Association between NDO-LID and PGL-1 for leprosy and class I and II human leukocyte antigen alleles in an indigenous community in Southwest Amazon

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

Association

between

NDO-LID

and

PGL-1

for

leprosy

and

class

I

and

II

human

leukocyte

antigen

alleles

in

an

indigenous

community

in

Southwest

Amazon

Stéfanie

Ferreira

Teles

_,

_Eliane

_Aparecida

_Silva

_,

_Rodrigo

_Medeiros

_de

_Souza

_,

Jane

Tomimori

_,

_Marcos

_Cesar

_Florian

_,

_Rogério

_Oliveira

_Souza

_,

Elaine

Valim

Camarinha

Marcos

_,

_Fabiana

_Covolo

_de

_{Souza-Santana}

_,

Mônica

Antar

Gamba

a_{UniversidadeFederaldoAcre,CentroMultidisciplinar,CruzeirodoSul,AC,Brazil}

b_{InstitutoLaurodeSouzaLima,Bauru/SP,LaboratóriodeImunologia,Bauru,SP,Brazil}

c_{UniversidadeFederaldoAcre,CentroMultidisciplinar,LaboratóriodeImunologiaeMicrobiologia,CruzeirodoSul,AC,Brazil}

d_{UniversidadeFederaldeSãoPaulo,EscolaPaulistadeMedicina,DepartamentodeDermatologia,SãoPaulo,SP,Brazil}

e_{InstitutoLaurodeSouzaLima,Bauru/SP,LaboratóriodeImunogenética,Bauru,SP,Brazil}

f_{UniversidadeFederaldeSãoPaulo,EscolaPaulistadeEnfermagem,DepartamentodeSaúdeColetiva,SãoPaulo,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received7February2020 Accepted31May2020 Availableonline24June2020

Keywords:

Leprosy

SouthAmericanindigenous populations HLA ClassIMHCgenes ClassIIMHCgenes Nursing

a

b

s

t

r

a

c

t

The frequencies of the Human leukocyte antigen (HLA) alleles in the Puyanawa indige-nousreservepopulationandtheirassociationwiththeNDO-LIDandELISAPGL-1rapid serologicaltestwasassessed. Thiswasacross-sectionalstudywithanepidemiological clinicaldesign conductedin two indigenous communitiesin the stateof Acre, Brazil. Bloodwascollectedina tubewithEDTAtoidentifyHLA allelesandperform serologi-caltests. DNAwasobtainedusingthe saltingoutprocedure.TheLabTypeTM _technique (One-Lambda-USA)wasusedforHLAclassI(lociA*,B*andC*)andII(lociDRB1*,DQA1* andDQB1*) typing.Allele frequency wasobtained by directcount,and the chi-square testwasusedtoassesstheassociationwiththeNDO-LIDandPGL-1tests.Themost fre-quentallelesinthetwocommunitieswere:HLA-A*02:01,HLA-B*40:02,HLA-DRB1*16:02, HLA-DQA1*05:05 and HLA-DQB1*03:01. The allele HLA-C*04:01 was the most common in the Barão community, and the allele HLA-C*07:01 in Ipiranga. Among individuals whopresentedseropositivitytotheNDO-LIDtest,theassociationwithallelesHLA-A*02 (43.18%vs24.8%,p=0.03,OR=2.35)andHLA-B*53(6.83%vs0.0%,p=0.03,OR=8.95)was observedintheBarãocommunity.HLA-B*15wasassociatedwithnon-seroconversionto theNDO-LIDtestinIpiranga.Inbothcommunities,HLA-B*40andHLA-C*03were associ-atedwithpositiveserologicalresponsetoELISAPGL-1.TheHLAclassIandIIallelesmost

∗ _{Correspondingauthor.}

E-mailaddress:stefanieftm@hotmail.com(S.F.Teles). https://doi.org/10.1016/j.bjid.2020.05.009

1413-8670/©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

frequently foundin thisstudy have already beendescribed amongTerena indigenous groups,andHLAclassIcontributestoseroconversiontoNDO-LIDandPGL-1testsin inhab-itantsoftheBarãoandIpirangacommunities.

