Measles, rubella, mumps and Toxoplasma gondii antibodies in saliva of vaccinated students of schools and universities in São Paulo City, Brazil

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

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Measles,

rubella,

mumps

and

Toxoplasma

gondii

antibodies

in

saliva

of

vaccinated

students

of

schools

and

universities

in

São

Paulo

City,

Brazil

Barbara

Carvalho

Fialho

Sampaio

a

_,

_Jaqueline

_Polizeli

_Rodrigues

a

_,

Luciana

Regina

Meireles

a

_,

_Heitor

_Franco

_de

_Andrade

_Junior

a,b,∗

a_Universidade_de_São_Paulo,_Instituto_de_Medicina_Tropical_de_São_Paulo,_Laboratório_de_{Protozoologia,}_São_Paulo,_SP,_Brazil

b_Universidade_de_São_Paulo,_Escola_de_Medicina,_Departamento_de_Patologia,_São_Paulo,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6August2019

Accepted10November2019

Availableonline19December2019

Keywords: Vaccine Prevalence Non-invasive Saliva IgG Toxoplasmosis

a

b

s

t

r

a

c

t

Introduction:Vaccinesarewell-establishedpublichealthinterventionswithmajorimpact

ontheprevalenceofinfectiousdiseases,butoutbreaksareoccurringfrequentlydueto

pri-maryandsecondaryfailures,despitehighcoverage.Surveillanceofefﬁcacyanddurationof

inducedimmunityisadifﬁculttaskasitrequiresinvasivebloodsamplinginchildrenand

teenagers.SalivacanbeanacceptablealternativesourceofIgGtoassessvaccineefﬁcacy

andtoxoplasmosisincidence.WeinvestigatedIgGresponseformeasles,mumps,rubella,

andT.gondiiinsalivasamplesofvaccinatedyoungpeople.

Methods:Salivawascollectedfrom249publicschoolsstudentsfromSãoPaulo,Brazil,aged

7to13yearsold,duringaninteractiveexhibitiononhygiene.WeusedS.aureusprotein

AsolidphasecaptureassayforIgGreactivetobiotinylatedpuriﬁedproteins.Pairedsaliva

andserum(47)weretestedfromyoungadultswithserumevidenceofT.gondiiinfection

andfromnegativechildrenlessthan12montholdforstandardization.Reproducibilitywas

greaterthan98%andsensitivityandspeciﬁcityofthesalivaassaysweregreaterthan95%,

aswellastheconcordanceofpairedsalivaandserumsamples.

Results:Salivafromhighschoolstudentsshowedaprevalenceof8.5%(95%CI:5.0–11.9%)

forantiT.gondiiIgG;96.8%(94.6–99%)ofanti-measlesIgG;59.1%(53–65%)ofanti-rubellaIgG,

and57.5%(51.3–63.6%)ofanti-mumpsIgG.

∗ _{Corresponding}_author_at:_Laboratório_de_{Protozoologia,}_Instituto_de_Medicina_Tropical_de_São_Paulo,_Universidade_de_São_Paulo,_Av._Dr.

EneasdeCarvalhoAguiar,470,1o

.andar,05403-000,SãoPaulo,SP,Brazil.

E-mailaddresses:bfcsampaio@gmail.com(B.C.Sampaio),jaquelinepolizeli@gmail.com(J.P.Rodrigues),lrmeirel@usp.br(L.R.Meireles),

hfandrad@usp.br(H.F.AndradeJunior).

https://doi.org/10.1016/j.bjid.2019.11.005

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Discussion: Theprevalenceofantibodiesagainstmumpsandrubella after6–8yearsof

vaccinationwaslowerthanagainstmeaslesamongstudents.Theﬁndingsofthisstudy

demonstratethefeasibilityofsalivasamplingforfollow-upofvaccineimmunestatusin

teenagers.ThisusefulapproachallowsforIgGdetectionforvaccinecontrolor

epidemio-logicalstudies.

