J. Pediatr. (Rio J.) vol.92 número6

(1)

www.jped.com.br

ORIGINAL

ARTICLE

Evaluation

of

the

Western

blotting

method

for

the

diagnosis

of

congenital

toxoplasmosis

夽

,

夽夽

Jaqueline

Dario

Capobiango

a,∗

,

Thaís

Cabral

Monica

b

,

Fernanda

Pinto

Ferreira

c

,

Regina

Mitsuka-Breganó

d

_,

_Italmar

_Teodorico

_Navarro

d

_,

_João

_Luis

_Garcia

d

_,

Edna

Maria

Vissoci

Reiche

e

a_Universidade_Estadual_de_Londrina_(UEL),_Centro_de_Ciências_da_Saúde,_Departamento_de_Pediatria_e_Cirurgia_Pediátrica,

Londrina,PR,Brazil

b_Universidade_Estadual_de_Londrina_(UEL),_Centro_de_Ciências_Agrárias,_Programa_de_{Pós-graduac¸ão}_em_Ciência_Animal,

c_Universidade_Estadual_de_Londrina_(UEL),_Departamento_de_Medicina_Veterinária_Preventiva,_Laboratório_de_Zoonoses_e_Saúde

Pública,Londrina,PR,Brazil

d_Universidade_Estadual_de_Londrina_(UEL),_Centro_de_Ciências_Agrárias,_Departamento_de_Medicina_Veterinária_Preventiva,

e_Universidade_Estadual_de_Londrina_(UEL),_Departamento_de_Patologia,_Análises_Clínicas_e_{Toxicológicas,}_Londrina,_PR,_Brazil

Received13October2015;accepted16February2016 Availableonline6August2016

KEYWORDS

Congenital Toxoplasmosis; WesternBlotting; Diagnosis; Serology

Abstract

Objective: ToevaluatetheWesternblottingmethodforthedetectionofIgGanti-Toxoplasma gondii(T.gondii)(IgG-WB)intheserumofchildrenwithsuspectedcongenitaltoxoplasmosis.

Methods: Weaccompanied 47 mothers with acquiredtoxoplasmosis inpregnancy and their children,betweenJuneof2011andJuneof2014.TheIgG-WBwasdoneinhouseandthetest wasconsideredpositive ifthechildhadantibodiesthatrecognizedatleastonebandonIgG blotsdifferentfromthemother’sorwithgreaterintensitythanthecorrespondingmaternal band,duringthefirstthreemonthsoflife.

Results: 15children(15.1%)metthecriteriaforcongenitaltoxoplasmosisand32(32.3%)hadthe diagnosisexcluded.Thesymptomswereobservedin12(80.0%)childrenandthemostfrequent werecerebralcalcificationin9(60.0%),chorioretinitisin8(53.3%),andhydrocephalusin4 (26.6%). IgMantibodiesanti-T. gondiidetected bychemiluminescence(CL) were foundin6 (40.0%)childrenandthepolymerasechainreaction(PCR)fordetectionofT.gondiiDNAwas

夽

Pleasecitethisarticleas:CapobiangoJD,MonicaTC,FerreiraFP,Mitsuka-BreganóR,NavarroIT,GarciaJL,etal.Evaluationofthe Westernblottingmethodforthediagnosisofcongenitaltoxoplasmosis.JPediatr(RioJ).2016;92:616---23.

夽夽

StudyconductedatUniversidadeEstadualdeLondrina,Londrina,PR,Brazil.

∗_{Corresponding}_author.

E-mail:jaquedc@uel.br(J.D.Capobiango). http://dx.doi.org/10.1016/j.jped.2016.02.014

(2)

positivein5of7performed(71.4%).ThesensitivityofIgG-WBwasof60.0%[95%confidence interval(CI)32.3---83.7%]andspecificity43.7%(95%CI26.7---62.3%).ThesensitivityofIgG-WB increasedto 76.0and89.1% when associated to theresearchofIgM anti-T. gondiior PCR, respectively.

Conclusions: TheIgG-WBshowedgreatersensitivitythanthedetectionofIgManti-T.gondii; therefore,itcanbeusedforthediagnosisofcongenitaltoxoplasmosisinassociationwithother congenitalinfectionmarkers.

