Exophiala asiatica , a new species from a fatal case in China

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Exophiala asiatica, a new species from a fatal case in China Dong Ming Li ab; Ruo Yu Li bc; G.S. De Hoog d; Yu Xin Wang e; Duan Li Wang bc a Department of Dermatology, Peking University Third Hospital, Beijing, P.R. China b Research Center for Medical Mycology, Peking University, c Department of Dermatology, Peking University First Hospital, Beijing, P.R. China d Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands e Department of Dermatology, Beijing Tongren Hospital, P.R. China First Published on: 19 December 2008

To cite this Article Li, Dong Ming, Li, Ruo Yu, Hoog, G.S. De, Wang, Yu Xin and Wang, Duan Li(2008)'Exophiala asiatica, a new

species from a fatal case in China',Medical Mycology,47:1,101 — 109 To link to this Article: DOI: 10.1080/13693780802538019 URL: http://dx.doi.org/10.1080/13693780802538019

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Medical Mycology February 2009, 47 (Special Issue), 101109

Exophiala asiatica, a new species from a fatal case in China

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DONG MING LI1,2, RUO YU LI2,3, G.S. DE HOOG4, YU XIN WANG5 & DUAN LI WANG2,3 1 Department of Dermatology, Peking University Third Hospital, Beijing, P.R. China, 2Research Center for Medical Mycology, Peking University, 3Department of Dermatology, Peking University First Hospital, Beijing, P.R. China, and 4Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, and 5Department of Dermatology, Beijing Tongren Hospital, P.R. China

We describe a new species, Exophiala asiatica, isolated from an infection of the pharynx in a 20-year-old, immunocompetent woman in Nanjing, China. The infection was initiated by a fishbone prick in the pharynx, soon developed with facial nodules but subsequently seemed to have disappeared. Tonsil ulceration with progressive soreness of the pharynx was observed 3 years later. Dysphagia, headache and paralysis occurred four years after first signs of infection. Hyphae and yeast-like cells were detected in tissue and a black fungus was recovered repeatedly from pharynx tissue. Despite antifungal therapy for more than one year, the patient died of apparent cerebral dissemination of the etiologic agent. On the basis of morphology, nutritional physiology, ribosomal small subunit DNA and ITS sequence data the strain could not be matched with any existing species. A new species, Exophiala asiatica, is therefore proposed. Keywords

Black yeast, laryngitis, brain, Exophiala, taxonomy

Introduction Exophiala infection has aroused worldwide attention because of the cerebral and disseminated infections in young, otherwise healthy Asian patients. Due to its progressive, highly mutilating and fatal disease process, Exophiala dermatitidis has been considered to be an emerging pathogen [15]. Sporadically fatal cases have been reported to be caused by other species of the genus Exophiala, such as E. spinifera, known from previously reported disseminated infections with cutaneous nodules which ultimately reached the bones [6,7]. Exophiala jeanselmei was recently described in a fatal case of a young female from China which started as an orbital infection [8]. Systemic infections by melanized fungi in previously healthy individuals have been recognized in China over the past decades, and given its high degree of morbidity and mortality this kind of disease deserves special attention. The situation is particularly pressing in the light of supposed association of opportunistic

Received 30 May 2008; Final revision received 5 September 2008; Accepted 8 October 2008 Correspondence: Ruo-yu Li, Department of Dermatology, Peking University First Hospital, No. 1 Xi’anmen Street, Xicheng, Beijing 100034, P.R. China. E-mail: [email protected]

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melanized fungi with environmental pollution by xenobiotics [9]. In the present paper we report a new species in the genus Exophiala recovered from a fatal case in a previously healthy female student. The presentation involved pain of the pharynx and dysphasia during 3 months, which were both traced back to a fishbone prick and subsequent recurrent episodes of facial nodules 3 years earlier. The infection was caused by a species of Exophiala that could not be identified as any of the known species and is therefore introduced as a new taxon. This was the seventh fatal infection with an Exophiala species reported from China.

