Medical Mycology, March 2010, 48, 318–327 The clinical spectrum of Exophiala jeanselmei, with a case report and in vitro antifungal susceptibility of the species Exophiala jeanselmei is clinically redefined as a rare agent of subcutaneous lesions of traumatic origin, eventually causing eumycetoma. Mycetoma is a localized, chronic, suppurative subcutaneous infection of tissue and contiguous bone after a traumatic inoculation of the causative organism. In advanced stages of the infection, one finds tumefaction, abscess formation and draining sinuses. The species has been described as being common in the environment, but molecular methods have only confirmed its occurrence in clinical samples. Current diagnostics of E. jeanselmei is based on sequence data of the Internal Transcribed Spacer (ITS) region of ribosomal DNA (rDNA), which sufficiently reflects the taxonomy of this group. The first purpose of this study was the re-identification of all clinical (n ⫽ 11) and environmental strains (n ⫽ 6) maintained under the name E. jeanselmei, and to establish clinical preference of the species in its restricted sense. Given the high incidence of eumycetoma in endemic areas, the second goal of this investigation was the evaluation of in vitro susceptibility of E. jeanselmei to eight conventional and new generations of antifungal drugs to improve antifungal therapy in patients. As an example, we describe a case of black grain mycetoma in a 43-year-old Thai male with several draining sinuses involving the left foot. The disease required extensive surgical excision coupled with intense antifungal chemotherapy to achieve cure. In vitro studies demonstrated that posaconazole and itraconazole had the highest antifungal activity against E. jeanselmei and E. oligosperma for which high MICs were found for caspofungin. However, their clinical effectiveness in the treatment of Exophiala infections remains to be determined. Keywords Exophiala jeanselmei, black yeasts, ITS rDNA, mycetoma, antifungal susceptibility testing Introduction Mycetoma is defined as a localized, chronic, granulomatous, suppurative and progressive inflammatory disease of subcutaneous tissue and contiguous bone after a traumatic inoculation of the causative organism [1]. Etiologic agents Received 9 March 2009; Received in final revised form 6 May 2009; Accepted 28 June 2009 Correspondence: G.S. de Hoog, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands. Tel: +31 30 2122 663; E-mail: de.hoog@cbs.knaw.nl  2010 ISHAM are recovered from humans as well as domestic animals [2]. In advanced stages of the infection tumefaction, abscess formation and draining sinuses arise. The hallmark of a mycetoma is the presence of the fungus in the form of grains. In Africa the infection is particularly common from Sudan to Senegal, an area known as the ‘mycetoma belt’. The main etiologic agents in that area are Madurella mycetomatis and Leptosphaeria senegalensis. Other recurrent fungal agents are Madurella grisea and several species of coelomycetes. DOI: 10.3109/13693780903148353 Downloaded from https://academic.oup.com/mmy/article/48/2/318/1014535 by guest on 02 September 2021 H. BADALI*†‡, M. J. NAJAFZADEH*†, M. VAN ESBROECK§, E. VAN DEN ENDEN§, B. TARAZOOIE*, J. F. G. M. MEIS^ & G. S. DE HOOG*† *CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, †Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam,The Netherlands, ‡Department of Medical Mycology and Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran, §Institute of Tropical Medicine, Antwerp, Belgium, and ^Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen,The Netherlands The clinical spectrum of Exophiala jeanselmei to the species level cannot be performed by molecular methods. It is possible that the etiological agent may often have been misdiagnosed. In this article, a case of eumycetoma caused by E. jeanselmei matching the original clinical concept of the species is presented. Current diagnostics of E. jeanselmei is achieved through the use of sequence data of the Internal Transcribed Spacer (ITS) region of ribosomal DNA (rDNA), which reflects the taxonomy of this group [14]. Therefore, the first purpose of this study was the re-identification of all clinical (n ⫽ 11) and