PALAVRAS-CHAVE

Toxoplasmose Congênita; Westernblotting; Diagnóstico; Sorologia

Avaliac¸ãodométodoWesternBlottingparadiagnósticodetoxoplasmosecongênita

Resumo

Objetivo: AvaliarométodoWesternBlottingparadetecc¸ãodeIgGanti-Toxoplasmagondii(T. gondii)(IgG-WB)nosorodecrianc¸ascomsuspeitadetoxoplasmosecongênita

Métodos: Acompanhamos47mãescomtoxoplasmoseadquiridanagravidezeseusfilhos, entre-junhode2011ejunhode2014.OIgG-WBfoifeitointernamenteeotestefoiconsideradopositivo quandoacrianc¸aapresentava anticorposque reconheciampelomenos umabandanas man-chasdeIgGdiferentedasbandasdamãeoucommaiorintensidadedoqueabandamaterna correspondente,duranteosprimeiros3mesesdevida

Resultados: Atenderamaoscritériospara diagnósticode toxoplasmosecongênita15crianças (15,1%) e 32 (32,3%) tiveram o diagnóstico excluído. Os sintomas foram observados em12 crianças (80%) e os mais frequentes foram calcificação cerebral em nove (60%), coriorre-tiniteem oito (53,3%) e hidrocefalia em quatro (26,6%). Os anticorpos IgM anti-T. gondii

detectadosporquimiluminescência(QL)foramencontradosemseiscrianças(40%)eareação em cadeiada polimerase(RCP) para detecçãodo DNA deT. gondiifoi positivaem cincode setereações(71,4%).AsensibilidadedoIgG-WBfoide60%[intervalodeconfiança(IC)de95%, 32,3a83,7%]eaespecificidadefoide43,7%(ICde95%,26,7a62,3%).Asensibilidadedo IgG-WBaumentoupara76e89,1%quandorelacionadaàpesquisadeIgManti-T.gondiiouàRCP, respectivamente

Conclusões: O IgG-WB mostrou maior sensibilidade do que a detecção de IgM anti-T. gondii;portanto,podeserusadoparaodiagnósticodetoxoplasmosecongênitaemassociação comoutrosmarcadoresdeinfecçãocongênita.

Introduction

Mostchildren withcongenitaltoxoplasmosis(CT) showno signs or symptoms at birth, yet there is a risk of devel-oping late sequelae, particularly ocular and neurological impairment.1

Allchildren areconsideredsuspect whosemothers had

acutetoxoplasmosisinthecourseofpregnancy;therefore,

thesechildren mustbesubjected toserological

investiga-tionwithantibodydetectionforanti-Toxoplasmagondii(T.

gondii).1,2

However,a confirmed serologicaldiagnosis ofT. gondii

infection through the detection of specific IgM and/or

IgA antibodies against the parasite does not occur in all

newborns.1,3---5_Therefore,_IgG_antibodies_against_T._gondii_in

serialserumsamplesmustbeanalyzedandthechildremains

underoutpatientfollow-up,whichcantakemonthsuntilthe

definitivediagnosis.1,6

In an infected fetus, IgGand IgM antibodies produced

againsttheantigenicdeterminants ofT.gondiimaydiffer

fromthoseanti-T.gondii IgGandIgMantibodies detected

in the maternal serum, suggesting a neosynthesis of

spe-cificantibodies.Thus,childrenwithCTwithnon-reactivity

inconventionaltestsforIgMdetectionhavebeendiagnosed

inthefirstmonthsoflifethroughtheWesternblotmethod

(WB).7---9

There is a need for early and rapid diagnosis using

a low complexity method that allows the reference

lab-oratories for toxoplasmosis to differentiate the dubious

results obtained using the routine conventional

serologi-cal methods, such as indirect immunofluorescence (IIF),

enzyme-linkedimmunoassay (ELISA), and immunoassay of

microparticles using chemiluminescence (CL). The

objec-tiveof thisstudy wastoevaluatetheWB methodfor the

detection of IgG antibodies against T. gondii (IgG-WB) in

(3)

toxoplasmosisin pregnancytoassistintheearly diagnosis

ofCT.

Methods

Subjects

The population was comprised of women with suspected acquired toxoplasmosis in pregnancy and their children, assisted at the Pediatric Infectious Disease Outpatient, betweenJuneof2011andJuneof2014.Thestudyincluded children whose mothers demonstrated reactivityfor anti -T.gondiiIgMduringthepre-natalcare.Thebloodsamples ofchild/motherpairswerecollectedsimultaneouslyduring thefirstthreemonthsofchild’slifeandstoredat−20◦_C_for WB.The control groupconsisted of childrenwhose moth-ersshowedsuspectedacutetoxoplasmosisduringpregnancy, butdidnotmeetthecriteriaforCT.2,10,11_In_this_period,₄₇

children andtheir mothers were monitored. Of these,15

(15.1%)childrenwerediagnosedwithCTandin32(32.3%),

thisdiagnosiswasexcluded.Pregnantwomenreceived

spi-ramycinorsulfadiazinepluspyrimethamineandfolinicacid

untilchildbirth. The suspected infected childrenreceived

sulfadiazineplus pyrimethamine andfolinicacid untilthe

definitivediagnosis.

