Mycopathologia (2013) 176:119–122 DOI 10.1007/s11046-013-9653-0

First Case of Tritirachium oryzae as Agent of Onychomycosis and Its Susceptibility to Antifungal Drugs Ali Naseri • Abdolmajid Fata • Mohammad Javad Najafzadeh

Received: 13 December 2012 / Accepted: 10 April 2013 / Published online: 17 April 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract The first case of Tritirachium oryzae isolated from an Iranian patient is reported. A 44-year-old woman with a lesion in her fingernail was examined for onychomycosis. Direct microscopic examination of the nail clippings revealed fungal filaments and inoculation of portions of the nail clippings on cultures media yielded T. oryzae after 8 days. The isolate was identified as Tritirachium spp. on the basis of gross morphological characteristics of the fungal colony and microscopic characterization of slide cultures. The diagnosis of T. oryzae was confirmed by PCR sequencing of the internal transcribed spacer domain of the rDNA gene. In vitro antifungal susceptibility test demonstrated that the fungus was susceptible to itraconazole and posaconazole. The patient was treated with oral itraconazole. A. Naseri (&) Department of Medical Mycology and Parasitology, Imam Reza Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran e-mail: [email protected]; [email protected] A. Fata Research Center for Cutaneous Leishmaniasis, Department of Parasitology and Mycology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran M. J. Najafzadeh Department of Parasitology and Mycology, Cancer Molecular Pathology Research Center, Ghaem Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

Keywords Tritirachium oryzae
Onychomycosis
Non-dermatophyte mold
Iran
Drug susceptibility test

Introduction Non-dermatophytic onychomycosis is a fungal infection of fingernails or toenails caused by molds, yeasts and occurs throughout the world [1, 2]. Prevalence of the disease ranges from 1.45 to 22 % according to the geographical area and diagnostic criteria used [3]. Non-dermatophytes fungi are filamentous fungi which are commonly found in nature as soil saprophytes and plant pathogens. It is not known whether non-dermatophyte infections occur as a primary ailment on healthy nails or exist as secondary invaders in already damaged nails by ischemia, trauma, or other diseases [4]. The major dermatophytic molds that identified as causative agents of onychomycosis are Scopulariopsiss spp., Fusarium spp., Aspergillus spp., Acremonium spp., and Scytalidium spp. [3–9]. Tritirachium is a mitosporic fungus commonly isolated from paper, jute, textiles, and adhesives. Its natural habitat is soil and decaying plant material [10]. This fungus has been reported to cause otomycosis, corneal ulcers, and scalp dermatomycosis [10–12]. We report here a case of onychomycosis due to Tritirachium oryzae. To the best of our knowledge, the fungus has not been described previously as an agent of nail infection.

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Case Report A 44-year-old woman with a history of a lesion in her right big fingernail for the past 3 months was referred to the Medical Mycology Laboratory of Imam Reza Hospital, Mashhad University of Medical Sciences. The lesion affected approximately half of the nail plate, with a subungual hyperkeratosis that caused nail color alteration to yellowish and damage to the free edge of the nail plate (Fig. 1). The patient’s history showed that she had a history of trauma to her nail since approximately 1 year ago. Subungual keratinus debris and nail clippings were collected from the deepest part of the affected nail. Direct microscopic examination was carried out using 20 % KOH preparations. Hyaline septate hyphae were detected in nail samples. Clinical materials were cultured on sabouraud glucose agar (Merck, Germany) with chloramphenicol and potato dextrose agar (Merck, Germany) and incubated in 25–27 °C aerobically. Following the appearance of colonies, they were subcultured in fresh medium to avoid any contamination. Several colonies yielded after 8 days on culture medium. The colonies were rose vinaceous to lilac, velvety on the surface, and brownish on the reverse (Fig. 2). Slide culture revealed hyphae hyaline to pale brown, smooth and thin walled. Conidiophores were thick walled, suberect, and in the upper part verticillate side branches. Conidia were hyaline, smooth, and spherical to ellipsoid (Fig. 3). The fungus was identified as Tritirachium spp. After PCR sequencing, the isolate was identified as T. oryzae. Viable cultures were deposited at the Centraalbureau voor Schimmel cultures, Urtrech, the Netherlands (CBS130019). GenBank accession numbers of the nucleotides used to confirm fungal identification were JF779663.1, JF779658.1, JF779657.1, JF779660.1, and GQ329853.1. The patient was treated with systemic itraconazole. Oral therapy consisted of itraconazole 400 mg daily for 1 week per month for a period of 4 months (pulsed therapy). Follow-up after 2 months showed that the lesion apparently improved. The patient was advised to come back to the laboratory after 2 months, but unfortunately, she did not present for final follow-up.

