Subcutaneous Mycetoma of Pseudallescheria boydii complexes Revealed by 18S rDNA sequencing

E

훈펜흐앤

[01 M혀ici삶 d UfεScien

∞

Vol. 1ι ~~lfJun .•)，~20_13] Subcutaneous Mycetoma of

Pseudall

낭

scheria boydii

complexes

Revealed by

18S

rDNA

sequencing

Ji Eun Park

，

Young Ree Kim

，

Sung Ha Kang

，

Sun Hyung Kim

，

.Jae. Wang Kim

，

Keun Hwa Lee Jeju National UniversitySCh

。이。

f Medicine.Jeju，Korea

(Received May 14，2013; Re띠sed May 21，2013; Accep떠d May 28，2013)

Abstract

Pseudallescheria ellips.α'dea，one 01 Ihe P. boydii complexes，is a ub띠uilously isolated saprophytic fungus from soil，pOuttry， cattle，sewage ，and palluted water. It is known 10 cause varlous diseases such as subcutaneous mycetoma in immunocompetent individuals and sepsis in immunocompromized patients. We experienced a case 01 cutaneous P. ellipsoidea infeclion in a 79 year-이d man. He was laking medicalion for diabeles mellilus and heart disease and visi!ed aur dermalologic ou1patient clinic wilh vi이el-colored ，nontender，f1uclualing，keral이c，conglomerated 미aques on the righl lorearm. Skin biopsy was consislenl with foreign body reaction to ruptured epidermal Cyst. After skin cullure，PCR and sequencing revealed P. boydii complexes ， ~ith forward sequencing showing 96% homology 01 P. boydii，and revere sequencing showlng homology 100% of P. ellipsoidea A

’

ler Ihe palien1 received f1uconazole for 5 weeks，discharged 씨lh improved condilions. (J Med Ufe SCi 2013;10(1):50-53) Key Words : Pseuda1lescheria boydii complexes，Mycetoma，Sequencing

Introduction

]

Case Report

PseudBJlescheria elJipsoidea is a saprophytic fungus ubiquitously isolated from

∞

11，po비kγ，cattle，sewage，and polluted water". Re엉1Uy.It is known to cause vanous dise，잃es

such as subcutaneous myceroma in immunocompetent individuals and disseminate sepsis in inununocompromized patients2.l. 80me study have proposed that P. eJJipsoidea，P. angusta. and P. fusoidea are the another name of P. boydii

with no significant difference in tJ1.e aspect of c1inical manifestation 뻐d rDNA sequencesll)‘But. other studies have

reve외ed that it is onc. of the P. boydii complexes which consists of at least six different species (P. boydii，P. e//ipsoidea. P. 81앵l.Jsta. P. aurantiacu，P. fusoide8 ，and P.

milJutispora) ‘에AI1hough the use of voriconazole，these

infections may have high mortality and need to furtber established treatmenfl. Until now，only one case of pulmonalγ fungal ball of F농euda1Jeschen'aboydii identified

by I.SU rDNA D2 region sequencing have been reported in our country"l. We intl'oduce our experience with a case of cutaneous P. boydii eomplex infeetion in a imrnunoeompetent patient revealed by 18S rDNA sequeneing

[CorrespOndingA띠hor] Young Ree Kim

j잉u NationalU애%성tv. SChCd01MeálCinea얘Depar1menl01l.abo<a1OlY

Medieine.Jeju Nalional Universily Hospital 1753-3，Ara-1 Dong Jeju-웅i，Jeju Special S잉I-Governing Province. Korea 690-716 E-mail. namu879αê>ieiunu.ac.kr

A 79-year-old m밍1 visited our dennatologic outpatient

clinic，complaining of violet-eolored ‘nontender‘fluetuaψ19‘

keratolie，

∞

n잉omerated. plaques on the ri앙1t fore밍m (Fig. 1)

Fi밍Jre 1. Skin lesion이d잉lt forearm.

He had taken the medieaüons .for diabetes mellitus and héart disease for sever외 years without any other similar

앙rnptoms. Physi

∞

1 examinaüon revealed no specific fmding

except above right fore81π lesion. 8kin biopsy done after admission was consistent with foreign body reaetion ω ruptured epidermal eyst. There was no evidence of tuberculosis irûeetion in acid fast s1ain and culbJ.re of his n빼t forearm lesion. On Gram s1ain. mold 와-e noted.，so，the

speeimen was cultured in Sabouraud's dex1rose ag밍 (SDA.

Har피 KOMED，Seoul. Korea) at 30't. On the 7th cul띠re day，the isolare started k> grow 까1e front surfaee of the

-50-Subcutaneous Mycetomaof Pseudallescheriabo찌ii complexesReve띠ed by 18S rDNAsequencing

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I

C

/‘’‘엇

ζ

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Fi밍ue 3. Micrα:;copicfir녕mgs허10Wing∞띠diophoresw삐1 conidia

of the fur핑us on l.ac1D-phenol

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φn 비ue stain. A QFCB s벼in.앙ID)

B.<r.PCB s떠in. x 400)

Based on the above results. the organism was suspected as F농eudalJescheria spp. DNAs of fungus were extracted using the bead beater-phenol ext.l"actionmethod. A loopful

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f culωre of each isolate was suspended in 200，uL of TEN buJTer (10m M까is - HCI，1 mM EDTA. 100 mM NaCl: pH 8이‘placed in a 2.0 rnL screw-cap microcentrifuge tube filled

