Medical Mycology April 2012, 50, 305–309
Trichophyton bullosum: a new zoonotic dermatophyte species
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EMILIE SITTERLE*, EMILIE FREALLE†, FRANÇOISE FOULET*, ODILE CABARET*§, GENEVIEVE CREMER*, JACQUES GUILLOT‡§, LAURENCE DELHAES† & FRANÇOISE BOTTEREL*§ *Laboratoire de Parasitologie-Mycologie, Groupe hospitalier Chenevier-Mondor, (AP-HP), †Laboratoire de Parasitologie-Mycologie, CHRU de Lille et Faculté de Médecine de Lille – BDEEP, Center for Infection and Immunity of Lille, Institut Pasteur de Lille - Inserm U1019 -CNRS UMR 8402 - Univ Lille Nord de France, Lille, ‡Service de Parasitologie-Mycologie, Ecole Nationale Vétérinaire d’Alfort, Maisons-Alfort, and §UMR BIPAR, UPEC, ANSES, ENVA, France
We report the first human case of dermatophytosis caused by Trichophyton bullosum in a 21-year-old male who had a skin lesion located on his forearm. The dermatophyte was isolated in culture and further identified by sequence analysis of internal transcripted spacer regions. The species T. bullosum is a zoophilic dermatophyte rarely isolated from the coat of horses in Africa and Asia. In the present case, it was probably transmitted by contact with an infected donkey in a rural area in France. Antifungal therapy led to remission of the lesion in the patient after 2 months of treatment. T. bullosum ITS region sequences were closely related to those of the African species of Arthroderma benhamiae and grouped in a zoophilic cluster with Trichophyton verrucosum, T. erinacei and the Trichophyton anamorph of A. benhamiae (zoophilic species of the T. mentagrophytes complex). Systematic molecular identification could contribute to an accurate identification of this unusual species. Keywords dermatophytosis, Trichophyton bullosum, zoonosis, ITS
Introduction Dermatophytosis remains one of the most common infectious diseases in the world. It can be caused by several dermatophyte species that may be acquired from humans (anthropophilic species), animals (zoophilic species) or soil (geophilic species). Identification of dermatophytes is time consuming and requires expertise due to their morphological similarity, variability, and phenotypic polymorphism. Recently, molecular tools have been described for the identification of dermatophytes that were sometimes misidentified with traditional methods [1]. Trichophyton bullosum is a zoophilic dermatophyte that was described for the first time in 1933 by Lebasque [2]. Received 12 April 2011; Received in final revised form 1 July 2011; Accepted 13 July 2011 Correspondence: Françoise Botterel, Laboratoire de ParasitologieMycologie, Hôpital Henri Mondor, 51 avenue du Maréchal De Lattre de Tassigny, 94010 Créteil, France. Tel: ⫹33 1 49 81 28 91. Fax: ⫹33 1 49 81 36 01. E-mail:
[email protected]
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It has been isolated from cutaneous lesions of horses in Tunisia, Sudan and Syria. Here, we report the first human case of dermatophytosis due to T. bullosum which was seen in a 21-year-old man who had direct contact with donkeys. Identification was accomplished through the use of molecular tools in which the internal transcribed spacer (ITS) region of the fungus was amplified with universal primers and PCR products were identified by sequence analysis.
Clinical case A 21-year-old male presented in dermatology clinics with a lesion on his left forearm that had been rapidly expanding for 2 weeks. Clinical examination revealed the presence of one erythemato-squamous lesion (about 4 cm in diameter), with a poorly circumscribed border. The lesion was pruritic but non inflammatory. The results of all other clinical studies were normal. Direct microscopic examination of the scrapings from the lesion revealed the presence of fungal septate hyphae. DOI: 10.3109/13693786.2011.605810
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Treatment was started with local antifungal therapy (ketoconazole cream 2%), followed by oral antifungal therapy with griseofulvin (1,000 mg per day) which led to the complete remission of the lesion after 2 months of therapy. Approximately 2 weeks prior to the occurrence of the cutaneous lesion, the patient, who had no pets at home, reported riding donkeys with suspected dermatophyte lesions on a farm for 1 week. Unfortunately, no samples could be collected from these animals.
