Isolation of Candida spp. from denture-related stomatitis in Pará, Brazil

brazilian journal of microbiology49(2018)148–151

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Clinical

Microbiology

Isolation

of

Candida

spp.

from

denture-related

stomatitis

in

Pará,

Brazil

Lurdete

Maria

Rocha

Gauch

_,

_Simone

_Soares

_Pedrosa

_,

_Fabíola

_{Silveira-Gomes}

_,

Renata

Antunes

Esteves

_,

_Silvia

_Helena

_{Marques-da-Silva}

a_{FederalUniversityofPará,ScienceHealthInstituteBelém,OdontologyCollege,Belém,PA,Brazil}

b_{FederalUniversityofPará,ScienceBiologicInstitute,PostgraduatePrograminBiologyofInfectiousandParasiticAgents,Belém,PA,}

Brazil

c_{EvandroChagasInstitute,BacteriologyandMycologySection,MycologyLaboratory,Belém,PA,Brazil}

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Articlehistory:

Received27November2013 Accepted2May2016 Availableonline29July2017 AssociateEditor:CarlosPelleschi Taborda Keywords: Candidaalbicans Candidanon-albicans Stomatitis

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TheaimofthisstudywastoisolateandidentifyCandidaspeciesfromtheoralcavityof den-turewearerswithdenture-relatedstomatitiswhowereattendedattheUniversityFederal ofPará(BelémCity,ParáState,Brazil).Atotalof36denturewearerswithdenture-related stomatitiswereincluded,andtypeI(50%),typeII(33%)andtypeIII(17%)stomatitiswere observed.Candidaspp.wereisolatedfrom89%ofthecasesandincludedfivedifferent

Candidaspecies.C.albicanswasthemostfrequentlyrecoveredspecies(78%ofthecases), followedbyC.famataandC.tropicalis.WeobservedasignificantassociationbetweenCandida

speciesisolationandunsatisfactorydenturecondition(p=0.0017).Ourresultsdemonstrated thehighlyfrequencyofCandidaspeciesisolationindenturewearerswithdenture-related stomatitisandshowedtherelationshipbetweenthesespeciesandpoordenture mainte-nance.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Approximately200Candidaspeciesareknown,and10%can causeinfectionsinhumans.C.albicansisthemostfrequently described species in cases of hospital infections, followed byC.parapsilosis,C.tropicalisandC.glabrata.1_Thefrequency atwhich these species are observed has significant impli-cationsforhumaninfections.Thesespeciesarecommensal

∗ _{Correspondingauthor.}

E-mail:[email protected](L.M.Gauch).

organismsthatconstitutepartofthenormaloralmicrobiota, and they are presentin 30–60%ofhealthy individuals and 60–100%ofpatientswithdentures.2_{Thelong-termuseof} den-tures isthe mostimportant riskfactor forCandidaspecies colonization of the mucosa surface and may be sufficient forthedevelopmentoforalcandidiasis.3_{Oralcandidiasisis} associatedwithmucosaltraumacausedbypoordenturefit, theincreasingageofthedenturewearers,theincreasedage ofthedentures,fungal infections(primarilyC.albicans)and

https://doi.org/10.1016/j.bjm.2017.07.001

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

brazilian journal of microbiology49(2018)148–151

149

poordentalhygiene.4–6_{Inthiscontext,theadherenceof} Can-didatothesurfaceofdenturematerials,suchaspolymethyl methacrylate,facilitatescolonizationbyCandida.7,8_The mech-anismsbywhichC.albicansadheretopolymericsurfaces(e.g., dentures)primarilyincludebiofilmformationand morpho-logicalswitching, whichfacilitatethe colonizationofthese materialsbythefungus.Colonizationisthe mainrisk fac-torforthedevelopmentofdenture-relatedstomatitis(DRS), whichisthemostcommonclinicalmanifestationofCandida

infectionindenturewearers.9

InfectionbyCandidaspp.isfrequentlyobservedinpatients withdenturesandmayleadtosecondaryorallesions,suchas lichenplanus,leukoplakiaandcarcinoma.10_{Candidaspecies} colonization11,12 _{and infections}9 _{inthe oral cavity of} den-turewearershavebeenreportedworldwide,andC.albicans

isparticularlyprevalent.13_{Moreover,theisolationofCandida} species other than C. albicans has been increasing, which is likely because of the misuse of antifungals.14 _{In Brazil,} fewstudieshavedemonstratedtheprofileofCandidaspecies relatedtocolonizationofthedenturesurfaceororalmucosa andtheincidenceoftheselesionsindenturewearers.15 There-fore,wefocusedthisstudy ontheisolatingandidentifying

Candidaspecies from the surface ofdentures and the oral mucosaofdenturewearerswithDRS.