Diagnosticcriteriainchildren

CTwasconsidered when the childpresented elevation of specificanti-T.gondiiIgGtitersinsequentialsamplesinthe firstmonthsofhislifeand/or thepersistenceofT.gondii IgGtitersafter12monthsoflifeand/orreactivityfor anti-T.gondiiIgMand/orchorioretinitisand/orlesionofcentral nervoussystem (CNS) withreactivity for IgGand/or posi-tivityfortheT.gondiiDNAusingpolymerasechainreaction (PCR).2,10,11

Serologicaltestforthedetectionofanti-T.gondii IgMandIgG

The serological tests performed during pre-natal care and on samples from children were performed by con-ventional routine laboratory methods. Anti-T. gondii IgG antibodies were determined by Indirect immunofluores-cence(IIF)12 _(Immunoblot,_{Biolab-Mérieux,}_Rio_de_Janeiro,

RJ, Brazil), with T. gondii obtained from ascitic fluid of

infectedmice,andchemiluminescence(CL)(Architect,

Sys-temAbbott---Wiesbaden,Germany)withp30(SAG1)andp35

(GRA8)recombinantantigensofT.gondii.Anti-T.gondiiIgM

antibodieswerealsodetectedusingCL(Architect,System

Abbott---Wiesbaden, Germany) with p30 antigen and total

lysateofT.gondii.

T.gondiiantigens

Fivealbinofemalemice,agedfrom45to60daysand weigh-ingbetween25and40gwereusedforobtainingRHstrain tachyzoitesof T. gondii. The animals were inoculated by intraperitonealroutewitha suspensionoflivetachyzoites (105_/mL)_in _sterile _saline_solution._Forty-eight_hours_after

theinoculation,theexudatewasobtainedbywashingthe peritonealcavitywith3.0mLofsterilesalinesolution.The sampleswere passedthrougha 27Gneedlefor rupture of the hostcells. Later,they werecentrifugedandthe sedi-ment wasstandardized at 109_{tachyzoites/mL}_by _counting inaNeubauerchamber.13

TheIgG-WBMethod

Theproteins ofT.gondiitobeusedinIgG-WBwere quan-tifiedusingtheLowryetal.method,14 _and_{polyacrylamide}

gel12%wasusedforelectrophoreticseparationofproteins

withsubsequenttransferoftheproteinstoanitrocellulose

membrane (iBlot® _Gel _Transfer _System, _California, _USA).

The nitrocellulose paper was stained with Ponceau and,

afterwashingwithdistilledwater,thenitrocellulosestrips

werecutandblockedwithskimmilk5%plustrisbuffered

saline (TBS)withTween®_. _Washes_were _subsequently

per-formed withTBSandskimmilk5%andthen, thepatient’s

serumwasaddeddilutedinTBSwithTween(Sigma---Aldrich,

MO,USA)andskimmilk5%,carriedoutwashes,andadded

conjugatedanti-humanIgG(Invitrogen,LifeTechnologies

---CA, USA) diluted in TBS with Tween (Sigma---Aldrich, MO,

USA) and skim milk 5%. After further washes the authors

added the chromogen substrate diaminobenzidine (DAB)

0.2%(AcrösOrganics,ThermoFisherScientific---Geel,

Bel-gium)andhydrogenperoxide(100␮L).Whenthebandswere

visualized, the reaction wasstopped withdistilled water.

Positiveandnegativecontrolsamplesforanti-T.gondiiwere

includedineachtest.

The test wasconsidered positive ifthe child produced

antibodiesthatrecognizedatleastonebandofprotein

dif-ferent fromthe motheror withhigher intensity than the

corresponding maternal band (Fig. 1), characterizing the

neosynthesisofanti-T.gondiiIgGantibodies.15---18_To_control

thesubjectivityofreading,twoindependentobserversread

theIgG-WBpatternsblindlyandwithout knowledgeofthe

previousserologicalresultsoftheconventionaltests;there

wasconcordancein100.0%oftheresults.

The specificity of the IgG-WBmethod was determined

with26serumsamplesobtainedfromindividuals

seroneg-ative for toxoplasmosis (anti-T. gondii IgG and IgM

non-reactivebyCL)andwithseroreactivitytoantibodiesagainst

other pathogens, such as Treponema pallidum (n=5),

Trypanosomacruzi(n=5),Leishmaniaspp.(n=2), Paracoc-cidioidesbrasilienses(n=5),orseroreactivitytoserological

markersofautoimmunitydisorders,suchasantinuclear

anti-bodies(n=5)andanti-DNAdoublestrandantibodies(n=4).