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Laboratory methods Cultures were grown on Sabouraud dextrose and Malt agars at 27°C for 3 weeks. The colonies were small, glabrous, regularly dome-shaped, and of cream colour (Fig. 1A). With age, colonies folded in the center and developed a tough leathery consistency with a cerebriform appearance around a circular edge with extension into the agar. No pigmentation was observed on the reverse. Microscopic examination revealed the presence of thick, irregularly fragmented and often wavy hyphae, with many intercalar or terminal chlamydospores (Fig. 1B and 1C).
Molecular identification was performed by DNA sequencing. Briefly, 1 ml of ATL Tissue Lysis Buffer (Qiagen, Courtaboeuf, France) was added to the culture tube and vortexed for 30 s. The suspension was transferred into a ceramic MagNa Lyser Green beads tube (Roche Diagnostics, Meylan, France). The suspension was homogenized twice with the MagNa Lyser Instrument (Roche Diagnostics) for 30 s at 5,000 rpm, and then centrifuged for 3 min at 3,000 rpm. Nucleic acids were extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions. Fungal ITS PCR amplification targeted a DNA sequence fragment that spanned the 3′ end of the 18S ribosomal RNA (rRNA) gene, the 5.8S rRNA gene, and the 5′ end of the 28S rRNA gene, including the ITS 1 and 2. The PCR was performed with primers ITS1 (5′TCCGT AGGTGAACCTGCGG3′) and ITS4 (5′TCCTCCGCTTAT TGATATGC3′) [3]. The amplification reaction mixture consisted of 1 ⫻ Fast Start Taq PCR Buffer, 2 mM MgCl2, 0.2 mM each deoxyribonucleotide triphosphate, 0.2 μM ITS1 primer, 0.2 μM ITS4 primer, 0.025 UI/μl of Fast Start Taq DNA polymerase (Roche Diagnostics), and 5 μl of
Fig. 1 (A) Trichophyton bullosum: colonies are glabrous, heaped or button-like, dome-shaped, cream-yellow coloured. (B) Hyphes of Trichophyton bullosum with many intercalar or terminal chlamydospores (magnification ⫻100). (C) Chains of Trichophyton bullosum chlamydospores (magnification ⫻400).
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Trichophyton bullosum, the first human case
revealed a 100% identity with ITS region sequences of T. bullosum strains CBS 363.35 and CBS 557.50 (GenBank accession number FM992675 and FM992676, respectively) [4]. The strain was deposited at the BCCM/ IHEM and CBS collections (number BCCM/IHEM 34321 and was identified by CBS). The ITS region sequence determined in this study was submitted to the GenBank nucleotide sequence database under accession number JF308286, as indicated in Fig. 2.
Phylogenetic analysis Twenty-three other ITS region sequences of T. equinum, T. tonsurans, T. interdigitale, T. simii, T. mentagrophytes, T. erinacei, Trichophyton of the Arthroderma benhamiae
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extracted DNA. After initial denaturation at 95°C for 7 min, samples were amplified for 35 cycles with denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. PCR products were verified by 4% polyacrylamide gel electrophoresis and purified with High Pure PCR Product Purification kit (Roche Diagnostics). PCR products were then sequenced using DiDeoxy Terminator cycle sequencing kit v1.1 protocol (Applied Biosystems, Courtaboeuf, France) on an ABI PRISM 310 genetic analyser (Applied Biosystems). For molecular identification at species level, the whole sequence (including the 5.8S region) was analyzed using the Basic Local Alignment Search Tool at the NCBI web site (http://blast.ncbi.nlm.nih.gov/Blast.cgi). This analysis
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Fig. 2 Rooted neighbor-joining (NJ) tree inferred from ITS sequences of Trichophyton spp. and Arthroderma benhamiae. ITS sequence accession numbers are given after each strain number. Bootstrap NJ values are given as percentages next to the individual nodes; values of ⬍50 % are shown as *. The evolutionary distances between organisms are indicated by the horizontal branch lengths (scale bar: 0.005 substitutions per base pair), which reflect the number of nucleotide substitutions per site along the branches from node to end point. Ab: A. benhamiae, As: A. simii, Tb: T. bullosum, Te, T. erinacei; Teq, T. equinum; Ti, T. interdigitale; Tm, T. mentagrophytes; Tr, T. rubrum; Tt, T. tonsurans; Tv, T. verrucosum.
