AntiPGL1 salivary IgAIgM, serum IgGIgM, and nasal Mycobacterium leprae DNA in individuals with household contact with leprosy

Anti-PGL1

salivary

IgA/IgM,

serum

IgG/IgM,

and

nasal

Mycobacterium

leprae

DNA

in

individuals

with

household

contact

with

leprosy

Paula

Brito

e

Cabral

a

,

Jose´ Evandro

Cunha

Ju´nior

a

,

Alexandre

Casimiro

de

Macedo

a

,

Alexandre

Rodrigues

Alves

a

,

Thially

Braga

Gonc

¸

alves

a

,

Tereza

Cristina

Brito

e

Cabral

b

,

Ana

Paula

Soares

Gondim

c

_,

_Maria

_Isabel

_Moraes

_Pinto

d

_,

_Karen

_Tubono

_Oseki

d

_,

Lilia

Maria

Carneiro

Camara

e

,

Silvia

Helena

Barem

Rabenhorst

e

,

Aparecida

Tiemi

Nagao-Dias

a,

*

a_{DepartmentofClinicalAnalysisandToxicology,FacultyofPharmacy,UniversidadeFederaldoCeara´,RuaCapita˜oFranciscoPedro,1210,CEP60430-370}

Fortaleza,Ceara´,Brazil

b_{MunicipalHealthSecretariat,Crato,Ceara´,Brazil}

c_{DepartmentofPharmacy,FacultyofPharmacy,UniversidadeFederaldoCeara´,Fortaleza,Ceara´,Brazil} d_{DivisionofPediatricInfectiousDiseases,UniversidadeFederaldeSaoPaulo,SaoPaulo,Brazil} e_{DepartmentofPathology,FacultyofMedicine,UniversidadeFederaldoCeara´,Fortaleza,Ceara´,Brazil}

1. Introduction

Leprosyremainsapublichealthchallenge,withapproximately 250000newcasesbeingreportedeachyearworldwide.1_Brazilhas

thesecondhighestnumberofcasesintheworld,with33955new cases registeredin 2011.2 _{In 2012, a total of 2066 cases were}

reported in the state of Ceara´, which represents a detection coefficientof24casesper100000inhabitants.3

Strategies for leprosy control include the administration of multidrugtherapytothepatients andvaccinationofhousehold

contacts with the bacille Calmette–Gue´rin (BCG) vaccine.4 _The

World Health Organization (WHO) also recommends clinical evaluation of leprosy householdcontacts and health education aspartofthediseasecontrolstrategy.1_{Physicalexaminationdoes}

not identify the early stages of the disease, when clinical manifestationsarerarelypresent.5,6_{Therefore,it isnecessaryto}

employmoresensitivetoolsinordertoinvestigateMycobacterium lepraeinfectionamonghouseholdcontacts.5_{SerologicalandPCR}

testshavebeenusedinseroepidemiologicalstudies.1,6

Phenolicglycolipid(PGL1)isanM.leprae-specificantigen,7_and

this dominant lipid in the cell wall is responsible for its immunologicalspecificity.8_{Themoststudiedantibodyisotypeis}

theserumanti-PGL1IgM.9–12_{Fewstudieshaveevaluatedsalivary}

anti-PGL1 IgA and IgM,13,14 _{or serum anti-PGL1IgG.}7,14–17_Our

previous work suggested that salivary anti-PGL1 IgA and IgM

ARTICLE INFO

Articlehistory:

Received19October2012 Receivedinrevisedform9May2013 Accepted21May2013

CorrespondingEditor:EskildPetersen, Aarhus,Denmark

Keywords:

MycobacteriumlepraeDNA Serumandsalivaryanti-PGL1 Leprosyhouseholdcontacts

SUMMARY

Objectives:Leprosyhouseholdcontactsrepresentagroupathighriskofdevelopingthedisease.Theaim ofthisstudywastodetectMycobacteriumlepraesubclinicalinfectioninthisgroupthroughserological andmolecularparameters.