PCRfordetectionofT.gondiiDNA

TheDNAwasextractedfromperipheralbloodcellsof chil-dren,collectedwithEDTAastheanticoagulantuptothree monthsafterbirth,usingthekitEasyPrepDNAMiniI (Easy-Gen, Favorgen Biotech --- Austria). The DNA amplification of T. gondii was performed using the method described byHomanetal.19 _Primers_Tox4

(CGCTGCAGGGAGGAAGAC-GAAAGTTG)and Tox5(CGCTGCAGACACAGTGCATCTGGATT)

wereusedfortheamplificationofafragmentof529base

pairs(bp;GenBankNo.AFI46527)ofT.gondiiDNA.PCRwas

(4)

55

kDa kDa

94

77

67

58

37 30

23

20

13 6 72

64

58

kDa kDa

68

50

45

40 27

16

14

M C M C M C PC NC

A

B

C

D

46 44

39 31

25 22

16

11

160 154 130 121

104 101 94 78 77 76 67 64 58 50 46 44 40 37 33 31 30

25 23

20 13

10

6

Figure 1 Pattern recognition ofRH strain proteins of Toxoplasmagondii by IgG antibodiesusing the Western blotting (IgG-WB)methodperformedwithserumsamplesfrommotherswithacquiredtoxoplasmosisduringpregnancyandtheirchildrenwith suspectedcongenitaltoxoplasmosis.(A)PositiveIgG-Westernblottingforcongenitaltoxoplasmosis.EqualbandsrecognizedbyIgG antibodies,withstrongerintensityinthechildsamplecomparedtomaternalsample.(B)PositiveIgG-Westernblottingforcongenital toxoplasmosis.DifferentbandsrecognizedbyIgGfromthechildsampleascomparedtothematernal sample.(C)Negative IgG-Westernblottingforcongenitaltoxoplasmosis.EqualbandsrecognizedbyIgGantibodiesfromchildandmothersamples.M,mother; C,child;PC,positivecontrol;NC,negativecontrol.

DNAextracted fromthesample;1.0␮Lof1.0mMforeach

primer,100mMofdNTP(Invitrogen,LifeTechnologies---CA, UnitedStates),60mMTris---HCl(pH9.0),15mM(NH4)2SO4, 2mM MgCl2, and0.25␮LTaqDNA polymerase(Invitrogen,

LifeTechnologies---CA,UnitedStates),alsocompletedwith 7.75␮Lof MilliQ water.The amplificationwasperformed

with35 cyclesin athermal cycler (PTC-100, MJ Research ---CA,UnitedStates),usingthefollowingcyclingcondition: 7minat94◦_C_for_denaturation_in_the_1st_cycle,_followed_by 33cyclesof1minat94Cfordenaturation,1minat55◦_C_for annealing,and1minat72◦_C_for_extension;_cycle₃₅_was fol-lowedbyafinalextensionof10minat72◦_C._Aliquots_of_each PCRproductweresubjectedtoelectrophoresisin2%agarose gel.RHstrainTachyzoites(107_/mL)_had_their_DNA_extracted tobeusedaspositivecontrol.The waterwasregardedas thenegativecontrol.Positiveandnegativecontrolsamples forT.gondiiwereincludedineachtest.

Statisticalanalysis

The statistical analysis was done on GraphPad Prism 5 (GraphPadSoftware,Inc.---SanDiego,UnitedStates). Cat-egorical variableswere expressed asabsolute number(n) and percentage (%) and analyzed by the chi-squared test orFisher’sexacttest.Parametersofsensitivity,specificity, positive predictive value (PPV), and negative predictive value(NPV)werecalculated,withaconfidenceinterval(CI) of95.0%.Valuesofp<0.05wereconsideredstatistically sig-nificant.

The study was approved by the Ethics Committee for ResearchInvolvingHumanBeings.Allmothersparticipated voluntarilyandsignedtheinformedconsent.

Results

Ofthe 15 childrenwith CT,12 (80.0%)were symptomatic andmettheclinicalcriteriaforthedefinitionofthedisease (ninewithcerebralcalcification,eightwithchorioretinitis, andfourwithhydrocephalus),six(40.0%)presented reactiv-ityfor anti-T.gondiiIgM,five(33.3%)showedelevationof anti-T.gondiiIgGinthefirstmonthsoflife,andone(6.7%) showedpersistenceofanti-T.gondiiIgGtitersinsequential samples. Five of the seven children (71.4%) who under-wentPCR todetect T.gondiiDNAshowed positiveresults (Table1).