Materials

and

methods

Ethicalaspects

Thestudywas approvedbythe Ethics Committeeof Evan-droChagasInstitute(CEP/IEC032/10)andconductedbetween MarchandOctober2012.Allpatientswereinformedaboutthe studyandprovidedwritteninformedconsent.

Population

Thirty-six(n=36)patientsfittedwithacrylic-baseddentures whopresentedwithdenture-relatedstomatitiswereincluded. Thepatientswerefittedwithcompletedentures(n=32)or par-tiallyremovabledentures(n=4).Alloftheparticipantswere attended atthe dental schoolclinic atthe University Fed-eralofPará (Belém,Pará,Brazil). Analyseswere performed ofthepatient demographicdata, whichincludedage, gen-der,hygiene habits(poor or not),mouthwash use, present denturecondition(satisfactoryorunsatisfactory)and quali-tativecharacteristic(newor olddentures).Thepresenceof DRSwasassessedaccordingtoamodifiedversion of New-ton’sclassification.16_{Theseverityofthepalatalinflammation} wasclassifiedas(1)nostomatitis,whichincludednoevidence ofpalatalinflammationorslightcolorchangeofthepalate mucosa;(2) stomatitistypeI, which includedpetechiae dis-persedthroughoutall orany partofthepalatal mucosain contactwith thedenture; (3)type II, which included mac-ular erythemawithout hyperplasia;and (4) typeIII, which included diffuse or generalized erythema with papillary hyperplasia.

The patient exclusion criteria included the presence of diabetes or autoimmune disease and the use of corticosteroids.

Isolationandidentification

Afteranexaminationoftheoralcavity,dentureandmucosal specimenswereharvestedbyscrapingsterileswabsacrossthe innersurfaceofthedenture(basisofprosthesis,BP)andthe oralmucosa(palatalmucosa,PM)incontactwiththedenture. Subsequently,thespecimenswereculturedinSabouraud dex-trose agar (Difco,Laboratories, Detroit,MI, USA),incubated at35◦Candobserveddailyfor7days.Whenthegrowthof yeastcolonieswasobserved,theGramstainmethodwasused toverifytheabsenceofbacterialcontamination.Theyeasts were identifiedvia carbohydrate assimilationprofiles using theVitek2System(BioMerieuxI’Etoile,France)accordingto themanufacturer’sinstructions.

Yeasts identified as C. dubliniensis were subjected to molecular confirmation because of the close phenotypical relationshipofthisspecieswithC.albicans.Briefly,genomic DNAwasextractedaspreviouslydescribed,17_andwhen nec-essary,molecularidentificationwasperformedasdescribed byMannarelliandKurtzman18_{(C.dubliniensis/forward:CDU2} – 5-AGT TAC TCT TTC GGG GGT GGC CT-3; C. dublinien-sis/reverse:NL4CAL–5-AAGATCATTATGCCAACATCCTAG GTA AA-3) and byLuo and Mitchell19 _{(C. albicans/forward:} CALB1 – 5-TTTATC AAC TTGTCA CAC CAG A-3; C. albi-cans/reverse:CALB2–5-ATCCCGCCT TACCACTACCG-3). Themixwaspreparedtoafinalvolumeof25␮Lasfollows: 10×MgCl2(2␮L),10mMdNTP(1␮L),10×PCRbuffer(2.5␮L),Q

solution(2␮L),TaqDNApolymerase(1U;InvitrogenLife Tech-nologies,Carlsbad,Calif.)andgenomicDNAtemplate(2␮L). Amplificationwasperformedinathermalcycler(TX96plus, Amplitherm, Axigen)asfollows:forC.dubliniensis: 98◦Cfor 3min;followedby35cyclesof95◦Cfor1min,52◦Cfor1.5min, and72◦Cfor10min;andthen72◦Cfor10min;andC.albicans:

96◦Cfor5min;followedby40cycles of94◦Cfor30s,58◦C for30s,and72◦Cfor30s;andthen72◦Cfor15min.ThePCR productswere submittedtohorizontalelectrophoresis.The amplifiedfragmentswere175bpand273bpforC.dubliniensis

andC.albicans,respectively.

Statisticalanalysis

Statistical inferences of the descriptive results were per-formed based on non-parametric tests, such as the G adherenceindependencetest,usingBioEstatversion5.3 (Insti-tutoMaumirauá,Belém,Pará,Brazil).Statisticalsignificance wasconsideredatp≤0.05.