Methods:Serumanti-PGL1IgG/IgMandsalivaryanti-PGL1IgA/IgMwasinvestigatedusinganELISA,and nasalcarriageofM.lepraeDNAwasdetectedbyPCR,inleprosyhouseholdcontactsofpaucibacillary(PB) andmultibacillary(MB)householdleprosypatients(n=135),theirindexcases(n=30),andinpersons livinginalowendemiccity(n=17).

Results:Salivaryanti-PGL1IgAandIgMandserumanti-PGL1IgGshowedgoodcorrelationcomparing contactsandindexcases(p<0.01,p<0.005,andp<0.0001,respectively).Thiswasnotobservedfor serumanti-PGL1IgM(p>0.05).Ahighfrequencyofanti-PGL1IgMpositivitywasfoundinIgG-negative samples(p<0.0001).ForIgG-positivesamples,IgMantibodieswerealsopositiveinmostofthesamples. Noneofthe17volunteerslivinginalowendemiccitypresentedseropositivityforIgG;however,twoof themshowedpositivityforanti-PGL1IgM.M.lepraeDNAwasfoundinthenasalswabsofnineoutofthe 85MBhouseholdleprosycontacts(10.6%)andinthreeoutofthe50PBhouseholdleprosycontacts (6.0%).

Conclusion:WestronglysuggestthatserumIgG/IgMandsalivaryanti-PGL1IgA/IgMmeasurementsare usedtofollowleprosyhouseholdcontacts.

ß 2013InternationalSocietyforInfectiousDiseases.PublishedbyElsevierLtd.Allrightsreserved.

*Correspondingauthor.Tel.:+558533668270;fax:+558533668292.

E-mailaddresses:[email protected],[email protected]

(A.T.Nagao-Dias).

ContentslistsavailableatSciVerseScienceDirect

International

Journal

of

Infectious

Diseases

j o urn a l hom e pa ge : ww w. e l s e v i e r. c om/ l o ca t e / i j i d

1201-9712/$36.00–seefrontmatterß2013InternationalSocietyforInfectiousDiseases.PublishedbyElsevierLtd.Allrightsreserved.

antibodiesmightbeindicativeofinfectionbutnotofdisease.17_Itis

knownthatindividualsseropositiveforanti-PGL1antibodieshave a7.5-foldgreaterriskofacquiringleprosycomparedto seronega-tive contacts.18 _{A prospective study}9 _{demonstrated that two}

seropositivecontactsprogressedtoclinical,borderlinetuberculoid (BT)leprosywithin1yearoffollow-up.

Several authorshave described methods to detectM.leprae

basedonnucleicacidamplificationtechniques,6,19–23_sincedirect

bacterialdetectiontestshavetoolowsensitivity.24

Inthepresentwork,weevaluatedserumIgG/IgMandsalivary IgA/IgMagainstPGL1inadditiontothedetectionofnasalcarriage ofM.lepraeinleprosyhouseholdcontactsandtheirindexcases.

2. Materialsandmethods

2.1. Patientsandcontacts

Thirty leprosy patients (median age 45 years) and 135 householdcontacts(median age26years) livinginthe cities ofCratoandMaracanau´,Ceara´ State,Brazil,wereincludedinthe study.Seventeenindividualslivinginacitywithalowincidence of leprosy (city of Sao Paulo)who reported no contact with leprosypatientswerealsoincludedintheprojectforserological evaluation(controlgroup– CTgroup).In2012,thedetection ratesofleprosyinthecitiesofCratoandMaracanau´ were42.8 and30.5per100000inhabitants,respectively.3_Thedetection

rateof leprosyinthecity ofSaoPaulowas2.21per 100000 inhabitantsin2011.25

Leprosy diagnosisand classificationof theindex cases were based on clinical assessment and on the detection of acid-fast bacilliinslitskinsmears.Theleprosypatientswereclassifiedas paucibacillary(PB)ormultibacillary(MB),accordingtothelesion count.26_{SeventeenwereclassifiedasMBpatients and13 asPB}

patients. Fifty out of 135 (37.0%) were contacts of PB leprosy patients,and85outof135(63.0%)werecontactsofMBleprosy patients.

