Salivary anti-PGL-1 IgM may indicate active transmission of Mycobacterium leprae among young people under 16 years of age

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

Brief

communication

Salivary

anti-PGL-1

IgM

may

indicate

active

transmission

of

Mycobacterium

leprae

among

young

people

under

16

years

of

age

Alexandre

Casimiro

de

Macedo

_,

_José

_Evandro

_Cunha

_Jr.

_,

Juliana

Navarro

Ueda

Yaochite

_,

_Clodis

_Maria

_Tavares

_,

_Aparecida

_Tiemi

_Nagao-Dias

a_{UniversidadeFederaldoCeará(UFC),FaculdadedeFarmácia,DepartamentodeAnálisesClínicaseToxicológicas,Fortaleza,CE,Brazil}

b_{UniversidadeFederaldeAlagoas,FaculdadedeEnfermagemeFarmácia(ESENFAR),Maceio,AL,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3January2017 Accepted11May2017 Availableonline9June2017

Keywords:

Leprosy

Salivaryantibodies

Phenolicglycolipid-1antigen

Mycobacteriumleprae

a

b

s

t

r

a

c

t

ConsideringthatthemainrouteofMycobacteriumlepraetransmissionistheupperrespiratory tract,detectionofsalivaryantibodiescanbeausefultoolfordiagnosingearlyinfection.The studyaimedtoanalyzesalivaryanti-PGL-1IgAandIgMantibodiesin169childrenaged4–16 yearsold,wholivednearbyorinsidethehouseofmultibacillaryorpaucibacillaryleprosy patientsintwoendemiccitiesinAlagoasState–Brazil.Salivaryanti-PGL-1antibodieswere quantifiedbymodifiedELISAmethod.Thefrequencyofcontactandclinicalformofthe indexcaseweresignificantlyassociatedwithsalivaryantibodylevels.Highfrequencyof IgMpositivitystronglysuggestsactivetransmissionofM.lepraeinthesecommunities.We suggestinthepresentworkthatsalivaryanti-PGLIgAandIgMareimportantbiomarkersto beusedforidentifyingcommunitieswithprobableactivetransmissionofM.leprae.

©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Introduction

Brazil isthe second country withthe highestincidence of leprosyintheworld.In2015,thecountrypresenteda detec-tionrateof14.06casesper100,000inhabitants.1_Althoughthe

numberofnewcasesseemstodecrease,itmay not repre-sentthereality.Forinstance,thehighincidenceofthedisease amongchildrenmeansthatanactivetransmissionoccursin

∗ _{Correspondingauthor.}

E-mailaddress:anagaodias@gmail.com(A.T.Nagao-Dias).

thecommunity.2_{In2015,thedetectionratesofleprosyamong}

people under15 years oldin SantanadoIpanema andRio Largo,twoBraziliancitieslocatedinAlagoasState,were13.77 and32.81per100,000inhabitants,respectively.3

Asthebacteriaarenotcultivable,secretoryantibodiescan beausefultooltodetectearlyinfection.Thenasopharynxis themainportalofentryforMycobacteriumleprae(M.leprae),

andthenasalepithelialcellsareanimportantreservoirofthe bacteria.4_{Asmucosalimmuneorgansandtissuescomposean}

integratedsystem,salivaisfrequentlyconsideredtobe repre-sentativeofmucosalhumoralimmuneresponse.Thepurpose ofthe present work was to evaluatesalivaryanti-phenolic

http://dx.doi.org/10.1016/j.bjid.2017.05.001

1413-8670/©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

5.0 4.0 3.0 2.0 1.0 0.0 p=0.03 p=0.0019 p=0.028 p=0.055 MB PB MB PB HH PD HH PD

IgM IgA IgM IgA

OD 405 5.0 4.0 3.0 2.0 1.0 0.0 OD 405

A

B

Fig.1–Levelsofsalivaryanti-PGL-1antibodiesin169youngcontactsofleprosypatients.(A)Medianandrangeofsalivary

anti-PGL1IgMandIgAincontactsofmultibacillary(MBcontacts,n=115)andpaucibacillary(PBcontacts,n=40)leprosy

patients.(B)Medianandrangeofsalivaryanti-PGL1IgMandIgAlevelsinhousehold(HH,n=57)andperidomiciliar(PD,

n=112)contacts.Salivaryanti-PGL-1antibodiesweredetectedbymodifiedELISAmethod.

glycolipid1antigen(PGL-1)IgAandIgMisotypesamong169 leprosycontactsaged4–16yearslivinginthemunicipalities ofSantanadoIpanemaandRioLargo(Alagoasstate,Brazil).