Case report A previously healthy 20-year-old woman living in Nanjing, P.R. China, presented with pharynx pain and dysphagia of 3 months duration. The patient recalled a severe fishbone prick of the pharynx 3 years prior to admission. Four months after the initial pharynx trauma, nodules appeared on her face, but they disappeared after she was treated for tuberculosis with isoniazid and rifampin. Three years after the infection apparently resolved, the patient reported tonsil ulceration with progressive sore throat, followed DOI: 10.1080/13693780802538019

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by dysphagia, progressive headache, and soon afterwards, paralysis of the legs. A biopsy of tonsil tissue revealed lymphocyte infiltration, septate, brownish hyphae and yeast-like cells. Re-examination of the slides of the facial nodules from her earlier infection showed similar features. On examination, the patient was dysphasic, febrile, and could feed orally only with liquid nutrients. Submaxillary lymph nodes were enlarged and haphalgesic, her palate was stiff, and her uvula was disfigured. Her right tonsil was absent as a result of a necrotic process. Palate, uvula, left tonsil, and epiglottis were inflamed, necrotic, and covered with a greyish-black pseudomembrane, which was subsequently found to be rich in black hyphae (Fig. 1A). A nodule 0.5 cm in diameter was present on the left pharyngeal recess. Laboratory results were as follows: leukocytes 13.1  109/Q with 79% band cells, 20% lymphoid cells; erythrocytes 4.55_1012/l, hemoglobin 131 g/l, platelets 284 109/l. Subgroups of lymphocytes were as follows: T4 cells 16.5%, T8 cells 29.5%, T3 80% [10]. Immunoglobulin types were normal and immunological tests for HIV were negative. Liver, kidney and bone marrow functions were normal. Radiography of the sinus revealed osteolysis and dense areas. Bronchofibroscopic examination showed pharyngeal necrosis. Examination of hematoxiline and eosin stain material from the laryngeal biopsy revealed lymphocyte infiltration, septate hyphae, and spores (Fig. 1B). Similar features were seen in the facial nodules. A black fungus was recovered (BMU 195) from the pharynx, and the patient was diagnosed as having a mycosis with cerebral dissemination which was caused by a pigmented mould. The patient was first given amphotericin B (from 2.5 mg/d gradually increasing to 25 mg/d; total amount given was 750 mg) and despite improvement in her condition, this medication was discontinued because of severe side effects and was switched to itraconazole 0.2 g once daily. Two months later, her clinical symptoms began to improve, but another black fungal strain, BMU 15, was

isolated from pharynx tissue. After being discharged from the hospital, her condition continued to aggravate with leg paralysis being noted one year later. Her infection continued and she died soon afterwards.

Materials and methods Mycology Tissues from the pharyngeal biopsy were inoculated onto Sabouraud glucose agar (SGA) with chloramphenicol and incubated at 258C. Strains BMU 00195 ( CBS 122848) and BMU 00015 (CBS 122847) were isolated from specimens before and 2 months after the introduction of therapy. These isolates were transferred to potato dextrose agar (PDA), cormeal agar (CMA), malt extract agar (MEA) and Czapek agar (CZA) and incubated at 258C for 14 days in darkness to assess growth rate and to assess colonial phenotypic features. Microscopic morphology was assessed by cutting 1 1 cm blocks from a PDA plate, mounting the blocks onto sterile microscope slides and pin-point inoculating them with the two isolates. The blocks were then covered with sterile cover slips and incubated in moist chambers for 14 d at 258C. The structure and branching pattern of conidiophores were observed at magnifications of 100, 200 and 400 on intact slide cultures under the microscope without removing the cover slips from the agar blocks. For higher magnifications the cover slips were carefully removed and mounted in lactic acid with aniline blue (Fig. 2A, B).

Physiology API 20C Yeast Identification System (bioMe´ rieux, Marcy l’Etoile, France) was used according to the manufacturer’s instructions with minor adaptations to accommodate slow growing black yeasts. Mature (7 day) cultures grown at 308C on PDA tubes were used to obtain inoculum suspensions in sterile distilled water which had a final turbidity equivalent to McFarland

Fig. 1 (A) Palate, uvula, tonsil and epiglottis covered with greyish-black pseudomembrane with inflammation and necrosis. (B) Tonsil tissue biopsy with Grocott silver staining exhibited lymphocyte infiltration, septate hyphae and spores. Magnification  400.