The study was approved by the Ethics Committee of the Universidade Federal do Ceara´, and each participant or his/her guardianprovidedwritteninformedconsent.

2.2. Samples

Serumsampleswerecollectedandstoredat 208C. Unstimu-latedsalivawascollected30minafterwaterconsumptionand2h aftersolid food intakeand kept at 208C. Using sterile swabs moistenedin0.9%salinesolution,nasalsecretionswerecollected frombothnostrilsbygentlyswabbingtheouternares.Eachswab tipwasplacedintoatubecontaining300

m

loflysissolutionand

keptat 208C.Thelysissolutionwascomprisedof100mMTris– HCl,pH8.5,with3%TritonX-100and1mg/mlproteinaseK.

2.3. Serumanalysis

Serologicalanalysisforanti-PGL1antibodieswasperformed in accordance with the procedure described by Nagao-Dias etal.14

Polystyrene microplates (Costar, USA) were coated with 10mg/l of native PGL1 in absolute ethyl alcohol, kindly donatedby Dr JohnSpencer, Colorado State University, USA. The plates were incubated for 18h at room temperature (RT). For serum IgG and IgM antibody measurements, the microplates were incubated with 1% bovine serum albumin (BSA,Sigma,USA) inphosphate bufferedsaline (PBS,pH7.4) for2h at RT. After four washes with PBS–0.01% BSA, serum samples previously diluted 1:50 in PBS–0.5% BSA were added to the plates in duplicate. The plates were then

incubated overnight at 48C. After washing, peroxidase-labeled anti-IgG or anti-IgM(Sigma, USA) previously diluted 1:1000 was added to the plates and incubated for 1.5h at RT. After washing, the plates were incubated for 30min with the substrate solution which contained 0.4mg ortho-phenylenediamine per milliliter of 0.01M citrate–phosphate buffer, pH5.0. Thereactionwas interruptedby adding 25

m

l

of 2.5N sulfuric acid.Theanalysis wasperformed at 492nm using an ELISA plate reader. An aliquot of pooled normal human serum was used as cut-off sample and tested in all assays. The results were expressed according to the following formula: optical density (OD) mean of the test sample (minus blank) divided by the OD mean of the normal human serum pool (minus blank). The cut-off was considered to be 1.0. Values above 1.3, which were 30% above the cut-off, were considered to be positive.14

2.4. Salivaryanalysis

For salivaryIgA andIgM measurements, PGL1-coatedplates were blocked with 1% BSA–Tris solution for 2h at RT. Saliva samples,previously centrifuged at 2600g for 15minat 48C, werediluted1:50with0.5%BSA–Tris,addedtotheplates,and incubatedfor18hat4₈C.Afterfourwasheswith0.01%BSA–Tris, alkaline phosphatase-conjugated anti-IgA or anti-IgM (Sigma, USA)wasdiluted1:1000in0.5%BSA–Trisandaddedtotheplates. Theplateswerethenincubatedfor2hatRT.Afterwashing,the plates were incubatedwith thesubstrate solution (1mg/ml p -nitrophenylphosphatein10%diethanolaminecontaining0.5mM MgCl2,pH9.8).After100min,absorbancereadingswererecorded

at405nmusinganELISAplatereader.Theresultswereexpressed as the OD mean of thevalues (minus blank). The cut-off was consideredtobethe97th_{percentileofnormalcontrols.}14_Results

consideredtobe30%abovethecut-offvaluewereconsideredtobe positive.Blanksamplescontainedallreagentsexceptforsalivaor serum.

2.5. MolecularM.lepraedetection

DetectionofM.lepraeDNAinnasalswabswasperformedin accordancewiththeproceduresdescribedbyTorresetal.21_andDe

Witetal.,27_{withsomemodifications.}

Thenasalswabsimmersedinlysisbufferwereincubatedat 55₈Cfor18hfollowedbyincubationfor15minat97₈C.After removingtheswabs,135

m

lof5MNaClsolutionwasaddedto

eachtubeandincubatedfor2hat 208C.Aftercentrifugationat 8117gfor5min at48C,DNAcontainedinthe supernatant fractionwasprecipitated byadditionof2volumesoficecold isopropylalcohol.Thesolutionwaskeptat 208Cfor24h.After a new centrifugation at 8117g for 5min at 48C, the supernatant was discarded and the tubes were left open overnightatRTtoallowthealcoholtoevaporate.Subsequently, 50

m

lofsteriledistilledwaterwasaddedtoeachtubeandleftin

awaterbathfor1hat37₈C.TheDNAsampleswerestoredat 208Cuntilfurtheranalysis.

