Korean J Clin Microbiol Vol. 13, No. 3, September, 2010 DOI: 10.5145/KJCM.2010.13.3.135 Fungemia due to Exophiala dermatitidis Eun Sun Jeong, Jong Hee Shin, Myung Geun Shin, Soon Pal Suh, Dong Wook Ryang Department of Laboratory Medicine, Chonnam National University Medical School, Gwangju, Korea We report a rare case of fungemia due to Exophiala (Wangiella) dermatitidis in a 4-month-old female infant who was admitted to an intensive care unit with sudden infant death syndrome (SIDS). E. dermatitidiswas repeatedly isolated from blood cultures (on the 28th and 32nd day of hospitalization) of the patient, who died on the 44th day of hospitalization. The fungus was identified by its morphological characteristics and DNA sequencing of both the D1/D2 domain and the ITS region of rdna. To our knowledge, this is the first reported case of E. dermatitidis fungemia in Korea. (Korean J Clin Microbiol 2010;13: 135-139) Key Words: Exophiala (Wangiella) dermatitidis, Fungemia, ITS, Sequencing 서 Exophiala dermatitidis는흑색효모양진균으로서흙, 물및인체대변등의환경에주로존재하며드물게인체감염을일으킨다 [1,2]. 이균은주로면역저하환자에서피부및피하조직을침범하는흑색진균증 (pheohyphomycosis) 을유발하며 [2], 드물게복막투석연관복막염 [3] 이나전신감염 [4] 등을유발한다. 특히이균은젊고건강한사람에서치명적중추신경계감염을유발함이보고되었는데, 이증례들은국내 1인을포함하여모두아시아인이었다 [4]. 국내에서의감염보고는매우드물어현재까지이균에의한중추신경계감염 1예 [4] 와피부감염 2예 [5], 간조직감염 [6] 등총 4예가보고된바있다. 저자들은중환자실에서입원중인생후 4개월된환아의혈액배양에서 2회연속분리된흑색진균을핵산염기순서분석법과형태학적방법으로 E. dermatitidis로동정하였기에국내에서발생한 E. dermatitidis 진균혈증의첫증례로보고하는바이다. 증 생후 138일된 6.3 kg의여아가영아급사증후군의임상진단하에타병원에서심폐소생술후혼수상태로내원하였다. 본원내원시기관삽관된상태였으며저산소성뇌증으로진단되어중환자실에서중심정맥관삽입및인공호흡기치료와함께보존적 Received 12 January, 2010, Revised 13 July, 2010 Accepted 20 August, 2010 Correspondence: Jong Hee Shin, Department of Laboratory Medicine, Chonnam National University Medical School, 671 Jebong-ro, Dong-gu, Gwangju 501-757, Korea. (Tel) 82-62-220-5342, (Fax) 82-62-224-2518, (E-mail) shinjh@chonnam.ac.kr 론 례 인치료를시행하였다. 내원시동맥혈가스검사상 ph 7.008, PCO 2 17.5 mmhg, PO 2 321 mmhg, HCO 2 4.2 mmol/l, 동맥혈산소포화도 99.3%, 총혈구계산상백혈구 25,300/mm 3,, 혈색소 12.0 g/dl, 적혈구용적백분율 35.7%, 혈소판 369,000/mm 3, AST 808 U/L, ALT 289 U/L, CRP <0.3 mg/dl 이었다. 내원당일발열이있어내원당일및 2일, 13일에시행한혈액배양에서는균이자라지않았으며, 내원 2일실시한요배양에서 Escherichia coli가 10 5 CFU/mL 이상분리되었다. 내원시시행한흉부방사선검사상에서는이상소견은없었다. 내원 2일부터 26일사이에 clindamycin, netilmicin 및 ceftizoxime 등의항균제를사용하였다. 그후지속적인항균제치료에도불구하고발열은지속되었고내원 28일과 32일에시행한혈액배양에서배양 4일후호기성배지에서균이자랐다. 배양된균은혈액한천배지에서흰색의작은집락이었고그람염색상효모균으로칸디다와감별이어려웠으며, potato dextrose agar (PDA) 에다시접종하여 30 o C에서배양한결과느리게증식되는양상으로처음엔흰색의작은집락을보였으나약 2주정도가되었을때점차점액성의진한검은색으로변하였고배지뒷면또한검은색으로변하였다 (Fig. 1). 이균은 nitrate assimilation 음성이었으며 42 o C에서성장하였다. 슬라이드배양후 lactophenol cotton blue 염색을한결과, 격벽이있는갈색의균사와동일색의효모균이다수관찰되었다. 분생자병은실린더모양으로끝이둥글었고여기에서생산된분생자는단세포성으로둥글거나난원형으로분생자가지의끝에공처럼모여있거나옆에쌓여있는특징적인모양을보였다 (Fig. 2). Clinical and Laboratory Standards Institute M27법에의한생체외항진균제검사를시행한결과, 각항진균제의 MIC는 amphotericin B 0.5μg/mL, fluconazole 8μg/mL, itraconazole 0.06μg/mL, voriconazole 135