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Exophiala asiatica in China


CBS118720| CBS83496|C CBS102227| CBS102237| dH12331|Fo CBS102230| CBS102594| CBS14784|C CBS109797| CBS17352|C CBS26937|F CBS27137N| CBS27237|F CBS28993|F CBS30694|C CBS55683|C dH12332|Fo 100 CBS 115144 Cladophialophora potulentorum CBS 112793 Cladophialophora australiensis CBS 602.96 Capronia moravica dH18907 Cladophialophora species CBS 454.82 Cladophialophora species



BMU00195 BMU00001 BMU00015 BMU00006

Exophiala asiatica CBS25983|C CBS85896|C CBS40996|C CBS114405| dH14614|Cl CBS84069|P CBS27337|P CBS26083|C CBS28647|P



CBS 126.86 Cladophialophora boppii CBS 491.70 Cladophialophora chaetospira CBS 102080 Exophiala calicioides UMBT 1229 Exophiala dopicola CBS 181.65 Ramichloridium anceps CBS48292|E 99 CBS14693|E dH13563|Ex CBS61796|C 100 CBS 609.96 Capronia pulcherrima CBS 618.96 Capronia acutiseta CBS 604.96 Capronia dactylotrichoides CBS 614.96 Capronia fungicola

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CBS89968|E CBS101534| CBS66876|E CBS50790|E CBS101538_ CBS72588|E CBS20450|E RN497|Meri CBS118157| CBS117642| BMU00003|E CBS54682|E UMBT1286|P CBS53594|E CBS52676|E CBS35352|E BMU00009|E



GHP968|Exo dH13436|Ex CBS15858N| CBS12333|E CBS23239|E CBS 123.88 Capronia parasitica




CBS52576|E KUA0052|Ex CBS29249|E CBS13188|E CBS20735|E CBS53494|E CBS23233|E CBS60696|C WUC7|Capro IFM46117|E CBS61596|C CBS10167|C CBS 650.93 Rhinocladiella mackenziei CBS 616.96 Capronia villosa CBS 520.82 Exophiala alcalophila CBS 520.76 Exophiala moniliae


CBS15767|E dH13448|Ex dH13753|Ex dH13221|Ex CBS16089|E CBS66176|E dH13077|Ex CBS19187|E CBS25692|E CBS66076|E CBS62782|E CBS53773|E dH14520|Ex CBS11923|E CBS110371_ 99 CBS 254.57 Veronaea botryosa dH 11917 Veronaea botryosa CBS 587.66 Exophiala brunnea



dH12534|Ph CBS11385|P TRN436|Mer CBS12996|P CBS116461| RN107|Ruib

99 98


CBS 119970 Exophiala crusticola TRN30 rock-inhabiting fungus

UPSC 2646 Ceramothyrium linnaeae


Fig. 2 SSU ML tree of selected members of Chaetothyriales. Bootstrapping performed with RAxML after 450 replicates with bootstrapping criterion. Bootstraps 70% are visible at the branches; Pearson average of 100 random splits: 0.992190. Ceramothyrium linnaeae, UPSC 2646 was selected as outgroup.

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standard #2. API 20C strips were placed in the incubation tray provided by the manufacturer, covered loosely with a lid, and incubated at 308C for 7 days. Cultures of Candida glabrata were used as control. Growth was read daily for 4 days. Thermotolerance was tested by incubating freshly inoculated cultures at 28, 37, 38, 39 and 408C.

In-vitro susceptibility tests

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Antifungal susceptibility tests were performed using the broth microdilution method according to NCCLS (National Committee on Clinical Laboratory Standards) [11] guidelines as described previously [12]. Amphotericin B, ketoconazole, miconazole, itraconazole, fluconazole, flucytocine and terbinafine were obtained commercially and tested separately.