DNAprimersS13(50_{-CTCCACCTGGACCGGCGAT-3}0_)andS62

(50_{-GACTAGCCTGCCAAGTCG-3}0_{) were selected based on the}

nucleotide sequence of the 531-bp fragment (proline-rich region)of aspecific gene thatencodesthe 36-kDaantigenof

M.leprae.21

The PCR technique was performed by adding5.2

m

l sterile

water,2

m

lTween20,10

m

lMasterMix(Promega,USA),0.4

m

l

primers,and2

m

lDNAtemplatetoeachmicrotube.Subsequently,

finalelongationstepof7minat728Ccompletedtheamplification. Fourmicrolitersoftheampliconweresubjectedtoelectrophoresis ina2%agarosegel.TheDNAwasvisualizedunderultravioletlight afterstainingthegelwithethidiumbromide.

2.6. Acid-faststainofnasalsmears

Nasalsmearswerecollected,spreadonaslide,heat-fixed,and stainedbyZiehl–Neelsenmethod.

2.7. Statisticalanalysis

Thedatawere analyzedusing non-parametric testsas the data did not follow a Gaussian distribution (Kolmogorov– Smirnov test, p<0.0001). The Wilcoxon and Spearman tests

were used to compare the antibody isotype titers between pairedgroups.TheMann–Whitney test wasused tocompare theisotype antibodytitersbetween unpairedgroups.Fisher’s test was used to calculate the probability of association between the clinical form of the leprosy disease and the frequency of positiveor negativeantibody/molecular results; also for comparingserum and salivaryantibody parameters. The software used for the statistical analysis was Prism GraphPad Instat version 3.10. The level of statistical signifi-cancewas consideredtobe0.05.

3. Results

3.1. Serologicalandmoleculardatainhouseholdleprosy(HHL) contacts

Table 1 presents the positivity of serological and molecular

parametersinHHLcontacts.Serumanti-PGL1IgGandIgMwere positive in 14 (10.4%) and 80 (59.3%) out of 135 samples, respectively.Salivaryanti-PGL1IgAandIgMtiterswerepositive in 41 (30.3%)and 14 (10.4%)out of 135samples, respectively. Twelveoutof135samples(26.7%)werepositiveforM.lepraeDNA. No statistical significance was found when the frequencies of positiveserumandsalivaryantibodiesortheM.lepraeDNAwere associatedwiththePBandMBHHLcontacts.

3.2. CorrelationbetweenHHLcontactsandindexcases

Figure1 demonstrates thecorrelationofserumand salivary

antibody titersbetween HHLcontacts andindex cases(n=97). Salivaryanti-PGL1IgAtitersshowedgoodcorrelationcomparing HHL contacts and index cases (r=0.25, p<0.01); a similar

observation was made for salivary anti-PGL1 IgM (r=0.34,

p=0.005)and serumanti-PGL1IgGlevels(r=0.39,p<0.0001).

The same wasnot observedfor serum anti-PGL1IgM (r=0.14,

p>0.05). Table1

Frequencyofpositiveserumandsalivaryanti-PGL1antibodytitersandnasalM.lepraeDNAamongleprosyhouseholdcontacts

Leprosycontacts—clinicalformoftheindexcase Anti-PGL1antibodies M.lepraeDNA

SerumIgG,n(%) SerumIgM,n(%) SalivaIgA,n(%) SalivaIgM,n(%) Nasal,n(%)

Paucibacillary(n=50) 8(16%) 26(52%) 11(22%) 5(10%) 3(6%)

Multibacillary(n=85) 6(7.1%) 54(63.5%) 30(35.2%) 9(10.5%) 9(10.6%)

