www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Aspergillus
in
endodontic
infection
near
the
maxillary
sinus
夽
,
夽夽
Cinthya
Cristina
Gomes
a,∗,
Larissa
Christina
Costa
Pinto
a,
Fernanda
Loretti
Victor
b,
Erlange
Andrade
Borges
da
Silva
a,
Apoena
de
Aguiar
Ribeiro
a,
Maria
Inês
de
Moura
Sarquis
c,
Isabel
Coelho
Gomes
Camões
baUniversidadeFederalFluminense(UFF),CampusNovaFriburgo,NovaFriburgo,RJ,Brazil
bUniversidadeFederalFluminense(UFF),Niterói,RJ,Brazil
cFundac¸ãoOswaldoCruz(FIOCRUZ),RiodeJaneiro,RJ,Brazil
Received21June2014;accepted21September2014 Availableonline21July2015
KEYWORDS
Aspergillus; Endodontics; Infection; Maxillarysinus
Abstract
Introduction:Diseasesofthemaxillarysinushavebeenassociatedwithdentalrootsnearthe maxillarysinusthathaveundergoneendodontictreatment.
Objective: Toinvestigatethepresenceoffilamentousfungiinpatientswithdentalrootsnear themaxillarysinuswhohadapicalperiodontitistreatedendodontically,andtoalert practition-ersthatthiscouldbeapossibleavenueofcontaminationofthesinusinpatientswhodevelop maxillarysinusinfection.
Methods:Cross-sectionalstudyin60palatalrootsofthefirstmaxillarymolarsnearthemaxillary sinus,thatunderwentendodontictreatmentforapicalperiodontitis.Afterremovalofthefilling material,dentinshavingswerecollectedandplacedintesttubescontainingSabourauddextrose agarandchloramphenicol. Thephenotype wasdeterminedby macroscopic andmicroscopic examinationofthecolonies.Forpolymerasechainreaction,theprimersITS-5andITS-4were used.ThesequencesobtainedwerecomparedwiththosedepositedatGenBankusingtheBasic LocalAlignmentSearchToolprogram.
夽 Pleasecitethisarticleas:GomesCC,PintoLCC,VictorFL,daSilvaEAB,RibeiroAA,SarquisMIM,etal.
Aspergillusinendodonticinfection nearthemaxillarysinus.BrazJOtorhinolaryngol.2015;81:527---32.
夽夽Institution:SchoolofDentistry,UniversidadeFederalFluminense(UFF),CampusNovaFriburgo,NovaFriburgo,RJ,Brazil. ∗Correspondingauthor.
E-mail:[email protected](C.C.Gomes). http://dx.doi.org/10.1016/j.bjorl.2015.07.013
Results:Filamentousfungiwereisolatedfrom6of60canals (10%):Aspergillusniger(6.7%),
Aspergillusversicolor(1.6%),andAspergillusfumigatus(1.6%).
Conclusion:Rootcanals nearthemaxillarysinuswithendodontictreatmentandapical peri-odontitismayexhibitpositiveculturesforfilamentousfungi.Interestedprofessionalsshouldbe alert,becausethesemicroorganismshavepathogeniccharacteristicsthatcancausediseaseof odontogenicorigininthemaxillarysinus.
© 2015Associac¸ãoBrasileira de Otorrinolaringologiae CirurgiaCérvico-Facial. Publishedby ElsevierEditoraLtda.Allrightsreserved.
PALAVRAS-CHAVE
Aspergillus; Endodontia; Infecc¸ão; Seiomaxilar
Aspergilluseminfecc¸õesendodônticaspróximasaoseiomaxilar
Resumo
Introduc¸ão:Doenc¸asdoseiomaxilartêmsidoassociadasàraízescomtratamentoendodôntico próximasaoseiomaxilar.
Objetivo:Investigarapresenc¸adefungosfilamentososemraízescomtratamentoendodôntico elesãoperiapical,próximasaoseiomaxilar,alertandoparaumapossívelcontaminac¸ãodoseio maxilarporviaodontogênica.
