Staphylococcus aureus in tonsils of patients with recurrent tonsillitis: prevalence, susceptibility profile, and genotypic characterization

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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

Original

article

Staphylococcus

aureus

in

tonsils

of

patients

with

recurrent

tonsillitis:

prevalence,

susceptibility

proﬁle,

and

genotypic

characterization

Veraluce

Paolini

Cavalcanti

a

_,

_Leandro

_Azevedo

_de

_Camargo

b

_,

_Fabiano

_Santana

_Moura

c

_,

Edson

Júnior

de

Melo

Fernandes

b

_,

_Juliana

_{Lamaro-Cardoso}

a

_,

Carla

Afonso

da

Silva

Bitencourt

Braga

a

_,

_Maria

_Cláudia

_Porﬁrio

_André

a,∗

a_Universidade_Federal_de_Goiás_(UFG),_Instituto_de_Patologia_Tropical_e_Saúde_Pública,_Departamento_de_Biociências_e_Tecnologia,

Goiânia,GO,Brazil

b_Universidade_Federal_de_Goiás_(UFG),_Hospital_das_Clínicas,_Goiânia,_GO,_Brazil c_Hospital_Pio_X,_Centro_Clínico_Diagnóstico_e_Imagem,_Ceres,_GO,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22May2018

Accepted29December2018

Availableonline6March2019

Keywords:

Tonsillitis Tonsillectomy

Staphylococcusaureus

Pulsedﬁeldgelelectrophoresis

a

b

s

t

r

a

c

t

Introduction:Bacterialtonsillitisisanupperrespiratorytractinfectionthatoccursprimarily

inchildrenandadolescents.Staphylococcusaureusisoneofthemostfrequentpathogensin

theetiologyoftonsillitisanditsrelevanceisduetoitsantimicrobialresistanceand

persis-tenceintheinternaltissuesofthetonsils.Tonsillectomyisindicatedincasesofrecurrent

tonsillitisafterseveralfailuresofantibiotictherapy.

Materialandmethods: Inthisstudyweevaluated123surgicallyremovedtonsilsfrompatients

whohadhistoryofrecurrenttonsillitis.Thetonsilsweresubmittedtomicrobiological

analy-sisfordetectionofS.aureus.TheisolateswereidentiﬁedbyPCRforfemAgene.Antimicrobial

susceptibilityoftheisolateswasdeterminedbydiskdiffusiontests.Allisolateswere

sub-mittedtoPCRtodetectmecAandPanton-Valentineleucocidin(PVL)genes.Thegenetic

similarityamongallisolateswasdeterminedbypulsedﬁeldgelelectrophoresis.

Results:Sixty-oneS.aureusisolateswereobtainedfrom50patients(40.7%)withmeanageof

11.7years.Theisolatesshowedhighlevelresistancetopenicillin(83.6%),9.8%hadinducible

MLSbphenotype,and18.0%wereconsideredmultidrugresistant(MDR).mecAgenewas

detectedintwoisolatesandthegenecodingforPVLwasidentiﬁedinoneisolate.Thegenetic

similarityanalysisshowedhighdiversityamongtheisolates.Morethanonegenetically

differentisolatewasidentiﬁedfromthesamepatient,andidenticalisolateswereobtained

fromdifferentpatients.

Conclusions:MDRisolatescolonizingtonsilsevenwithoutinfection, demonstrate

persis-tence of the bacterium and possibility of antimicrobial resistance dissemination and

recurrenceofinfection.AspeciﬁccloneinpatientscolonizedbyS.aureuswasnot

demon-strated.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

∗ _{Corresponding}_author.

E-mailaddress:mcporﬁrio@hotmail.com(M.C.André).