©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

Leprosyisachronicgranulomatousinfectioncausedbythe

Mycobacteriumleprae(M.leprae)1_{thathasaffectedhumanityfor}

centuries.Evenaftertheintroductionofpolychemotherapy,it remainsapublichealthprobleminBrazil.2_{Theinfection is}

transmittedmainlyamongfamilymemberswhosharegenetic andenvironmentalfactors,besidesbeingconsiderably influ-encedbythevariationingenesrelatedtoimmunity.3–7

Manydiseases–includingleprosy–havebeenrelatedto theHLAsystemduetoits importanceinadaptiveimmune response.4,8–10_{Studiesdesignedasfamilyaggregation,}

com-plex segregation, linkage, association, and genome scans suggestthatleprosyphenotypeslargelydependonthehost’s geneticcharacteristics.Numerouspositiveassociationshave linkedclassIandIImoleculeswithsusceptibilitytoleprosy, itsclinicalformsand,leprosyreactions.3,11–14

Anexperimentalstudy conductedwithVietnamese and Indian individuals showed a strong association of HLA-C (HLA-C*15:05)inthesusceptibilitytoleprosy.11_Thepresence

ofHLA-A*28, in Mexico’s mixed-race population, has been definedas apredisposition marker tolepromatous leprosy (LL).13_{InBrazil,apositiveassociationwasfoundbetween}

HLA-A*11,HLA-B*38,HLA-C*12,andleprosy,12_{andallelesA*03and}

A*11were associatedwithsusceptibility todevelop border-line leprosy(BL).3 _{The protectiveassociation was observed}

betweenclinicalborderlineleprosyandHLA-C*05.6

Regarding class II molecules, studies indicate a more consistentassociationbetweenHLA-DRB1/DQB1 allelesand susceptibilitytoleprosy,suggestingthatthesecouldbethe maingenes associated with leprosysusceptibility.8–10

Sev-eralstudieshavereportedassociationofallelesHLA-DRB1*04, DRB1*10,DRB1*12,DRB1*15andDRB1*16withsusceptibilityor resistancetoleprosyinseveralpopulationsworldwide.8–10,14

Studies show that alleles HLA-DRB1*15, and DRB1*16 are associatedwithleprosy,especiallyinIndian, Japanese,and Brazilianpopulations,5,8,9,15,16 _{whereas alleles HLA-DRB1*04}

andHLA-DRB1*07areassociatedtoprotection,5,17andthe lat-terwithBL.6

SerologicalmethodsareindicatorsofthespreadoftheM.

lepraeinfectioninthegeneralpopulationandcancontribute

positively asanepidemiologicalsurveillance instrument of leprosy.18–20 _{These methods use techniques based on the}

detectionofspecificanti-M.lepraeantibodies.Thus,thosewith positiveresponsestothetestshavehigherdiversityof anti-bodiesagainstthebacillus, aresponsepresentedmainlyin multibacillarycases(MB).18,21,22

Seropositivityto phenolic glycolipid 1(PGL-1) estimates a three-fold greater chance of developing the disease.23,24

Wambieretal.,25_{observedthatthePGL-1seropositivityrate}

wasaffectedbykinship,beinghigheramongsiblings,followed byparents,spouses,andchildren,suggestingthat transmis-sion of the disease occurs mainly among individuals who sharegeneticandenvironmentalaspects.