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

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

Introduction

Theuse ofsafeand effectivevaccines are well-established

and cost-effectivepublic healthinterventions overthe last

threedecades and with amajor impacton the prevalence

ofinfectiousdiseases.1_Usually_restricted_to_children,

bene-ﬁtsofimmunizationareincreasinglyextendedtoadolescents

andadults,providingprotectionagainstlife-threatening

dis-easessuchasinﬂuenza,meningitis,andcancersthatoccur

inadulthood.2_Despite_this_progress,_{vaccine-preventable}

dis-easescontinuetobeamajorcauseofmorbidityandmortality.

Culturaland fakenewsonassociationofvaccinesand

dis-easeshave induced higherrate ofnon-vaccinated children

duetoparentalconcern.3_Some_authors_have_demonstrated

thatalackofresponsetothevaccine,primaryvaccine

fail-ure (PVF), may occur in about 2–5% formeasles, 3–7% for

mumps,and2–5%forrubella.4,5 _Among_the_main_causes_of

PVFare the presenceofmaternal antibodiesand improper

vaccinestorageandmanagement(handling,administration,

coldchain).Secondaryvaccinefailure(FVS),whichisthedrop

inimmunityovertimeexpressedasloworevenundetectable

antibodylevels,thusfailingtoconferprotection.6–8_For_these

reasons,evenwithaneffectivechildrenimmunization

pro-gram,asreportedinourregion,9_a_growing_number_susceptible

individualscanbefoundafterafewyearsasaresult.

There-fore,vaccinationcoveragemaynotbeequivalenttopopulation

immunity.10,11_The_triple_viral_vaccine_(MMR)_is_prepared_from

liveattenuatedmeaslesvirus(Schwarz,Moratenand

Edmon-stonZagrebstrains),mumps(JerylLynn,L-3ZagrebandUrabe

AM9strains)andrubella(WistarstrainRA27/3).Astudy

con-ductedinFinlandshowedthatantibodylevelsinducedbythe

MMRvaccine decreaseprogressively afterthesecond dose.

Theprotectioninducedbythis vaccineseems topersistat

leastuntilthebeginningofadulthood;however,thesituation

requires constant vigilance.12 _Antibodies _against _measles,

mumps and rubella decrease on average 3%per year and

showahighdegreeofindividualvariation.Theantibodydecay

ratevariessubstantiallybetweenindividualsandbetweenthe

threegroupsofantigenspresentintheMMRvaccine.However,

higherrateofdeclinealongwithhighvariationisobserved

withmumps.13_Prevalence_studies_of_measles,_mumps,_and

rubellaantibodiesarenotonlyaimedatmeasuringthe

pro-portionofthe immunepopulation,but ratherprovide data

tosupportnewimmunizationstrategiestobeintroducedto

controlpossiblevaccinefailuresandpredicttheoccurrenceof

outbreaks,ascommonlyseeninourcountry.14

Becauseoftechnologicalandlaboratoryadvancesinoral

ﬂuidstudies,salivatestsbesidesdetectingdiseasesrelatedto

oralhealthmaybeusedasmarkersofthegeneralhealthof

theindividual.15,16

Withtheseadvancements,earlydisease monitoringand

detection17,18_have_played_a_key_role_in_the_success_of_therapy

andmayreducetheseverityandcomplicationsofdiseases.