environmental strains (n ⫽ 6) preserved under the name E. jeanselmei in the collection of Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, and to establish the species clinical manifestations. Given the high incidence of eumycetoma in endemic areas, the second goal of this study was the evaluation of the in vitro susceptibility of E. jeanselmei to eight conventional and new generations of antifungal drugs to improve the antifungal therapy in patients. New antifungal agents with a better activity may help to improve the management of eumycetoma as sufficient knowledge is gained as to the in vitro activity of new antifungal agents. Materials and methods Fungal strains Strains used in this study were obtained from the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (Table 1), deposited and phenotypically identified E. jeanselmei. Stock cultures were maintained on slants of Table 1 Summary of source and identification of strains tested. CBS number Other collection CBS 122239 CBS 119095 CBS 109635 CBS 148.97 CBS 507.90 CBS 116.86 CBS 677.76 CBS 664.76 CBS 528.76 CBS 814.95 CBS 634.69 CBS 538.76 CBS 537.76 CBS 642.82 CBS 527.76 dH 18920 dH 13453 dH 12305 dH 10808 dH 15933 (T) dH 15309 dH 16163 ATCC 34123 ATCC10224 dH 16266 dH 16108 dH 15984 dH 15981 dH 16117 dH 15966 CBS 102241 CBS 663.76 dH 11605 dH 16149 Source Man, mycetoma Man, foot skin Man, arm lesion Man, subcutaneous cyst Man, mycetoma Man, chromomycosis Man, mycetoma Man, skin infection Man, hand skin lesion Soil biofilter Wood, ship resting Man, bronchus Man, eye infection Soft rot of preservative-treated wood Culture contaminant of Hyphodontia breviseta Soil under coffee plant Wood Country Reference Final name Thailand USA, Dallas USA, San Antonio Japan, Ibaraki France Japan Pakistan None None Netherlands, Delft Baltic Sea None Italy Australia, Lucia Sweden, Bohuslan Current paper None [13] [22] [20] [10] [21] [23] None [13] None None [24] None None Exophiala jeanselmei Exophiala jeanselmei Exophiala jeanselmei Exophiala jeanselmei Exophiala jeanselmei Exophiala jeanselmei Exophiala jeanselmei Exophiala jeanselmei Exophiala jeanselmei Exophiala oligosperma Exophiala oligosperma Exophiala oligosperma Exophiala oligosperma Exophiala oligosperma Exophiala xenobiotica Brazil, Paraná None None [23] Exophiala bergeri Capronia pilosella Abbrevations used: ATCC ⫽ American Type Culture Collection, Manassas, U.S.A.; CBS ⫽ Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; DH ⫽ G.S. de Hoog working collection; IFM ⫽ Research Institute for Pathogenic Fungi, Chiba, Japan; IHM ⫽ Laboratory of Mycology, Faculty of Medicine, Montevideo Institute of Epidemiology and Hygiene, Montevideo, Uruguay; BMU ⫽ Beijing Medical University. T ⫽ ex-type culture.  2010 ISHAM, Medical Mycology, 48, 318–327 Downloaded from https://academic.oup.com/mmy/article/48/2/318/1014535 by guest on 02 September 2021 Classically, the black yeast Exophiala jeanselmei has been reported to be involved in subcutaneous lesions of traumatic origin, eventually causing eumycetoma [3,4]. Members of the ascomycete order Chaetothyriales, are remarkably frequently encountered and reported as agents of disease. Many of these species are oligotrophic [5] and are therefore found in uncommon habitats. This property also seems to be a predisposing factor for human infection, since a large number of species are found as etiologic agents of disease. Several members of Chaetothyriales have been reported involved in eumycotic mycetoma in humans and animals. Werlinger et al. [6] and Bonifaz et al. [7] described cases of mycetoma caused by Cladophialophora bantiana. Guillot et al. [8] reported on a case in a dog caused by the same species. Cladophialophora mycetomatis is a novel species recently described by Badali et al. [9] as an agent of eumycetoma in Mexico. Black yeasts identified as E. jeanselmei have also been reported from many other types of infection, such as chromoblastomycosis or mild cutaneous disease [10], disseminated infections, endocarditis and arthritis [11]. However, recent molecular studies have shown that the species is very heterogeneous. The original morphological varieties have now been raised to species level [12], and additional, morphologically nearly indistinguishable