Results

Thirty-six (n=36)denture wearers withDRS were included inthisstudy.Thepatientsrangedinagefrom40to83years (mean age=62 years) and included 12 males (33%)and 24 females(67%).AccordingtoNewton’sclassification,theDRS casesweredistributedasfollows:TypeI(50%),TypeII(33%), and Type III (17%) among the cases. Candida specieswere isolatedfromtheBPonly(17%),FMonly(5%)andBPandFM simultaneously (67%). In four cases (11%), Candida species werenotisolated.Basedonbiochemicalorbiochemicaland molecular identification,weobservedfivedifferentCandida

150

Table1–PresenceofCandidaspeciesintheoral specimensfrom36denturewearerswith denture-relatedstomatitis.

Patient registerno.

Specimencollectionlocation Stomatitis type

BP PM

5 Candidaalbicans WCG TypeI

7 C.albicans C.famata TypeI

8 C.albicans WCG TypeI

10 WCG C.famata TypeI

11 C.albicans C.albicans TypeI 12 C.albicans C.famata TypeI

13 C.albicans WCG TypeII

18 WCG WCG TypeI

19 C.albicans WCG TypeII

22 WCG WCG TypeI

24 C.albicans WCG TypeII

26 C.albicans C.albicans TypeII 27 WCG WCG TypeII 33 C.albicans C.albicans TypeI 34 WCG WCG TypeIII 41 C.parapsilosis C.albicans TypeI 55 C.albicans C.tropicalis TypeI 56 C.albicans C.albicans TypeIII 63 C.albicans C.albicans TypeIII

66 C.tropicalis WCG TypeI

70 C.albicans C.albicans TypeI 74 C.tropicalis C.albicans TypeII 78 C.albicans C.albicans TypeII 81 C.famata C.albicans TypeI 82 C.albicans C.famata TypeI 83 C.albicans C.albicans TypeII 85 C.albicans C.albicans TypeII 86 C.albicans C.albicans TypeII 87 C.albicans C.albicans TypeIII 90 C.famata C.albicans TypeIII 91 C.albicans C.albicans TypeI 92 C.tropicalis C.tropicalis TypeII 94 C.krusei C.parapsilosis TypeIII 96 C.albicans C.albicans TypeI

97 WCG C.albicans TypeII

98 C.tropicalis C.albicans TypeI BP,basisofprosthesis;PM,palatalmucosa;WCG,withoutCandida

growth.

species.C.albicanswasthemostfrequentlyobservedspecies (78%ofthecases),anditwasobservedaloneorin simultane-ousisolationswithC.famata,C.tropicalisorC.parapsilosis.The restrictedisolationofCandidaspeciesotherthanC.albicans

was observedin 11% ofthe cases. All of theseresults are summarizedinTable1.

According to the demographics data, an unsatisfactory dentureconditionwasaninfluencingfactorfortheisolation ofCandida species (p=0.0017); however, gender(p=0.7015), mouthwashuse(p=0.6514),hygienehabits(p=0.3897), con-tinuousdentureuse(p=0.4011)andolddentures(p=0.2502) werenotrelatedtoCandidaspeciesisolation.

Discussion

DRS exhibits a multifactorial etiology and is associated withdenture use, indicating that disease presentationcan be affected by both endogenous and exogenous factors.7,9

Acriticalriskfactor,however,isthecolonizationoftheoral mucosabyCandidaspecies.20_{Inthepresentstudy,Candidaspp.} wereisolatedin89%ofthecasesofdenturewearerswithDRS (accordingtoNewton’sclassification),therebyreinforcingthe relationshipbetweenCandidaspp.andDRS.InBrazilian den-turewearers,DRSwaspreviouslyobservedasthemajorlesion intheoralcavity,21_{whichisconsistentwithourresults.In} thepresentstudy,thefactorthatinfluencedwhetherCandida

wasisolatedwasanunsatisfactorydenturecondition.The fre-quentmaintenanceofdenturesallowsforimprovementsto certainfunctionalcharacteristics,suchastheRVD(rest verti-caldimension)andVDO(verticaldimensionofocclusion)of thepatient,theadhesionofthedenturetothemucosaandthe diminutionoftheresin roughness.Improving thesefactors may decrease microorganismcontamination ofthe surface denture,whichwillinfluencetheoralhealthofdenture wea-rers. Inthis context,Pereira-Cenci etal.22 _{and Parketal.}23 observedthatthecolonizationofdenturesbyCandidaspecies iscausedbysurfaceroughness,whichfacilitatesthe adhe-sionoftheyeasttoresindentures.Becausetheadherenceof

C.albicans toacrylicsurfaceresinsisrelatedtothesurface porosityandroughness,denturesurfacescanbeconsidered aninfection source,and thesefindingsare consistentwith thesignificantcolonizationobservedindenturewearers pre-sentingwithDRSbecauseallpatientswerefittedwithacrylic resindentures.Moreover,theresultssupporttherelationship betweenunsatisfactorydentureconditionandCandidaspecies isolation.

