ww w . r e u m a t o l o g i a . c o m . b r
REVISTA
BRASILEIRA
DE
REUMATOLOGIA
Original
article
Systemic
and
localized
infection
by
Candida
species
in
patients
with
rheumatic
diseases
receiving
anti-TNF
therapy
Nadia
E.
Aikawa
a,b,∗,
Daniela
T.A.
Rosa
c,d,
Gilda
M.B.
Del
Negro
c,d,
Julio
C.B.
Moraes
b,
Ana
C.M.
Ribeiro
b,
Carla
Gonc¸alves
Saad
b,
Clovis
A.
Silva
a,b,
Eloisa
Bonfá
baUniversidadeSãoPaulo,FaculdadedeMedicina,UnidadedeReumatologiaPediátrica,SãoPaulo,SP,Brazil
bUniversidadeSãoPaulo,FaculdadedeMedicina,DisciplinadeReumatologia,SãoPaulo,SP,Brazil
cUniversidadeSãoPaulo,FaculdadedeMedicina,LaboratóriodeMicologiaMédica(LIM53),SãoPaulo,SP,Brazil
dInstitutodeMedicinaTropicaldeSãoPaulo,SãoPaulo,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received18July2014 Accepted1March2015
Availableonline8September2015
Keywords:
Systemiccandidiasis Candidaspp. Anti-TNF
Rheumatoidarthritis Ankylosingspondylitis
a
b
s
t
r
a
c
t
Objective:ToevaluatetheprevalenceofsystemicandlocalizedinfectionbyCandidaspecies anditspossibleassociationwithdemographic,clinicalandlaboratorymanifestationsand therapyinpatientswithrheumaticdiseasestakingTNFblockers.
Methods:Consecutive patientswith rheumatic diseasesreceiving anti-TNFagents were included.Thefollowingriskfactorsuptofourweekspriortothestudywereanalyzed:use ofantibiotics,immunosuppressantdrugs,hospitalizationandinvasiveprocedures.All sub-jectswereevaluatedforclinicalcomplaints;specificbloodcultureswereobtainedforfungi andbloodsampleswerecollectedforCandidaspp.detectionbypolymerasechainreaction. Results:194patients[67withrheumatoidarthritis(RA),47withankylosingspondylitis(AS), 36withjuvenileidiopathicarthritis(JIA),28withpsoriaticarthritisand16withother con-ditions]wereincluded.Theaverageageofpatientswas42±16years,with68(35%)male andmeandiseasedurationof15±10years.Sixty-four(33%)patientswerereceiving adal-imumab,59(30%)etanerceptand71(36%)infliximab.Eighty-onepercentofpatientswere concomitantlytakingimmunosuppressantdrugs.Atthetimeofthestudy,onlyone(0.5%) patienthadlocalizedfungalinfection(vaginalcandidiasis).Noneofthepatientsincluded hadsystemiccandidiasiswithpositivebloodculturesforfungiorPCRpositiveforCandida spp.inperipheralbloodsample.
Conclusions:Thiswasthefirststudytoassesstheprevalenceofinvasiveandlocalized fun-galdiseasebyCandidain asignificant number ofpatientswith rheumaticdiseases on anti-TNFtherapy,anddemonstratedlowriskofcandidiasis,despitethehighprevalence ofimmunosuppressivedruguse.
©2015ElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mails:[email protected],[email protected](N.E.Aikawa). http://dx.doi.org/10.1016/j.rbre.2015.08.004
Infecc¸ão
sistêmica
e
localizada
por
Candida
spp
.
em
pacientes
reumatológicos
em
terapia
anti-TNF
Palavras-chave: Candidíasesistêmica Candidaspp. Anti-TNF
Artritereumatoide Espondiliteanquilosante
r
e
s
u
m
o
Objetivo: Avaliaraprevalênciadeinfecc¸ãosistêmicaelocalizadaporCandidaspp.esua possívelassociac¸ãocomdadosdemográficos,manifestac¸õesclínicaselaboratoriaise ter-apêuticaempacientescomdoenc¸asreumatológicasemusodeanti-TNF.