species have been described [13]. Hence, the clinical predilection of E. jeanselmei has to be re-evaluated on the basis of correctly identified material. With obsolete mycological procedures, identification of black yeasts remains difficult [3]. Since the original isolates no longer are available for most cases published in older literature, their identification down 319 320 Badali et al. 2% Malt Extract Agar (MEA; Difco) and Oatmeal Agar (OA; Difco) and incubated at 24°C for two weeks [15]. The identification of 17 strains was verified with sequence data of the internal transcriber spacer regions (ITS) of the rDNA. An isolate from a recent case of mycetoma was added (CBS 122339). Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands. If the similarity of sequences of the ITS region was more than 99% between a studied strain and its nearest neighbor, the strain concerned was considered to be the same species as its nearest neighbor. In vitro susceptibility DNA extraction and sequencing Alignment and phylogenetic reconstruction Sequence data obtained in this study were adjusted using the SeqMan of Lasergene software (DNAStar Inc., Madison, Wisconsin, USA). ITS sequences were aligned manually using BioNumerics version 4.61 (Applied Maths, Kortrijk, Belgium). The program RAxML-VI-HPC v.7.0.0 [17], as implemented on the Cipres portal v.1.10, was used for the tree search and the bootstrap analysis (GTRMIX model of molecular evolution and 500 bootstrap replicates). Bootstrap values equal or greater than 70% were considered significant [18]. Newly generated sequences were subjected to a BLAST search of the NCBI databases, sequences with high similarity (ⱖ98%) were downloaded from GenBank and sequences were compared based on the alignment using a black yeast molecular database maintained at the MICs (minimum inhibitory concentration) and MECs (minimum effective concentration, echinocandins only) were determined as described in the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) document M38-A2 [19]. The eight antifungal agents evaluated: amphotericin B (AMB, Bristol-Myers Squib, Woerden, The Netherlands); fluconazole (FLU, Pfizer Central Research Sandwich, U.K.); itraconazole (ITC, Janssen Research Foundation, Beerse, Belgium); voriconazole (VOR, Pfizer); posaconazole (POS, Schering-Plough, Kenilworth, USA); isavuconazole (ISA, Basilea, Basel, Switzerland); caspofungin (CAS, Merck Sharp & Dohme, Haarlem, The Netherlands) and anidulafungin (ANI, Pfizer) were provided by the manufacturers as reagent-grade powders. As per the CLSI guidelines, stock solutions of the drugs were prepared in the appropriate solvent [19]. The drugs were diluted in the standard RPMI1640 medium (Sigma Chemical Co.) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma) with L-glutamine without bicarbonate to yield two times their concentrations were as follows: amphotericin B, itraconazole, voriconazole, posaconazole and caspofungin 0.016–16 μg/ml; fluconazole 0.063–64 μg/ml; isavuconazole 0.002–2 μg/ml and anidulafungin 0.008–8 μg/ml. Plates were stored at −70°C until they were used. Briefly, inoculum suspensions were prepared from 7 to 14 days potato dextrose agar (PDA; Difco) cultures by adding sterile saline solution with Tween 40 (0.05%) and slightly scarping the surface of mature colonies with sterile cotton swab. If large aggregates existed, they were allowed to settle for several minutes, the homogenous conidial suspensions were then transferred to sterile tubes and the supernatants were performed spectrophotometrically at 530 nm wavelength to optical density (OD) that ranged from 0.17–0.15 (68 to 71 T%). Therefore, the final size of the stock inoculum suspensions of the isolates tested ranged from 0.4 ⫻ 104–3.1 ⫻ 104 CFU/ml as performed by quantitative colony count on sabouraud glucose agar (SGA; Difco) to determine the viable number of colony forming units per milliliter [19]. The inoculum suspensions which consisted primarily of non-germinated conidia were diluted 1:50 in RPMI 1640 medium. Microdilultion plates were incubated at 35°C and examined optically and spectrophotometrically at 420 nm after 72 h (if insufficient growth was found, plates were incubated longer, until 96 h) for MICs and MECs