、

η1h2oo μL (packed volurne) of glass beads (diameær，

0.1 mm: Biospec Products，B양Uesville，OkJa.l and 200 μL of

Phenol:Chloroform: Isoamyl alcohol (25:24:1) (SIGMA chemical co. P-2069). To disrupt Ihe fnngus，the lube was

。

scillaæd on a Mini-8ead Beaær (8iospec Prcducts) for 1 min. To separate the phases the tube was centrifuged (15.000 rpm. 15 min). After the aqueous phase was transf erred inω another c1ean tube. 10 μL of 3 M sodium acetaæ and 250 μL of ice-cold ethanol were added to enable the DNAto precipitaæ‘the mixture was kept at - 20t

for 30 min 끼1e DNA pellet was washed with 70% ethanol. dissolved in 60 μL of TE buJTer (10 mM Tris - HCI，1 mM EDTA，100 mM NaCI: pH 8이없d used as a template for

PCR. PCR was peñormed with a set of prirners (Forw와d

primer (0817) 5'-TTAGCATGGAATAATRHAATAGGA-3’and

Heverse primer (1536) 5’-ATTGCAATGCYCTATCCCCA-3') for amplification of the partial 188 rDNA sequences Templaæ DNA (3 μ1) and 20 μL of each primer were added to a PCR mixture ωbe (ACCl피Power PCR PreMαBioneer

Daejeon. Korea) ，which contained 1 U of l'aq DNA polymerase. each deoxynucleoside triphosphate at a concentration of 250 μL，50 111MTris-HCI (pH 8.3)，40 mM

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、

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윈gure 2. Features on Sabornuá5dextro!영ea뿜r

A Front image after 7 d맹s incubation

B. Reverneimage 7 days incubation C. Fìuntimage외ler 14 days incubation D.He

‘

lt!JSeimage 14 days incubation

、

Lacto-phenol cotton blue stain for microscopic examination showed septated hyphae with simple long or short conidiophores wi당1 conidia having unicellular and oval，

and had the larger end toward the apex. and cut off at base (F

、

g. 3. A，B)

agar grew cotton like white mycelium. which later tumed slightJy brown afær 14 d앙5incubations (Fig. 2. A，C)

π

，

e

reverse surface were sli앙1t yellow at f1rst，then became smokybroWn brown afær 14 d뻐s incubations (Fig. 2. B‘D)

-에

Ji Eun Park. YoungRee Kim.Sung Ha Kang.Sun HyungKim‘Jae、，vang Kim.KeunHwa Lee

KCI. 1.5 mM MgC12，and gel loading dye. The reacLion mixture was sub.iected to 35 eycles of amplification (2 min at 94t ，10 s at 56t ，and 30s at 72t) ，followed by a 5-min extension at 72t (model 9600 thennocycler: Perkin E띠ler Cews). PCR products were electrophoresed on a 1.2%

agarose gel and were purified with a QIAEX II gel extraction kit (QIAGEN，Hilden，Germany). For the 188 rDNA analysis，we used Applied Biosystems rnodel 373A automa

“

c sequencer and a BigDye Terminator Cycle Sequencing kit (Perkin-Elmer Applied Biosystems. W밍πn밍on，United Kingdom). Sequencing revealedP. boydil

complexes，with forward sequencing showing 96% homology

。

f P. boydii，and revere homology 100% of P. eJJipsoidea

After the patient received f1uconazole for 5 weeks. discharged with improved conditions

Discussion

The Pseudallescheria/Scedosporium species complex are about to be emerging opportunistic fungal pathogens in some coun띠es. Especially，in Austra.lia，the survey of soil

samplings revealed an abundance of the Pseudallesche끼al

Scedosporium in locations associated with high human activitù-'밍ld strnins of S. aurantiacum were most frequently IS이ated (54.6%)，followed by S. prolificans (43%)，P. boydil

(2.1%) and S. deho앵ii (Q.3%)~1.ln some cases，radiographic or histopathologic tests cannot differentiate Scedosporium from Aspergil1us. and the diagnostic confusion lead t

。

complicate the management of Scedosporium infecl10nl

“’

because the drug of choice for those species is voriconazole，

not amphot.ericinB in case of Aspergillus infection. Also，the prevalence of S. apiopsermum (or P. boydW infection， especially the mycetomatous form could have been underestimatedl1

’

80，timely and accurate laboratory diagnosis is essential for proper σ-eatrnent. RecentJy，hi양비’ species-specific multiplex PCR assay offers a rapid and simple method of detection of the most c1inicallyimp

。

π밍lt

Scedosparium species in respiraωry Íl'act specimensl2l.Buι even though the sequencing of LSU rDNA 02 region identified 비e organism as P. elJipsoidea，U1e suggest.ed

spe잉es inP. boydii complex co비d not be difTerentiatedfrom

each oLher，so. further studies with ß -tubulin genes， calmodulin genes，and the ITS region sequencing might be recommended81. We presented a case of cutaneous P. eJlipsoidea infection in an immunocompetent pat.ient with underlying diabet.es mellitus and heart disease，initially suspected by 8DA and LPCB stain밍1d revealed by PCR and

188 rDNA seqüencing as .P.boydü complexes. But. Further

studies with sequencing of o~er' regions such as intemal transcribed spacer ，actin. ‘heé.t shock protein ，and topoisomerase II musL be needed' t

。

이fferentiate the

species inP. b。얘ïicomplex. .•.

Acknowledgement

This research was supported by the 2013 scientific promotion program funded by J~u National University

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-Subcutaneous Mycetoma of Pseudaliescheria boydii complexes Revealed by 188 rDNA sequerícmg

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