Amongthe15childrenwithCT,theIgG-WBtestwas

pos-itiveinnine(60.0%),andamongthe32childrenwithoutCT,

thetestwaspositivein18(56.3%),whichresultedina

sen-sitivityof60.0%(95% CI:32.3---83.7%),specificity of43.7%

(95%CI: 26.4---62.3%), PPVof 33.3% (95% CI:16.5---54.0%),

andNPVof70.0%(95%CI:45.7---88.1%)(p=0.05875).Among

thechildren withCT,twopresented negativeIgG-WBand

werepositiveforanti-T.gondiiIgM;fivechildrenpresented

positiveIgG-WB andwere negativefor anti-T.gondii IgM,

fourpresented both tests aspositive, andfour presented

bothtestsasnegative.

Themolecularweight(MW)oftheproteinrecognizedby

(5)

Table1 Clinicalandlaboratorycharacteristicsof15childrenwithcongenitaltoxoplasmosis,fromJune2011toJune2014.

Case Anti-T. GondiiIgM

PCR IgG1stsamplea _Following IgGb

Western blottingb

Signsandsymptomsin the1stmonthoflife

IFI CL

1 Negative NR 1:512,000 NR ↓ Negative Chorioretinitis+calcification+

hydrocephalus

2 Positive NR 1:32,000 926.8 ↑ Positive Chorioretinitis+calcification

3 Positive NR 1:512,000 NR ↓ Positive Vitritis(chorioretinitis

after)+calcification+hydrocephalus

4 Negative NR <1:16 NR ↑ Positive Chorioretinitis

5 Negative NR 1:1024 162.0 ↔ Positive Calcification

6 Negative Positive NR 200.0 ↑ Positive Asymptomatic

7 Negative Positive 1:32,000 147.5 NR Negative Hydrocephalus

8 Negative Negative 1:8000 312.0 ↑ Negative Asymptomatic

9 Negative Positive 1:512,000 175.6 ↓ Negative Asymptomatic

10 Positive Positive 1:512,000 188.1 ↓ Positive Chorioretinitis

11 Negative Positive 1:32,000 139.8 NR Positive Calcification

12 Positive Negative NR 84.8 ↑ Negative Vitritis+calcification

13 Positive NR NR 200.0 NR Positive Chorioretinitis+calcification

14 Negative NR NR 173.9 ↓ Positive Chorioretinitis+calcification+

hydrocephalus

15 Positive NR NR 1655.0 ↓ Negative Chorioretinitis+calcification

PCR,polymerasechainreactionforDNAdetectionofToxoplasmagondii;IFI,indirectimmunofluorescencefordetectionofIgGanti-T. gondii;CL,microparticlechemiluminescenceimmunoassayforthedetectionofanti-T.gondiIgG,expressedinUI/mL;NR,unrealized;

↑,increasedantibodylevelsofanti-T.gondiiIgGinserialserumsamples;↔,persistenceoflevelsofanti-T.gondiiIgGinserumsamples duringfollow-up;↓,declineofantibodies.

a_Sample_collected_in_the_1st_month_of_life.

b _Sample_collected_within_the_first₃_months_of_life,_with_all_children_in_treatment.

from2to94kDa,andregardingtheproteinsrecognizedby IgGantibodies in the serumof the non-infected, the MW rangedfrom1to160kDa.Amongtheninechildrendiagnosed throughIgG-WB,sevenpresenteddifferentbandsfromthose observedin thematernal serumandtwopresentedbands ofgreaterintensitythanthosepresentedbytheirmother’s sample.The dominantbands thatwererecognizedby IgG antibodiesfromsamplesof childrenwithCTandthe non-infectedaredemonstratedinFig.2.

Asforthe26samplesfrompatientswithotherdiseases,

twoshowednoreactivityand24showedreactivityfor IgG

antibodiesthatrecognizedproteinswithMWranging from

17to118kDa.Thosemostfrequentlyrecognizedwerep22

(11/40.7%),p34 (9/33.3%), p38(8/29.6%), p94(6/22.2%),

p56(5/18.5%),andp30(5/18.5%).

The analysis of the results of anti-T. gondii IgM using

CLshowedsensitivityof40.0%(95%CI:16.3---67.6%),

speci-ficityof100.0%(95%CI:89.1---100.0%),PPVof100.0%(95%

CI:54.1---100.0%), and NPV of 78.1% (95% CI: 62.4---89.4%;

p=0.0005).For the detectionof the parasite’s DNA using

PCR,thesensitivitywas71.4%(95%CI:29.0---96.3%),

speci-ficityof100.0%(95%CI:69.2---100.0%),PPVof100.0%(95%

CI:47.8---100.0%), and NPV of 83.3% (95% CI: 51.6---98.0%;

p=0.0034).

ThesensitivityandspecificityofIgG-WBwhenassociated

withothermarkersofCT---suchasthepresenceofanti-T.

gondiiIgM,positive PCR, andtheclinical symptoms--- are

showninTable2.

