Subcutaneous infection by Graphium basitruncatum in a heart transplant patient

(1)

w w w . e l s e v ie r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Case

report

Subcutaneous

infection

by

Graphium

basitruncatum

in

a

heart

transplant

patient

Analía

L. Fernández

a,b

_,

_Patricia

_O.

_Andres

a

_,

_Cecilia

_H.

_{Veci ˜no}

b

_,

_Claudia

_B.

_Nagel

c

_,

María

Teresa

Mujica

b,∗

a_Hospital_{Universitario}_Fundación_Favaloro,_Laboratorio_Central_Servicio_de_{Microbiología,}_Buenos_Aires,_Argentina

b_Universidad_de_Buenos_Aires,_Facultad_de_Medicina,_Departamento_de_{Microbiología}_y_{Parasitología}_Médica,_Buenos_Aires,_Argentina c_Hospital_{Universitario}_Fundación_Favaloro,_División_{Infectología,}_Buenos_Aires,_Argentina

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3May2017

Accepted14August2017

Availableonline18September2017

Keywords: Graphiumbasitruncatum Phaeohyphomycosis Hearttransplant Subcutaneousinfection

a

b

s

t

r

a

c

t

Graphiumbasitruncatum,a synanamorphofPseudoallescheriahasbeenrarelyreportedin

humaninfections.Wereportacaseofsubcutaneousphaeohyphomycosiscausedbythis

fungusinahearttransplantrecipient.Wealsodescribethephenotypic,molecularmethods

andmatrix-assistedlaserdesorption/ionizationtime-of-ﬂightmassspectrometry

(MALDI-TOFMS)usedtoachieveisolateidentiﬁcation.

openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/

by-nc-nd/4.0/).

Introduction

MembersofthegenusPseudallescheria,producingScedosporium

andeventuallyGraphiumsynanamorphs,arefrequentlyfound

insubcutaneousandsystemicinfections.1_These_species_are

alsoencounteredaspersistentcolonizersoftherespiratory

tractinpatientswithcysticﬁbrosis.ThegenusGraphiumsensu

latohasbeenidentiﬁedbyusuallywell-developeddark

syn-nemata,producingsingle-celled conidiainslimy massesat

theirapices.MonomorphicGraphiumspecieshavebeen

iso-latedfromsoil,plantdebris,woodysubstrateandgalleriesof

barkbeetlesinconiferwood.2_However,_a_few_human_cases3,4

∗ _{Corresponding}_author.

E-mailaddress:[email protected](M.T.Mujica).

and onedogcase5 _of_infection_with_Graphium_species_have

beenreportedpreviously.

The epidemiology of invasive fungal infections is also

changing with many species not previously described as

pathogens now contributing to the disease burden.6 _We

describe a case offungal infection in subcutaneoustissue

byGraphiumbasitruncatuminahearttransplantpatient.

Fur-thermore, phenotypicand molecularmethods usedforthe

identiﬁcationoffungiarealsodiscussed.

Case

report

A48-year-oldwhitemanwasadmittedatFundación

Faval-oro,UniversityHospital,BuenosAiresinDecember2015.He

had undergoneheart transplantation in2011 and received

tacrolimus(2mg/12h)anddeltisone(6mg/days)as

immuno-suppressive regimen.Physical examinationshowedneither

http://dx.doi.org/10.1016/j.bjid.2017.08.001

(2)

brazj infect dis.2017;21(6):670–674

671

Fig.1–K(OH)40%P/Vsmearfromscrappedlesionintherightpalm,illustratingthepresenceofseptatehypha(400×).