136 Korean J Clin Microbiol 2010;13(3):135-139 Fig. 1. Two weeks after incubation at 30 o C on the potato dextrose agar, the colonies appeared as black, reverse black, wet, and mucoid. 서와 100% 일치하는결과를얻었다. Neighbor-joining algorithm을이용한계통분석도 (pyelogenetic analysis) 상으로도 E. dermatitidis (AY663828.1) 과 100% 일치하는결과를보였으며 Exophiala의유성생식형 (teleomorph) 인 Capronia epimyces와도 100% 일치하는결과를보였다 (Fig. 3) [9]. 환아는내원시부터전신상태가매우좋지않아중심정맥관제거나항진균제치료등의적극적인치료를시행하지않았고내원 44일째다장기부전으로사망하였다. 고 찰 Fig. 2. Microscopically, septate, and pale olivaceous hyphae and black yeast synanarmorphs were found. Conidiogenous cells were cylindrical, with rounded apeces producing one-celled conidia. Round to ovoid, pale brown conidia accumulated in balls or slipped down the side of conidiophores (Lactophenol cotton blue stain, 400). 0.125μg/mL, caspofungin 2μg/mL, micafungin 8μg/mL이었다. 원인균은 Vitek II (biomeŕieux, Marcy l Etoile, France) 에서는 Stephanoascus ciferrii (87.47%) 로, API 20C (Biomerieux, France) 에서 Candida lusitaeniae (98.2%) 로동정되었으며, 정확한진균동정을위하여균집락에서핵산을추출한후핵산염기순서분석을실시하였다. 염기순서분석은 ITS (internal transcribed spacer, 5.8S rrna gene 포함 ) 부위와 large-subunit rrna gene (26s rrna region) D1/D2 domain 부위의두부위를목표로하여두가지의 primer 쌍 pits-f와 pits-r[7] 및 NL1과 NL4[8] 를이용하여시행하였는데, Basic Local Alignment Search Tool (BLAST) 데이터베이스를통해모두 E. dermatitidis (GenBank Accession number AY663828.1) 의염기순 E. dermatitidis는드물게진균혈증을유발하는데현재까지국내에서는이균에의한균혈증이보고된바없고, 외국의경우총 7예의진균혈증이보고되어있다 [10]. E. dermatitidis에의한 7예의진균혈증을살펴보면기저질환은백혈병, 유방암, 폐암등의악성질환과화상, 장절제술후, HIV 감염등이었고나이는 1.5세부터 61세까지다양한분포 ( 소아 4명, 성인 3명 ) 를보였다. 현재까지보고된총 7예의진균혈증의경우모든환자들이공통적으로중심정맥관을사용하고있었고일부는중심정맥관연관진균혈증으로보고된바있다 [10]. 본증례의경우도중심정맥관을사용하고있었으나중심정맥관을통한혈액정량배양을따로시행하지않았고, 환자가사망한관계로중심정맥관 tip 배양이이루어지지않아중심정맥관연관진균혈증유무를판단할수없었으나 [11] 장기간의입원과항균제치료, 중심정맥관사용과영아급사증후군으로인한면역저하상태가진균혈증을일으킨요인으로생각되었다. E. dermatitidis는인체처럼따뜻한곳에잘생존하여내이 (inner ears), 폐, 기관지혹은장내에집락형성 (colonization) 할수도있으며, 또낭성섬유증 (cystic fibrosis) 환자의폐나설사환자의변에서증상없이규칙적으로분리될수도있다 [12]. 또한
Eun Sun Jeong, et al. : Fungemia due to Exophiala dermatitidis 137 Fig. 3. A pyelogenetic tree of Exophiala and neighboring species, constructed by using neighbor-joining algorithm. 이균은최근소아에서간침범으로인한간경화가보고되었으며 [6] 오염된정맥주사제를투여한후노인에서병원감염성가유행 (pseudoepidemics) 이보고된바있다 [13]. 따라서본환자의경우중환자실에입원한동안환자체내에집락형성한균이혈액성으로퍼져진균혈증을유발하였거나외인성으로환경에있던균이중심정맥관을통해혈류감염을유발하였을가능성도있다. 현재까지보고된총 7예의진균혈증의경우중심정맥관을제거하거나동시에항진균제를투여함으로써모두회복되었다. 그러나본증례의경우전신상태가입원당시부터좋지않아적극적치료를하지않았고항진균제치료나중심정맥관제거를하지않았다. 따라서본증례는 E. dermatitidis에의한진균혈증이적극적치료를하지않는경우인체에치명적일수도있음을처음으로보여주었다. 흑색효모균은생활환중일정시기에효모처럼발아세포를생산할수있는다양한흑색진균을일컫는다. 