DNA sequencing About 0.1 g mycelium grown on PDA was transferred to a 2 ml Eppendorf tube containing a 2:1 (w/w) mixture of silica gel and Celite (Merck, Amsterdam, The Netherlands); DNA was extracted according to methods described previously [13]. We designed a pair of primers, NCS1/D12 (NCS1 5?-GTA AGC GCA AGT CAT CAG CTT GCG-3?; D12 5?-GAG CTG CAT TCC CAA ACA ACT CGA C) targeted at the lower end of 18S rDNA, ITS1, 5.8S rDNA, ITS2 and upper end of 28S rDNA to amplify the internal transcribed spacer (ITS) region of rDNA; N1/N9 for small subunit (18S) ribosomal gene [14]. PCR was performed on 50 ml volumes of a reaction mixture containing 10 mM Tris HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 ×6H2O, 0.01% gelatin, 200 mM of each deoxynucleotide triphosphate, 25 pmol of each primer, 10100 ng rDNA, and 0.5 U Taq DNA polymerase (Bioline, GC Biotech, Alphen a/d Rijn, The Netherlands). Thermal cycling parameters were set according to their annealing temperature with the methods described elsewhere. Amplicons were purified using the Wizard DNA clean up kit† (Promega, Madison, USA) or with Sephadex G-50 Superfine to remove remaining primers and dNTPs. Sequencing was performed in Bioasia Co. Ltd, Shanghei, China, using sets of primers, N1/N9 for 18S rDNA and ITS1/ITS4 for ITS rDNA. The D1/D2 regions were sequenced at CBS. Sequence PCR was performed as follows: 958C for 1 min, followed by 30 cycles consisting of 958C for 10 sec, 508C for 5 sec, and 608C for 2 min. BigDye terminator cycle sequencing RR mix v 1.1 (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) was used according to the manufacturer’s instructions.

Sequence analysis and taxonomy Sequences of ITS were aligned in a database using BioNumerics software v. 4.61 (Applied Maths, Kortrijk, Belgium) and 18S sequences were aligned with ARB beta-package (v. 22-08-2003) developed by W. Ludwig et al. (2004). ITS trees were reconstructed using neighbor-joining algorithm with Kimura 2 correction with 100 bootstrap replications methods in Treefinder [15]. SSU tree was reconstructed using Maximum Likelihood in RaxML.

Results Morphology and physiology The two isolates of the black fungus recovered from the patient’s tonsil tissue before and during treatment showed good growth at 258C. The maximum temperature tolerated was 408C. Colonies on MEA and PDA grew faster but their morphologic characteristics (below) were similar to those of colonies grown on CMA and CZA. The isolates showed identical cultural and microscopic morphologies and had similar physiological characteristics, except that BMU 15, isolated during therapy, was able to assimilate adonitol, while BMU 00195 could not.

Susceptibilities to antifungal agents The minimum inhibited concentrations (MIC) against strains BMU 00195/00015 of the tested antifungals were as follows: amphotericin B 0.25/1 mg/ml, ketoconazole 0.25/0.25 mg/ml, miconazole 2/2 mg/ml, itraconazole 0.25/0.5 mg/ml, fluconazole 16/64 mg/ml, 5flucytocine 4/4 mg/ml, terbinafine 0.03/0.03 mg/ml.

SSU phylogeny and ITS sequence data PCR-based amplification of SSU rDNA with primers NCS1/D12 yielded products of about 1,500 bp with strains BMU 00015 and 00195, as well as strains BMU 00001 (E. alcalophila), IFM 4869 CDC B-1785 (Exophiala sp.) and 00009 (E. bergeri) and IFM 40919. Analysis of aligned sequences revealed the presence of 539 bp insertions one base pair upstream of the universal primer ITS1. Amplicons of strains lacking the intron were about 1,000 bp. An SSU tree with collapsed branches was constructed with hundreds of strains (Fig. 3); BMU 00015 and 00195 were identical to each other and could be confidently aligned with members of the order Chaetothyriales. The sequences showed 100% identity to strains BMU 00001 (Exophiala cf. alcalophila) and 00006 (Exophiala sp). Strains BMU 00015 and 00195 did not belong to any of the – 2009 ISHAM, Medical Mycology, 47, 101109