LID-1 (Leprosy IDRIDiagnostic),derivedfrom the fusion betweenrecombinationalproteinsML0405 andML2331 and the PGL-1 antigen, represent important antigens specific to leprosy serology, and studies have suggestedthat their associationimprovesserologicalsensitivitytodiagnose pau-cibacillary leprosy (PB) and early disease.26–28 _{Such good}

serologicalreactivityledtothedevelopmentofasimpleand rapidtestincorporatingtheLID-1andPGL-1antigens, immo-bilized in a nitrocellulose membrane for the detection of antibodies,aidingintheleprosydiagnosis.27

ThestateofAcre,anendemicregionforleprosyinBrazil, maintainsahighdetectioncoefficientof15.07per100 thou-sandinhabitants.29

TheoccurrenceofMBcasesinIpirangacommunity, the possibledetectionfailureinearlydisease,30_{contactwiththe}

regionalpopulation,andculturalandsocioeconomichabitsof indigenouspeoples,aresubstratesthatpredisposetoahigher riskandexposuretothedisease,justifyinganinvestigation.

To analyze genetic and serological characteristics in an indigenouscommunityisnotacommonandeasytask.Given thiscontext,thisstudywasanopportunitytogather knowl-edgeaboutleprosy,HLAgenotypes,andthe contributionof thesemoleculestoseroconversiontoNDO-LIDandELISA PGL-1testsinthisnever-studiedethnicgroup.

Thus, assuming the participation of HLA alleles in the immunological response inleprosy and the importanceof serologyintheidentificationofsubclinicalinfectionandasa diagnosticaid,thisstudyproposestoidentifythefrequencies ofHLAallelesintheindigenouspopulationofthePuyanawa reserve,andtoassesstheirassociationwiththeresultsofthe rapidlateralflowserologicaltestNDO-LIDandELISAPGL-1.

Material

and

method

Thiswasacross-sectionalstudywithepidemiological clini-caldesignconductedintwoindigenouscommunities,Barão andIpiranga,inthemunicipalityofMâncioLima,Acre,Brazil. Thesecommunitiesarepartoftheterritoryoftheindigenous ethnicgroupPuyanawawithestimatedpopulationof576 indi-viduals–320inBarãoand256inIpiranga,accordingtodata fromtheIndigenousHealthCareInformationSystem.

Thesampleconsistedof284individuals,over18yearsof age,whohadlivedformorethanoneyearinthecommunities withoutpriordiagnosisofleprosy.Amongthestudied individ-uals,145(51%)weremen,meanage36,rangingfrom18to31 years.

Samples

ToidentifyHLAallelesandconducttheserologicaltests,10mL peripheralvenousbloodwascollectedinatubewith ethylene-diaminetetraacetic acid (EDTA). Of the obtained material, 5mLwereusedtoextractplasma,whichwasseparatedinto aliquotsandstoredat−20◦_{C.Therestofthematerial(5mL)}

wasstored at2–8◦C.Onealiquot wasused toperformthe NDO-LIDtest,andtheremainingsamplewassenttotheLauro deSouzaLimaInstituteinBauru/SPforidentificationofthe HLAallelesanddetectionofELISAPGL-1.

Serologicaltests

DetectionofIgMantibodies against PGL-1ofM. lepraewas performed byELISA technique, developedaccording tothe methodology described by Brett et al., 1983. All sera were testedinduplicateandtheELISAresultswereconsidered pos-itivewhenopticaldensity(OD)wasequaltoorgreaterthan 0.150(themeanabsorbancesplusthreestandarddeviations of100healthyindividuals).

ThekitmanufacturedbyOrangeLifeinBrazilwasusedfor immunochromatographytechnique(NDO-LID),adding 10␮L ofplasmafollowedby100␮Lrunningbuffer(2–3drops).The testresultwasconsideredpositivewhentheypresentedcolor inthetestandcontrollines.Theresultwasconsidered neg-ativewhennocolorwasseeninthetestline.Thetestswere photographedusingaDSC-HX9Vcamera(Sony,Tokyo,Japan) 20minaftertheadditionofthebuffersolution.

DNAextraction

ThesaltingouttechniquewasusedtoobtainDNAfromthe leukocytes.Theconcentrationofthesamplewasadjustedat 20ng/mLandstoredat−80◦_{Cinafreezeruntiluse.}

HLAtyping

TypingofclassI(lociA*, B*andC*) andclassII(lociDRB1*, DQA1*,DQB1*)HLAalleleswasconductedusingtheLabTypeTM

technique (One-Lambda, USA), which applies LuminexTM

technologytothereverseDNA-SSOtypingmethod.