https://doi.org/10.1016/j.bjid.2018.12.003

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Introduction

Tonsillitisisaninfectionofthepalatinetonsilsthatoccurs

mostlyinchildrenandyoungadults,andrecurrenttonsillitis

isamongthemostcommonchildhooddiseases.1,2_Much_has

beenwrittenabouttheetiologyofrecurrenttonsillitis,butit

remainsacontroversialtopic.Whileasinglemicrobialspecies

maycauseacutetonsillitis,ithasbeensuggestedthat

recur-renttonsillitisisaconsequenceofapolymicrobialinfection.2

MicroorganismsotherthanGroupABetaHemolytic

Strepto-coccus(GABHS)maybethecauseofchronictonsillitis.1

Bacterial bioﬁlms are recognized as the main factor

involved in the chronicity of infections and resistance to

antibiotictreatment.Therefore,theseinfectionshavea

con-siderablynegativeimpacton patients’quality oflifeand a

signiﬁcantburdenonpublichealth.Bacterialbioﬁlmmayplay

a role in various recurrent/chronic upper respiratory tract

infections, including chronic tonsillar disease. Bioﬁlm has

been foundinthe tonsillar tissueofchildrenwithchronic

infections.3

Especially in recent years, the presence of the

beta-lactamaseproducing bacteria suchas Staphylococcus aureus

andHaemophilusinﬂuenzaeintonsilsmicrobiotacanpromote

penicillinresistance.Several researchershaveclaimedthat

failure of antibiotic therapy may be due to the

underesti-mation of resistant microorganisms,1 _which _could _also _be

explainedbylowconcentrationofantibioticsinthe

tonsil-lartissue,potentiallycombinedwiththepresenceofresident

bacteriaproducingprotectiveenzymes,orspeciﬁcantibiotic

resistancepatternsoftheinvolvedpathogenicbacteria.4

Thepresenceofthe bacterium inthe internaltissue of

the tonsil may be responsible for its persistence in this

site.Tonsillarsurfacecommonlypresentsbacteriabelonging

tothenormaloralmicrobiota,andinternaltissuecontains

pathogenicmicroorganisms.S.aureushavebeendetectedin

bothexternalandinternaltissuesofthetonsils.4

Inthisstudy,weinvestigatedtheprevalenceofS.aureus

fromtonsilsthatwereremovedduetorecurrenttonsillitisas

wellastheantimicrobialsusceptibilityproﬁleoftheisolates.

Inaddition,virulencegeneswereinvestigated,andthegenetic

similarityamongtheisolateswasalsodetermined.

Material

and

methods

Studysettingandethicsstatement

Thiscross-sectionalstudywascarriedoutatatertiary

hospi-tal,intheDepartmentofSurgicalClinicofaTeachingHospital

inGoiania,amunicipalityinmidwestBrazil.Thestudy

pro-tocolwasapprovedbythe localEthicsCommittee(Protocol

CEP/HC/UFGnumber071/2011),andwritteninformedconsent

wasobtainedfromthechildren’sparentsorlegalguardians.

Studysubjects

For twoyears,outpatientsaged0.8–48 yearsold who were

referredtotheDepartmentofSurgicalClinictoperform

tonsil-lectomywererecruited.Thepatientshadahistoryoftonsillar

hyperplasiaand/orrecurrenttonsillitisafterrepeatedfailures

ofantimicrobialtherapy.

Followingtonsillectomy,thetonsilswereplacedina

ster-ilecontainerandpromptlytakentotheMedicalBacteriology

LaboratoryoftheFederalUniversityofGoias,wheretheywere

processed.

Samplesprocessing

All of the sampleswere homogenized inBuffered Peptone

Water0.1%andinoculatedinMannitolSaltagarandSheep’s

Bloodagar(5%)forbacterialisolation.Theisolateswere

iden-tiﬁed according to standard methods.5 _Isolates _presenting

coccusmorphology,Grampositivestain,betahemolysis,and

producingcatalase,coagulase,andDNAseweresubmittedto

PCRforidentiﬁcationofS.aureusbydetectionoffemAgene.

DNAextration

GenomicDNAwasextractedfromculturesgrownonTryptic

Soyagarplatesandwasthenusedasatemplatefor

ampliﬁ-cationaccordingtoAiresdeSouzaetal.6

PCRfordetectionoffemAgene

TheisolateswerescreenedbyPCRforthepresenceoffemA

gene,whichisspeciﬁcforS.aureus,accordingtoMehrotraetal.

(2000).7_The_primers_used_for_gene_{ampliﬁcation}_were_femA-F:

5AAAAAAGCACATAACAAGCG3andfemA-R:5

GATAAAGAA-GAAACCAGCAG3,toobtaina132bpamplicon.