Methods

Subjectsandsamplecollection

Thecontacts(n=169)includedinthestudywereclassifiedas paucibacillary(PBcontacts,n=40)ormultibacillary(MB con-tacts,n=115)contacts,accordingtoclinicalformoftheindex case.Fourteencontactswerenotclassifiedbecausethe infor-mationwasnotavailableinthepatients’medicalrecords.The participantswerealsoclassifiedashouseholdcontacts(HH,

n=57)orperidomiciliarcontacts(PD, n=112).Peridomiciliar contactswerethosewhowererelativesoftheindexcasebut didnotliveinthesamehouseorthosewholivedclosetothe indexcase’shouse(uptofivehousesapart).Theprojectwas approvedbytheNationalCommitteeforEthicsinResearch. Unstimulatedsalivasampleswerecollectedintotubes,which weretransportedwithicepackstothelaboratory,wherethey werekeptat−20◦Cuntiltesting(uptothreeweeksafter col-lection).Thepresenceoflesionsandnerveenlargementwere investigatedatthemomentofsamplecollection.Cases sus-pectedofhavingthedisease werereferredtoadoctorand excludedfromthestudy.

Detectionofsalivaryanti-PGL-1antibodies

MicroplateswerecoatedwithnativePGL-1at5mg/Lin abso-lutealcoholfor2hat37◦C(protocol modifiedfromBrito e Cabraletal.,2013).5_{Afterblockingwith1%fetalbovineserum}

(FBS,LGCBio,Brazil)-Trissolutionfor2hat37◦C,thewells were incubated with previously cenrifuged saliva samples (dilutedto1:50with1%FBS-Tris).After18hat4◦Cand wash-ingwith0.05%FBS-Trissolution,anti-humanIgAoranti-IgM alkalinephosphatase antibodies (Sigma, USA,1:1000 in1% FBS-Tris)wereleft on theplates for2h at37◦C.Afternew incubationfor2hat37◦C,andwashing,thesubstratesolution

(1mg/mL p-nitrophenyl phosphate in 10% diethanolamine containing 0.5mM MgCl2, pH 9.8) was added to the wells.

After100minatroomtemperature,absorbancereadingswere recorded at405nm using an ELISA microplate reader. The resultswereexpressedastheODmeanofthevalues(minus blank).Thecut-offwasbasedonthe97thpercentileofnormal controls.6_{Results30%abovethecut-offvaluewereconsidered}

tobepositive.

Analysisofdata

Thedatawereanalyzedusingnonparametrictestsasthedata didnotfollowaGaussiandistribution(Kolgomorov–Smirnov test).All statisticalanalysiswasperformedusingGraphPad Prismversion5.0.Thelevelofstatisticalsignificancewas5% (p<0.05).

Results

Salivaryanti-PGL-1IgMpresentedgoodcorrelationtosalivary IgAtiters(Spearmancorrelation,r=0.71,p<0.0001).No statis-ticalsignificancewasfoundregardingtheagerange,eitherfor IgMorIgA(Kruskall–Wallistest,p=0.149andp=0.312, respec-tively,Table1).Nosignificantdifferenceswereeitherfoundin IgMorIgAtitersinrespecttothedegreeofrelationshipwith the indexcase(p=0.325andp=0.590,respectively,Table1). Contactswhoreportedhavingweeklycontactwiththeindex casehadhigherIgMantibodytitersthanthosewithdaily con-tact(p=0.04,Table1).MBleprosycontactspresentedhigher levelsofsalivaryanti-PGL-1IgMandIgA(Mann–Whitneytest,

p=0.03 and p=0.05, respectively)than PB leprosycontacts (Fig.1A).Interestingly,PDcontactshadhigherlevelsof sali-varyIgMandIgA(Mann–Whitneytest,p=0.019andp=0.028, respectively)thantheHHcontacts(Fig.1B).