Theprincipleofthetechniqueconsistsinthe amplifica-tion of the HLA region by PCR using a specific primer for eachlocus.ThePCRproductisbiotynilated,allowingittobe detectedusingstreptavidinconjugatedwithR-phycoerythrin (SAPE).ThePCRproductis denaturedandre-hybridized in a specific sequence oligonucleotide probes immobilized in fluorescence-codedmicrospheres.Thefluorescenceintensity ofPE(phycoerythrin)ineachmicrospherewasidentifiedby aLabScanTM _{100 flowanalyzer. An analysissoftware (HLA}

Fusion)was usedtodeterminethe HLAgenotyping,which isbased onthe reaction pattern comparedtothe patterns associatedtoalreadyidentifiedHLAgenesequences.

Ethicalaspects

This study was approvedby the ResearchEthics Commit-teeon HumanBeingsand bythe National ResearchEthics

Commission (CONEP) under opinion nos. 940.163 and 1.018.673,respectively.

Statisticalanalysis

Thefrequencyofalleleswasobtainedbydirectcount. The analysis of the association between HLA alleles and NDO-LID results was conducted using the chi-square or Fisher’s exact test using the Graph Pad program (http://www.graphpad.com/quickcalcs/contingency1.cfm). Statisticalsignificancewasconsideredforp-valuesless0.05. OddsRatio(OR)witha95%confidenceinterval(95%CI)was cal-culated using SISA(http://www.quantitativeskills.com/sisa/) whenpwasstatisticallysignificant.

From the HLA genotypes for the different loci, Hardy–Weinberg equilibrium calculations were performed andthepopulationstructurewasevaluatedbyapplyingthe Gtest.

GiventhevariabilityofHLAspecificities,HLAalleleswere presentedinallelicgroupstoavoidtheoverstratificationof thesampleandtoperformthecomparativeanalysesbetween HLAalleles,NDO-LID,andPGL-1.

Results

GeneticaspectsofHLA

TypingofHLAidentified21allelesonlocusA,52onlocusB, 22onlocusC,42onlocusDRB1,13onlocusDQA1,and15 onlocusDQB1.Tables1and2listthemostfrequentallelesin thispopulation,andotherallelesarepresentedin supplemen-taryTables(S1–S6).ThemostfrequentclassIHLAallelesin theBarãocommunitywereA*02:01(29.93%),B*40:02(14.28%), and C*04:01 (15.65%); and for class II: DRB1*16:02 (23.81%), DQA1*05:05(26.19%),andDQB1*03:01(34.7%).Thesame alle-les were observed atIpiranga community, namely, A*02:01 (29.7%), B*40:02 (18.04%), DRB1*16:02 (21.43%), DQA1*05:05 (22.18%)andDQB1*03:01(34.21%);exceptforlocusC,inwhich themostfrequentallelewasHLA-C*07:01(14.28%).

Regarding the Hardy–Weinbergequilibrium calculations, theBarãosampleshowedsignificantvaluesforHLA-A,HLA-B andHLA-Cloci,whileintheIpirangasampleonlyHLA-Alocus wasinequilibrium.

TheresultsoftheGtestshowedtheexistenceof popula-tionstructureandlowgeneflowintheanalyzedpopulations, andsimilarlytheFstatisticelements(frequencyofallelesand presenceofexclusivealleles)corroboratedthesefindings.Due tothisstructure,allelefrequencieswerepresentedseparately ineachpopulation.

AssociationbetweenNDO-LIDandHLA

A significant association between positive NDO-LID test wasfoundwithallelesHLA-A*02(43.18%vs.24.5%,p=0.03, OR=2.35)andHLA-B*53(6.83%vs.0.0%,p=0.03,OR=8.95)in residentsofBarãocommunity.ForIpirangacommunity,a sig-nificantassociationwasfoundbetweenalleleHLA-B*15(3.85% vs.16.25%,p=0.05,OR=0.19)withnegativeresultforthe NDO-LIDtest.Whenanalyzingthe twopopulationsjointly,there

Table1–MostfrequentallelesofHLAclassI(lociA*,B*eC*)inBarãoandIpirangacommunities.