Determinationofantimicrobialsusceptibilityproﬁle

Theantimicrobialsusceptibility proﬁleoftheS.aureus

iso-lates to penicillin, cefoxitin, erythromycin, clindamycin,

quinupristin–dalfopristin, linezolid,

trimethoprim-sulfamethoxazole, amoxicillin-clavulanate, ciproﬂoxacin,

ceftriaxone, tetracycline, gentamicin, and rifampin was

determined by disk diffusiontest accordingto the Clinical

Laboratory Standards Institute (CLSI) guidelines.8 _The _S.

aureusstrainATCC25923wasusedascontrol.Todetermine

theinducibleorconstitutiveresistanceproﬁletomacrolides,

lincosamides,andstreptograminsB(MLSb),theD-zonetest

wasperformedaccordingtoCLSI.8

Allmethicillin-resistantS.aureus(MRSA)andtheisolates

thatshowedresistancetothreeormoreantimicrobialclasses

wereconsideredmultidrug-resistant(MDR).9

PCRfordetectionofmecAgene

The detection of mecA gene was considered as MRSA

marker.TheprimersusedtoamplifythemecAgene(310bp)

were P1: (5-TCCAGATTACAACTTCACCAGG-3 and P2: 5

-CCACTTCATATCTTGTAACG-3)aspreviouslydescribed.10

PCRfordetectionofPanton-ValentineLeukocidin

Theprimers weredesignedtoobtain ampliﬁcationof

lukS-PVandlukF-PVgenesaccordingtothepublishedsequences

(3)

TheampliﬁedPCRproductswereelectrophoresedin1.5%

agarosegelsinTBE(TrisBorateEDTA)bufferat120Vfor1h,

stainedwithethidiumbromide,andphotographedunderUV

MolecularImager® GelDocTM _XR_system_(Bio-Rad

Laborato-ries,Hercules,CA,USA).

Pulsedﬁeldgelelectrophoresis–PFGE

Pulsed-ﬁeldgelelectrophoresis(PFGE)ofSmaI-digestedmacro

fragments of the respective chromosomal DNA was used

to ﬁngerprint the isolates according to methods described

previously,12 _using ₂₀_U _SmaI _restriction _enzyme.

Elec-trophoreticrunwasperformedin0.5XTBEbuffer(90mMTris,

90mMBoricacid,and2mMEDTA)underthefollowing

condi-tions:6V/cmfor22h,pulsetimesfrom5to35sat14◦C,using

CHEFDRIISystem(Bio-RadLaboratories,Hercules,CA,USA).

The chromosomalDNA restriction patterns were analyzed

withBionumericssoftware(version5.0;AppliedMaths,Ghent,

Belgium)usingthe UPGMAalgorithmwithDicecorrelation

coefﬁcients (0.8% optimizationand 1.0% tolerancesetting).

IdenticalPFGEproﬁles(100%ofsimilarity)weredeﬁnedasa

pulsotype(PT),andacut-offof80%wasappliedtoclusterthe

strains.

Statisticalanalysis

ThedescriptivestatisticswereanalyzedwithStatisticalPackage

forthe SocialSciences (SPSS)version 17.0 (SPSS, Chicago,IL,

USA)andEpiInfo softwareversion 2000(CDC,Atlanta,GA,

USA).Categoricalvariableswerecomparedbychi-squareor

Fisher’sexact test,whenappropriate.Differencesinmeans

wereassessedbytheStudentT-test.p≤0.05wasdeemed

sta-tisticallysigniﬁcant.

Results

Tonsilsfrom123patients,aged0.8–48years,were analyzed

overtwoyears.Themeanageofthepatientswas11.3years,

ofwhom53.7%(n=66)weremaleand46.3%(n=57)female.

Ofthe123tonsilsanalyzed,61isolates(49.6%)were

iden-tiﬁedasS.aureusbyfemAgenedetection.Itshouldbenoted

thatatthetimeofthetonsillectomy,thepatientshadnoacute

inﬂammatoryprocess.

S.aureuswasisolatedin50outof123(40.7%)patientsaged

0.8–36years(meanage=11.7years),inwhom76.0% (n=38)

hadrecurrentpharyngotonsillitisand88.0%(n=44)presented

tonsillar hypertrophy, with degrees of obstruction varying

betweenIIIandIV.In12.0%(6/50)ofthepatients,S.aureus

wastheonlyagentfound,andin18.0% (9/50)twoormore

S.aureusisolateswithdifferentgenotypicproﬁleswere

iden-tiﬁed.Ofthese50patients,78.0%reportedantimicrobialuse

beforetonsillectomy.Thedrugofchoicetotreatthe

pharyn-gotonsillitiswasamoxicillin,whichwasusedby44.0%(22/50)

ofthepatients.