Método: Estudotransversalemsessentaraízespalatinasdeprimeirosmolaressuperiores próxi-masaoseiomaxilar,comtratamentoendodônticoelesãoperiapical.Apósremoc¸ãodomaterial obturador,raspasdedentinaforamcoletadas einseridasem tubosdeensaiocontendoAgar
SabouraudDextroseeClorafenicol. Ofenótipofoideterminadopelaanálisemacroscópicae microscópicadascolônias.ParaoPCRutilizou-seiniciadoresITS-5eITS-4.Assequenciasobtidas foramcomparadasasdisponíveisnoGenBankutilizandoBasicLocalAlignmentSearchTool.
Resultados: Fungosfilamentosos foramisolados de 6dos 60 canais (10%):Aspergillus niger
(6,7%),Aspergillusversicolor(1,6%)eAspergillusfumigatus(1,6%).
Conclusão:Raízespróximasaoseiomaxilarcomtratamentoendodôntico elesão periapical, podem apresentar culturapositivapara fungosfilamentosos.Profissionais afinsdevemestar alerta,poisestemicro-organismopossuemcaracterísticasdepatogenicidadepodendocausar doenc¸asnoseiomaxilardeorigemodontogênica.
©2015Associac¸ãoBrasileira deOtorrinolaringologiaeCirurgiaCérvico-Facial.Publicadopor ElsevierEditoraLtda.Todososdireitosreservados.
Introduction
Approximately 10---12% of cases of maxillary sinusitis are caused by odontogenicinfection, due tothe proximity of the roots of posterior maxillary teeth with the maxillary sinuscavities.1Apicalandmarginalperiodontitisconstitute
83%ofodontogenicfactorsthatmaycausesignsinmaxillary sinuses.2,3Luetal.3 showedadirectrelationshipbetween
presenceof apicalperiodontitis andmaxillarysinus mem-branethickening,atarateof100%.Theauthorsbelievethat microorganismsandtoxinspresentinendodonticinfections canseepintothemaxillarysinusdirectly orthroughtheir countlessvascularanastomoses,alveolarbonemarrow,and lymphvessels.
Althoughbacteriaarethemostextensivelystudied eti-ologicalagents inendodonticinfections,fungi alsocanbe isolatedfromroot canals.4---9 The presence of filamentous
fungiinrootchannelsofteethwithpulpnecrosisand api-cal periodontitis was first detected by Gomes et al. in 2010.10
Thereareseveral articlespublishedin themedical lit-eraturethatlink thepresence offilamentous fungi inthe maxillarysinus withendodontically treatedroot canalsin closecontactwiththe maxillarysinus.11---16 Recent studies
havereportedthatendodontictreatmentinposterior max-illaryteethisastrongriskfactorforonsetofafungusball withinthemaxillarysinus.12,13
However,theroleofendodontic infectionsinmaxillary sinuses is notvery clear.Based onthesereports, the aim of this study was toinvestigate the presence of filamen-tousfungiinendodonticallytreatedrootcanalswithapical periodontitis,locatednearthemaxillarysinus.
Methods
ThisresearchwasapprovedbytheResearchEthics Commit-tee (CEPCMM/HUAP 134/08CAAE 0101.0.258.000-08). All participantscompletedahealthquestionnaireandsignedan informedconsent.Forthisstudy,60(24femaleand36male) patients aged 18---65 years were selected; these patients wereundertreatmentattheSchoolofDentistry.
Studydesign
Criteriaforinclusion
Palatine roots of first maxillary molars that were radio-graphically in close contact with maxillary sinus, with endodontictreatmentperformedovera3-yearperiodprior tothestudyandwithevidenceofapicalperiodontitis.
Exclusioncriteria
Teethwithpulpchamberexposedtooralcavity,with symp-tomsofpain,presenceofmobility,andperiodontalpocket. Patientstreatedwithoralantibioticsand/orantifungalsin thelastsixmonthsandthosewithclinicalmanifestationsof oralcandidiasiswerealsoexcluded.