IgG-WBwaspositiveinsixofninepatients(66.7%)with

eyeinjuriesandinthreeofsixpatients(50.0%)withCT

with-P 94 0 10 20 30 40

P 78 P 58

With congenital toxoplasmosis Without congenital toxoplasmosis P 44

Antigenic bands (kDa)

F

requency

, %

P 31 P 30 P 13

Figure2 DistributionofthemostfrequentToxoplasmagondii

proteinsrecognizedbyIgGantibodiesintheserumofchildren withcongenitaltoxoplasmosisandchildrenwithoutthedisease, accordingtotheir molecularweight(kDa)(p>0.05forallthe recognizedproteins).

(6)

Table 2 Sensitivity and specificity of the Western blot-tingmethodforthedetectionofanti-ToxoplasmagondiiIgG (IgG-WB),anti-ToxoplasmagondiiIgMtest,thepolymerase chainreaction(PCR)toToxoplasmagondii,andthepresence ofclinicalsymptomscompatiblewithcongenital toxoplas-mosis,assessed inseriesandinparallel,according tothe presenceorabsenceofcongenitaltoxoplasmosis.

Test Sensitivity

(%)

Specificity (%)

IgG-WB+ 60.0 43.7

IgMT.gondii+ 40.0 100.0 IgManti-T.gondiiorIgG-WB+ 76.0 43.7 IgManti-T.gondiiandIgG-WB+ 24.0 100.0

PCR+ 71.7 100.0

PCR+orIgG-WB+ 89.1 43.7

PCR+andIgG-WB+ 42.6 100.0

Symptoms+ 80.0 100.0

Symptoms+orIgG-WB+ 92.0 43.7

Symptoms+andIgG-WB+ 48.0 100.0

IgG-WB, Western blotting for diagnosis of congenital infec-tionwithdetectionofIgG antibodies;anti-Toxoplasma gondii

IgM detected bychemiluminescence; Symptoms, presence of symptomsand/orclinicalsignsconsistentwithcongenital toxo-plasmosis;+,positivetest.

Discussion

The diagnosis of CT is a challenge in clinical practice, becausethesensitivityof anti-T. gondiiIgMusing conven-tional serological methods varies widely, from 48.3% to 75.0%.3---5_The_absence_of_anti-_T._gondii_IgM_can_be_justified

bythefactthatfetalinfectionoccurredatthebeginningof

pregnancy or asa result of maternal treatment for

toxo-plasmosis carried out during the first two trimesters of

pregnancy, which would cause blockage or delay of the

immuneresponse.20 _Moreover,_the_delay_or_the_absence_of

detectableimmuneresponsebystandardmethods(IgGand

IgM dosage or WB) couldbe related todifferences in the

individualimmune response.Another disadvantageof this

markeristhedelayinthesamplecollection,withdecreased

positivityofIgMafterthefirst30daysoflife.21_Therefore,_it

isoftennecessarytomonitortheserologicalresultswiththe

identificationofstableorincreasingtitersofanti-T.gondii

IgG,whichdelaysthediagnosisandcausesuncertaintytothe

family.16_In_this_context,_the_IgG-WB_can_be_used_to_compare

thepatternsofantibodiesagainstT.gondiiinserumsamples

fromthemothersandtheirchildren,anddetermineifthe

antibodiesaretransmittedpassivelyorsynthesizedby the

fetusorinfantincasesofCT.15

Otherauthorshaveobservedahighercontributionof

IgG-WB to thediagnosis of CT in the first months of life.22,23

In addition, the association of IgG-WB with other

sero-logical methods can increase theprobabilityof diagnosis.

In a cohort of children, the sensitivity of IgG-WB was

82.4% and increased to 85.7% when IgG-WB was

associ-ated with the presence of IgM and/or IgA anti-T. gondii

usingELISA.16 _Another_study _showed_that_the_combination

ofIgAandIgMimmunocapturetests,theanalysisofIgGan

IgMWB patterns, andthecombination ofboth techniques

allowedthedetectionof94.0%,94.0%,and100.0%ofcases,

respectively.24

The IgG-WB results obtained in the present study are

closetothosefound byother authors, whoreported

sen-sitivityof 73.5%.18 _These _same _authors _reported_that _the

combinationofIgG-WBandIgM-WBincreasedthesensitivity

to86.5%.18

The lower sensitivity obtained in this study can be

explained,inpart,becauseallthepatientswithsuspected

CT were treated with sulfadiazine, pyrimethamine, and

folinicacidduringtheinvestigation,whichcouldinhibitthe

neosynthesisofantibodies.20

Anotherstrategytoimprovethesensitivity ofthis

pro-posedmethod is the repetition of IgG-WBthroughout the

firstthreemonthsoflife.8_In_a_study_with_samples_collected

atbirth,thesensitivityofanti-T.gondiiIgMbyconventional

methods(ISAGAandELISA)was52.0%andforWB(withIgG

and IgM) was67.0%. By combining the two methods, the

sensitivityincreasedto78.0%atbirthandto85.0%atthree

monthsoflife,withthedetectionof94.0%ofthecasesof

CT.17

Thepresentstudyanalyzedsomesequentialsamplesof

IgG-WB.Amonginfectedchildren,withfalse-negativeresult

in the first sample by IgG-WB, two of six (33.3%)