The maintoolfor the surveillanceand monitoring of

vac-cinated childrenisrelated to the increaseof expedite and

non-invasive diagnostic tests and help preventing possible

outbreaks.19,20_The_advent_of_oral_ﬂuid_testing_has_transformed

measlessurveillanceinEnglandandWales.Withasensitivity

of92%andaspeciﬁcityofatleast98%intheIgMtestwith

oralﬂuid,itissuitableforsurveillance.21_It_is_known_that_the

measlesvaccinationfailureraterangesfromtwoto10%,andit

isanimportantissueintheepidemiologyofthedisease,since

thetransmissioncouldonlyoccurduetofailuresinchildhood

vaccinecoverage.22_Recent_studies_have_shown_that_children

aretheprimarytargetsformeaslesand,inthisnon-invasive

technical contextfordiagnosis,salivacan beavery useful

toolinearlydetectionofmeasles.Theresultsconﬁrmthat

bothserologicalandmolecularassaysoforalﬂuidare

suit-ableforroutineuse.23 _The_variable _amount_of_IgG _in_saliva

isthemainproblemforthedetectionofspeciﬁcIgGwhich

mayresultinfalsenegativeresults,butwehavedevelopedan

effectiveassayforthedetectionofanti-T.gondiiIgG,usingthe

captureofaﬁxedamountofIgGinthesolidphaserevealed

by biotinylatedantigen.24 _In_the _present _study, _we

investi-gated thepresenceofspeciﬁcIgGagainstmeasles,mumps

andrubellaortoxoplasmosisinsalivaofteenagersfrompublic

schools.UndergraduatestudentsfromourUniversityprovided

pairedsamplesofbloodandsalivatostandardizethemethod.

Material

and

methods

Studiedpopulation

A total of 249 samples of saliva from elementary school

studentsand47seraand47saliva(paired)from

undergrad-uatestudents werecollected.In addition,50 serafrom our

biorepository knowntobepositiveforthe diseasesstudied

and 40negativesamplesfrom 3to5pooleddiscardedsera

from infantsbetweeneightandtwelvemonthsofagefrom

theChildren’sInstituteoftheHospitaldasClínicasoftheSão

PauloMedicalSchool(IC-HCFMUSP)werealsoused.

All volunteers,highschooland undergraduatestudents,

were adequately informed of all procedures to be

devel-oped, with signed individual or parental consent form,

accordingtoResolutionNo.196/96,whichregulatesresearch

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informedandconsentedinindividualparticipation,asideto

approvedparentalconsent.Thisstudywassubmittedtothe

ResearchEthicsCommitteeandapprovedunderprocess no

23109613.1.0000.0065.

Salivacollection

Salivettecommercialtubeswithoutastringentwereusedfor

salivacollectioncontaininganabsorbentcottonrolleranda

tubesuitableforsalivacentrifugationaftercollection.The

stu-dentswere instructed tomouthwash with25–40mloforal

rinse (Listerine®), followedby mouthwasheswith drinking

water,insertionofthecottonrolleroftheannotateddevicein

mouthwithouttouching,chewinguntilappearscompletely

moistenedand reinsertedinthe tube,whichwas storedin

ice bathuntil centrifugation. Thetube was centrifuged at

2000gfor10minat4◦Candtheclearedsalivatransferredto

a2mlconictube,withadditionof1volumeof−20◦_C

abso-luteethanol.Thesolutionwasvortexedandmaintainedover

nightat−12◦_C._The_precipitated_IgG_was_recovered_by

cen-trifugationat10,000gforthreeminutesat-−2◦_C,_drained_and

dissolvedin1/5oftheoriginalsalivavolumewithsalineborate

buffer.

Immunoenzymaticassays

We have developed IgG immunoenzymatic assays using

captureofIgGbysolidphaseproteinAandsubsequent

reac-tionwithin-housebiotinylatedrecombinantviralantigens,24

revealedwithavidin-peroxidase(supplementarydata).96-or

384-wellpolystyreneplateswellswereabsorbedwithaﬁxed

amountofproteinAallowingforaﬁxedamountofIgGpresent

insalivaor serum to bindevenly through its Fcfragment,

independentofits concentrationinsaliva. Thesubsequent

reactionwithviralorT.gondiiantigensconjugatedtobiotin

allowsthedetectionoftheproportionofspeciﬁcIgGinthe

sample, with avidin-peroxidase and development by TMB.

AdequatecontrolswereusedineachplateandIgGlevelswere

expressedasArtiﬁcial Units, asaratio ofobservedO.D. in

sampleandcut-offO.D.

Statisticalanalysis

AlldatawereanalyzedusingaGraphPadPrism5.0software.

ROC curves and serological indexes were calculated using

negative and positive standards conﬁrmed by other tests.

Betweengroupsdifferenceswereconsideredsigniﬁcantwhen

p<0.05.Allfrequencydatawerepresentedwithits95%

con-ﬁdenceinterval.Reproducibilityassaysand sera and saliva

comparisonswereperformedindifferentdayswithdifferent

microplateswiththesamesamplesandcomparedbyPearson

correlation.