Métodos: Foramincluídospacientesconsecutivoscomdoenc¸asreumatológicasemusode agentesanti-TNF.Foramanalisadososseguintesfatoresderiscoatéquatrosemanasantes doestudo:usodeantibioticoterapia,imunossupressores,hospitalizac¸ãoeprocedimentos invasivos.Todosforamavaliadosparaqueixasclinicas,coletaramhemoculturaespecífica parafungoseamostrasdesangueparapesquisadeCandidaspp.porreac¸ãoemcadeiade polimerase.
Resultados: Foramincluídos194pacientes[67comartritereumatoide(AR),47espondilite anquilosante (EA), 36 artrite idiopática juvenil (AIJ), 28 artrite psoriásica e 16 outros]. Amédiadeidadeerade42±16anos,com68(35%)dosexomasculinoemédiadedurac¸ão dedoenc¸ade15±10anos;64(33%)pacientesusavamadalimumabe,59(36%)etanerceptee 71(36%)infliximabe;81%faziamusoconcomitantedeimunossupressores.Nomomentodo estudo,apenasum(0,5%)pacienteapresentouinfecc¸ãofúngicalocalizada(candidíase vagi-nal).Nenhumdospacientesincluídosapresentoucandidíasesistêmicacomhemocultura positivaparafungosouPCRpositivaparaCandidaspp.emamostradesangueperiférico. Conclusões: Estefoioprimeiroestudoqueavaliouprevalênciadedoenc¸afúngicainvasivae localizadaporCandidaemumexpressivonúmerodepacientesreumatológicosemterapia anti-TNFedemonstroubaixoriscodecandidíase,apesarda altaprevalênciadeusode imunossupressores.
©2015ElsevierEditoraLtda.Este ´eumartigoOpenAccesssobumalicenc¸aCC BY-NC-ND(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Anti-TNF therapy has been widely used in patients with
rheumaticdiseaseswhodonotrespondtodisease-modifying antirheumaticdrugs(DMARDs),withasignificant
improve-ment in prognosis. However, at the same time, concerns
relatedtoimmunosuppressionduetosuchtherapyemerge, generatingincreasingreportsofinfections byopportunistic fungalagentssuchasCandidaspp.1
Thisfunguscanproduceawidespectrumofclinical pre-sentations,rangingfromsuperficialmucocutaneousdisease to severe invasive infections. Systemic candidiasis affects
mainly patients treated with immunosuppressive drugs,
includingglucocorticoidsandbroad-spectrumantibiotics,and submittedtoinvasiveprocedures,withhighmortalityrates.2–5 Candidemiahaspreviouslybeenreportedinsmallseriesof patientsonbiologicaltherapywithTNFblockers,butaclear causalrelationshiphasnotbeenestablished.6,7 Inaddition, therearerarereportsofsystemicinfectionbyCandidaspp.in patientswithrheumaticdiseases.8,9
Theaimofthisstudywastodeterminetheprevalenceof systemicandlocalizedcandidiasisanditspossibleassociation withdemographics, clinical and laboratorymanifestations, and therapyin patientswith rheumaticdiseases in use of anti-TNFagents.
Materials
and
methods
Consecutivepatientswithadiagnosisofrheumatoidarthritis (RA),10ankylosingspondylitis(AS),11psoriaticarthritis(PsA),12 juvenileidiopathicarthritis(JIA),13andwithotherconditions (Behcet’sdisease,Crohn’sdisease,reactivearthritis,Still’s dis-ease,idiopathicuveitisandTakayasuarteritis)wereregularly followedintheRheumatologyOutpatientClinicatthe Hos-pitaldasClinicas,MedicalSchool,UniversidadedeSãoPaulo. Allpatientsweretreatedwithanti-TNFagents(adalimumab, etanerceptorinfliximab)attheCenterofDispensationofHigh CostMedications(CEDMAC)andwereinconcomitantuseof disease-modifyingantirheumaticdrugs(DMARDs).
This study was approved by the Institutional Ethics
Committee,andinformedconsentwasobtainedfromall par-ticipants.
Evaluationofrheumaticdiseases
Thediseaseactivitywasassessedusingstandardized
instru-ments, including the Disease Activity Score (DAS28) for
(mm/1sthour)and C-reactiveprotein(CRP) levelsby neph-elometry(mg/L).