Barãocommunity(N=147) Ipirangacommunity(N=133)

HLA-A* n % HLA-A* n % 02:01 88 29.93 02:01 79 29.7 31:01 72 24.49 31:01 62 23.31 68:01 35 11.9 68:01 32 12.03 HLA-B* n % HLA-B* n % 40:02 42 14.28 40:02 48 18.04 35:01 36 12.24 39:01 35 13.16 39:01 32 10.88 07:02 21 7.89 HLA-C* n % HLA-C* n % 04:01 46 15.65 07:01 38 14.28 08:03 28 9.52 08:03 36 13.53 02:02 26 8.84 03:04 34 12.78

N,numberofindividuals;n,numberofalleles(2n);%,allelefrequency;boldface,mostfrequentallele.

Table2–MostfrequentallelesofHLAclassII(DRB1*,DQA1*,DQB1*)inBarãoandIpirangacommunities.

Barãocommunity(N=147) Ipirangacommunity(N=133)

HLA-DRB1* n % HLA-DRB1* n % 16:02 70 23.81 16:02 57 21.43 07:01 28 9.52 04:11 24 9.02 03:01 19 6.46 14:02 19 7.14 HLA-DQA1* n % HLA-DQA1* n % 05:05 77 26.19 05:05 59 22.18 03:01 47 15.99 03:01 45 16.92 01:02 41 13.94 04:01 37 13.91 HLA-DQB1* n % HLA-DQB1* n % 03:01 102 34.7 03:01 91 34.21 03:02 44 14.96 04:02 40 15.04 02:01 22 7.48 03:02 38 14.28

N,numberofindividuals;n,numberofalleles(2n);%,allelefrequency;boldface,mostfrequentallele.

was an association between seropositivity to the test and theHLA-B*57allele (6.25%vs.1.15%,p=0.03,OR=5.73),and seronegativitywithallelesHLA-B*15(6.25%vs.14.37,p=0.005, OR=0.39)andHLA-C*02(2.08%vs.10.92%,p=0.008,OR=0.17) (Table3).

AssociationbetweenPGL-1andHLA

Alleles HLA-B*40(31.25%vs. 15.15%, p=0.04,OR=2.56) and HLA-C*03(40.62%vs.20.43%,p=0.021,OR=2.66)were asso-ciatedwithapositiveserologicalresponsetotheELISAPGL-1 testatBarãoandIpirangacommunities(Table4).Thereduced numberofparticipantswithpositivePGL-1(16individuals)did notenabletheassessmentoftheassociationineach popula-tionseparately.

Discussion

TheknowledgeofHLAallelesisanimportantinstrumentto understandtheoriginofpopulations,andtheirdetermination contributestotheunderstandingofmechanismsassociated withsusceptibilityorresistancetocertaindiseases.7,31,32

This study showed class I and II HLA alleles found in the population of the Puyanawa indigenous reserve. The mostfrequentalleles were HLA-A*02:01,HLA-B*40:02, HLA-C*04:01,HLA-C*07:01,HLA-DRB1*16:02,HLA-DQA1*05:05,and HLA-DQB1*03:01.

The finding of lackof equilibriumfor the different loci in both populationswas not apparently due to inbreeding processes,but,incontrast,theHLA-AlocusattheBarão com-munityandHLA-DQB1attheIpirangacommunityindicateda slightincreasednumberofheterozygotes.

Althoughthestructuringwasobserved,aswellasalow geneflowamongtheanalyzedpopulations(GtestandF statis-tic),therehasbeennolossofallelediversityorcrossbreeding that would directthe populationto eventualhomozygosis. Evenbeingofthesameethnicity,marriagebetween individu-alsfromthesetwovillagesisnotveryfrequent,whichexplains thelowgeneflowobserved.