According to the antibiogram, the S. aureus isolates

presented greater resistance to penicillin (83.6%)

fol-lowed by cefoxitin (26.2%), ciproﬂoxacin (24.6%) and

amoxicillin-clavulanate(13.1%).Sixisolates(9.8%)presented

inducibleMLSbphenotypeandwere,therefore,resistant to

Table1–ResistanceproﬁleofS.aureusisolatesobtained

fromtonsilsofpatientssubmittedtotonsillectomy.

Antimicrobial Resistance n % Penicillin 51 83.6 Cefoxitin 16 26.2 Ciproﬂoxacin 15 24.6 Erythromycin 10 16.4 Amoxicillin-clavulanate 08 13.1 Tetracycline 07 11.5 Clindamycin 06 9.8 Q/D 06 9.8 Ceftriaxone 03 4.9 Rifampin 01 1.6 Linezolid 01 1.6 Gentamicin 00 0.0 Trimethoprim-sulfamethoxazole 00 0.0

erythromycin, clindamycin, and quinupristin–dalfopristin.6

All isolates were susceptible to gentamicin and

sulfamethoxazole-trimethoprim (Table 1). Of the 61 S.

aureusisolates,11(18.0%)wereconsideredMDR.9

AccordingtotheCLSI,8_cefoxitin_is_used_to_predict_oxacillin

resistancemediatedbymecAgeneexpression.Inthepresent

study,26.2%(16/61)oftheS.aureusisolateswereresistantto

cefoxitinandthemecAgenewasdetectedintwoisolates.The

genecodingforPVLwasfoundinoneMDRisolate,susceptible

tomethicillin.

Theisolatesshowedhighgeneticdiversityaccordingtothe

similarityanalysisperformedbyPFGE.OnlyClusters5and8

groupedgeneticallyidenticalisolatesfromdifferentpatients.

Inninepatients(ID:17,36,85,90,111,124,129,133,134),more

thanonegeneticallydifferentisolatewasidentiﬁedcolonizing

the tonsil,and inoneofthem (ID: 133),four different

iso-latesweresimultaneouslyobtained(Fig.1).Cluster5grouped

nineisolatespresentingmorethan80%ofsimilarity,withtwo

clonalpulsotypes,obtainedfromdifferentpatients(Fig.1).

No clonal relationship was observed between the two

MRSAisolates,whichshowsthatthesephenotypesare

dis-persedamongthedifferentisolatesobtainedfromthetonsils.

Discussion

Bacterialpharyngotonsillitisisaninfectionthataffectsmainly

childrenandadolescentsbetweenﬁveand15yearsold.13,14

Inthisstudy,themeanageofthepatientsinvolvedwas11.3

years,inlinewiththeliteraturedata.Duringchildhood,dueto

thevariouscontactsofchildrenathome,school,anddaycare

centers,thereisanincreaseinvariationoforalmicrobiota,

leadingtoanincreaseinthefrequencyoftheseinfections.15

Therewasnosigniﬁcantsexdifferenceamongthose

under-goingtonsillectomy,asobservedinotherstudies.14

Recurrent pharyngotonsillitis was reported in 75.5% of

the patients, showing its importance forthe indicationof

tonsillectomy.Accordingtosomestudies,recurrent

pharyn-gotonsillitisand/ortonsillar hypertrophy,withsleep apnea,

nocturnalsnoringandrespiratorypausesarethemain

(4)

Dice (Opt:0.80%) (Tol 1.0%-1.0%) (H>0.0% S>0.0%) [0.0%-100.0%] ID 136 145 66 72 77 17 24 85 125 127 150 135 49 90 96 111 15 88 2 2 3 3 4 4 5 5 5 5 5 5 5 5 6 6 6 7 7 7 7 7 7 8 8 9 9 134.1 128 138 21 139 140 112 144 133 111.1 148 26 137 133.1 133.2 133.3 124.1 100 129.1 124 129 87 134 116 85.1 95 90.1 20 82 29 63 123 50 54 27 75 22 38 36.1 36 61 PVL + MDR MDR MDR mecA+/MDR mecA+/MDR MDR MDR MDR MDR MDR MDR 86 17.1 1 1 1 CI ViF ResP

Fig.1–GeneticsimilarityofStaphylococcusaureusisolatedfromtonsilsofpatientssubmittedtotonsillectomy,established withSmaIPFGEanalysis.ID:sampleidentiﬁcation;CL:cluster;VF:virulencefactor;RP:resistanceproﬁle.