Fisher’stestp-value >0.1 >0.1 >0.1 1.0 >0.5

Serum anti-PGL1 I

gG

0 2 4 6 8

0 2 4 6

r=0.39,p<0.0001

Patients

Co

n

ta

c

ts

salivary anti-PGL1 I

gM

0 1 2 3 4

0 1 2 3 4

r=0.34,p=0.005

Patients

C

ont

a

c

ts

Salivary anti-PGL1 IgA

0.0 0.5 1.0 1.5 2.0 2.5

0 1 2 3

r=0.25,p=0.007

Patients

0 5 10 15 20

0 5 10 15 20

r=0.14,p>0.05

Patients

Co

n

ta

c

ts

r=0.14, p>0.05

r=0.34, p=0.005

r=0.39, p<0.0001

r=0.25, p<0.01

Index cases

Index cases

Index cases

Serum anti-PGL1 IgM

Serum anti

-PGL1 IgG

Sali

vary

anti

-PGL1 Ig

A

Salivary anti

-PGL1 IgM

Contacts

Contacts

Contacts

Index cases

3.3. Controls(CTgroup)

SalivaryIgA/IgM and serum IgG antibodytitersfrom theCT group were below the cut-off values (Figure 2a and 2b, respectively). Two serum samples showed IgM antibody titers above2.0(Figure2b).Levelsofsalivaryanti-PGL1IgAweremuch lowerthanthoseoftheothergroups(p<0.0001).Inrespectto

salivaryIgM, titers werelower than those of the HHL contact group;however,astatisticaldifferencewasonlyfoundwhentheir levelswerecomparedtothoseofthepatients(p=0.02).Inregard toserumantibodies,levelsoftheIgGisotypedidnotdifferfrom thoseof the contacts,and weremuchlower than those of the patients(p<0.05).Serumanti-PGL1IgMlevelsweremuchlower

than those of the HHL contacts (p=0.01) and the patients (p=0.0009).

3.4. SerumantibodyisotypesinHHLcontacts

A statistically significant correlation was found between serum IgM and IgG isotypes for both the MB (r=0.39,

p<0.0001) and the PB (r=0.39, p<0.005) HHL contacts.

However,theserumIgMandIgGlevelswereverydifferentfrom one another(p<0.0001). ConsideringserumIgG-negativeand

IgG-positivesamplesseparately(Figure3),ahighfrequencyof anti-PGL1IgMpositivity(71outof121samples;58.6%)wasfound inIgG-negativesamples(p<0.0001).ForIgG-positivesamples,

IgMantibodieswerealsopositiveinmostofthesamples(10outof 14samples;71.4%)andtheirlevels didnot differsignificantly fromtheIgGtiters(p>0.1).

3.5. SalivaryantibodyisotypesinHHLcontacts

Astatisticallysignificantpositivecorrelationbetweensalivary IgAandIgMwasfoundinbothPB(r=0.54,p<0.0001)andMB

(r=0.61; p<0.0001) HHLcontacts.Twenty-fourout of45 HHL

contacts who demonstrated positivity for salivary antibodies (53.3%)werenegativeforserumantibodies.Conversely,64outof 90 HHL contacts who demonstrated negativity for salivary antibodies(71.1%)werepositive for serumantibodies(Table 2,

p<0.01).

3.6. Directandmoleculardetection

NoHHLcontactspresenteddirectnasalsmeardetection.M. lepraeDNAwasfoundinthenasalswabsofnineoutofthe85 MB HHL contacts (10.6%) andin three outof the 50 PBHHL contacts(6.0%).Amongthe12contactswhowerepositivefor

M.lepraeDNA(Table3),fivewerepositiveforserumanti-PGL1 IgM,sevenwerepositiveforsalivaryanti-PGL1antibodies,and onewaspositiveforalloftheisotypes,exceptforsalivary anti-PGL1IgM.Thisindividualwastheonlyonewhodidnothavea BCG scar.

Serum anti-PGL1

P

C

_CT

P

C

_CT

0

5

10

15

20

Inde

x

_p<0.05

p=0.0009 p=0.01

N.S.