Others factorshavebeenfrequentlyreportedas influen-tial in thedevelopment ofDRS. For example,Bulad et al.4 mentioned that the continuous use of dentures facilitated denture stomatitis because extended mucosal exposureto dental plaques may intensify certain injuries. Additionally, Darwazehetal.24_{observedastrongassociationbetweenpoor} dentalhygieneandCandidacolonizationandsuggestedthat plaqueaccumulationonthedenturesurfacemaycreatean idealenvironmentforyeast.However,anassociationbetween poor hygiene, continuousdenture use or old dentures and the isolation of Candida species was not observed in this study.

Among the colonized denture wearers in our study, C. albicans was the major isolated species, followed by C. famata and C. tropicalis, which were the most frequent non-C.albicansspecies.Theseresultscorroboratedthe find-ings of other studies,11,14,21,25–27 _{including other Brazilian} studies.16,28–30 _{Additionally, Sant’Ana et al.}31 _{reported that} the presence of two or more species in the same patient can predispose the patient to recurrent stomatitis, and double isolations were also observed in denture wearers with DRS in the present study. Moreover, C. famata was observed inthePM ofdenturewearers who exhibitedDRS and found in association with C. albicans. These results demonstratedthepotentialforemergentpathogensincases ofDRS.

Weobserved asignificant frequencyofDRSinBrazilian patientscolonizedbyCandidaspecies,andtheinfectionwas primarilycausedbyC.albicans.Theresultsindicatethatinthe observedpatients,DRSdevelopedalongapathwaythatwas dependentontheunsatisfactorystateoftheprosthesisand thecolonizationoftheoralcavitybyCandidaspecies.

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Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Funding

Thepreparationofthismanuscriptwassupportedbythe Post-graduatePrograminBiologyofInfectiousandParasiticAgents, FederalUniversityofPará(EditalPAPQ/2013-UFPA).

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s

1.MalucheME,SantosJI.Candidasppeinfecc¸õeshospitalares:

aspectosepidemiológicoselaboratoriais.RBAC.

2008;40:65–67.

2.LosterBW,LosterJ,WieczorekA,RyniewiczW.Mycological

analysisofthecavityofpatientsusingacrylicremovable

dentures.GastroenterolResPract.2012:951572.

3.AbaciO,OzturkB,BoyaciogluH.DeterminingCandidaspp.

incidenceindenturewearers.Mycopathologia.

2010;169:365–372.

4.BuladK,TaylorRL,VerranJ,McCordJF.Colonizationand

penetrationofdenturesoftliningmaterialsbyCandida

albicans.DentMater.2004;20:167–175.

5.JeganathanS,LinCC.Denturestomatitis–areviewofthe

etiology,diagnosisandmanagement.AustDentJ.

1992;37:107–114.

6.WebbBC,ThomasCJ,WillcoxMDP.Candida-associated

denturestomatitis.Aetiologyandmanagement:areview.

PartI.FactorsinfluencingdistributionofCandidaspeciesin

theoralcavity.AustDentJ.1998;43:45–50.

7.SilvaS,HenriquesM,HayesA,OliveiraR,AzeredoJ,Williams

DW.CandidaglabrataandCandidaalbicansco-infectionofan

invitrooralepithelium.JOralPatholMed.2011;40:421–427.

8.WatersMG,WilliamsDW,JaggerRG,LewisMA.Adherenceof

Candidaalbicanstoexperimentaldenturesoftlining

materials.JProsthetDent.1997;77:306–312.

9.GendreauL,LoewyZG.Epidemiologyandetiologyofdenture

stomatitis.JProsthodont.2011;20:251–260.

10.SpolidorioLC,MartinsVRG,NogueiraRD,SpolidorioDMP.

FrequênciadeCandidasppembiópsiasdelesõesdamucosa

oral.PesqOdontBras.2003;17:89–93.

11.NavasEAFA,InocêncioAC,DiasJ,etal.Oraldistributionof

CandidaspeciesandpresenceoforallesionsinBrazilian

leprosypatientsundermultidrugtherapy.JOralPatholMed.

2009;38:764–767.