Samplecollection
Theselectedteethwereaccessedafterreceivingabsolute isolation, antisepsiswith 5.25% sodium hypochlorite, and neutralization with5% sodiumthiosulfate. Access to root canals wasperformed withthe use of a high-speed, car-bide,rounddrillbit,andthentheoperativefieldwasagain cleaned with the aforementioned solutions. The palatine root canal was irrigated with sterile saline; according to a pre-established length calculated in an initial radiogra-phy,gutta-perchawasremovedwithoutusingsolvent,using Gates-Gliddenburs#2and#3andHedströenfiles#25,#30 and#40.Then,aKerrfileofcompatiblediameterandlength fortherootcanalwaspassivelyintroduced,andthe odon-tometry wasestablished with theuse of an apex locator, withradiographicconfirmation.
FollowingtheprotocolrecommendedbyGomesetal.,10
thechannelwasirrigatedwithsterilesalineandaHedströen file #40wasused toremovedentinshavings. Then,three sterile absorbentpaperpointswereinserted intothe root canal,onebyone,1mmshortoftherootapex,and main-tainedthereforoneminute.Thesepaperpointswerethen transferredtoatesttubecontainingSabouraudagar(Difco Laboratories, Detroit, MI, United States) with additionof 0.5%chloramphenicol(Medleyk Campinas,SP, Brazil),in a two-lampisolatedfield.An openPetridish containingthe sameculture medium (Sabouraudagarplus 0.5% chloram-phenicol) wasplaced withinthe fieldisolated bythe two lampsinordertoverifytheaccuracyofisolationofthe oper-ativefield(negativecontrol).Atthesametime,apositive control,includingaPetridishcontainingthesameculture mediumbutwhichremainedopenoutsidetheisolatedfield duringthecollectionofsamples,wasconducted,inorderto checkthegrowthofpotentialenvironmentalfungi,verifying whethertheseorganismscoincidedwiththoseisolatedfrom rootcanals.Petridishesandtesttubeswerekeptatroom temperature for 7---14 days; during this period, mycelial growthwasobservedforsubsequentisolateanalysisatthe FungalCultureCollectionLaboratory.
Directexaminationofcolonies
Fromamonosporicculture,acolonyfragmentwascollected andplacedonaslide.AdropofAmann’slactophenol-cotton blue cytoplasmic stain was added (Difco Laboratories,
Detroit, MI, UnitedStates) and an opticalmicroscope (YS 100;NikonCorporation,Japan)wasusedtoobserve vegeta-tiveandreproductivemyceliumofthefungi.
Macroscopicidentification
Aftercheckingthecharacteristicsofthefungi, thestrains weresubcultured withtheaid of a sterile platinumloop, and one fragment of fungus was inoculated on a Petri dishcontaining20mLofaspecificmediumtofacilitateits growthand sporulation. Depending on the characteristics presented,a specific culture medium wasused, including maltextractagar,Czapekagar,or potatoagar(Difco Lab-oratories, Detroit, MI, United States). The colonies were incubatedat roomtemperature andobserved every 24h, beingaware that each strain has a specific growth time, dependingonitstaxonomicgroup.Then,thecolonieswere analyzed and classified macroscopically with respect to texture,color(frontandbackside),diameter,pigment pro-duction,growthtime,andproductionor nonproductionof exudate.