The current treatment (concomitant toanti-TNF agent)
with prednisone, DMARDs and immunosuppressant drugs
(methotrexate,azathioprine,leflunomide, antimalarial, sul-fasalazineand/orcyclosporine)wasalsoevaluated.
Evaluationofcandidiasisandriskfactors
Patientswereclinicallyevaluatedonthedayofapplicationof anti-TNFtherapyforsigns andsymptomsofinfection with Candidaspp., includingoralcandidiasis,genitalcandidiasis, orcharacteristicdysuriaorleucorrhea.Uptofourweeksprior tothestudy,thefollowingriskfactorswereanalyzed:useof antibiotics,useofimmunosuppressantdrugs,hospitalization andinvasiveprocedures.
1. Fungalperipheralbloodculture:Fungalperipheralblood cul-tureinthepopulationusinganti-TNFagentwascarriedout
withBDBACTECTM-MYCO/FLyticMedium(Becton
Dickin-son,USA)bloodculturesflasks.
2. PCRforCandidaspp.inperipheralblood:DetectionofCandida spp.DNAwasperformedbypolymerasechainreaction.16 Theanalysisofthecollectedmaterialwasheldatthe Medi-calResearchLaboratoryofMycology(LIM-53)ofHC-FMUSP inpatientsundergoinganti-TNF-alphatherapy.
DNA extractions from whole blood samples of those
patientsincludedinthisstudywereperformedaccordingto the technique described byLoeffler et al.17 Blood samples (3–5ml) were collected inEDTA tube and subjected to ini-tialhemolysiswith1.55MNH4Cl,100mMKHCO3,and10mM
EDTApH 7.4. After obtainingthe erythrocyte-freecell pel-let, 3ml of a solution containing 100mM Tris–HCl pH 8.0,
4M NaCl, and 20mM EDTA pH 8.2 were added. Next, the
samples were vigorously stirred, and 600L of a solution containingTRIS50mM, 10mMEDTA,and lyticase250U/ml (L-4276,Sigma,USA)and5%-mercaptoethanolwereadded. Thetubeswerekeptat37◦Cfor2:30handafterthe incuba-tionperiod,theextractionoftotalDNAwasperformedwith QIAamp®DNAMinikit(QIAGEN,Germany),accordingtothe
protocoldescribedbythesupplier.
Aspositivecontrolsinamplificationreactions,DNA sam-plesofCandidaalbicans,C.glabrata,C.parapsilosis,C.tropicalis, C.krusei,C.lusitaniaeandC.pelliculosawereobtained.
For amplification ofDNA extracted from blood samples frompatientsandsamplesofCandidaspp.,thetechniqueof nested-PCRwasused.Inthefirstamplificationstep,ITS1and ITS4primerswereused,andthesecondstagewasdesigned fortwoamplificationsystems,usingspecies-specificprimers: system 1 comprises primers for C. albicans, C. tropicalis, C. glabrataand C. lusitaniae; and the system2, primers for C.pelliculosa,C.parapsilosisandC.krusei.
Detection of the amplified systems was carried out in agarose gels, at 2.5%, electrophoresed in horizontal tanks Horizon M-58 (Life Technologies, USA), in 1× TAE buffer (TRIS–aceticacid–EDTA pH8.0), 80Vfor45min. After elec-trophoresis,thegelswerestainedwithGelRedTM(Biotium,
USA),withresultsrecordedonaphotodocumentationsystem (UVITEC,England).18
Statisticalanalysis
Theresultswerepresentedasmean±standarddeviation(SD) ormedian(range)forcontinuousvariables,andaspercentage forcategoricalvariables.
Results
Amongthe194patientsincluded,67hadRA,47hadAS,36 had JIA, 28 had psoriatic arthritis, and 16 had other diag-noses.Themeanageofpatientsatthetimeofthestudywas 42±16 years, with 68 (35%) male; mean disease duration wasof15±10yearsandmeandurationofanti-TNFtherapy wasuse1.9±1.6years.Astoethnicity,85%wereCaucasian, 8%black,7%brownand1%yellowsubjects.Sixty-four(33%) patients were receiving adalimumab, 59 (30%) etanercept, and71(36%)infliximab.Eighty-onepercentofpatientswere
concomitantlytakingimmunosuppressantdrugs. Regarding
concomitantmedications,96(49%)usedprednisone,88(45%), methotrexate,46(24%)leflunomide,24(12%)sulfasalazine,10 (5%)cyclosporine,6(3%)azathioprine,and11(6%)chloroquine diphosphate(Table1).