Results

Highschoolsamplesvaccinationstatusanddemography

TheprevalenceofantibodiesagainstT.gondiiand the

anti-gensofthetripleviralvaccine instudentsfromthecity of

São Paulo was assessed after adecade from two doses of

liveMMR vaccine.Parental consentwasobtainedfrom 263

students, but14studentsrefusetoparticipateinthestudy,

despite parental consent. All students participated in the

hygiene exposition, regardlessof parental consentor their

ownconsenttoparticipateinthestudy.Vaccinationcoverage

ofthestudysamplewasexcellent,accordingtothe

vaccina-tioncardrecoveredfrom82.3%ofthestudents,averyreliable

instrument.However,asigniﬁcantfractionhadnotcompleted

vaccination,althoughallhadtakenatleastonedoseofthe

vaccine recorded inthe card.Thisistheusual vaccination

coverageinSãoPaulo,astatewithover95%MMRcoverage.

Therewere160girlsand89boys,aratiosimilartothe

stu-dentpopulationwithgirlsmoreadherenttohighschoolthan

boys,afactcausedbytheirsocioculturalbackgroundand

fam-ilyeconomicstatus.Regardingtoxoplasmosisriskfactors,38%

referedingestionofraworcrudemeat,withonlyafew

vege-tarians(3.2%),mostlyingestingonlywell-cookedmeat(57.8%).

Standardizationofsalivatests

Atotalof49pairedserumandsalivasamplesandadequate

undergraduateadultstudentscontrolsinourassay,usingall

thebiotinlabelledantigens(supplementaryFig.S1),wereused

toverifythereliabilityofsalivaassays.Asitcanbeseenin

Fig.1,therewasnosigniﬁcantquantitativedifferencebetween

serumandsalivaIgGwiththecaptureproteinAassay.The

qualitativeresultsobtainedforserumandsalivasamples

pre-sentedthesimilarproportions,butasmallnumberofnegative

vaccinated samples wasdetectedwith someantigens, like

mumps(2/47)andrubella(3/47),butthesesampleswerealso

of lowreactivity with serum,with border-lineresults. The

mostimportantresultistheabsence ofdifferenceand the

reliabilityofbothIgGsamples,showingthattheuseofcapture

proteinAIgGassaysisafeasiblemethodfordetectingspeciﬁc

IgGforthestudiedantigensinsalivasamples.Dataonantigen

quality,reproducibility,andquantitativecomparisonbetween

serumandsaliva,canbeseeninthesectionofSupplementary

data.Allreactionshadasigniﬁcantcorrelationbetweenserum

andsaliva(SupplementaryFig.S2).Therewashighreactivity

ofmeaslesandrubellasamples,withhighODandconsequent

blurring beyondtheaccuracyofmicroplate reader, causing

alessintensecorrelation,butallpositivesamplespresented

qualitativeconcordanceregardingthepresenceofspeciﬁcIgG,

independentofthesource.

HighschoolsalivasamplesincaptureproteinAELISA

Highschoolstudents’ salivasamplesweretestedonlyafter

conﬁrmingsalivaasareliablesourceofspeciﬁcIgG.The

gold-standardforcomparisoninthisdetectionwasundergraduate

students’saliva,whichhadpairedserum-salivasamples,used

aspositivecontrols.Asnegativecontrols,weusepoolsoffour

negativesera(75)ofunvaccinated8–12month-oldchildren.

Weevaluatedtheefﬁciencyandreproducibilityofourprotein

Acaptureassayandbiotinylatedantigenbycomparingthe

qualitativereactionbytwodifferentreactions,performedwith

thesamesalivadilutionatdifferentdays(SupplementaryFig.