Microscopicidentification
Mycelial fragments of each isolate were carefully exam-ined in preparations obtained from microcultures in a specific medium, according to the Rivalier and Seydel technique.17 This methodology shows the arrangement
of conidia in situ, allowing the morphological visualiza-tionof reproductive and/or propagation structuresunder optical microscopy (YS 100; Nikon Corporation, Japan), after slide preparation and application of a cytoplasmic stain,Amann’slactophenol-cottonblue(DifcoLaboratories, Detroit,MI,UnitedStates).Frommorphologicalmacroscopic andmicroscopicstudiesandwiththeaimofthespecific lit-erature,thetaxonomicidentificationofisolatedfungiwas performed.18---20
Molecularidentification---DNAextraction
Aimingtoconfirmthetaxonomicidentification,sequencing ofisolateswasperformed.DNA extractionwasperformed accordingYamakamietal.21Approximately50mLofculture
medium were inoculatedwith a conidium suspension and incubated at 30◦C×72h. The mycelium was transferred
toa 50-mLpolypropylene vialcontainingsix glassspheres (diameter4mm). The tube was immersedin liquid nitro-genfor10sandagitatedinavortexfor20s.Then,2mLof DNAextractionbuffer(containing10mMTris---HCl[pH8.0], 10mMEDTA,0.15MNaCl,2%sodiumdodecylsulfate[SDS], and0.5mg proteinaseKper mL [Sigma Chemical Co.,St. Louis,MO,UnitedStates])wereadded.Then,themixture wasallowedtostandfor thawing. This mixturewas incu-batedat55◦C
×2h.ProteinaseKwasinactivatedbyheating
themixtureat 95◦C×10min,andavolumeof0.7mLwas
transferredtoa1.5-mLmicrocentrifugetube;andanequal volumeofphenol---chloroform(1:1)wasadded.Themixture wascentrifugedat10,000gat4◦C
×5min.Thesupernatant
DNAwasprecipitatedwithtwovolumesofethanolat−20◦C
andcentrifugedat12,000×gat4◦C×20min.Thus,the
pel-letobtainedwasdried.Afterwashingwith70%ethanolat 4◦C,theextractedDNAwasdissolvedinto50Lofdistilled
waterand5Lofsuspension,andusedforpolymerasechain
reaction(PCR).
AmplificationofITSregionbyPCR
Amplificationreactions were performed in a final volume of25Lcontaining10pmolofeachprimer;1.5mLofMgCl2 (25mM);1.0LofdNTPs(5mM);2.5Lof10×buffer(Taq);
1unitofTaqDNApolymerase,and20ngofDNA(Fermentas Inc.).PrimersITS5 and ITS4(ITS5 --- 5′-GGA AGTAAAAGT
CGT AAG AAC G-3′, ITS4 --- 5′-TCC TCC GCT TAT TGA TAT
GC-3′) were used. PCRs were performed in a T100
ther-mocycler(Bio-RadLaboratories BrasilLtda.), programmed withthefollowingsteps:pre-denaturationat94◦C
×5min,
30 cycles (denaturation at 94◦C
×30s), 40---55◦C with an
increaseof0.5◦C/s(totaling30s)(annealing),andextension
(72◦C/1min
×30s).The finalextensionwascarriedoutat
72◦C×4min(29).Theamplificationreactionproductswere
detected using agarose gel electrophoresis in 1% 1× TBE
buffer(Tris---borateEDTA),and an electrophoresisanalysis wasperformedat36V×2h.A1-KbDNAmolecularmarker
wasused.Thegelwasstainedwithethylbromide0.5g/mL
for 15min, visualized with the aid of an ultraviolet (UV) transilluminator, and photographed with an image detec-tion and analysis automatic system (Gel Doc XR+System with Software; ImageLab, Bio-Rad Laboratories Brasil Ltda.).
Sequencing
RCPproducts(6L)weretreatedwiththeenzymes
exonu-cleaseI(USB;3.3L)andshrimpalkalinephosphatase(SAP, USB;0.66L)inordertoeliminateremnantsofprimersandof dNTPs.The tubescontainingthemixtureweremaintained at37◦C×45minandthenat80◦C×15min.Inthe
sequenc-ingreaction,fluorescentdideoxynucleotidewasusedwith anABIPrism377automaticsequencermodel.Thereactions wereperformedinafinalvolumeof10Lcontaining0.5mL
ofprimersDYEnamicETTerminatorSequencePremix (DYE-namicETDyeTerminatorCycleSequencingKit; Amersham Bioscience).The T100 thermocycler (Bio-RadLaboratories BrasilLtda.)was programmedin 30 cyclesof 96◦C×30s,
40◦C
×30s, and 60◦C×1min. An aliquot (20L) of the
sequencingreactionwastransferredtoasteriletube,with additionof20LofMilliQ®waterand60Lofisopropanol.
Themixturewashomogenizedbyvortex,leftfor20minat roomtemperature,andthen centrifuged.Thesupernatant wasremovedandtheprecipitatewashedtwicewith250L
of70%ethanolanddriedinanovenat37◦C.TheDNAwas
dissolvedin4Lof FormanideLoadingDye andsubjected
toelectrophoresisinanABIPRISM377automaticsequencer (Applied Biosystems). The obtained sequences were ana-lyzedusingtheBasicLocalAlignmentSearchToolprogram (BLASTn)andthesequencesobtainedwerecomparedwith thosedepositedatGenBank.