Discussion

Withtheadventofmultidrugtherapythereportofnewcases ofleprosyhad asharp decrease.However,thisdeclinehas

Table1–Titersofsalivaryanti-PGL1IgAandIgMin169youngcontactsofleprosypatientsaccordingtotheagerangeof leprosycontacts,theirdegreeandfrequencyofrelationshipwiththeindexcase.

Salivaryantibodyisotype

Agerange(years) IgM IgA

4–6 7–11 12–16 4–6 7–11 12–16 n 26 84 59 26 84 59 Antibodytiters (median) 0.032 0.070 0.050 0.087 0.115 0.102 Antibodytiters (interquartile range) 0.010–0.109 0.026–0.428 0.018–0.270 0.050–0.155 0.062–0.226 0.06–0.183 Kruskall–Wallis test,p 0.149 0.312 Familyorsocial relationship withtheindex case Son/daughter Brother/sister Grand son/Grand daughter Nephew/ niece/cousin/ cousine Others Son/daughter Brother/sister Grand son/Grand daughter Nephew/ niece/cousin/ cousine Others n 32 18 26 93 32 18 26 93 Antibodytiters (median) 0.035 0.100 0.036 0.062 0.095 0.132 0.125 0.107 Antibodytiters (interquartile range) 0.010–0.088 0.023–0.250 0.010–0.483 0.024–0.324 0.050–0.165 0.059–0.194 0.042–0.263 0.067–0.200 Kruskall–Wallis test,p 0.325 0.590 Frequencyof relationship withtheindex case

Daily Weeklya _{Monthly N.m. Daily Weekly Monthly N.m.}

n 72 27 5 65 72 27 5 65 Antibodytiters (median) 0.037 0.25 0.087 0.050 0.100 0.130 0.076 0.110 Antibodytiters (interquartile range) 0.010–0.190 0.030–0.550 0.038–0.399 0.02–0.232 0.058–0.185 0.076–0.260 0.062–0.362 0.060–0.199 Kruskall–Wallis test,p 0.04 0.499 N.m.=notmentioned.

a _{p<0.05inrelationtodailyrelationship.}

becomelesssteepinrecentyears;onthecontrary,therehas beenariseinleprosycasesincludingchildren.6_Thismakes

thegoalofeliminatingleprosyimpossibletobeachievedinthe nextfewyears,2_{rememberingthattherearepossible}

undiag-nosedcasesthatarehiddensourcesofbacterialtransmission. Inaddition,therearemanyunknownaspectsregardingthe ecology of M. leprae.6 _{Strategies are necessary to interrupt}

transmission,suchasthedevelopmentofbiomarkersto iden-tifycontactsand/ortoidentifythoseatriskofdevelopingthe disease.7

Mucosal immunity in leprosy is poorly understood, although it is known that the nasal cavity is one of the firstsitesinfectedbyM.leprae,andthe oralcavitycanalso be affected, as observed in late-diagnosed patients.8

Sali-varyantibodies ofthe IgAisotypehavebeenconsidered as biomarkersofinfection,and alsoofimmunity,considering that they may play a role in inhibiting cell adhesion and

in opsonophagocytosis.9 _{Smith et al.(2004), in a follow-up}

studyofpeopleresidinginendemicregionsforleprosy,found aninitialpositivityof1.6%forpolymerase-chainreactionof nasalswaband67.7%forsalivaryanti-M.lepraeIgA.10_Avery

interesting aspect observedin the study was that the fre-quencyofpositivitywashigherincertainseasonalperiods, especially inthepresenceofhumidity, suggestingthat the bacillus remains in the community but not necessarily in theindividual.10_{Inaccordancewiththishypothesis,Mohanty}

andcolleaguesdetectedviablestrainsofM.lepraein environ-mentalsamplesobtainedfromaroundthehousesofleprosy patients in Ghatampur (India). The prolonged presence of bacillicould playanimportantrole inthecontinued trans-missionofleprosy.11