determinations. Paecilomyces variotii (ATCC  2010 ISHAM, Medical Mycology, 48, 318–327 Downloaded from https://academic.oup.com/mmy/article/48/2/318/1014535 by guest on 02 September 2021 Mycelia were grown on 2% MEA plates for 2 weeks at 24°C [15] and a sterile blade was used to scrape off the mycelium from the surface of the plate. DNA was extracted using an Ultra Clean Microbial DNA Isolation Kit (Mobio, Carlsbad, CA 92010, USA) according to the manufacturer’s instructions. DNA extracts were stored at −20°C until used [9]. Internal Transcribed Spacers rDNA (ITS) were amplified using primers V9G and LS266 and sequenced with the internal primers ITS1 and ITS4 [16]. PCR reactions were performed on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, USA) in 50 μl volumes containing 25 ng of template DNA, 5 μl reaction buffer (0.1 M TrisHCl, pH 8.0, 0.5 M KCl, 15 mM MgCl2, 0.1% gelatine, 1% Triton X-100), 0.2 mM of each dNTP and 2.0 U Taq DNA polymerase (ITK Diagnostics, Leiden, The Netherlands). Amplification was performed with cycles of 5 min at 94°C for primary denaturation, followed by 35 cycles at 94°C (45 s), 52°C (30 s) and 72°C (90 s), with a final 7 min extension step at 72°C. Amplicons were purified using GFX PCR DNA and gel band purification kit (GE Healthcare, Ltd., Buckinghamshire UK). Sequencing was performed as follows: 95°C for 1 min, followed by 30 cycles consisting of 95°C for 10 s, 50°C for 5 s and 60°C for 2 min. Reactions were purified with Sephadex G-50 fine (GE Healthcare BioSciences AB, Uppsala, Sweden) and sequencing was done on an ABI 3730XL automatic sequencer (Applied Biosystems, Foster City, CA, USA). The clinical spectrum of Exophiala jeanselmei Case report A 43-year-old Thai man was admitted in 2004 to the Institute of Tropical Medicine, Antwerp, Belgium, due to a complaint of a tumefaction that localized and developed on the left leg at the dorsum of the foot affecting the fourth and fifth toe (Fig. 1A,B). The patient did not remember any history of trauma or puncture at the site of the lesion. First surgical resection biopsy was performed in 2005 and initial clinical diagnosis was that of chromoblastomycosis. Histopathologically there was no evidence of presence of muriform cells. Subsequently, the patient underwent surgical excision of the medial mass with skin graft closure and no antifungal drugs were prescribed. Two years later, he was referred again to the Institute of Tropical Medicine, presenting with a relapse of the lesion which consisted of a deformed tumorous area, with nodules and multiple draining sinuses. Sinuses discharged small amounts of sanguineous fluid without visible grains. The patient limped due to discomfort at the site of infection. Radiography of the foot was normal with no visible bone destruction or osteomyelitis, and no significant regional lymphadenopathy was noted. Results of bacteriological (syphilis) and parasitological (leishmaniosis) evaluations, and serological test for human immunodeficiency virus (HIV) were all negative. Laboratory investigations including full blood count, blood chemistry and renal tests were within normal limits, whereas liver functions were slightly disturbed, i.e., SGOT 46 (normal ⬍59), SGPT 82 (normal ⬍72), GGT 166 (normal ⬍73). Therapy with oral itraconazole at 400 mg/day for 6 months yielded good clinical response. Fig. 1 (A,B) Clinical signs of mycetoma caused by Exophiala jeanselmei, deformed tumorous area of the foot, with nodules, draining sinuses with discharging and ulcers. (C,D) Section of the biopsy material stained with hematoxylin and eosin (H&E) exhibiting pigmented granules; some were sickle-shaped, and surrounded by a dense inflammatory infiltrate consisting of numerous neutrophils (H&E stain ⫻ 400).  2010 ISHAM, Medical Mycology, 48, 318–327 Downloaded from https://academic.oup.com/mmy/article/48/2/318/1014535 by guest on 02 September 2021 22319), Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) were used for quality control. The MICs endpoints for amphotericin B, itraconazole, voriconazole, posaconazole and isavuconazole were determined with the aid of a reading mirror as the lowest concentration of the drug that prevented any recognizable growth (100% inhibition) and for fluconazole as a prominent reduction of growth, i.e., ⱖ50% inhibition compared to drug-free growth control. The MEC of the echinocandins (caspofungin and anidulafungin) was defined microscopically as the lowest concentration of the drug that led to the growth of small, rounded, compact hyphal forms as compared with the long, unbranched hyphal clusters that were seen in the growth control [19]. 