Fig. 1 Clinical feature of the big fingernail caused by T. oryzae

Fig. 2 Colony of T. oryzae on sabouraud glucose agar after 8 days

Antifungal Susceptibility Test The in vitro antifungal drug susceptibility to the isolate (CBS 130019) was investigated by the microdilution

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Fig. 3 Conidia and conidiophores of T. oryzae

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method. The broth microdilution method was performed according to CLSI document M38-A2 guidelines. Minimal inhibitory concentrations (MICs) and minimum effective concentrations (MECs) for the clinical isolate of the unknown species toward seven antifungal agents were determined. Methods for sporulation and preparation of suspensions were according to Najafzadeh et al. [13]. Amphotericin B (Bristol-Myers-Squib, Woerden, the Netherlands), fluconazole (Pfizer Central Research, Sandwich, UK), itraconazole (Janssen Research Foundation, Beerse, Belgium), voriconazole (Pfizer), posaconazole (Schering-Plough, Kenilworth, USA), caspofungin (Merck Sharp & Dohme, Haarlem, the Netherlands), micafungin (Astellas Pharma), and terbinafin (Novartis Pharmaceuticals) were obtained as reagent-grade powders from their respective manufacturers. Antifungal agents were dissolved as prescribed by the CLSI. Antimycotics were diluted in RPMI 1640 medium (GIBCO BRL, Life Technologies, Woerden, the Netherlands) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) (SigmaAldrich Chemie GmbH, Steinheim, Germany) and dispensed into 96-well microdilution trays at the following concentration ranges: amphotericin B, itraconazole, voriconazole, posaconazole: 0.016–16 lg/ml; terbinafin 0.004–4 lg/ml; fluconazole: 0.063– 64 lg/ml; caspofungin; and micafungin: 0.008–8 lg/ml. Microdilution trays were stored at -80 °C prior to use. Conidial suspensions in physiological saline containing Tween 20 (0.05 %) were adjusted spectrophotometrically (530 nm) to optical densities in the range between 0.15 and 0.17 and then diluted 1:25 in buffered RPMI 1640 medium; final inoculum suspensions contained 0.5 9 104–4 9 104 CFU/ml, as verified by colony counts on sabouraud glucose agar. Microtiter plates inoculated with the fungus under study were incubated at 25 °C. After 96–192 h, the plates were examined visually and MICs and MECs determined. Candida parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), and Paecilomyces variotii (ATCC 22319) were used for quality controls. The MICs of five of the seven antifungal drugs used in this study were amphotericin B ([16 lg/ml), fluconazole (16 lg/ml), itraconazole (0.5 lg/ml), voriconazole (2 lg/ml), and posaconazole (0.5 lg/ml). MECs for two echinocandin agents, caspofungin and micafungin, were [1 lg/ml.

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Molecular Identification DNA extraction and quality tests were performed using glass beads (Sigma G9143) according to protocols described previously by Najafzadeh et al. [14]. rDNA ITS were chosen for species identification. ITS amplicons were generated with primers V9G and LS266 and were sequenced with primers ITS1 and ITS4 [15, 16]. PCR amplification and sequencing were conducted as previously described [17]. rDNA internal transcribed spacer (ITS) sequences were compared with GenBank and aligned with voucher strains maintained at CBS. Based on DNA sequencing, the etiologic agent was identified as T. oryzae. The sequence data for the isolate were deposited in GenBank with accession number KC 283187.

Discussion Non-dermatophyte onychomycoses are caused by a heterogeneous group of filamentous fungi and yeast. Most of these have been found in dystrophic nails [18]. Non-dermatophytic molds are an increasing cause of onychomycosis. The etiologic agents of molds onychomycosis vary according to the geographical region and the diagnostic methods used [3, 8]. Several authors have reported onychomycosis due to nondermatophytic fungi in Iran [2, 19, 20]. On the basis of the studies of Chadeganipour et al. [21], Zarei Mahmoudabadi and Zarrin [2], Hashemi et al. [22], Bassiri-Jahromi and Khaksar [20], and Aghamirian and Ghiasian [23], among the non-dermatophytic molds, Aspergillus spp. was the most causative agent of onychomycosis in Iran, whereas Khosravi and Mansouri [19] reported Scopulariopsis brevicaulis as the most common etiologic agent of onychomycosis in Tehran (Iran). Other species described as agents of onychomycosis in Iran are Fusarium and Acremonium [20]. T. oryzae is an airborne fungus [24] and has been isolated from insulation materials, wood, and ceiling tiles [10]; however, there are few reports of infections with this fungus. From a clinical point of view, there is no general agreement about differences in the manifestations of onychomycosis caused by dermatophytes and non-dermatophytes [25]. As described in different reports, the most frequent clinical presentation has been distal subungual onychomycosis [3, 20, 25]. In

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our study, the patient had also this clinical feature. Therefore, it is important to identify the etiologic agent by accurate mycological examination. The analysis of the associated predisposing factors is important, since traditionally molds were considered exclusively as contaminating fungi or secondary pathogens that affected only the nails that had a history of trauma or disease [3]. We were unable to exactly correlate the disease with patient’s previous history, but the patient’s history showed that she had a history of trauma to her nail since approximately 1 year ago; therefore, nail trauma has been probably the predisposing factor for disease and we assumed that there is a possibility of contact with the fungus some months prior to the disease presenting. In other studies, no antifungal susceptibility test was done for this fungus, but the isolate was susceptible in vitro to itraconazole and posaconazole. Although few cases of other infections due to T. oryzae have been reported [10–12], there have been no records of this fungus as the etiologic agent of onychomycosis. In conclusion, T. oryzae is reported for the first time as the cause of onychomycosis. Acknowledgments The authors would like to thank the personnel of the Medical Mycology and parasitology Laboratory of Imam Reza Hospital, Mashhad University of Medical Sciences for their help and reviewer for review of the manuscript.

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