S3),whichshows excellentreproducibilitywithanR2_>_0.95

(4)

5 4 3 2 1 0 Rubella (serum) Ar tificial Units (U A) Rubella (Saliva) Measles (Serum) Measles (Saliva) Mumps (Serum) IgG Assay (Sample)

Mumps (Saliva) T.gondii (Serum) T.gondii (Saliva)

Fig.1–PairedserumandsalivasamplesonplateadsorbedwithproteinAandwithbiotinylatedantigens.Thetypeof antigenisexpressedatthebottomofthegraphwithsampletypebetweenparentheses.Opendotsareserumsamplesand closeddotssalivasamples.Artiﬁcialunits(UA)representstheratioofthereactivityofeachpointand95%cutoffofnegative samplesinthesameplate.Theerrorbarsrepresentthemeanandstandarddeviationofeachpopulationstudied.

TheassaysfordeterminationofIgGspeciﬁcityby

biotiny-latedantigensaftersolidphasecapturewithproteinAshowed

acleardistinctionbetweenpositiveandnegativeseraforthe

fourtypesofantigensanalyzed.Thisallowedustoinferthe

prevalenceoftheseantibodiesinthestudiedpopulationof

preadolescentchildren.QuantitativeanalysisshowninFig.2

depictsexcellentquantitativediscriminationamongstudents,

usingsalivaasthesourceofantibodies.Asformeasles,the

studentsmaintainedahighfrequencyofpositivityfor

anti-measlesIgGantibodies,whichwasnotthecasefortheother

viruses,wherethepositivityratewaslower,aswasthe

fre-quencyofdetectablelevelofspeciﬁcIgG(p<0.001).Therewas

nodifferenceinthefrequencyofpositivitybetweenrubella

andmumps.Fortoxoplasmosis,therewasamuchlowerrate

ofpositivityamongthe students, andtherewas noreason

tocomparethisinfectionwiththeviralantibodiesresulting

fromvaccination,whichonlyrepresentsthecontactofthose

students with that pathogen. Aside from this quantitative

analysis,weperformedaROCcurveanalysisfordeﬁningthe

bestcut-off(SupplementaryFig.S4).Usingthiscut-off,itwas

possibletoestimatethefrequencyofpositivesamplesamong

highschoolstudents,asshowninTable1.

Discussion

Inthiswork,weaimedtostudyifsalivacouldbeusedasa

sourceofspeciﬁcIgGinavaccinatedsampleofhighschool

students.Adolescent peoplehaveadequate comprehension

but not legal responsibility for participation, demanding

parentalconsent.Thisapprovaldemandsseveralcontactsfor

informationand,despitetheefforts ofteachersand school

staff,therewaslowresponseratesinpublicschools,asthe

parentshad no interest or were busy precludingadequate

Table1–Qualitativefrequencyofpositivityinschool salivaintheprotein-speciﬁcIgGcaptureassayand reactivitywithbiotinylatedantigens,afterROCcurve analysis.

Disease Positives Negatives %positives(95% conﬁdence interval) Measles 241 8 96,8%(93,8–98,4) Mumps 143 106 57,4%(51,2–64,4) Rubella 147 102 59%(52,8–65) Toxoplasmosis 21 228 8,4%(5,6–12,5)

analysis. Adhesion to the study was compromised by the need ofbloodcollection. As anstrategyto increase partic-ipation, wepromoted an interactiveexpositionon hygiene andinfectionstransmissionwaystofostervoluntary partic-ipation.Salivacollectionismuchmorefriendlyandcheaper, butpreviousstudieshadreportedsmallfractionsoffalse neg-ativeresultsduetolowIgGcontentinsaliva24_or_proteolysis

of antibodies.24 _Towards _reducing _those _problems, _we _had

reportedaproteinAcaptureassaytorevealspeciﬁcIgGagainst

T.gondiiinsaliva,24_which_minimized_false_negative_results.

Thisresultisexpectedduetothebiologicalactivityof

pro-teinA,whichbindstotheFcoftwoIgGs,presentingtothe

antigenfourbindingFABs,thusresultinginasensitiveassay

withlowIgGlevels,asexpectedinsaliva.Theassaywasalso

performedwithclearedandethanolprecipitatedsaliva,

bring-ing theIgG concentrationtoone-ﬁfth ofthe originalsaliva

volume.