AmongpatientswithRA,meanDAS28was3.6±1.5,HAQ 1.1±0.6, ESR18±16mm/1sth and CRP 10.1±15.4mg/L. In patientswithAS,meanBASDAIwas3.2±2.6,BASFI43±32, ASQoL6.7±5.4,ESR8±8mm/1sth,andCRP4.3±5,2mg/L.
Table1–Demographicsandtreatmentdataofpatients withrheumaticdiseasesonanti-TNFtherapy.
Variable n=194
Demographics
Currentage,years 42±16
Malegender,n(%) 68(35)
Race
White,n(%) 165(85)
Black,n(%) 16(8)
Browns,n(%) 14(7)
Yellow,n(%) 2(1)
Diseaseduration,years 15±10
Timeofanti-TNFtherapy,years 1.9±1.6
Treatment Anti-TNF
Adalimumab,n(%) 64(33)
Etanercept,n(%) 59(30)
Infliximab,n(%) 71(36)
Glucocorticoids,n(%) 96(49)
Meandose,mg/day 7.8±4.6
Methotrexate,n(%) 88(45)
Averagedose,mg/week 22.2±8.6
Leflunomide,n(%) 46(24)
Sulfasalazine,n(%) 24(12)
Cyclosporine,n(%) 10(5)
Azathioprine,n(%) 6(3)
Chloroquine,n(%) 11(6)
Table2–Clinicalandlaboratoryparametersofdisease inpatientswithrheumaticdiseasesreceivinganti-TNF therapy.
Variables
Rheumatoidarthritis(n=67)
DAS28 3.6±1.5
HAQ 1.1±0.6
ESR,mm/1sthour 18±16
CRP,mg/L 10.1±15.4
Ankylosingspondylitis(n=47)
BASDAI 3.2±2.6
BASFI 43±32
ASQoL 6.7±5.4
ESR,mm/1ahora 8±8
CRP,mg/L 4.3±5.2
JuvenileIdiopathicArthritis(n=36)
DAS28 3.4±1.3
ESR,mm/1sthour 15±18
CRP,mg/L 19.5±55.1
Psoriaticarthritis(n=28)
DAS28 2.5±1.3
BASDAI 3±1.7
ESR,mm/1sthour 10.3±7.8
CRP,mg/L 4.2±4.7
DAS28,DiseaseActivityScore28;HAQ,HealthAssessment Ques-tionnaire; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; BASDAI, Bath Ankylosing Spondylitis Disease Activity Index;BASFI,BathAnkylosingSpondylitisFunctionalIndex. Dataarepresentedasnumber(%)andmean±standarddeviation.
Among patients with JIA, mean DAS28 was 3.4±1.3, ESR 15±18mm/1sthandCRP19.5±55,1mg/L(Table2).
Atthetimeofthestudy,onlyone(0.5%)patienthad local-izedfungalinfection(vaginalcandidiasis)andnonehadoral thrush.Noneoftheincludedpatientshadcandidemiawith positivebloodculturesforfungiorPCRpositiveforCandida spp.inperipheralbloodsamples.
TheanalysisofpossibleriskfactorsforCandidainfection revealedthatonly14(7.2%)had receivedantibiotictherapy uptofourweeksbeforetheevaluation,9forrespiratorytract infection,3forcutaneousinfection,1forurinarytract infec-tionand1forodontogenicinfection.Furthermore,nopatient washospitalizedorwassubjectedtoinvasiveproceduresup toonemonthpriorthestudyentry.
Discussion
Thiswasthefirststudytoassesstheprevalenceofinvasive andlocalizedfungaldiseasecausedbycandidainasignificant numberofpatientswithrheumaticdiseasestreatedwith anti-TNFtherapy,showinglowriskofcandidiasis,despitethehigh prevalenceofimmunosuppressivedrugsuse.