이들은임상검체에서매우드물게분리되며, 생체외배양에서처음에는효모균모양을보이다가점차균사형진균으로변하기때문에검사실에서이들균종을신속하고정확하게동정하고감별하기는쉽지않다. 여기에속하는균종으로는 Aureobasidium pullulans, Hormonema dematioides, Exophiala jeanselmei, Exophiala moniliae, Exophiala spinifera, Phaeoannelomyces werneckii, Phaeosclera dematioides, Sarcinomyces phaeomuriformis 및 Exophiala dermatitidis 등이있다. 이들흑색효모균중 E. dermatitidis 는각종인체감염과연관되어가장흔하게임상검체에서분리될수있는흑색효모균이다 [14]. 본증례의경우원인균은염기순서분석결과와형태학적특성이외에도 42 o C에서자라고, nitrate assimilation 음성이면서집락이매우점액성인 3가지특징으로다른 Exophiala 균종과감별할수있었다. 최근에는많은검사실에서진균의동정에유전자염기순서분석법을사용하기시작하였다. 이는기존의생화학적및형태 학적동정법을보완하고결과가지체되는것을방지하는등유용성이있다 [15]. 본증례에서혈액배양에서 2회분리된효모균은초기에칸디다로생각되었으나 Vitek II에의해매우드문 S. ciferrii (87.47%) 로동정되었고이는 API 20C 동정결과 (C. lusitaeniae) 와일치하지않았다. 따라서진균동정에가장유용하다고알려진 ITS 부위와 D1/D2 domains 부위의염기순서분석을실시하였다 [7,8]. 염기순서분석결과, 본균주는두부위모두에서 E. dermatitidis와 100% 일치한결과를보여쉽게동정되었다. 또이균은배양 2주정도후점차검은색의사상형진균으로변하여슬라이드배양을실시하였고형태학적으로도 E. dermatitidis임을확인할수있었다. E. dermatitidis는항진균제내성이드물며 itraconazole, voriconazole과 terbinafine 등의항진균제에낮은 MIC를보이는것으로알려져있다 [16]. 본증례에서분리된원인균의항진균제감수성검사소견은 amphotericin B (MIC, 0.5μg/mL), voriconazole (0.125μg/mL) 등에감수성이나 fluconazole에대해감수성이저하되어있었고 (8μg/mL), caspofungin (2μg/mL), micafungin (8μg/mL) 등의 echinocandin 항진균제에는생체외내성을나타낼수있음을보여주었다. 요약하면, 본증례는중환자실에서입원중인생후 4개월된환아에서발생한 E. dermatitidis 진균혈증의국내첫증례로서, 혈액배양에서분리된진균이일반적인효모균동정제품으로잘동정이되지않는경우흑색효모균등의가능성을고려하여신속하고정확한균동정을위해서유전자염기순서분석이필요하며진균혈증의심시에조기에적극적인치료가환자의예후에중요한영향을미침을보여주었다. 참고문헌 1. Nachman S, Alpan O, Malowitz R, Spitzer ED. Catheter-associated
138 Korean J Clin Microbiol 2010;13(3):135-139 fungemia due to Wangiella (Exophiala) dermatitidis. J Clin Microbiol 1996;34:1011-3. 2. Hohl PE, Holley HP Jr, Prevost E, Ajello L, Padhye AA. Infections due to Wangiella dermatitidis in humans: report of the first documented case from the United States and a review of the literature. Rev Infect Dis 1983;5:854-64. 3. Vlassopoulos D, Kouppari G, Arvanitis D, Papaefstathiou K, Dounavis A, Velegraki A, et al. Wangiella dermatitidis peritonitis in a CAPD patient. Perit Dial Int 2001;21:96-7. 4. Chang CL, Kim DS, Park DJ, Kim HJ, Lee CH, Shin JH. Acute cerebral phaeohyphomycosis due to Wangiella dermatitidis accompanied by cerebrospinal fluid eosinophilia. J Clin Microbiol 2000; 38:1965-6. 5. Kim DS, Yoon YM, Kim SW. Phaeohyphomycosis due to Exophiala dermatitidis successfully treated with itraconazole. Korean J Med Mycol 1999;4:79-83. 6. Hong KH, Kim JW, Jang SJ, Yu E, Kim EC. Liver cirrhosis caused by Exophiala dermatitidis. J Med Microbiol 2009;58:674-7. 7. Sugita T, Nishikawa A, Ikeda R, Shinoda T. Identification of medically relevant Trichosporon species based on sequences of internal transcribed spacer regions and construction of a database for Trichosporon identification. J Clin Microbiol 1999;37:1985-93. 