Figure1 Aspergillusspp.macroscopicimageinmaltextract agarmediumat37◦C.
Results
The growth of filamentous fungi was observed in six out of60analyzedpalatineroots (10%).Macroscopic examina-tionofisolatesshowedgrowthofcoloniescompatiblewith
Aspergillusspp.(Fig.1).Microscopicexaminationofisolates showed growth of hyaline, septate, and globous conidio-phorehyphae,characteristicsofAspergillusgenus(Fig.2). The homology with sequences of filamentous fungi, ana-lyzed by BLAST and compared with sequences deposited at GenBank, was: 100% for Aspergillus niger (GenBank No.AJ223852)infoursamples,100%forAspergillus versi-color(GenBank No.JX232271)inonesample, and99%for
Aspergillusfumigatus(GenBankNo.AB025434)inone sam-ple.
Afterphenotypicandgenotypicanalyzes,thefilamentous fungiisolatedwereclassifiedasA.niger(6.7%),A.versicolor
(1.6%),andA.fumigatus(1.6%).
Discussion
Althoughseveralstudieshavealreadypointedtothe pres-enceofyeast4---9,22---24andfilamentousfungi10 inendodontic
infections,nostudysofarhadcheckedthepresenceof fil-amentous fungi in root canals withendodontic treatment andapicalperiodontitis,althoughtherearereportsin the literatureshowingthatlowzincconcentrations,asfoundin mostendodonticsealers,provideanidealenvironmentfor thedevelopmentofthesepathogens.25
The present study resorted to palatine roots of first maxillary molars because the apexes of these roots are anatomicallyclosertothefloorofthemaxillarysinus.3,26,27
Thus,thehypothesiswasthatteethwithendodontic treat-ment, withapical periodontitis and in close contactwith themaxillarysinus,couldresultinapositiveculturefor fila-mentousfungi,sincethesepathogensarecommonlyisolated fromthemaxillarysinus.
The results of this study may serve as a warning to dentistsandotolaryngologists,sinceAspergillusorganisms are also isolated from the maxillary sinus in cases of chronicsinusitis,andaerogenicandodontogenicpathways aredescribed asthemain routesof entry.26 Several
stud-ies have emphasized that the low concentration of zinc found in root canal sealers, based on zinc oxide plus eugenol,can facilitatethe fungusinstallationinthe max-illarysinus membrane.11---13,15,16 Leakage offilling material
into the maxillary sinus during endodontic treatment has alsobeen suggestedasacauseofchronic sinusitisby for-eign body.2,11---13 Moreover,the amount of eugenol usedin
endodonticsealershasbeendirectlyrelatedtothegrowth oftheAspergillusorganism,causativeofmycetomawithin themaxillarysinus.15,16
Thisproximityofthepalatalrootsofuppermolarscould encouragethemigrationofthesefilamentousfungiisolated fromrootcanalstothemaxillarysinus;ontheotherhand, existingfungiinthemaxillarysinuscouldhavetheirgrowth facilitatedby a propitious environment inthe root canal, withthepresenceofnutrientsandsealingmaterials essen-tialforgrowthandspreadingofthesepathogens.Interested professionals should be aware of this scenario, as these organismsexhibitcharacteristicsofpathogenicitythatmay cause maxillarysinus disease fromodontogenic origin, or viceversa.Thus,furtherstudiesshouldbeconductedto cor-relatethepresenceoffilamentousfungiinrootcanalsand thepossibilityofinfiltrationofthesepathogenstowardthe maxillarysinus,alsotoinvestigatethepossibilitythat the maxillarysinuscanactasapathwayforcontaminationthat mayleadtoendodontictreatmentfailure.
Conclusion
Under the experimental conditionsin which this research wasconducted,andbasedontheseresults,itcanbe con-cluded that dental roots in close proximity to maxillary sinuses undergoing endodontic treatment for apical peri-odontitismayexhibitpositiveculturesforfilamentousfungi.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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