fevernorsepticemicsigns.Thepatientpresentedwithalesion

inthepalmofhisrighthandwhichhadevolvedforsixmonths,

involvingthecutaneousplanetothefasciawithoutaffecting

theﬂexorlevel.Thelesionwasanon-tender,erythematous

skinnodule withcentral softening. Computed tomography

(CT)scansofthorax,paranasalsinusesandabdomenrevealed

no lesions. The right hand lesion was punctured and the

aspiratedmaterialwas sent tothe laboratory. It wasfresh

mountedwithand without KOH 10%.Neither bacteria nor

acid-fastbacteriawereobservedinGramandZiehl–Neelsen

staining. Thesmearsshowed septate hyphae with slightly

unevenwidths (2–4␮m),irregularbranching andthin walls

withfocal bulbousdilation (Fig. 1). Itwas inoculated onto

bloodagar(BioMerieux®),chocolateagar(BioMerieux®),and

Sabouraud dextrose agar (Britania®) and incubated at 28

and37◦C.Upondermatomycosisdiagnosis,voriconazolewas

empiricallyadministeredwithaloadingdoseof300mgtwice

adaytheﬁrstday,andsubsequently200mgevery12hfor30

days.Surgerywasscheduledtobeperformedattheendof

January2016.Theexcisedmaterialwasprocessedasinthe

previoussampleanddemonstratedthesamefungalelements

observedinFig.1.Theisolatesweresubculturedontoan

in-housepotatodextroseagarmediumandincubatedat28◦C

for10daystoassessthemacroscopicmorphologyand

micro-scopicfeaturesformedinslidecultures.Colonieswereﬂuffy,

relativelyﬂat,andwhiteinitially,becominggray-brown

cen-trallywithaninvisiblemargin(Fig.2).Distinctivemicroscopic

features included hyphae in prominent fascicles (Fig. 3a),

“sausageshaped”orcurved(allantoid)hyalineconidiawith

truncatebases(2.5–5.0mmlong by1.5–2.5mm wide)borne

from single annellatedconidiogenouscells (Fig.3Aand B),

larger,brown,ovalconidia(4–6.5mmlongand3–4mmwide)

(Fig.3A)andprominentsynnemata(Fig.3C).Basedupon

mor-phologicfeatures,theisolateswerepresumptivelyidentiﬁed

asGraphiumspp.

Asinourcase,itisoftenchallengingtoidentifysuchrare

fungalpathogensbyconventionalmethods.However,

deﬁni-tiveidentiﬁcationispossibleusingDNAsequencingmethods.

MolecularidentiﬁcationwasundertakenusingaPCR

(poly-merase chain reaction) assay. TheITS region ofthe rDNA

genewasampliﬁedbyPCRfollowingtheprotocoldescribed

byWhiteetal.7_{Subsequently,}_the_amplicon_was

bidirection-allysequenced.TheampliﬁcationoftheITSregionresultedin

afragmentof536bp.Thenucleotidesequencewascompared

withthoseavailableintheGenBankdatabaseusingBLASTN

[National Center for Biotechnology Information (NCBI)

Internethomepage,(https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Sequences ofthe ITS region of Graphium isolatesobtained

inthisstudy weresubmittedtotheGenBankdatabase and

assignedthefollowingAccessionNo.:KY552920.TheGraphium

culturehasbeendepositedattheMycologyCenterinthe

Uni-versityofBuenosAires,SchoolofMedicine.

Alignment of over 536bpcorresponding toITS1 (partial

sequence),the5.8Sribosomalgene(completesequence),and

ITS2 (partial sequence) showed 100% similarity by partial

(3)

b r a z j i n f e c t d i s . 2 0 1 7; 2 1(6) :670–674

(4)

brazj infect dis.2017;21(6):670–674

673

Fig.3–MicroscopicmorphologyofGraphiumbasitruncatum

froma10-dayold,28◦Cpotatodextroseagarslideculture demonstratingdistinctivemicroscopicfeaturesincluded hyphaeinprominentfascicles,“sausageshaped”orcurved (allantoid),hyalineconidiawithtruncatebases(2.5–5.0mm longby1.5–2.5mmwide)bornefromsingleannellated conidiogenouscells(AandB)(1000×)orsimilarlyindark, prominentsynnemata(C)(200×),andlarger,brown,oval conidia(4–6.5mmlongand3–4mmwide)(A)(1000×).

KT828733.1) and 99% was obtained with Graphium

penicil-lioides(AccessionNo.KJ780752.1),Graphiumjumulu(Accession

No. NR137980) and Graphium carbonarium (Accession No.

KM245114).Thequerycoveragewas99%withanE-valueof

0.0ascomparedtothesequencesabove.

Thefungalspecieswasalsoidentiﬁedbymatrix-assisted

laserdesorption/ionizationtime-of-ﬂightmassspectrometry

(MALDI-TOFMS).Theisolatewastestedwithasimple“short

extraction”protocol.8,9 _The_analysis_on_the_MALDI_BioTyper

system with the BioTyper 3.0 SR software (Bruker

Dalton-icsGmbH,Bremen,Germany)resultedinG.penicillioideswith

score<1.7.

Discussion

In thepresent report,the fungal infectionwas localizedin

the subcutaneous tissuewithout evidence ofother lesions

suggestingmicrobiologicalinvestigation.Thissingleﬁnding

wasinlinewithhisgoodgeneralconditionandtheresults

obtainedbyCTscans.Itisunknownhowourpatientacquired

theinfection.Heregularlyusedtoprunetreesinhis

birth-place,provinceofMisiones(Argentina).However,therewas

no signofarecent woundbythornor splinter andhedid

notrememberanyinjury.ThefactthatG.basitruncatumwas

isolatedtwicefromsamplescollectedatdifferenttimes

con-ﬁrmeditsroleastheetiologicalagentofthisdisease.