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BMU00048 E.spinifera dH12312 E.spinifera BMU00049 E.spinifera dH12302 E.spinifera CBS00048 E.spinifera BMU00054 E.spinifera BMU00051 E.spinifera CBS269.28 E.spinifera CBS425.92 E.spinifera 89 CBS119098 E.spinifera BMU00013 E.oligosperma BMU00033 E.oligosperma ATCC16637 E.oligosperma BMU00222 E.oligosperma CBS579.76 E.oligosperma 100 CBS725.88 E.oligosperma CBS507.90 E.jeanselmei CBS528.76 E.jeanselmei BMU00014 E.jeanselmei CBS116.86 E.jeanselmei 75 99 CBS148.97 E.jeanselmei BMU00043 E.jeanselmei CBS677.76 E.jeanselmei BMU00012 E. xenobiotica BMU00025 E. xenobiotica 96 CBS118157 E.xenobiotica IFM4855 E.xenobiotica CBS116372 E.xenobiotica CBS353.52 E.bergeri 99 CBS526.76 E.bergeri BMU00016 E.bergeri BMU00034 E.dermatitidis BMU00037 E.dermatitidis dH13106 E.dermatitidis CBS120473 E.dermatitidis CBS207.35 E.dermatitidis 79 CBS581.76 E.dermatitidis CBS686.92 E.dermatitidis BMU00039 E.dermatitidis 99 BMU00040 E.dermatitidis BMU00036 E.dermatitidis BMU00044 E.dermatitidis CBS109154 E.dermatitidis 99 BMU000100 E.heteromorpha CBS633.69 E.heteromorpha CBS116.97 E.heteromorpha 77 CBS101.67 Capronia mansonii CBS109146 E.phaeomuriformis CBS109150 E.phaeomuriformis CBS109137 E.phaeomuriformis CBS102400 E.lecanii-corni 98 CBS232.39 E.lecanii-corni 85 CBS158.58 E.castellanii CBS402.95 E.mesophila 100 CBS12307 E.attenuata IFM46115 E.attenuata CBS157.67 E.attenuata CBS520.82 E.alcalophila 100 CBS521.82 E.alcalophila CBS122256 E.alcalophila BMU00001 E.alcalophila 100 CBS120272 E.angulospora CBS441.92 E.angulospora 100 BMU00015 Exophiala asiatica BMU00195 Exophiala asiatica CBS100429 Cladophialophora bantiana CBS173.52 Cladophialophora bantiana CBS101158 Cladophialophora bantiana CBS260.83 Cladophialophora carrionii


spinifera clade

dermatitidis clade

mesophila clade

alcalophila clade pisciphila clade Exophiala asiatica bantiana clade


Fig. 3 Phylogenetic tree of Exophiala asiatica and allied black fungi based on the completed ITS 1-2 domain including the 5.8S rDNA gene, generated with the Treefinder package using the Neighbor-joining algorithm and Kimura correction. The tree was subjected to 100 bootstrap replications; Cladophialophora carrionii, CBS 260.83 was selected as outgroup.

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previously recognized chaetothyrialean clades centred around Cladophialophora bantiana, C. carrionii, Exophiala angulospora, E. spinifera, E. mesophila, E. dermatitidis, E. pisciphila and Phialophora europaea. Lengths of ITS regions of BMU 00015 and 00195 were 578 bp. ITS rDNA sequences revealed no close match when applying BLASTn in GenBank. The nearest neighbour of strains BMU 00015 and 00195 in a dedicated black yeast data base at CBS containing about 11,000 entries was Exophiala sp. IFM 4869 (Fig. 4). The following, sorted by similarity, was found with other Exophiala sp.: E. moniliae (89.7%), E. dermatitidis (86.5%), E. alcalophila (86.4%), E. bergeri (86.5%) and E. lecanii-corni (86.5%). In addition, similarities with species of the genus Exophiala, as well as the above mentioned members of Phialophora, Cladophialophora and Fonsecaea never exceeded 80 85%. The species was consequently regarded to represent a hitherto undescribed taxon, which is introduced below. Exophiala asiatica Dong Ming Li, Ruo Yu Li, de Hoog & Duan Li Wang, sp. nov.  MycoBank MB 511857, Figs. 4 and 5. Coloniae in agaro farina avenae confecto (OSD) vel agaro PDA dicto 258C 7 mm diam post 14 dies, primum leves et zymoideae, deinde elevatae, velutinae, griseo-olivaceae; reversum olivaceo-nigrum. Hyphae leves, dilute olivaeo-brunneae, intervallis regularibus septatae, cellulas germinantes, ellipsoideas, 4.56.045 mm, proferentes. Mycelium torulosum quasi absens. Conidiophora vix distinguenda, ramose vel simplicia, terminalia vel intercalaria; nonnumquam cellulae gemmantes etiam conidiogenae. Annelloconidia haud septata, late ellipsoidea, levia, 3.04.512 mm, cicatrice basilari vix distinguenda. Temperatura maxima crescentiae 388C. Teleomorphe ignota. Ex-type strain CBS 122847BMU 15 (dried culture holotype in CBS herbarium CBS-H-20111), isotype culture BMU 195, isolated from tonsil tissue of a patient, China, D.L. Wang. Cultures grown on oatmeal agar (OA) and potato dextrose agar (PDA) at 258C attain 7 mm diam. in 14 days, initially smooth and yeast-like, becoming raised, velvety, greyish olivaceous; reverse olivaceous black. Hyphae smooth-walled, pale olivaceous brown, regularly septate, forming ellipsoidal germinating cells measuring 4.5 6.045 mm, especially in young cultures; budding cells similar but smaller. Torulose mycelium nearly absent. Conidiophores poorly differentiated, branched or unbranched, terminal or intercalary; sometimes isolated budding cells functioning as conidiogenous cells are present. Conidiogenous cells bear a terminal or subterminal area from which successive conidia are formed by percurrent growth resulting in a peg-like extension with inconspicuous annellations which are discernible by scanning electron microscopy (Fig. 4J). Annelloconidia non-septate, broadly ellipsoidal, smooth-walled, 3.0 4.512 mm, with inconspicuous basal scars. Maximum growth temperature 388C. Teleomorph unknown. CBS 122847BMU 15 (dried culture holotype in CBS herbarium CBS-H-20111); CBS 122848BMU 195, both isolated from tonsil tissue of a human patient, China, D.L. Wang.