AccordingtodatafromRedeBrasildeImunogenética(Brazil ImmunogeneticsNetwork), allelesHLA-A*02, HLA-B*40,and HLA-DRB1*16:02areamongthemostfrequentinthe popula-tionofAcre.Theseresultsdemonstratethesimilaritybetween thePuyanawaindigenouspopulationandthepopulationof

Table3–SignificantassociationsbetweentheNDO-LIDtestandHLAallelesofindigenousresidentsofBarãoand

Ipirangacommunities(n=135),Acrestate,Brazil.

HLA Barãocommunity

NDO-LID p-value OR 95%CI

Positive(N=22) Negative(N=47)

n % n %

A*02 19 43.18 23 24.8 0.03 2.35 1.1–5.01

B*53 03 6.83 0 0 0.03 8.95 0.97–82.54

HLA Ipirangacommunity

NDO-LID p-value OR 95%CI

Positive(N=26) Negative(N=40)

n % n %

B*15 2 3.85 13 16.25 0.05 0.19 0.04–0.92

HLA BarãoandIpirangacommunities

NDO-LID p-value OR 95%CI

Positive(N=48) Negative(N=87) n % n % B*15 6 6.25 25 14.37 0.005 0.39 0.15–1.0 B*57 6 6.25 2 1.15 0.03 5.73 1.13–28.99 C*02 2 2.08 19 10.92 0.008 0.17 0.04–0.76 NDO-LID.

HLA,humanleukocyteantigen.

N,numberofindividuals;n,numberofalleles(2n);%,allelefrequency;p,Fisher’sexacttest(p≤0.05). OR,oddsratio.

95%CI,95%confidenceinterval.

Table4–AsignificantassociationbetweentheELISAPGL-1testandHLAallelesonresidentsofBarãoandIpiranga

communities,MâncioLima,Acre,Brazil,2018(n=109).

HLAalleles BarãoandIpirangacommunities

aPGL-1 p-value OR 95%CI

Positive(N=16) Negative(N=93)

n % n %

B*40 10 31.25 28 15.05 0.04 2.56 1.1–5.99

C*03 13 40.62 38 20.43 0.021 2.66 1.21–5.87

a-PGL-1,Anti-PGL-1ELISA. HLA,humanleukocyteantigen.

N,numberofindividuals;n,numberofalleles(2n);%,allelefrequency;p,Fisher’sexacttest(p≤0.05). OR,oddsratio.

95%CI,95%confidenceinterval.

thestateofAcre,showingthemiscegenationbetweenthese populations.

The history of the Puyanawa people is marked by a long process of domination, enslavement, and disappear-ance of their culture and language. Like many peoples of Acre, they suffered from the growth of the extraction of natural rubber in the region in the early 20th century. Sincethefirstcontactswithnon-indigenouspeoples,many

have died in conflicts or from diseases acquired in this process.33

TheHLAclassIandIIallelesmostfrequentlyfoundinthis studyhadalreadybeendescribedinTerenaindigenous popu-lations,whomostlyinhabitthestateofMatoGrossodoSul34

except forallele HLA-DQA1*05:05, which ispresent among GuaraniandKaingangindigenouspeople31_{andCayapapeople}

AllelesDRB1*16:02andDQB1*03:01arefrequentamongthe indigenouspopulationsofXavantes,GuaraniKaiowá,Guarani Mbya,GuaraniNandeva,Kaingang,andTicunainBrazil,31_and

MayanpeoplesinGuatemala.36

TheBrazilianpopulationhasagreatallelicdiversity result-ing from the miscegenation among Caucasians, Africans, andindigenouspeoples.37,38_{TheidentificationofHLAalleles}

showsItalianimmigrationtothe southernregionofBrazil, Dutch to the Northeast, and Moroccan Jews in the Ama-zonregion.38_{During theperiodknownastherubbercycle,}

the stateofAcrereceived many migrantsfrom the North-eastregion –mainlyfrom Ceará–due toadroughtinthe late19thcentury,aswellasindividualsfromSãoPauloand Syria/Lebanon.39