We must point out that the patient showed neither

adherence to treatment nor medical controls. Only once,

voriconazolecouldbedosed,andadequateserumlevelswere

achieved(3885ng/mL).Atsurgery,thelesionwascompletely

excisedandafterayeartherewasnoevidenceofdisease.

Graphium species belong to the Ascomycota, which are

foundinsoilandplants.Manyspeciesincludedinthegenus

Graphiumareknownasplantpathogens.Theanamorphgenus

Graphium Corda (1837) was originally described by Corda10

based on a specimen collected in Prague on Populus nigra

var italica(black poplar). Ellis11 listed the fungusas

occur-ring inPopulus woodinEuropeandNorthAmerica.Sutton

andLaut12_and_Sutton13_described_specimens_identiﬁed_as_G.

penicillioidesasacommon secondarycolonizer ofbark

bee-tle tunnels in Ulmus trees killed by Dutch elm disease in

ManitobaandSaskatchewan,Canada.Recently,Graphiumwas

isolated from ambrosiabeetle Megaplatypusmutatus in our

country, a drill ofwood that attacks numerous species of

native and exotictrees, aplagueofpoplarsinArgentina.14

However,thebiologyanddistributionofG.basitruncatumare

poorlyknown.Thefungushasbeenisolatedtwicefromsoil,

including its original location in the SolomonIslands, and

Japan(JCM8083).DescribedoriginallyasStilbumbasitruncatum

byMatsushima,15_the_species_was_later_regarded_as_a_synonym

ofG.penicillioidesbySutton.13

The morphology of the fungus was in line with that

of Graphiumspecies reportedpreviously.6 _In_addition, _DNA

sequenceanalysisoftheITSregionidentiﬁedthefungiasG.

basitruncatum.16

Our isolate had an acceptable percent identity, and

the query coverage with G. basitruncatum (Accession No.

KT828733)isolatebyCeriani-Nakamurakareetal.14_also

(5)

theaggregatenameofG.penicillioides.However,in2000,Okada

et al. re-evaluatedGraphium-like anamorphs that had long

beenconsideredG. penicillioidesand determinedthatbyITS

sequencingG.penicillioideswasaspeciesaggregateconsisting

ofatleastfour species,oneofwhich isG. basitruncatum.16

It is often difﬁcult to identify such rare fungal pathogens

byconventional methods.Hence,deﬁnitiveidentiﬁcation is

possible using DNA sequencing methods. This case report

highlightstheimportanceofcomparingphenotypicfeatures

withmolecularresultstoreachanaccurateidentiﬁcation.

Severalstudieshavereportedtheperformanceof

MALDI-TOF MS for the identiﬁcation of clinically relevant fungal

species.Theuse ofMALDI-TOF MSindiagnostic mycology

islimited so far, especiallyforthe identiﬁcation ofmolds,

mainlydue tothepoor fungal coverageofthe commercial

databasesandtherequirementofextendedsample

prepara-tionformolds toachievegood-quality massspectra.17 _The

Biotypersoftwarecompareseachsamplemassspectrumto

thereferencemassspectrainthedatabase,calculatesan

arbi-traryunitscorevaluebetween0and3reﬂectingthesimilarity

betweensampleandreferencespectrum,anddisplaysthetop

10matchingdatabaserecords.Scoresof2.0wereacceptedfor

speciesassignment,andscoresof1.7but2.0wereaccepted

foridentiﬁcationatthegenuslevel.Scoresbelow1.7were

con-sideredunreliable.18_However,_mass_spectra_of_dematiaceous

moldshadfewerpeaks,andspectrumqualitywasoftennot

sufﬁcienttocreatereferenceentries,suggestingthatsample

preparationforthesefungiismoredifﬁcult.Inaddition,

fun-gal pigmentsmay have inhibitedthe acquisition ofMALDI

TOFmassspectra.19_As_previously_speciﬁed,_the_fungal

iso-latedwasidentiﬁedatgenuslevelbyMALDITOFwithacutoff

valuewasbetween1.6and1.518_such_as_G._{penicillioides,}_the

onlyGraphiumspeciesinthedatabaseinvestigated.In

addi-tion,G.penicillioidesiscomposedofatleastfourspecies,one

ofwhichisG.basitruncatum.14

Therehavebeenscarcehumancasesandonedogcaseof

infectionwithGraphiumspeciesreportedelsewhere.3–5_Kumar

etal.4_reported_a_case_of_G._{basitruncatum}_fungemia_in_a_patient

withacute leukemia, the onlyexample of aclinical strain

inthe clade withmonomorphic Graphiumspecies showing

associationwithbarkbeetlecommunities.ElFeghalyetal.3

reportedacaseofGraphiumbasitruncatumfungemiaina

two-year-oldchild withdyskeratosis congenita who underwent

stemcelltransplantationtwomonthspriortoinfection.