321 322 Badali et al. The slight liver tests abnormalities were considered no contraindication for itraconazole treatment, but the patient was requested to avoid alcoholic beverages. Additional local heat therapy (hot footbath, local heated dry pillow, heat bag and/or infrared lamp) was suggested, but it remained unclear how far this was implemented, since verbal communication was rather difficult. On January 2008, there was a clear improvement of the wound, but no complete healing. Liver tests had normalized since he stopped alcoholic drinking. Afterwards, the patient did not return for follow up. The entire biopsied tissue was used for mycological and histopathological investigations. Direct examination with KOH (10%) revealed black granules and hyphal elements but were not obviously clear. Microscopic examination of biopsy sections stained with hematoxylin and eosin (H&E) revealed pigmented granules, some sickle-shaped, and surrounded by a dense inflammatory infiltrate consisting of numerous neutrophils and to the periphery chronic inflammation with fibrosis (Fig. 1C,D). The histopathological observations led to the diagnosis of a eumycotic mycetoma caused by an Exophiala species. Clinical specimens were cultured on Sabouraud glucose agar (SGA: Difco) and SGA supplemented with chloramphenicol (0.5 μg/ml) and incubated at 27–30°C for 7 days. Growth of dematiaceous fungi was observed and these were morphologically classified as Exophiala species. Stock cultures were maintained on slants of 2% MEA and OA at 24°C [15], and a voucher strain was deposited in the CBS culture collection as CBS 122339. Microscopic studies using the Results Seventeen strains from different geographical locations maintained as Exophiala jeanselmei in the CBS culture collection were sequenced using the ITS region (542 characters) and compared with alignable members of genus Exophiala. The ex-type strains of E. jeanselmei (AY156963), E. spinifera (AY156976), E. exophialae (AY156973), E. dermatitidis (FJ974060), E. oligosperma (AY163551), E. xenobiotica (DQ182587), E. bergeri (EF551462) and E. lecanii-corni (FJ974061) were used. The species Fig. 2 (A) Culture on Malt extract agar (MEA, Difco) produced dark, moist, olivaceous-black yeast-like colony at room temperature. (B,C) Conidia clustered at the apices of the tapered annellides and long, thick-walled, septate conidiophores. Scale bar ⫽ 10 μm.  2010 ISHAM, Medical Mycology, 48, 318–327 Downloaded from https://academic.oup.com/mmy/article/48/2/318/1014535 by guest on 02 September 2021 Mycological identification slide culture techniques with PDA or OA were conducted. These media were selected because they readily induce sporulation and suppress growth of aerial hyphae [3]. After two weeks, slides were prepared from these cultures in lactic acid or lactophenol cotton blue and light micrographs were taken using Nikon Eclipse 80i microscope with a Nikon digital sight DS-Fi1 camera. Colonies were moderately expanding and initially moist (yeast-like), forming velvety, olivaceous-green aerial hyphae; colony reverse was olivaceous-black (Fig. 2A). Numerous sub-spherical to ellipsoidal budding cells were present in young cultures, giving rise to short torulose hyphae that gradually changed into un-swollen hyphae. Conidia were often cohering in long chains and converted into hyphae. Conidiogenous cells were intercalary or lateral, then rocket-shaped, brown, with inconspicuous, slightly tapering annellated zones, producing smooth, narrow, thin-walled, broadly ellipsoidal conidia 2.6–5.9 ⫻ 1.2–2.5 μm (Fig. 2B,C). Cardinal growth temperatures of strain CBS 122339 were growth between 9–37°C, optimum at 27°C, no growth was observed at 40°C. The clinical spectrum of Exophiala jeanselmei Fig. 3 Consensus tree of ITS rDNA obtained from a ML analysis using RAxML. Bootstrap support values were estimated based on 500 replicates, and are shown above the branches. The Exophiala dermatitidis Clade was taken as outgroup.  