8. Kurtzman CP and Robnett CJ. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol 1997;35:1216-23. 9. Untereiner WA. Fruiting studies in species of Capronia (Herpotrichiellaceae). Antonie Van Leeuwenhoek 1995;68:3-17. 10. Al-Obaid I, Ahmad S, Khan ZU, Dinesh B, Hejab HM. Catheterassociated fungemia due to Exophiala oligosperma in a leukemic child and review of fungemia cases caused by Exophiala species. Eur J Clin Microbiol Infect Dis 2006;25:729-32. 11. Lim WH, Shin JH, Suh SP, Ryang DW. Diagnosis of central venous catheter-related sepsis using differential quantitative blood cultures. Korean J Clin Pathol 1998;18:208-14. 12. de Hoog GS, Matos T, Sudhadham M, Luijsterburg KF, Haase G. Intestinal prevalence of the neurotropic black yeast Exophiala (Wangiella) dermatitidis in healthy and impaired individuals. Mycoses 2005;48:142-5. 13. Woollons A, Darley CR, Pandian S, Arnstein P, Blackee J, Paul J. Phaeohyphomycosis caused by Exophiala dermatitidis following intra-articular steroid injection. Br J Dermatol 1996;135:475-7. 14. de Hoog GS and Vitale RG. Bipolaris, Exophiala, Scedosporium, Sporothriz, and Other Dematiaceous Fungi. In: Murray PR, Baron EJ, Jeorgensen JH, Landry ML, Pfaller MA, eds. Manual of Clinical Microbiology. 9th ed, Washington D.C.; ASM Press, 2007: 1898-917. 15. Petti CA. Detection and identification of microorganisms by gene amplification and sequencing. Clin Infect Dis 2007;44:1108-14. 16. Vitale RG, De Hoog GS, Verweij PE. In vitro activity of amphotericin B, itraconazole, terbinafine and 5-fluocytosine against Exophiala spinifera and evaluation of post-antifungal effects. Med Mycol 2003;41:301-7.
Eun Sun Jeong, et al. : Fungemia due to Exophiala dermatitidis 139 = 국문초록 = Exophiala (Wangiella) dermatitidis 에의한진균혈증 1 예 전남대학교의과대학진단검사의학교실정은선, 신종희, 신명근, 서순팔, 양동욱 Exophilala (Wangiella) dermatitidis는생체외배양에서처음에는효모균모양을보이다가점차균사형진균으로변할수있는흑색진균으로통상적검사방법으로는동정이매우어렵다. 국내에서는이균에의한뇌척수막염 1예및피부감염 2예, 간조직감염 1예등이보고된바있으나아직균혈증의보고는없다. 저자들은영아급사증후군으로병원에입원중인생후 4개월된여아에서 E. dermatitidis에의한진균혈증을경험하였기에보고하는바이다. 원인진균은입원후 28일과 32일에시행한호기성혈액배양에서두번분리되었는데, API 20C (Biomerieux, France) 에서 Candida lusitaenia (98.2%) 으로, Vitek II (Biomerieux, France) 에서는 Stephanoascus ciferrii (87.47%) 로동정된반면, D1/D2 domain (26s rrna region) 과 ITS 부위유전자염기순서분석에서둘다 E. dermatitidis와 100% 일치하는결과를얻었다. 이균은배양초기에는칸디다와감별이어려운효모균이었으나 2주이상배양후점차검은색의사상형진균으로변하였고슬라이드배양에서격벽이있는갈색균사와특징적인실린더모양의분생자병에서생성된분생자가공처럼모여있는 E. dermatitidis 의특징을보였다. 환아는항진균제치료가불가능하였으며전신상태의악화로내원 44일째다장기부전으로사망하였다. 본증례는국내에서 E. dermatitidis 진균혈증에관한첫증례보고이다. [ 대한임상미생물학회지 2010;13:135-139] 교신저자 : 신종희, 501-757, 광주시동구제봉로 671 전남대학교병원진단검사의학과 Tel: 062-220-5342, Fax: 062-224-2518 E-mail: shinjh@chonnam.ac.kr