G. basitruncatum had not been previously reported as a

humanpathogeninourcountry.Thus,thisistheﬁrst

doc-umentedcaseofhumaninfectioninArgentinacausedbythis

pathogen.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

Thisresearch was supportedbyresearch grants from

Uni-versityofBuenosAiresUBACyT20020150100158BA. Weare

grateful to Mr. Santiago Pola for his excellent technical

assistance.

r

e

f

e

r

e

n

c

e

s

1.GuarroJ,KantarciogluAS,HorréR,etal.Scedosporium apiospermum:changingclinicalspectrumofa

therapy-refractoryopportunist.MedMycol.2006;44:295–327.

2.JacobsK,KirisitisT,WingﬁeldMJ.Taxonomicre-evaluationof threerelatedspeciesofGraphium,basedonmorphology, ecologyandphylogeny.Mycologia.2003;95:714–27.

3.ElFeghalyRE,SuttonDA,ThompsonEH,etal.Graphium basitruncatumfungemiainanimmunosuppressedchildpost stem-celltransplantation.MedMycolCaseRep.2012;1: 35–8.

4.KumarD,SiglerL,GibasCFC,etal.Graphiumbasitruncatum

fungemiainapatientwithacuteleukemia.JClinMicrobiol. 2007;45:1644–7.

5.KäuferI,WeberA.Graphiumfructicolaasacausefora systemicmycosisinadog.Mykosen.1977;20:39–46.

6.deHoogGS,GuarroJ,GeneJ,FiguerasMJ.Atlasofclinical fungi.2nded.Utrecht,TheNetherlands:Centraalbureauvoor Schimmelcultures;2000.

7.WhiteTJ,BrunsS,LeeS,TaylorJ.Ampliﬁcationanddirect sequencingoffungalribosomalRNAgenesforphylogenetics. In:InnisMA,GelfandDH,SninskyJJ,WhiteTJ,editors.PCR protocols:aguidetomethodsandapplications.SanDiego: AcademicPress;1990.p.315–22.

8.TheelE,SchmittB,HallL,etal.Formicacid-baseddirect, on-platetestingofyeastandCorynebacteriumspeciesby BrukerBiotypermatrix-assistedlaserdesorption

ionization-timeofﬂightmassspectrometry.JClinMicrobiol. 2012;50:3093–5.

9.RellosoMS,NievasJ,FaresTaieaS,etal.Evaluacióndela espectrometríademasas:MALDI-TOFMSparala identiﬁcaciónrápidayconﬁabledelevaduras.RevArgent Microbiol.2015;47:103–7.

10.CordaAC,PublishedbyIconesfungorumhucusque cognitorum.1.Prague,CzechRepublic:TheAuthor;1837.

11.EllisMB.Dematiaceoushyphomycetes.Kew,Surrey,UK: CommonwealthMycologicalInstitute;1971.

12.SuttonBC,LautJG.GraphiumpenicilloidesonAmericanelmin ManitobaandSaskatchewan.Bi-MonthResNotesCanForest Servo.1970;26:25–6.

13.SuttonBC.HyphomycetesfromManitobaandSaskatchewan. MycolPap.1973;132:1–143.

14.Ceriani-NakamurakareE,SlodowiczM,Gonzalez-AudinoP, DolinkoA,CarmaránC.Mycobiotaassociatedwiththe ambrosiabeetleMegaplatypusmutatus:threattopoplar plantations.Forestry.2016;89:191–200.

15.MatsushimaT,editor.MicrofungioftheSolomonIslandsand Papua-NewGuinea.Kobe,Japan:MatsushimaT;1971.p. 15–20.

16.OkadaG,JacobsK,KiristisT,etal.EpitypiﬁcationofGraphium penicilloidesCorda,withcommentsonthephylogenyand taxonomyofgraphium-likesynnematousfungi.StudMycol. 2000;45:169–88.

17.BaderO.MALDI-TOF-MS-basedspeciesidentiﬁcationand typingapproachesinmedicalmycology.Proteomics. 2013;13:788–99.

18.SchulthessB,BrodnerK,BloembergGV,ZbindenR,Böttger EC,HombachM.IdentiﬁcationofGram-positivecoccibyuse ofmatrix-assistedlaserdesorption-timeofﬂightmass spectrometry:comparisonofdifferentpreparationmethods andimplementationofapracticalalgorithmforroutine diagnostics.JClinMicrobiol.2013;51:1834–40.

19.BuskirkAD,HettickJM,ChipindaI,etal.Fungalpigments inhibitthematrixassistedlaserdesorption/ionization time-of-ﬂightmassspectrometryanalysisofdarkly pigmentedfungi.AnalBiochem.2011;411:122–8.