A very low number ofstudies refer to the presenceof anti-PGL1IgMinsaliva,5,9,12,13_{whichpossiblyindicatesrecent}

is only five days, and their levels may be correlated with bacillaryload.12 _{Abeetal.(1984)foundafrequencyof}

pos-itivitycorresponding to4.5%(fiveout of110patients).9 _We

foundmuchhigherpositivityofsalivaryanti-PGL1IgM iso-typeamongleprosycontacts,i.e.17outof47samples(36.1%) inRioLargo,and15 out of122samples(12.3%)ofchildren fromSantanadoIpanema.Likewise,inapreviousstudy car-riedoutinCratoandMaracanaúcities,stateofCeara,Brazil, 13outof135samples(9.6%)turnedoutpositiveforsalivary anti-PGL1IgM.5 _{Inthisway,onecouldinferthe magnitude}

ofactive transmission in the community. In fact, in 2013, thecasedetectionrateamongyoungpeopleunder15years old was 13.25 casesper 100,000 individuals in Santana do Ipanema,whilenocasewasdetectedinRioLargo.In2014, nocasewasdetectedinthetwocities.In2015,thecase detec-tionrateinSantanadoIpanemawas32.81casesper100,000 individuals and 13.77 cases per 100,000 individuals in Rio Largo.1

Asleprosyinfectionrequiresprolongedcontacttime,those wholiveinthesamehouse oftheindexcaseisbelievedto beatriskfordevelopingthedisease;however,recentreports demonstrated that those who live nearby the index case shouldalsobeinvestigated.14

Itisanintriguing factfound inourpresent work those wholived nearbytheindexcasepresentedhigherlevelsof salivaryantibodiesthan thehouseholdcontacts. The para-doxtolerance/activationmakesthemucosalimmunesystem a challenging task. The mucosal immune response may be affected by various factors,such as soluble or particu-lateantigens,chemicalnatureandconcentrationofantigen, frequencyof exposition,gut microbiota composition, envi-ronmentalantigenicexposure,nutritionalstatus(deficiency of vitamin A), chronic infections with helminths or other parasites.15_{PGL-1,forinstance,facilitatesbacterialadhesion,}4

modulatesmacrophagecytokineandchemokineproduction, andmayleadTcellstoanergy.16_{Inthisrespect,itis}

proba-blethatPGL-1exertssometypeoforaltoleranceonmucosal immuneresponse.

Natural killer T cells recognize glycolipid antigens pre-sented byCD1d moleculeand may alsoplayan important roleinoraltolerancebyinducingtolerogenicdendriticcells andregulatoryTcells,orbydeletingantigen-specificTcells.17

Thesemechanismscouldpartlyexplainwhatmaybe occur-ringinchildrenwithprolongedandsustainedcontactwith theindexcase.

DetectionofpositivesalivaryIgMamongyoungpeople sug-geststhatM.lepraetransmissionisactiveinthecommunity. For this reason, a strategy atmunicipal level isextremely urgentinordertoreducethedisseminationofthebacillus. Finally,wesuggestinthepresentworkthatsalivaryanti-PGL IgAand IgMare importantbiomarkerstobeusedfor iden-tifyingcommunitieswithprobableactivetransmissionofM. leprae.

Funding

information

This research was financially supported by the MCTI/CNPq/MS-SCTIE[Process403461/2012-0].

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Wewould liketothankMrs. AnaLúciaCarneiroLeal, Mrs. GilvâniaFranc¸aVilela,andMrs.AndreaMárciaCostadeFarias forhelpfulassistance.

r

e

f

e

r

e

n

c

e

s

1.Brasil.SaladeapoioàgestãoestratégicadoMinistérioda Saúde(SAGE).Indicadoresdemorbidade:hanseníase;2017 [online].Availablefrom:http://sage.saude.gov.br/#/[Accessed March31,2017].

2.SalgadoCG,BarretoJG,daSilvaMB,FradeMA,SpencerJS. Whatdoweactuallyknowaboutleprosyworldwide?Lancet InfectDis.2016;16:778.