Astudy on the frequency ofdifferent HLA-DRB1alleles inindividuals fromendemicareasformalariaintheNorth regionofBrazil,including amunicipalityinAcre,identified thatthemostfrequentalleleswere HLA-DRB1*04, DRB1*08, DRB1*07,andDRB1*13andthatthehighprevalenceofallele HLA-DRB1*04reflectsthecontributionfromindigenous peo-plestothegeneticcompositionoftheBrazilianpopulation– especiallyinthenorthofthecountry40_{–sincethisalleleis}

characteristicofindigenouspeoplesoftheAmericas.41_These

resultswere alsoobservedingroupevaluatedinourstudy. Consideringbothpopulationstogether,thefrequencyofthe HLA-DRB1*04allelicgroupwas34.35%,withHLA-DRB1*04:11 being the most frequent. The frequencies of alleles HLA-DRB1*07, DRB1*08, and DRB1*13 were 15.91%, 17.29%, and 19.34%,respectively.

ThedistributionofHLAalleles andhaplotypesinethnic groupsthatcomposetheBrazilianRegistryofVoluntaryBone MarrowDonors(REDOME)presentsfrequentallelesin indige-nouspeoples,suchasHLA-A*03:04,A*23:02,A*29:03,A*31:28, HLA-B*35:21, and states where the allele HLA-B*40:15 was onlyobservedintheindigenousethnicgroup.42_Weverified

thepresenceofHLA-B*40asthemostfrequentallele; how-ever, the genotype foundinthe Puyanawa populationwas HLA-B*40:02.

ThemostfrequentalleleinthePuyanawapopulationin this study was HLA-DRB1*16:02. Alleles HLA-DRB1*15 and DRB1*16 have been associated with leprosy, especially in Indians, Japanese, and Brazilians.5,8,16 _{HLA-DRB1*16:01was}

associatedwithsusceptibilitytoBL,5_{whereasHLA-DRB1*16:02}

toLL.43_{AlthoughthestudywiththeBrazilianpopulationwas}

conductedinasampledifferentfromthatofourstudy,and sinceleprosycaseshaveoccurredintheindigenousreserve in the past, we can hypothesize that the identification of HLA-DRB1*16:02could suggestthattheseindividualswould bethemostpredisposedtothemanifestationofleprosy. Stud-ieswithgreatercontrolmustbeconductedtoconfirmthese results.

Todate,therearenostudiesassessingtheassociationof positiveserology(NDO-LIDandPGL-1)forleprosywithHLA alleles.However,therearepositiveassociationslinkedtothese moleculeswithleprosyanditsclinicalformsandreactions duetotheirimportanceinthepresentationofantigensand triggeringhumoralimmuneresponse.Knowingthat serologi-calmethodsusetechniquesbasedonthedetectionofspecific anti-M.lepraeantibodiesandthatseropositivitytothetests mainlyshowresponsestriggeredbyhumoralimmunitywith

ahigherpresenceofantibodiesagainstthebacillus,18,21,22_our

studyshowedthatHLAallelescontributetoseroconversionto NDO-LIDandELISAPGL-1tests.

Barretoetal.,44 _{statedintheirstudythatthechancesof}

developingleprosyinpeopleseropositivetothesurface

pro-teinoftheM.Lepraewere2.7-foldhigherthaninseronegative

people(p<0.01),indicatingthatinafollow-upof10 seropos-itive individuals,there isa greater than 90% probabilityof detectingatleastonenewcaseintwoyears.