2010 ISHAM, Medical Mycology, 48, 318–327 infections were considered to belong to E. oligosperma and E. xenobiotica [24]. Table 2 summarizes the results of in vitro antifungal susceptibility testing of eight antifungal drugs against E. jeanselmei (n ⫽ 9) and E. oligosperma (n ⫽ 5) employing the methods described in the CLSI guideline (M38-A2). The MIC90 was not determined due to insufficient numbers of isolates. Amphotericin B MICs for most non-dermatophyte opportunistic filamentous fungi isolates are clustered between 0.5–2 μg/ml. However, there is very little data available regarding the correlation between MICs and outcome of treatments with AmB for filamentous fungi. Results have shown that amphotericin B Downloaded from https://academic.oup.com/mmy/article/48/2/318/1014535 by guest on 02 September 2021 E. jeanselmei was unambiguously distinguished in a robust branch with ⬎98% bootstrap support (Fig. 3). Nine out of 17 strains showed 98% identity with the E. jeanselmei ex-type strain (CBS 507.90) which had originally been isolated from a true mycetoma-like infection. Five strains were re-identified as E. oligosperma, and one strain each was confirmed as E. bergeri (CBS 102241), E. xenobiotica (CBS 527.76) and Capronia pilosella (CBS 663.76) (Table 1). Strains confirmed to be E. jeanselmei all originated from subcutaneous infections in human patients [current case,10,13,20-23], whereas strains which originated from environmental samples like soil, wood and plant or from clinical relevant eye or disseminated 323 324 Badali et al. Table 2 In vitro susceptibility of Exophiala jeanselmei and Exophiala oligosperma for eight antifungal drugs expressed in μg/ml. Source (no. of strains) and drugs MIC50 0.25–2 8–32 0.031–0.25 0.25–2 0.016–0.063 0.25– ⬎ 2 2–8 0.063–4 1 16 0.125 1 0.031 2 4 0.5 1–2 16–32 0.031–0.25 1–4 0.016–0.125 0.5–2 0.25–4 0.016–2 1 32 0.063 1 0.031 1 4 0.5 Note. MIC90 not determined due to insufficient numbers of isolate tested. MICs ranged from 0.25–2.0 and 1–2 μg/ml for E. jeanselmei and E. oligosperma, respectively. Itraconazole and posaconazole showed potent activity against all E. jeanselmei and E. oligosperma isolates. Interestingly in terms of MIC50 (0.031 μg/ml) posaconazole was more active against E. jeanselmei than itraconazole (MIC50 0.125). Filamentous fungi are usually not susceptible to fluconazole and most MICs are ⬎64 μg/ml for these isolates. In our study, we found that fluconazole had the widest range and the highest MICs against E. jeanselmei and E. oligosperma, ranging between 8–32 and 16–32 μg/ml, respectively. Voriconazole and isavuconazole had highest MICs with complete inhibition end points with MIC50 (1 μg/ml and 2 μg/ml) against E. jeanselmei and MIC50 (both 1 μg/ml) against E. oligosperma. Posaconazole demonstrated the lowest MIC50 (0.031 μg/ml) of all azoles. Concerning the echinocandin drugs caspofungin and anidulafungin, both exhibited low in vitro activity against E. jeanselmei and E. oligosperma. Exophiala jeanselmei showed higher MEC50 (4 μg/ml) with caspofungin than to anidulafungin (MEC50⫽0.5 μg/ml). Overall, in terms of MEC50 caspofungin had no activity against E. jeanselmei and E. oligosperma. There was no statistically significant difference when comparing the susceptibilities of E. jeanselmei and E. oligosperma isolates for all antifungal drugs (P⬍0.05). Discussion In our previous studies, phylogenetic analysis of nucSSU, nucLSU and RPB1 genes have shown that the ex-type strain of E. jeanselmei (CBS 507.96) is a member of the  2010 ISHAM, Medical Mycology, 48, 318–327 Downloaded from https://academic.oup.com/mmy/article/48/2/318/1014535 by guest on 02 September 2021 Exophiala jeanselmei (9) Amphotericin B Fluconazole Itraconazole Voriconazole Posaconazole Isavuconazole Caspofungin Anidulafungin Exophiala oligosperma (5) Amphotericin B Fluconazole Itraconazole Voriconazole Posaconazole Isavuconazole Caspofungin Anidulafungin MIC range order Chaetothyriales, within the family Herpotrichiellaceae [9,25]. Identification of this and related fungi with standard mycological procedures is difficult because of their high numbers of budding