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complex, T. verrucosum and T. rubrum were retrieved from GenBank and aligned with the ITS region sequences of T. bullosum using CLUSTALW as contained in the BioEdit version 7.0.0 software [5]. Accession numbers of the sequences included in the dataset are listed in Fig. 2. The ITS region phylogenetic tree was constructed with unambiguously aligned sequences (490 sites) using the neighbor-joining (NJ) method with Kimura-2 parameter as substitution model and maximum parsimony (MP) analysis, in the MEGA program [6]. The reliability of internal branches was assessed using the bootstrap method in MEGA, with 1000 (NJ) and 100 (MP) replicates. T. bullosum ITS region sequences were closely related to the African race of A. benhamiae (strain RV 30000) sequence, and grouped in a zoophilic cluster (Clade II, Fig. 2), with T. verrucosum, T. erinacei and the Trichophyton anamorph of A. benhamiae (zoophilic species of the T. mentagrophytes complex) sequences. They were more distantly related to the other species of the T. mentagrophytes complex (the zoophilic T. mentagrophytes sensu stricto, phylogenetically related to the teleomorph species A. simi, and the zoophilic and anthropophilic T. interdigitale sequences). These sequences clustered in Clade I, with the anthropophilic T. tonsurans and the horse specific T. equinum sequences (Fig. 2). T. bullosum ITS region sequences exhibited a 96.8% identity with the African A. benhamiae sequence and a 95.7% identity with T. verrucosum sequences. In the ITS1 regions, which was the most polymorphic one, the African A. benhamiae and the T. verrucosum sequences were characterized by 10 (7 substitutions, 2 insertions, 1 deletion), and 12 (9 substitutions, 3 deletions) mutations, respectively. In the 5.8S, both sequences had substitutions at positions 314 and 319. In the ITS2 region, 4 substitutions and 7 mutations (with 6 substitutions, 1 deletion) were found, respectively.
Discussion In the present report, we described the first case of T. bullosum infection in a human. The species is a zoophilic dermatophyte, which was isolated for the first time in 1933 during a survey of the occurrence of equine dermatophytosis in different countries [1]. Seven strains were collected from the coats of horses in Tunisia, Sudan and Syria. The cutaneous lesions were located on the withers and breast. Hairs were slightly erected at the beginning of the infection with subsequent small alopecic lesions. Confluence of primary lesions in large plaques sometimes occurred. The dermatophyte T. bullosum grows very slowly on Malt and Sabouraud agars at 27°C. After 4 weeks, colonies appear waxy or glabrous, heaped or button-like, domeshaped, cream-yellow coloured, and similar to an air bubble
in a viscous liquid (hence, the name T. bullosum). The colony dome rises directly from the agar and beneath the dome, protruding into the agar, appears a root-like network of hyphae. The reverse is colorless. Microscopically, there are chains of intercalary and terminal chlamydospores. According to the first description by Lebasque in 1933, the KOH mount of infected hairs showed endo-ectothrix invasion by T. bullosum. Specimens could be inoculated onto specialized isolation media, like barley and wheat grain media at 26–28°C for 4 weeks. On these media, Lebasque described the presence of aleuria and rat tail-shaped macroconidia with a thin wall, which ranged in size from 35–80 μm by 4–10μm. Dermatophytosis due to zoophilic or geophilic dermatophytes is often very inflammatory and the duration of treatment is usually longer than infections caused by anthropophilic species. This was not the case in our patient where the lesion was not inflammatory. For initial therapy, a local antifungal treatment is recommended, to be complemented with an oral treatment in case of tinea capitis and/or in cases of persistence of pruritus or marked inflammatory lesions. For our patient, the unusually long delay in healing may be related to the high amount of hair on the patient’s forearms. The modes of transmission of T. bullosum from animal to human and/ or