Serological studies from different regions of Brazil confirmed the anti-LID-1 and anti-NDO-LID tests as diag-nostic tools for the detection of multibacillary leprosy and identification of individuals with subclinical M. leprae

infection.20,26

TheincreaseinHLA-A*02andHLA-B*53frequencies sug-gest,respectively,atwo-andnine-foldhigherriskofhavinga positiveserologicalresponsetotheNDO-LIDtest,respectively. Ontheotherhand,individualswhopresentedtheHLA-B*15 allelewerelesslikelytorespondtothetestand,consequently, havelower chancesofdeveloping MB,or evengettingsick. Inadistinctpopulation,Santanaetal.,6_observedthe

pres-enceofHLA-B*53(p=0.05andpc>0.05)inpatientswithBL, andassociationoftheHLA-B*15allele(p=0.05andpc>0.05) with the manifestationofthe type 1reaction. In the joint analysisofthetwopopulations,therewasassociationwith HLA-B*57increasingthechancebyalmostsix-foldofhaving positiveserologicalresultstoNDO-LID,whereastheHLA-B*15 andHLA-C*02alleleswouldreducethispossibility.

Seronegativitydoesnotruleoutthepossibilityofdisease development.Thechanceismuchhigherinseropositivethan inseronegativeindividuals.21,44_{Accordingtoafollow-upstudy}

inschoolchildren,22.3%and9.4%ofseropositiveand seroneg-ativeindividuals,respectively,developedleprosy.44

TheanalysisofHLA moleculesofthepopulation ofthe two communitieswithELISA PGL-1showedthat HLA-B*40, andHLA-C*03alleleswereassociatedwithanalmost3-fold greaterchanceofpresentingpositiveserologicalresponse.Ina reviewpublishedbyJardulietal.,8_{HLA-B*40antigenand}

HLA-A*02-B*40,HLA-A*11-B*40andHLA-A*24-B*40haplotypeswere frequentinIndianswithleprosy.

StudieshaveshownthatseropositivitytoELISA anti-PGL-1associated withthe riskofdeveloping leprosy,especially inintra-domiciliarycontactsofMBcases18,19,21,45 _isdirectly

related to genetic susceptibility, frequent exposure to the bacillus, and leprosy endemicity.25,45,46 _{Moreover, evidence}

alsoshowsthatincreasedage,healthproblems,and socioe-conomic conditions are associated with increased disease risk.47

Thus,establishingtheHLAfrequencyofthis indigenous communitywascrucialtocharacterizethegeneticprofileof thispopulation,contributingtothedistributionofHLAalleles intheBrazilianpopulationandestablishingtheHLA associ-ationwithserologicaltests(NDO-LIDandPGL-1)usedinthe diagnosisandmonitoringofleprosy.TheassociationofHLA allelesandseropositivitytosurfaceproteinsofM.lepraemay beageneticindicationforthepredispositiontoleprosy. How-ever,morestudiesareneededtofurtherestablishtheroleof HLAallelesinseroconversiontotherapidNDO-LIDandELISA PGL-1tests,andsoappropriatelytargetedpublichealth poli-ciesareestablished.

Funding

Thisresearchreceivedaspecificsubsidyfromthefollowing fundingagencies:Fundac¸ãoPaulistacontraHanseníaseand Fundac¸ãodeAmparoàPesquisadoestadodoAcre/Programa PesquisaparaoSUS(PPSUS).Theentireresourcewasusedto carryoutdatacollectionandstatisticalanalysis.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Wewouldlike tothankthe studentsand professorsofthe undergraduatecourseinNursingofFederalUniversityofAcre (UFAC)whoparticipatedindatacollectionandthegroupof theLaboratoryofImmunologyandMicrobiologyofUFACfor theirsupportinthepreparationofthematerialforanalysis. TotheresearchgroupoftheLaboratoryofImmunologyand ImmunogeneticsoftheInstitutoLaurodeSouzaLimafortheir supportandassistanceinlaboratoryanalysesandsupplyof antigens.TotheProgramadePesquisaparaoSUS(PPSUS)ofthe stateofAcre,andtheFundac¸ãoPaulistacontraHanseníasefor thefinancialsupport.

Appendix

A.

Supplementary

data

Supplementary data associated with this article can be found, in the online version, at https://doi.org/ 10.1016/j.bjid.2020.05.009.

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