Thus,a10ngbindingofproteinAresultsinthebindingof

90ngofIgGtothemicroplatesurfaceandincreasedbyalmost

10 timesthe absorptionofIgGinthesolidsupport.Several

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4 4 3 3 2 2 1 1 1.0 1.0 2.0 1.5 1.0 1.0 0.5 0.0 0.4 0.6 0.8 1.0 1.0 1.2 1.4 1.6 1.8 2.0 0.8 _0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0

High school students saliva Seropositive saliva

Ar tificial U A Ar tificial U A Ar tificial U A Ar tificial U A

Negative sera pools _{High school students saliva} _{Seropositive saliva} _{Negative sera pools}

High school students saliva Seropositive saliva Negative sera pools High school students saliva Seropositive saliva Negative sera pools

A

B

C

D

Fig.2–DistributionofIgGdetectionresultsbycaptureELISAinsalivasamplesfromhighschoolstudents,comparedto salivafrompositivepairedsamplesandnegativesera.CaptureIgGELISAreactivityexpressedasartiﬁcialunits(U.A.). Verticalaxisinterruptedat95%cut-offofnegativesamples.A–Measles,B–Rubella,C–Mumps,andD–T.gondiiSTAG.

tosupportsolidsuchasthoseusedinplasmonresonanceand

otherdetectionmethods,25_but_the_capture_proposed_in_this

studyprovidescomparativelymoreFABsperareaforspeciﬁc

detection,whichseemsidealfordetectionofspeciﬁcIgG.

Non-captureofantigensbymonoclonalantibodiesdemandsmore

stepsandreagents.24

Ourdatawereequallyefﬁcientandreproducibleforall

anti-gens.Initially, theassays weretested withserum thathad

excess IgG compared to the amount of IgG in saliva.23 As

expected,salivary IgGreactivity wasvery similar toserum

IgGreactivity,asevidencedinallofourassays.Thisﬁnding

wascertainlyrelateddirectlytotheuseofproteinAcapture

inthesolidsupport,whichstandardizedthe IgGsupplyfor

reactionwiththebiotinylatedantigen.Infact,weare

mea-suringtheproportionofIgGreactivetotheantigen,notthe

amountofspeciﬁcIgGinserum,similarlytonon-quantitative

ELISA.

TheIgGcaptureapproach,inadditiontoensuringa

uni-formamountofIgG,alsodecreasedtheriskofcompetition

with other immunoglobulins more frequently present in

saliva,suchasIgA,whichcouldleadtofalsenegativeresultsby

blockingtheIgGreactioninthepresenceoflargespeciﬁcIgA

titers.Antibodycompetitionwasmentionedbyauthorswho

workedwithdengueserologyonseveralbiologicalmaterials.26

Thelowprevalenceoftoxoplasmosisfoundinourstudywas

expectedaspreviousstudiessuggestedthattheincidenceof

thediseaseinourareaisdecayingamongtheyouth.24

Formumpsantigen,thefailureoftheoriginalbiotinylation

wasduetothisrecombinantantigenbeinglesssolubleand

ureawasusedintheoriginalpreparation,whichresidual

con-taminationpreventedcorrectlabelling.Theoptionfoundwas

biotinylating in dilutedsolutions aftermolecular exclusion

chromatographytoremovealllowmolecularweight

contam-inants inwhich wewere successful.However,it prevented

Western blot executiondue tothe extreme dilutionof the

biotinylatedantigen.Inthisway,allofourantigensshowed

excellentmarkingandallowedcontinuityoftheassay

devel-opment.

Ourdatashowed severalimportantaspectsofthetriple

viral vaccination. Mostimportantly,measles vaccine

cover-age inour groupis relativelymaintained, whileprotection

forrubella and mumpswas lessdurable witha signiﬁcant

fractionoflostprotection,whichmightexplaintherecent

(6)

needforadditionalvaccinationdosesduringadolescence,an

importantsanitarymeasureforthepreventionofcongenital

rubella.27

Ourdatacorroboratepreviousreportsofpreferentiallossof

immunityagainstmumpsandrubellacomparedtomeasles.