among humans have not been specifically investigated [7–9]. In horses, we imagine that transmission occurs similarly to that of Trichophyton equinum (the main causative agent of dermatophytosis in horses) [10], i.e., the infection may be readily transmitted from horse to horse by infected saddle girths on which arthroconidia can survive for months. Phenotypically, T. bullosum may be misidentified as T. verrucosum. However, the preferential hosts are different in that T. bullosum is usually isolated from horses [2] whereas T. verrucosum is clearly associated with ruminants, especially cattle. Moreover, T. bullosum is responsible for non-inflammatory cutaneous lesions, whereas T. verrucosum usually produces a highly inflammatory disease. In some European countries, the incidence of human T. verrucosum infections has declined dramatically over the past few decades. This is thought to be due to the high uptake rate of the T. verrucosum vaccine in cattle, in connection with other hygienic and preventive measures [11]. The incidence of T. bullosum infections in donkeys or horses is unknown and it would be of interest to conduct epidemiological surveys to determine the relative importance of the different causative agents of equine dermatophytosis in France. Since sequence-based methods are more accurate and reliable for the identification of some dermatophytes than microscopic or macroscopic examination [12], molecular analysis (such as ITS sequencing) should be part of the diagnosis for dermatophytosis, especially in © 2012 ISHAM, Medical Mycology, 50, 305–309
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Trichophyton bullosum, the first human case
reference laboratories with pre-existing molecular facilities, which would improve our knowledge of dermatophyte epidemiology [13]. As previously described [1,14,15], the nucleotide sequences of ITS regions, in particular the ITS1 region, appear to be the most discriminating tool for the identification of dermatophytes at the species level exhibiting more than 10% for ITS region polymorphism within genus/clade. Our phylogenetic analysis based on ITS region of several dermatophyte strains confirmed the close relationship between T. bullosum and the African species of A. benhamiae as recently described [1]. Although these two species have distinct host animals (horses vs hedgehogs) this close relationship could be consistent with their common restricted geographic localization in Central Africa (Sudan and Kenya) and Asia (Syria and Japan). Mating experiments between the known T. bullosum and American-European or African A. benhamiae tester strains should be performed to assess their teleomorphic status and determine if T. bullosum could be another A. benhamiae anamorph [16]. In conclusion, all skin lesions in patients with direct or indirect contact with horses or donkeys should be processed for mycological examination. Ideally, a similar examination should be performed on the animals. Fungal identification by sequencing the ITS region can lead to faster and more reliable diagnosis than with conventional tests. Moreover this molecular method is becoming essential for correct identification of the dermatophyte species, especially to determine the correct therapeutic management, identify the source of infection, and provide a better knowledge of the epidemiology of unusual dermatophyte species. Regarding T. bullosum, a systematic multicenter mycological study including molecular analysis should be recommended in donkeys and horses for further analysis of this fungus, in particular to evaluate its true prevalence among the other dermatophytes.
Acknowledgements The authors would like to thank Franziska A. Stressmann for her careful review and useful advice for writing this manuscript and Damian Rivett for his English assistance. This paper was first published online on Early Online on 9 September 2011.
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Declaration of interest: The authors have no conflicts of interest. The authors are responsible for the content and writing of the paper.
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