12.ReichartPA,SamaranayakeLP,BendickCH,

Schmidt-WesthausenAM,JayatilakeJA.Prevalenceoforal

CandidaspeciesinleprosypatientsfromCambodiaand

Thailand.JOralPatholMed.2007;36:342–346.

13.MartinsM,HenriquesM,RibeiroAP,etal.OralCandida

carriageofpatientsattendingadentalclinicinBraga,

Portugal.RevIberoamMicol.2010;27:119–124.

14.SnydmanDR.Shiftingpatternsintheepidemiologyof

nosocomialCandidainfections.Chest.2003;123:500S–503S.

15.SanitáPV,PavarinaAC,GiampaoloET,etal.Candidaspp.

prevalenceinwellcontrolledtype2diabeticpatientswith

denturestomatitis.OralSurgOralMedOralPatholOralRadiol

Endod.2011;111:726–733.

16.NewtonAV.Denturesoremouth:apossibleetiology.BrDent

J.1962;112:357–360.

17.SambrookJ,FritschEF,ManiatisT.MolecularCloning:A

LaboratoryManual.2nded.ColdSpringHaborLaboratory

Press;1986.

18.MannarelliBM,KurtzmanCP.RapididentificationofCandida

albicansandotherhumanpathogenicyeastsbyusingshort

oligonucleotidesinaPCR.JClinMicrobiol.1998;36:1634–1641.

19.LuoG,MitchellTG.Rapididentificationofpathogenicfungi

directlyfromculturesbyusingmultiplexPCR.JClinMicrobiol.

2002;40:2860–2865.

20.ZomorodianK,HaghighiNN,RajaeeN,etal.Assessmentof

Candidaspeciescolonizationanddenture-relatedstomatitis

incompletedenturewearers.MedMycol.2011;49:208–211.

21.FerreiraRC,MagalhãesCS,MoreiraNA.Oralmucosal

alterationsamongtheinstitutionalizedelderlyinBrazil.Braz

OralRes.2010;24:296–302.

22.Pereira-CenciT,CuryAA,CenciMS,Rodrigues-GarciaRC.

InvitroCandidacolonizationonacrylicresinanddenture

liners:influenceofsurfacefreeenergy,roughness,saliva,

andadheringbacteria.IntJProsthodont.2007;20:308–310.

23.ParkSE,BlissettR,SusarlaSM,WeberH-P.Candidaalbicans

adherencetosurface-modifieddentureresinsurfaces.J

Prosthodont.2008;17:365–369.

24.DarwazehAMG,HammadMM,Al-JamaeiAA.The

relationshipbetweenoralhygieneandoralcolonizationwith

Candidaspeciesinhealthyadultsubjects.IntJDentHyg.

2010;8:128–133.

25.DagistanS,AktasAE,CaglayanF,AyyildizA,BilgeM.

Differentialdiagnosisofdenture-inducedstomatitis,Candida

andtheirvariationsinpatientsusingcompletedenture:a

clinicalandmycologicalstudy.Mycoses.2008;52:266–271.

26.GePY,HeXG,LinT,LuXG,ShenNY,LiuWD.Firstisolationof

Candidadubliniensisfromoralcavitiesofdermatological

patientsinNanjing,China.Mycopathologia.2011;172:465–471.

27.KadirT,PisiricilerR,AkyüzS,YaratA,EmekliN,IpbükerA.

Mycologicalandcytologicalexaminationoforalcandida

carriageindiabeticpatientsandnon-diabeticcontrol

subject:thoroughanalysisoflocaletiologicandsystemic

factors.JOralRehabil.2002;29:452–457.

28.LyonJP,MoreiraLM,CardosoMAG,SaadeJ,ResendeMA.

AntifungalsusceptibilityprofileofCandidaspp.oralisolates

obtainedfromdenturewearers.BrazJMicrobiol.

2008;39:668–672.

29.MenezesEA,CavalcanteMS,FariasRB,etal.Frequencyand

enzymaticactivityofCandidaalbicansisolatesfromthe

buccalmucosaofchildrenofaday-carecenterofthecity

hallofFortaleza,Ceará,Brazil.JBrasPatholMedLab.2005;41:

9–13.

30.PiresFR,SantosEB,BonanPR,DeAlmeidaOP,LopesMA.

DenturestomatitisandsalivaryCandidainBrazilian

edentulouspatients.JOralRehabil.2002;29:1115–1119.

31.Sant’AnaPL,MilanEP,MartineR,etal.MulticenterBrazilian

StudyoforalCandidaspeciesisolatedfromAidspatients.