cells and poorly differentiated conidial structures. Nishimura et al. [26] studied the conidiogenesis of E. dermatitidis, E. jeanselmei, and E. spinifera and reported that the conidial ontogenesis of these three species is annellidic and may sometimes be difficult to differentiate. Masuda et al. [27] and Dixon et al. [28] showed that the morphological variants of E. jeanselmei distinguished earlier by de Hoog [29] are quite distinct on the molecular level and subsequently segregated into a number of individual species. Masuda et al. used DNA-DNA hybridization to classify E. jeanselmei into six groups. They suggested that DNA similarities between one group and another seem too low for these groups to comprise a single species [27]. Kawasaki et al. classified strains of E. jeanselmei into 18 types based on mitochondrial restriction profiles and believed that the organism constitutes a complex [30]. Wang et al. [31] used the DNA and amino acid types of the mitochondrial cytochrome b. They proposed that on the basis of mtDNA RFLP typing and similarities of nucleotide and amino acid sequences, that some E. jeanselmei strains should be re-identified. Moreover, Haase et al. suggested E. jeanselmei var. lecanii-cornii should be a distinct species, Exophiala lecanii-corni, based on investigation of ribosomal DNA [32]. In the present study, considerable sequence differences were found in nine out of 17 strains in the CBS culture collection. The isolate from the present case report exhibited ⱖ98% ITS similarity to the ex-type strain of E. jeanselmei (CBS 507.90) and formed a single cluster of E. jeanselmei and consequently should be regarded as identical to this species. The intraspecific variability within two strains ITS1 region was 2 bp (in the nucleotide position of 82 and 107) and 4 bp in ITS2 region (in the nucleotide position of 492, 519, 528 and 531), positions of ITS1 and 2 are counted after ATCATT. The ex-type strain originated from a case of mycetoma in a patient in France who was an immigrant from Martinique [20]. Strain CBS 116.86 represents the etiologic agent of chromoblastomycosis-like infection [10] with muriform cells in tissue. However, the clinical manifestation and the tissue form are different from that of the grains described by Langeron [20]. CBS 677.76 originated from a black-grain mycetoma in a patient from Pakistan [21] and formed grains in vivo that were morphologically identical to those formed by the ex-type strain that also originated from mycetoma. Sequence data of CBS 677.76 proved it to be close to E. jeanselmei. CBS 148.97 caused phaeohyphomycotic cysts in a 59-year-old Japanese male with enlarging nodules on his finger and forearm associated with wooden splinters [22]. It was The clinical spectrum of Exophiala jeanselmei  2010 ISHAM, Medical Mycology, 48, 318–327 voriconazole against molecularly confirmed Exophiala species. In the first, 188 clinical strains were analyzed and most appeared to be susceptible to the four widely used antifungal agents, except Exophiala attenuata which was resistant to amphotericin B. MIC50s of all E. jeanselmei isolates relative to amphotericin B, itraconazole, voriconazole and posaconazole were 0.5, 0.03, 0.125 and ⱕ0.015 μg/ml, respectively, whereas the MIC50s for E. oligosperma were 0.25, 0.06, 0.25, and 0.03 μg/ml respectively [35]. Fothergill et al. [36] studied the in vitro susceptibility to amphotericin B, itraconazole, posaconazole and voriconazole of 160 Exophiala strains including E. jeanselmei (n⫽8, originating from subcutaneous and deep infections) and E. oligosperma (n⫽40, from cutaneous, subcutaneous and deep infection). Concerning E. jeanselmei, the MIC50s were determined for amphotericin B (0.5 μg/ml), itraconazole (0.03 μg/ml), posaconazole (⬍0.015 μg/ml) and voriconazole (0.125 μg/ml) but posaconazole was more active than others antifungals [36]. Similar results were found in our series, posaconazole showed high activity against E. jeanselmei and E. oligosperma in terms of MIC50. In vitro testing of isavuconazole and voriconazole exhibited broad-spectrum activity against the majority of the opportunistic and pathogenic fungi [37]. In the present study, low in vitro activities isavuconazole (MIC50 2 μg/ml) and caspofungin (MIC50 4 μg/ml) were found with E. jeanselmei suggesting that these drugs might not be the optimal choice for treatment. In contrast, posaconazole demonstrated potent in vitro activity (MIC50 0.031 μg/ml) against E. jeanselmei that cause mycetoma. Itraconazole has been used clinically in cases of mycetoma due to E. jeanselmei, with successful outcome [38]. Voriconazole has only been employed in some cases of cutaneous infections [39]. Isavuconazole until now has not been used clinically for Exophiala infections, but itraconazole was the only clinically active antifungal drug in the treatment of mycetoma due to E. jeanselmei. Infections by Exophiala species may require a combination of surgical and medical treatment. Although amphotericin B and itraconazole, with or without additional flucytosine, are currently regarded to be efficacious against cutaneous and subcutaneous lesions, the newer triazole agents, itraconazole and posaconazole, expand the therapeutic options for these mycoses. In conclusion, posaconazole and itraconazole demonstrated the highest in vitro antifungal activity against E. jeanselmei and E. oligosperma that had high MICs for caspofungin. Therefore posaconazole and itraconazole seem to be the most active drugs for treating Exophiala infections. We did not investigate the relation between MIC and clinical response to treatment; their clinical effectiveness in the treatment of Exophiala infections remains to be determined. Downloaded from https://academic.oup.com/mmy/article/48/2/318/1014535 by guest on 02 September 2021 identified as E. jeanselmei by morphological and molecular analysis. In contrast, the remaining strains (CBS 119095, CBS 109635, CBS 528.76 and CBS 664.76) from unpublished cases were maintained as originating from cutaneous infections (arm, hand or foot). Environmental strains formerly identified as E. jeanselmei were considered to be distinct from E. jeanselmei s. str. They were within 1% differences with ex-type strains of E. oligosperma (CBS 725.88), E. xenobiotica (CBS 118157) and E. bergeri (CBS 353.52) (Fig. 3). Although E. oligosperma is phenotypically similar to E. jeanselmei and has mostly been confused with that taxon, genetically the species are clearly distinct. In our case report on CBS 122339, granules, the hallmark of mycetoma, were seen histopathologically in tissue (Fig. 1C,D). Parts of the grains were reniform, and clinical features of mycetoma, such as tumefaction, abscess formation, and fistulae were observed. In summary, nine strains confirmed as E. jeanselmei originated from mycetoma or subcutaneous phaeohyphomycosis, as well as in one instance from chromoblastomycosis according to histopathological examination. Strains with identity (100%) or near-identity (approximately 99%) with the ex-type strain of E. jeanselmei appeared to be very consistent in clinical behavior. The case of chromoblastomycosis is remarkable. The switch between grain and muriform cell as a response to host factors needs to be studied. Over the past two decades, more serious black yeast infections have been recognized. Different antifungal agents have been used in the treatment of mycetoma, such as amphotericin B, fluorocytosine, itraconazole, fluconazole, and voriconazole [33]. In the past, amphotericin B was the most potent antifungal drug for severe invasive fungal infections, but its use is associated with severe side effects, particularly nephrotoxicity and low efficacy and its use has been replaced by newer azoles and echinocandins. Unfortunately, little data are available on the in vitro antifungal susceptibility of conventional and new antifungal agents against confirmed strains of E. jeanselmei. In a comparative overview of in vitro activities of posaconazole, itraconazole, voriconazole, and amphotericin B against over 19,000 clinically yeasts and moulds, just 14 Exophiala species were included, without molecular identification [34]. Data have shown that posaconazole has good activity against agents that cause chromoblastomycosis, mycetoma, and phaeohyphomycosis, including Exophiala species and posaconazole was generally more active than itraconazole and amphotericin B against these organisms [34,35]. 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