ThehighprevalenceofS.aureusfound(40.7%)inthisstudy

indicatesbacterialpersistenceinthetonsilsafteran

inﬂam-matoryprocessandtreatmentwithantimicrobials.Thethroat

regionandtheanteriornostrilregionareconsideredthe

pri-marycolonizationsites ofS.aureus.16 _The_{identiﬁcation} _of

S. aureusas the mainagent of tonsillitis or as a probable

co-pathogenhasbeenreportedbyseveralauthors,with

preva-lenceashighas83.0%.3,4,17,18

ThepresenceofS.aureusintonsilinfectionsandits

persis-tenceintonsillartissueevenaftertheinﬂammatoryprocess

mayberelatedtoitsabilitytoformbioﬁlm.Thepresenceof

(5)

recurrenceoftheinfection,andrepresentanimportant

ele-mentofchronicity,evenintheabsenceofacuteinﬂammatory

process.3

Theresistanceoftheisolatestopenicillinwas83.6%and

13.1%totheassociationofamoxicillinandclavulanate.This

isevidencethatthehighrateofresistancefoundwasdueto

␤-lactamaseenzymeproduction,whichlimitstheuseofthis

commontherapeuticoptioninclinicalpractice.19

Thetherapeuticfailuresobservedwithpenicillinledtoan

increaseintheuseofotherantimicrobials,suchasthe

asso-ciationwith␤-lactamaseinhibitorsandcephalosporins.19

AnotherconcernregardingresistanceproﬁlesofS.aureus

isthe emergence ofMRSAstrains, bothin thecommunity

andinthe hospitalenvironment.6,20 _In_this _study,_two

iso-lates(3.3%)were consideredMRSA,inlinewiththe results

ofZautneretal.4_who_found_isolation_of_MRSA_in_just_one_out

of76isolates(1.3%)frompatientswithrecurrenttonsillitisin

Germany.InJapan,Hirakataetal.21_identiﬁed_9.1%_of_MRSA

isolatesinpatientswithpharyngotonsillitissymptoms,and

intheUSA,Brook&Foote22_found_16.0%_of_MRSA_in_tonsils_of

patientssubmittedtotonsillectomyduetorecurring

tonsilli-tis.Thesedatashowthatthereseemstobenorelationship

betweenMRSAandpharyngotonsillitisundertheconditions

ofourstudy,consideringthatonlyoutpatientswereincluded.

AlthoughcefoxitinresistanceisthebestmarkerforMRSA

screening,8 _only _two _of_the ₁₆ _isolates _(12.5%)_resistant _to

cefoxitinwere identiﬁedasMRSAbydetection ofthe mecA

gene. In thesecases, methicillin resistancemay be due to

mutationingenesencodingnormalPBPorbyoverproduction

of␤-lactamases.23_Mutations_in_genes_coding_for_PBPs_can

gen-eratestructuralmodiﬁcations,whichalterthebindingofthese

proteinsto ␤-lactams antibiotics by decreasing their

afﬁn-ityanddeterminingantimicrobialresistance.24_Mutations_can

also lead to overexpression of PBPs and produce a small

butsigniﬁcantincreaseinresistanceto␤-lactamantibiotics.

Thesemutationscanbegeneratedbytheselectivepressure

exertedbyexcessiveuseof␤-lactams.25

In addition to the changes in PBPs, S. aureus may

developresistanceto␤-lactamsduetohyperproductionof

␤-lactamases.23_A_study_of_McDougal_and_Thornsberry26_shows

thatS.aureusproducinglargeamountsof␤-lactamasescan

also inactivate more slowly penicillin resistant to

penicil-linases. In this study, the production of large amounts of

␤-lactamasesmayhaveinﬂuencedthecefoxitindisktestand

determinedphenotypicresistance.