Discussion We document a fatal Exophiala infection characterized by facial nodules, sore pharynx, tonsil necrosis, paralysis and osteolysis accompanied by fever in an apparently immunocompetent college student. The 20-yearold girl had experienced a trauma caused by a fishbone prick of her pharynx 3 years earlier, which is presumed to be the initial cause of the infection. Over a 20-month follow-up period, exacerbations were always accompanied by discontinuing antifungal therapeutic interventions, whereas improvements after therapy were followed by increased antifungal resistance. The MICs to most of the antifungal agents tested proved the species to be susceptible. However, after a period of treatment, strain BMU 00015 showed higher in vitro values to amphotericin B (1 mg/ml vs. 0.25 mg/ml) and fluconazole (64 mg/ml vs. 16 mg/ml). This suggests that during management of this type of severe fungal infection, antifungal regimens should be adjusted according to broth MIC results and patients’ reactions. In the present patient, medication was discontinued several times because of financial problems. This may have contributed to increasing resistance of the etiologic agent to antifungal agents and finally to the death of our patient. The black fungus recovered from the affected pharynx tissue appeared to be an undescribed species of the genus Exophiala. Exophiala asiatica is the fourth Exophiala species causing fatal infections in China, which includes E. dermatitidis, E. spinifera, and E. jeanselmei [7,8,16,17]. Assignment of the new species to the existing taxonomic system was problematic. Sequences analysis of SSU rDNA classified the fungus in the order Chaetothyriales, with close association to the teleomorph genus Capronia. Though the anamorph genus Exophiala is polyphyletic judging from SSU rDNA phylogeny data, the present species clearly represented a new clade, together with a clinical strain from the USA, Exophiala sp. (IFM 4869 CDC B-1785) and Exophiala alcalophila (Fig. 3). A 539 bp insertion was noticed in the SSU gene on position 1768 as compared to Saccharomyces cerevisiae, which is one base upstream of the ITS1 primer location. An intron characteristic for the neurotropic species Cladophialophora bantiana was noted at the same location [18,19]. Similar insertion locations were listed by Haase et al. [20] in Exophiala moniliae and Fonsecaea pedrosoi. Though it is unconfirmed whether these insertions play a role in pathogenesis and evolutionary process, their existence and distribution among

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Fig. 4 Exophiala asiatica, CBS 122847. (A) Colony on CMA (2 weeks). (B) Conidia and hyphae (magnification 100). (CI) Conidiogenous cells and one-celled conidia (magnification 1000). (J) Scanning electron microscopy of conidiogenous cells showing annellations.