IgG

IgM

N.S.= not significant, P=paents, C= leprosycontacts, CT= controls

Saliva

ry anti-PGL1

P

C

_CT

P

C

_CT

0

1

2

3

4

O

p

ti

c

a

l de

ns

it

y

IgA

IgM

p<0.0001

p=0.02 N.S.

p<0.0001

N.S.= not significant, P=paents, C= leprosycontacts, CT= controls

a

b

Figure2.(a)Salivaryand(b)serumantibodytitersinleprosypatients(P,n=30),in leprosyhouseholdcontacts(C,n=135),andinthecontrolgroup(CT,n=17).The dashedlinesindicatethecut-offvalues.

IgG IgM IgG IgM 0

5 10 15 20

IgG-negativesamples (n = 121)

IgG-positive samples (n=14)

p< 0.0001

p> 0.1

Ti

te

rs

Figure3.Serumanti-PGL1IgMandIgGtitersinIgG-positiveandIgG-negative samples.Thedashedlineindicatesthecut-offvalue.

Table2

Frequencyofpositiveandnegativeserumandsalivaryanti-PGL1antibodytitersin leprosyhouseholdcontacts

SerumIgGand/orIgMantibodies

SalivaryIgAand/or antibodies

Positive,n(%) Negative,n(%) Total,n(%)

Positive 21(46.7%) 24(53.3%) 45(100%)

Negative 64(71.1%) 26(28.9%) 90(100%)

4. Discussion

EarlydetectionofthediseaseisastrategytointerruptM.leprae

transmissionandtopreventtheoccurrenceofphysicaldisabilities, asevereconsequenceoflatediagnosis.1_{Inendemicareas,sensitive}

and specificserological tests associatedwithmolecular param-eterscouldbeofgreatuseforfollowingcontactsathighriskof developingthedisease.11

Whenaclinicalexaminationofaleprosycontactisperformed, suspectedcasescan beregistered.Bazan-Furiniet al.9 _detected

newleprosycasesin3.3%ofpatientcontacts(co-prevalentcases). Similarresultshavebeenfoundbyothers,4,28whoobservedarate ofco-prevalentcasesof5.2%.Inourstudy,wedidnotfindany co-prevalentcaseamongthecontacts.

Manyauthorsrefertoanti-PGL1IgM asthemainserological parameterinleprosy.10,29–31_{Itisknownthattherheumatoidfactor}

isoneofthemaincausesoffalse-positivevaluesinIgMdetection. The false-positivity occurs in toxoplasmosis32 _{and other}

dis-eases.33,34_{Serumanti-PGL1IgGcouldbeanimportantparameter}

forevaluationbecauseonlyafewcontactspresentit.Inthepresent study,58%oftheIgG-negativesamplesshowedpositiveIgM.On the other hand, 71.4% of the IgG-positive samples were also positiveforIgM.Moreover,among17volunteerslivinginalow endemiccity,whoreportednocontactwithleprosypatients,none presentedseropositivityforIgG;however,twoof themshowed anti-PGL1 IgM titers above 2.0. This meansthat both isotypes shouldalwaysbemeasured.Webelievethatthoseindividualsthat arepositiveforbothoftheisotypesshouldbefollowedeveryyear. Additionally,thosewhopresentwithanti-PGL1IgMlevelswith titersabove3.0(regardlessofIgG)shouldbefollowedaswell.We aredoubtfulabouttheusefulnessofimmunochromatographytests forthispurpose,sincetheyarenotabletoquantifyantibodiesin samples.