3.Brasil.Diretrizesparavigilância,atenc¸ãoeeliminac¸ãoda Hanseníasecomoproblemadesaúdepública:manual técnico-operacional[online].MinistériodaSaúde,Secretaria deVigilânciaemSaúde,DepartamentodeVigilânciadas Doenc¸asTransmissíveis–Brasília:MinistériodaSaúde;2016. Availablefrom:http://portalsaude.saude.gov.br/images/pdf/ 2016/fevereiro/04/diretrizes-eliminacao-hanseniase-4fev16-web.pdf

4.SilvaCA,DanelishviliL,McNamaraM,etal.Interactionof

Mycobacteriumlepraewithhumanairwayepithelialcells: adherence,entry,survival,andidentificationofpotential adhesinsbysurfaceproteomeanalysis.InfectImmun. 2013;81:2645–59.

5.BritoeCabralP,JúniorJE,deMacedoAC,etal.Anti-PGL1 salivaryIgA/IgM,serumIgG/IgM,andnasalMycobacterium lepraeDNAinindividualswithhouseholdcontactwith leprosy.IntJInfectDis.2013;17:e1005–10.

6.SmithWC,vanBrakelW,GillisT,SaundersonP,RichardusJH. Themissingmillions:athreattotheeliminationofleprosy. PLoSNeglTropDis.2015;9:e0003658.

7.SmithWC,AertsA.Roleofcontacttracingandprevention strategiesintheinterruptionofleprosytransmission.Lepr Rev.2014;85:2–17.

8.CostaMRSN.Considerac¸õessobreoenvolvimentoda cavidadebucalnahanseníase.HansenInt.2008;33:41–4.

9.AbeM,YoshinoY,MinagawaF,etal.Salivary

immunoglobulinsandantibodyactivitiesinleprosy.IntJLepr. 1984;52:343–50.

10.SmithWC,SmithCM,CreeIA,etal.Anapproachto

understandingthetransmissionofMycobacteriumlepraeusing molecularandimmunologicalmethods:resultsfromthe MILEP2study.IntJLeprOtherMycobactDis.2004;72: 269–77.

11.MohantyPS,NaazF,KataraD,etal.ViabilityofMycobacterium lepraeintheenvironmentanditsroleinleprosy

dissemination.IndianJDermatolVenereolLeprol. 2016;82:23–7.

12.Nagao-DiasAT,AlmeidaTL,OliveiraMeF,SantosRC,LimaAL, BrasilM.Salivaryanti-PGLIgMandIgAtitersandserum antibodyIgGtitersandaviditiesinleprosypatientsandtheir correlationwithtimeofinfectionandantigenexposure.Braz JInfectDis.2007;11:215–9.

13.BritoeCabralP,AlvesAR,MedeirosKBA,Castelo-BrancoES, deAlencarCP,Nagao-DiasAT.Empregodemarcadores

séricosesalivaresanti-PGL-1comoparâmetrosdeexposic¸ão ocupacionalaoMycobacteriumleprae.RevPanamInfectol. 2009;11:21–6.

14.BarretoJG,BisanzioD,GuimarãesLdeS,etal.Spatialanalysis spotlightingearlychildhoodleprosytransmissionina hyperendemicmunicipalityoftheBrazilianAmazonregion. PLoSNeglTropDis.2014;8:e2665.

15.LevineMM.Immunogenicityandefficacyoforalvaccinesin developingcountries:lessonsfromalivecholeravaccine. BMCBiol.2010;8:129.

16.DagurPK,SharmaB,UpadhyayR,etal.Phenolic-glycolipid-1 andlipoarabinomannanpreferentiallymodulateTCR-and CD28-triggeredproximalbiochemicalevents,leadingtoT-cell unresponsivenessinmycobacterialdiseases.LipidsHealth Dis.2012;11:119.

17.KimHJ,HwangSJ,KimBK,JungKC,ChungDH.NKTcellsplay criticalrolesintheinductionoforaltolerancebyinducing regulatoryTcellsproducingIL-10andtransforminggrowth factorbeta,andbyclonallydeletingantigen-specificTcells. Immunology.2006;118:101–11.