Epidemiological studies have shown a considerable
increase in infections in immunocompromised patients,
includingCandidaspp.,particularlyinnosocomialsepsis,with ahighmortalityrate.Candidaalbicansisconsideredasa com-mensal member ofthe normal floraof the digestive tract and its pathogenicity is the result of alterations in host
defensemechanismsthatinducebehavioralchangesofthe fungus.19,20
Patients with rheumatic diseases are often exposed to severalriskfactorsassociatedwithasignificantincreasein fungalinfectionincidenceinrecentdecades.Theuseof broad-spectrum antibiotics can cause changes in mucosal flora, leading to the proliferation ofcandida; corticosteroids can affecttheactivityofpolymorphonuclearcells,macrophages andTcells;surgicalproceduresandtheuseof immunosup-pressivedrugs, particularlybiologicals,facilitatethespread of opportunistic pathogens.2,21 Actually, in the published literature,in98%ofinvasivefungalinfectionssuchas histo-plasmosis,candidiasisandaspergillosis,theuseofatleastone immunosuppressiveagent,particularlycorticosteroids, asso-ciatedwithanti-TNFtherapy,wasreported.1
Innate immune response is the dominant mechanism
responsible for yeast clearance after an initial systemic
infection. Monocytes and macrophages are the cells most
associated with response against systemic infection with C.albicans.Thepathwaysbywhichthiseventoccursarenot entirelyclear,butappeartoinvolverecognitionof pathogen-associatedmolecularpatterns(PAMPs)ontheyeastmediated byphagocyticcellreceptors,resultinginactivationandrelease
of inflammatory cytokines.19 Furthermore, the spectrum
ofdefenses againstmucocutaneous and systemiccandidal
infectionincludescell-mediatedimmunitywhichis charac-terizedbythereleaseofcytokinesbylymphocytesandbythe activationofNKcellsandlymphocytesbyinterleukins. How-ever,thereisevidencetosupporttheroleofhumoralresponse intheprotectionagainstinvasiveCandidainfection.19,20
It isknown that, in casesofsystemic infection with C. albicans,TNFproductionisstimulatedbythepathogen.Louie etal.22 demonstrated,inananimalmodelofsystemic can-didiasis,thatthiscytokinehasaprotectiveroleininfection. TheneutralizationofTNFactivitywouldleadtosuppression oftheproductionofIFN,promotion ofmonocyteapoptosis andpreventionofmaintenanceofgranuloma,allowing fun-gusgrowthinseveralorgans.1Indeed,inareviewofstudies oninvasivefungalinfectionsassociatedwithanti-TNF ther-apy(infliximab,adalimumab,etanercept)invariousdiseases (graftvs.hostdisease,inflammatoryboweldiseaseand RA) between1966and2007,Tsiodrasetal.1found281cases. Can-didiasiswasresponsiblefor23%ofinfectionsand,ofthese, only3occurredinpatientswithRA.Althoughevidenceinthe literaturesuggests that theremay be differencesregarding the riskofinfection amongdifferent anti-TNFdrugs,23 the presentstudyshowedlowfrequenciesofcandidiasisforthe threeagentsused.
Moreover,amongpatientswithrheumaticdiseasesthere areonlytwosepticarthritisreportsbyCandidaspp.inpatients withrheumatoidarthritisinanti-TNFtherapy.8,9However,in bothcases,theconcomitantuseofotherimmunosuppressive agentsmayhavecontributedtothedevelopmentofcandida infection.
thefungal isolationincultureofsterilefluidsuchasblood andperitoneal fluidisassociatedwithalowrecoveryrate, reachingonlyapositivityof40–60%inpatientswithsystemic candidiasisconfirmedbyautopsy.26
From this study, weconcluded that invasive and local-izedfungaldiseasebycandidadidnotrepresentacommon infectioninpatientswithrheumaticdiseaseswith immuno-suppressivetherapyassociatedwithanti-TNFagents.
Funding
Thisstudy wasfundedbyFundac¸ãode AmparoàPesquisado EstadodeSãoPaulo(FAPESP2009/51897-5forEBandCAS), Con-selhoNacionaldoDesenvolvimentoCientíficoeTecnológico(CNPq 302724/2011-7 to CAS and 301411/2009-3 for EB), Federico FoundationforCASandNúcleodeApoioàPesquisa“Saúdeda Crianc¸aedoAdolescente”,USP(NAP-CriAd).
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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