The percentage of leprosy contacts with positive titers of salivaryanti-PGL1IgAantibodieswas32.5%,muchlowerthanthe frequencyreportedbySmithetal.(68%).13

Wefoundthat24 outof45HHLcontacts(53.3%)whowere positive for salivaryIgAand/or IgM antibodiesdidnot present serumantibodies.Assuming a diagnosticspecificity of approxi-mately 91% for the salivary anti-PGL1 antibody test,17 _{we can}

hypothesizethat48%oftheindividualswereinfectedbutdidnot develop serum antibodies. This probably means that they are infectedwithM.lepraebutthattheywillnotnecessarilyacquire thedisease.Conversely,64 outof90HHLcontacts(71.1%)who werenegativeforsalivaryanti-PGL1antibodiespresentedserum IgG and/or IgM antibodies. It is already known that the maintenanceoflong-termimmunologicalmemoryinthemucosal surfaces is a great challenge.35 _{After mucosalcolonization, the}

bacteriacantranslocateintothesystemiccirculation,reducingthe bacterialloadinthenasalsurfaces.Inthiscase,itisreasonableto assumethatsecretoryantibodieswilldecrease,meanwhileserum antibodylevelswillincrease.

M.lepraeDNAwasfoundin8.9%ofthenasalsamples.Other authorshavefoundpercentagesofpositivityvaryingfrom1.6%to 19.35%.10,12,13,36,37

One person showedboth positive molecular and serological parameters,exceptforsalivaryanti-PGL1IgM,anddidnotshowa BCGscar.AccordingtoLobatoetal.,11_{individualswithoutaBCG}

scarareatahigherriskofdevelopingleprosy.Webelievethatthis personshouldbefollowedup.

Westronglysuggestthatserumanti-PGL1IgG/IgMandsalivary anti-PGL1IgA/IgMmeasurementsshouldbeusedtofollowleprosy householdcontacts.ThedetectionofnasalM.lepraeDNA isnot essentialtofollowleprosycontactsbecauseofthehighpercentage of negative results in the contacts, even those with positive serology.

Acknowledgement

Thisresearchwassupported by theCNPq(process472.471/ 2007-4).

Conflict of interest: The authors report that there are no disclosuresrelevanttothispublication.

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2.WorldHealthOrganization.WeeklyEpidemiologicalRecord;87.2012;p.317– 28.

3.Informeepidemiolo´gicohansenı´ase.Brazil:Ceara´ StateDepartmentofHealth; February2013.

4.SalesAM,PoncedeLeonA,Du¨ppreNC,HackerMA,NeryJAC,SarnoEN,etal. Leprosyamongpatientcontacts:amultilevelstudyofriskfactors.PLoSNegl TropDis2011;5:e1013.

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8.ScollardDM,AdamsLB,GillisTP,KrahenbuhlJL,TrumanRW,WilliamsDL.The continuingchallengesofleprosy.ClinMicrobiolRev2006;19:338–81.

9.Bazan-FuriniR,MottaACF,Sima˜oJCL,Tarquı´nioDC,MarquesJrW,Barbosa MHN,etal.Earlydetectionofleprosybyexaminationofhouseholdcontacts, determinationofserumanti-PGL-1antibodiesandconsanguinity.MemInst OswaldoCruz2011;106:536–40.

Table3

Serumandsalivaryanti-PGL1titersinmultibacillary(MB)andpaucibacillary(PB)leprosyhouseholdcontactswhosenasalswabswerePCR-positiveforM.lepraeDNA.The presenceorabsenceoftheBCGscarwasalsoobserved.Cut-offvalueswere1.3and0.5forserumandsalivaryantibodies,respectively.

Leprosyhouseholdcontact Clinicalformofindexcase Age(years) BCGscar Serum Saliva

IgG IgM IgA IgM

C49.3 MB 19 Yes 0.7 3.1 0.64 0.01

C13.1 MB 73 Yes 0.1 0.4 0.14 0.05

C13.2 MB 55 Yes 0.3 0.7 0.58 0.02

C32.1 MB 26 Yes 0.6 2.2 0.18 0.03

C1.3 MB 27 Yes 0.3 0.4 0.50 0.02

C1.6 MB 52 Yes 0.3 0.8 1.70 3.00

C9.4 MB 30 Yes 1.1 1.1 1.10 0.03

C8.4 MB 29 Yes 0.3 0.6 0.60 0.04

C53.1 MB 34 Yes 0.8 1.7 0.08 0.00

C46.2 PB 13 Yes 0.9 3.1 0.10 0.02

C12.4 PB 20 Yes 0.3 1.0 0.13 0.05

C6.1 PB 39 No 1.4 2.1 1.10 0.14

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