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Fig. 5 Microscopic morphology of Exophiala asiatica, CBS 122847; slide culture on PDA, 14 d.

organisms and genes have given rise to a number of theories of intron origin and evolution. Homology among introns indicates horizontal transfer in BMU 00001 (E. alcalophila), 00003 (E. xenobiotica), 00006 (Exophiala sp.) and 00009 (E. bergeri) and other pathogenic black yeast-like fungi, as is known for other fungal species [21]. The corresponding ITS tree (Fig. 4) of the SSU E. asiatica clade showed considerable diversification among species. Its similarity to the nearest described species (E. alcalophila) was less than 90%. Comparing the ITS spacer lengths, E. dermatitidis (582 bp) is the longest in Exophiala, the new species is the second longest (578 bp), while the shortest one is E. attenuata (533 bp); the total range is 99 bp. Multiple alignment showed the presence of a CT-rich region in the 5? end of ITS2, which accounts for most of the variation. A distance tree constructed on the basis of ITS sequences with species allowing confident partial alignment shows that BMU 00015/000195 is truly unique, corresponding with the result of SSU analysis with Exophiala alcalophila being nearest neighbor at considerable distance.

Molecular identification of 20 clinical black yeasts from China revealed six Exophiala species, i.e., E. dermatitidis (n 7), E. spinifera (n5), E. xenobiotica (n 1), E. jeanselmei (n 2), and E. oligosperma (n1), in addition to E. asiatica. Seven isolates, involving four different species, concerned fatal infections in otherwise healthy patients. The most important pathogenic species is E. dermatitidis, followed by E. spinifera and E. jeanselmei. Remarkably, the same species are known to be involved in fungal infections in the United States, but always in debilitated patients [22]. Similar to other East Asian countries, the neurotropic species E. dermatitidis is the predominant species in China. Clinical strains of this species belong to a single ITS group within E. dermatitidis, referred to as genotype A [24]. The average clinical course of infections caused by this fungus varied from five days to twelve months. Cerebrospinal fluid rapidly changed to black and brain tissue showed extensive necrosis [16,17,23]. Exophiala spinifera is the second main pathogenic black yeast species in China. Two patients died of E. spinifera infection in China, while two more fatal cases were reported from the rest of the world [25,26]. All patients had nodular lesions, hematogenous dissemination to multiple organs and ultimately reaching the bones [7,27]. In contrast to Exophiala dermatitidis, E. spinifera is therefore presumed to be osteotropic. Environmental strains of the species have been reported, e.g., from apple juice [6] and fresh pineapple [24], suggesting a possible route of infection involving food. Since black yeasts are susceptible to most current antifungals, with sufficient medication patient should recover. For example, an Argentinean patient [28] had developed symptoms of disseminated E. spinifera infection with lymphadenopathy and fever for 3 years, without response to standard antifungal agents among which was amphotericin B. After switching to posaconazole (800 mg/day, for 13 months), however, she successfully recovered from her infection. The Chinese patient with the E. asiatica infection could not even afford itraconazole therapy, not to mention the newer antifungal agents like posaconazole. Exophiala jeanselmei usually causes mycetoma [29], with systemic infection having been recorded after traumatic inoculation in debilitated patients [30]. A systemic case in an otherwise healthy, pregnant young woman was reported from China [8]. The infection began at the orbital fossa and spread into the brain glossolalia, leading to right leg paralysis and death within three days despite amphotericin B therapy. Exophiala asiatica is now added to the list of species potentially causing fatal infections. Taking into account the clinical spectrum of black yeasts and the recurrence – 2009 ISHAM, Medical Mycology, 47, 101109

Exophiala asiatica in China

of fatal infections in China, it is puzzling why these infections are particularly  and for some species exclusively  found in East Asia, and what kind of preventive measures can be taken. Medical workers in this region should pay more attention to the potentially severe infections of black fungi.


Downloaded By: [Li, Dong Ming] At: 14:50 29 December 2008

This work was supported partly by grants from the National Natural Science Foundation of China (C30570003), the China Exchange Programme of the Netherlands Academy of Sciences, and the Foundation WOTRO for scientific research in the tropics. We thank Xiaohong Wang for taking some of the pictures and Bert Gerrits van den Ende for making the trees. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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This paper was first published online on iFirst on 19 December 2008. – 2009 ISHAM, Medical Mycology, 47, 101109


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