chil-drenhave differentrecognizedproteins inrelation tothe

maternal sample in the second collection, characterizing

neosynthesisofanti-T.gondiiIgG,whichjustifiestheneed

forrepetitionofIgG-WBinserialsamples.

Inapreviousanalysis,thetimeofmaternalsample

col-lectioninfluencedtheoutcomeoftheIgG-WBtest,because

motherswhoreceivedtreatment for toxoplasmosisduring

pregnancymaynolongerreacttotheantigensofT.gondii

inthelateafterbirthperiod,withfalse-negativeresult.16

Inthepresentstudy,thisphenomenonwasobservedinone

motherofaninfectedchild.However,themajorityof

moth-ersshowedanincreaseinthenumberofbandsrecognized

inthesamplescollectedimmediatelyaftertheinterruption

oftreatmentatdelivery.

Inapreviousstudy,theMWofantigenicbandsthatwere

recognizedby IgGof neonates withCT variedfrom 21 to

116kDa.18 _In _the _present _study, _similar _to _that _found _by

otherauthors,15,16_the_antibodies_found_with_high_frequency

ininfectedchildrenwerethoseagainsttheproteinswithMW

higherthan30kDa.ThedifferencesintheMWofrecognized

proteinsreportedbyprevious studiescanbeexplainedby

differentmethodologicalrequirements,suchasthe

prepara-tionoftheT.gondiiantigen,conditionsofelectrophoresison

acrylamidegel,andtheserumdilutions.20_Another

explana-tionisthegeneticdiversityamongstrainsofT.gondii,which

canlead tothe recognition of different proteins, besides

thefactthatsomeindividualspresent IgGthatrecognizes

multipleproteins,whileothers,asingleprotein.25

Inthepresent study,IgG-WBwasalsousefulin

demon-stratingtheactivesynthesisofanti-T.gondiiIgGinpatients

withIgGnon-reactive byconventionalmethods.InPatient

4,withanti-T.gondiiIgGtiter<1:16byIIFinthefirst

sam-pleandaslightincrease in sequentialsamples (maximum

of 1:64), disappearance of IgG antibodies was observed

whenassayedbyCLandIIFmethodsaftertreatment.

How-ever,bytheIgG-WBmethod,itwaspossibletodemonstrate

anti-T.gondii IgG,which recognizedtheproteins p22and

(7)

monthsoflife,thischild,whoshowedchorioretinitis

scar-ring,remainednon-reactivetoanti-T.gondiiIgGantibodies

evaluatedbyCLandIIF;however,positivitywasshownfor

IgG-WB.

CTwithnon-reactivityforanti-T.gondiiIgGandIgMmay

result from toxoplasmosis treatment on the mother and

neonate.20 _The_treatment_of_the_child_for_a_year_after_birth

mayalsocauseantibodynon-reactivity.However,inmostof

thesecasesthereisdetectionofanti-T.gondiiIgGsoonafter

treatmentinterruption,calledthereboundeffect.1,7,20

Inthispresentstudy,Patient1showednon-reactivity,but

presenteddetectableanti-T.gondiiantibodiesafter

inter-ruptionofthetreatment.SimilarlytoPatient4,Patients6,

8,and9alsoshowedadrop inantibodies uptothepoint

of non-reactivity and remained thus after interruption of

treatment.Itispossiblethatthesepatientsnolonger

rec-ognizetheproteinsp30andp35presentintheCLtest,which

justifiesthenon-reactivityinthistestwiththeevolutionof

theinfection.

Therefore,insteadofevaluatingonlythepersistenceof

anti-T. gondii IgG by CL, the authors suggest the use of

IgG-WBinserological monitoring,since theinfected child

canproduceantibodiesagainstotherproteinsofthe

para-siteandthusremainspositiveafter12monthsage.Inthis

case,the absenceof anti-T.gondii IgGinthe CLmaynot

excludeCT,whichwouldalsojustifythelowerspecificityof

IgG-WBfoundduringthepresentstudycomparedto

previ-ousstudies.16---18,22,23_However,_more_studies_should_be_done

beforeusingIgG-WBinserologicalmonitoring.

