (performed in five cases) varied according to the sample type. Hairs were suspended in mineral oil on a microscope slide. Samples including abundant keratinous material were cleared in 20% NaOH before microscopy. 6 The tape pieces were placed on a microscope slide over a drop of stain [blue solution of the Hemacolor kit (Sigma-Aldrich; Waltham, MA, USA) or lactophenol blue]. Cultures were performed on Mycobios Selective Agar (Biolife; Milan, Italy) at 25°C. Results of laboratory exams are reported in Table 1, with some examples shown in Figures 3 and 4. Human contagion was never reported. Available information on therapies is summarised in Table S1 in Supporting information.
Fungal identification was achieved by sequencing the internal transcribed spacer region (ITS), one of the molecular markers most employed in dermatophyte taxonomy studies. 2,3,5 DNA was extracted using a commercially available kit (NucleoSpin Tissue, Macherey-Nagel; Düren, Germany). PCR was performed with the primer pair V9G and LR3. 7 PCR products were sequenced using ITS5 and ITS4 primers 8 through a commercial service (Macrogen Europe). Using Mega 11 software (https://www.megas oftwa re.net/), ITS sequences were aligned with those of the species belonging to the T. benhamiae complex 3,9 deposited in GenBank (https://www.ncbi.nlm.nih.gov/ genba nk/), and a phylogenetic tree was constructed by the neighbour-joining analysis. The robustness of the branches was assessed by bootstrap analysis with 1,000 replicates. The tree (shown in Fig. S1), allows subdivision of the complex into three clades/lineages (T. benhamiae, T. erinacei and T. bullosum) and eleven species. 3,9,10 Our isolates were identified as T. benhamiae var. luteum (six cases) and T. europaeum (three cases). Representative sequences concerning our fungal isolates were deposited in GenBank (Table 1).

DISCUSSION
Our findings confirm that in Europe, T. benhamiae var. luteum is the most prevalent species from the T. benhamiae clade to cause guinea pig infections, 3 and adds the chinchilla to the list of rodents that may harbour this fungus. Another recognised host is the common degu (Octodon degus). 3 To our knowledge, the cases caused by T. europaeum are the first ever to be reported in dogs. This fungus has been isolated predominantly from guinea pigs in previous reports. 3,10 Other proven infection cases have included rabbits and foxes. 3,11,12 Not unexpectedly, we did not identify other species of the clade. Trichophyton benhamiae var. benhamiae occurs mainly in the USA; it seems to have a broader range of primary host including dogs, foxes and the North American porcupine (Erethizon dorsatum), a close relative of the guinea pig. 3,5 Trichophyton japonicum, responsible for most rabbit and guinea pig dermatophyte infections in Japan, also occurs in Europe and with low prevalence. 3 Trichophyton concentricum is the lone anthropophilic species of the entire complex and is distributed in Oceania, Southeast Asia, and Central and South America. 3 Trichophyton persicum was identified in several human cases and a cat, mainly in southern and western Iran. Trichophyton spiraliforme is known only from a case in a Czech person who probably contracted the infection from a dog. 9 The lesions in the guinea pigs described herein (alopecia with scaling and crusting, located principally on the head) correspond to what has been reported previously. 3 The presentation in chinchillas was very similar to that of the guinea pigs. The two dogs showed highly inflammatory forms that recall those reported during infections due to "sylvatic" dermatophytes, such as T. erinacei, T. mentagrophytes and Nannizia (formerly Microsporum 2 ) persicolor. 1 Such diseases derive from digging activity in soils contaminated by fungal elements or direct contact with wildlife. 1 Lesions occur on the muzzle and extremities, as in the cases presented here. Contact with guinea pigs (the primary host of T. europaeum 3 ) was not recorded in the case histories for the dogs in our report, yet it cannot be ruled out, especially for Case 3 that was free to roam in a rural context. Other sources of infection may have been foxes and rabbits. 3,11,12 Concerning direct microscopic examination results, the fungal elements were abundant and easily visualised in samples acquired from the guinea pigs and chinchillas. The sticky tape technique represented a practical and straightforward way of sampling scales and hairs. This technique makes it possible to stain the sample, which improves the visualisation of hyphae and arthroconidia (Figure 3b). For samples that include Yellowish white, pale orange reverse (Figure 4c,d) abundant keratinous material (as in Case 1), use of a clearing agent (NaOH) allows for easier interpretation (Figure 3a). A direct diagnosis was more challenging in Case 3 (a dog) owing to the absence of arthroconidia and the position of the hyphae, which were hardly visible within the hair shaft ( Figure 3d). All fungal isolates grew easily in culture, confirming what is known for these species. 3 A detailed mycological description of the T. benhamiae clade can be found elsewhere. 3,9 To summarise, the more widely distributed zoophilic species (T. benhamiae var. benhamiae, T. europaeum and T. japonicum) have a "T. mentagrophytes-like" appearance ( Figure 4). Colonies are white, velvety, powdery to granular, with a brown, orange or brown/red reverse pigment. Microscopically they show sparse to abundant pyriform microconidia, and usually also cigar-shaped macroconidia and spiral hyphae, especially in older colonies. Trichophyton benhamiae var. luteum has a yellow pigmentation of the colony reverse. It typically exhibits poor sporulation and slower growth compared with the other species. It produces pyriform microconidia and no macroconidia, and spiral hyphae. Molecular differentiation of the two varieties of T. benhamiae can be achieved by the use of microsatellite markers. 3 Notably, the yellow pigmentation may make it challenging to differentiate T. benhamiae var. luteum from Microsporum canis, also because the latter strains sometimes do not produce conidia in culture. 3 A further complication in distinguishing between these species is that the morphological traits may vary as a function of culture conditions. For example, yellow pigmentation is not always evident in primary cultures (Figure 4c,d). Therefore, definitive identification is possible only through molecular analyses. 3 Despite the zoonotic potential of these pathogens, human involvement was not reported in our cases. One reason may be the clear evidence of lesions ( Figure 1) that discouraged contact with the animals. Moreover, the guinea pigs and the chinchillas were confined to cages most of the time, while the dogs were housed outdoors. Although further studies are necessary for confirmation, dogs most likely represent "accidental" hosts for these fungi, which may be another explanation for the lack of human contagion.

C ON FLI C T OF I NTE R EST
None declared.