Therearedifferentmumpsvirususedinvaccineswith

vary-ingdegreesofefﬁcacy.28 _The_worldwide_triple _viral_vaccine

hasvariableratesofsuccessandtherehavebeenfewrecent

qualitycontrolinvestigations.Inarecentreview,Plotkinetal.

discussseveralaspectsoftheconcomitant immunogenicity

ofviralinfectionspreventablebyMMR.Thereisacorrelation

betweenantibodies andprotectionformeaslesandrubella,

butfewstudieshavebeenconductedwithmumps,andeven

inthisreview,therearenoreportsofefﬁcacyevaluationofthis

vaccine.29

Ourstudyshowsapossibleshorterdurationofprotection

againstthetwolessdominantviruses(rubellaandmumps)

inourpopulationand theexplanation forthatiscomplex.

Inanaturalinfection,measlesvirusinduceshost

immuno-suppression, asassessed bylung histologyinchildren.29,30

Incontrast,measlesvaccineappearstoaffecttheimmunity

of vaccinated persons by increasing resistance to

infec-tions.Interestingly,thedisappearanceofimmunityhasbeen

reportedforonlytwovaccinesecondaryviruses,31_as_found_in

ourstudy.

Therearenoreportsofconstantqualitycontrolof

com-mercialpreparationsofthevaccineandtheremaybemore

environmentalresistanceofthemeaslesvirus.Thus,itwas

notunexpectedthatimmunitytosecondaryvaccineviruses

would decline, as short-term reports had already

demon-stratedthisproblem.32_The_need_for_prevention_of_congenital

rubellasyndromehasbeen ofmajorconcernandtherefore

revaccination in with the triple viral vaccine is

recom-mended in adolescence,30 _as _has _been _conducted _in _our

state.

Resurgentoutbreaksofvaccinepreventablediseases

pre-viouslycontrolledoreliminatedhavebeenobservedinmany

contexts.Vaccinationcampaignscanpreventorevencontrol

outbreaksbut must beworkedwithconsiderable certainty,

andreal-timesurveillancecanprovidevaluableinformation

aboutthepopulationatriskandwhowouldbetheprimary

targetsforvaccinationtoblockpotentialoutbreaks.However,

wearefacedwithpreventivediagnosticlimitations,since

ide-allytheimmunologicalstatusshouldbeconﬁrmedforeach

individual,beforeanunnecessaryrevaccination,32,33_in_areas

withverygoodimmunizationcoverage.9

Ourapproachwasefﬁcientinallaspects,fromthe

inter-activeexhibitionforbettercompliancewithsalivacollection

tothedevelopmentofreliablecaptureELISAtobeusedwith

salivafor determinationofvaccine immunestatus or

tox-oplasmosis immunity among public high school students.

Inconclusion,wecanproperlyplanadequatepublichealth

measuresbothindividuallyor asgroup, suchasindividual

revaccinationincaseoflossofvaccineimmunityorestimating

realvaccinecoverageinlargergroups.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

This experimental work was supported by Fundac¸ão a

Pesquisa no Estado de São Paulo/SP/Brazil(FAPESP Process

13/04676-9). Thiswork was the experimentalpart ofPh.D.

thesis inTropical Medicine/USPofBarbara CarvalhoFialho

Sampaio. B.C.F.Sampaio and J.P. Rodrigues were fellowsof

Coordenac¸ãodeAperfeic¸oamentodePessoaldeNivelSuperior

(CAPES)forthePh.D.PrograminTropicalMedicine/USP.L.R.

MeirelesandJ.P.Rodriguesleadedtheinteractiveexposition

withparticipationofGabrielaMarquesandYugoGushiken.

ThisexpositionwasﬁnancedbythePró-ReitoriadeCultura

eExtensãoUniversitária,USP,Brazil.LIMHCFMUSPpartially

supportedinfrastructureofthisresearch.

Appendix

A. Supplementary

data

Supplementary material related to this article can be

found, in the online version,at doi:https://doi.org/10.1016/

j.bjid.2019.11.005.

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