Anotherpossibilityistheexistenceofagenehomologous

tothemecAgene,themecALGA251,ormecCgene.Thisgene

has63% homology attheaminoacid level and 70%atthe

DNAlevelwiththemecAgeneencodingthePBP2aprotein.27

Sofar,isolateswiththemecCgenehaveshownphenotypic

resistanceto␤-lactams,buthavenotbeenidentiﬁedin

con-ventionalPCRsforthemecAgene.8 _An_explanation_for_this

phenomenonwouldbethatPBP2aencodedbythemecCgene

couldhaverelativelyhighafﬁnitytooxacillinbutalowafﬁnity

tocefoxitin.28

The resistance rate of 24.6% to ciproﬂoxacin is

worri-some. Thisdrug has been reported to be effective against

severalpathogenic agents oftonsillitis, including S.aureus,

and increased resistanceleaves fewtherapeutic optionsto

treatthiscondition.29_Inducible_MLSb_resistance_proﬁle_was

detectedin9.8%oftheisolates.Thisresistancemaybe

medi-atedbythepresenceofermgene,whichproducesmodiﬁcation

atthedrugbindingsiteinrRNAandcausestherapeutic

fail-uresandrelapses.30_Clindamycin_is_a_widely_used_therapeutic

alternative for staphylococcal infections, well tolerated by

children,andthemostfrequentoptionforpatientsallergicto

penicillin.31_Therefore,_the_iMLSB_proﬁle_(inducible_resistance

toclindamycin)shouldbeconsidered,whenperformingthe

susceptibilitylaboratorytests,inordertoprescribethe

ade-quateantimicrobialtherapytothepatient.Oneofthemajor

concernswhenanalyzingresistanceproﬁlesofS.aureusisthe

presenceofMDRstrains.Inthisstudy,11 (18.0%)MDR

iso-lateswereidentiﬁed.Thepatientsinvolvedinthestudywere

treatedasoutpatients.Mainlybecausetheyarechildrenand

adolescentswhoareinconstantcontactwithotherchildren

andtheirrelatives,thespreadofMDRcanovercomehospital

barriers,makingtherapyanddisseminationofthesestrains

achallenge.18Itisnoteworthythatoneisolatepresentedthe

genecodingforPVL.Thisisanimportantvirulencefactorof

S.aureusthatcauses seriousinfections suchasnecrotizing

pneumonia.PVL-positiveS.aureusaremoreassociatedwith

childrenandyoungadults,andmaycarrydifferentlysogenic

phagethatcontaingenesforPVL.Thesecanbeeasily

trans-mittedhorizontallyandinfectotherPVL-negativestrainsofS.

aureus.32,33

The similarity analysis of the isolates demonstrated

genetic diversity between them. The presenceof multiple

isolatesinthesamepatientfavorsthetransferofgenetic

infor-mationbetweenthem.34_This_demonstrates_the_dynamics_of

colonizationand thepossibility ofgeneticalterationofthe

isolatesthat persistcolonizingthe tonsil,eveninclinically

healthypatients.

Thisdiversityofgenotypicproﬁleswasalsoobservedby

Zautneretal.,4_where₇₆_S._aureus_isolates_were_grouped_into

24differentproﬁles.Thisdemonstratesthatunderthestudy

conditions,thereisnocloneassociatedwithinfectionsand

thatisolatesaredispersedinthispopulation.Probably,the

fac-torsassociatedwithcolonizationandpersistenceofS.aureus

inthetonsilsarecommoninthespecies.

Theclusteringofsimilarisolatesfromdifferentpatients

suggeststhatthesemayhaveacommonoriginandare

asso-ciatedwiththepathogenesisofrecurrentpharyngotonsillitis.

Futureinvestigationsarenecessarytodeterminethecommon

geneticcharacteristicsoftheseisolatesthatleadto

dissemi-nationinpatientswiththesameclinicalproﬁle.

Theresultspointtoachangeintheparadigmof

diagno-sisandtreatmentofrecurrentpharyngotonsillitisinorderto

allowthecorrectuseofantimicrobialsandtoreduce

recur-rence,whichisthemaincauseoftonsillectomy.

Funding

ToNationalCouncilforScientiﬁcandTechnological

(6)

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

ToNationalCouncilforScientiﬁcandTechnological

Develop-ment(CNPq)forﬁnancialsupport.

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