According toa studycarried outin a Brazilian

popula-tion, patients with IgM reactive by WB method (IgM-WB)

showedgreaterriskforactivemacularinjurythanpatients

withnegativeIgM-WB.18 _However,_no_significant_difference

wasfound betweenpositivityof IgG-WBandthepresence

of macular injury, asin the present study. It is not clear

whethertherearespecificproteinsofT.gondiistrains

asso-ciatedwitheyeinjuriesandiftheycouldexplainthegreater

frequencyofeyeinjuryonBrazilianchildrenwithCTthan

in other countries.26 _The _main _limitation _to _this _present

study wasthe absence of evaluation of the samples with

IgM-WB,which couldimprovethe diagnostic sensitivityof

thetest.

ThetechnicalprocedureforIgG-WBisoflowcomplexity

andwithavailablecost,which makesitviable inthe

lab-oratoryroutine. The drawback of the in-house method is

itsslowness,which couldbereducedwithstandardization

of a set of reagents to be produced in Brazil. The

semi-automationofthemethodwiththeuseofaprogramforthe

readingofbandintensityviewedthoughWBwouldfacilitate

thereadingandthereproducibilityofresults.20

The results reinforce the usefulness of the IgG-WB

methodforserologicalevaluationofpatientswithCT,with

greatersensitivitythanthedetectionofanti-T.gondiiIgMby

conventionalmethods.Therefore,IgG-WBcanbeusedfor

theearlydiagnosisofCTincombinationwithothermarkers

ofthecongenitalT.gondiiinfection.

Funding

ProgramofSupporttotheUniversityExtensionofthe Min-istryof Education ofBrazil (PROEXT---MEC/SESu) andthe

National Council of Scientific and Technological Develop-ment(CNPq).

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Toallthepatientsandfamilymemberswhocontributedto thefulfillmentofthiswork,tothePostgraduateProgramin Health Sciencesof the UniversidadeEstadual deLondrina (UEL),andtothePostgraduatePrograminAnimalSciences oftheUEL.

References

1.RemingtonJS,McLeodR,WilsonCB,DesmontsG. Toxoplasmo-sis.In:RemingtonJS,KleinJO,WilsonCB,NizetV,Maldonado YA,editors.Infectiousdiseaseofthefetusandnewborninfant. 7ed.Philadelphia:ElsevierSaunders;2011.p.918---1041.

2.AmericanAcademyofPediatrics.Reportofcommitteeon infec-tiousdiseases. Toxoplasmagondii infections(Toxoplasmosis). In:Americanacademyofpediatrics.Redbook.28ed.Illinois: ElkGroveVillage;2009.p.667---72.

3.Lebech M, Andersen O, Christensen NC, Hertel J, Nielsen HE, Peitersen B, et al. Feasibility of neonatal screening for toxoplasma infection in the absence of prenatal treat-ment.DanishCongenitalToxoplasmosis StudyGroup.Lancet. 1999;353:1834---7.

4.LagoEG,Neto EC,MelamedJ, RucksAP,Presotto C,Coelho JC,etal.Congenitaltoxoplasmosis:latepregnancyinfections detectedbyneonatalscreeningandmaternalserologicaltesting atdelivery.PaediatrPerinatEpidemiol.2007;21:525---31.

5.CapobiangoJD, Mitsuka-BreganóR, NavarroIT,RezendeNeto CP,CasellaAM,Lopes-MoriFM,etal.Congenitaltoxoplasmosis inareferencecenterofParaná,SouthernBrazil.BrazJInfect Dis.2014;18:364---71.

6.RodriguesIM,CastroAM,GomesMB,AmaralWN,AvelinoMM. Congenitaltoxoplasmosis:evaluationofserologicalmethodsfor detection of anti-Toxoplasma gondii IgMand IgA antibodies. MemInstOswaldoCruz.2009;104:434---40.

7.PinonJM,DumonH,ChemlaC,FranckJ,PetersenE,LebechM, etal.Strategyfordiagnosisofcongenitaltoxoplasmosis: evalua-tionofmethodscomparingmothersandnewbornsandstandard methodsforpostnataldetectionofimmunoglobulinG,MandA antibodies.JClinMicrobiol.2001;39:2267---71.

8.Tissot-DupontD,FrickerHH,PinchartMP,Bost-BruC,Thomas PA,PellouxH.UsefulnessofWesternBlotinserological follow-upofnewbornssuspectedofcongenitaltoxoplasmosis.EurJ ClinMicrobiolInfectDis.2003;22:122---5.

9.RemingtonJS,ThulliezP,MontoyaJG.Recentdevelopmentsfor diagnosisoftoxoplasmosis.JClinMicrobiol.2004;42:941---5.

10.Brasil. Ministério da Saúde. Secretaria de Atençãoà Saúde. DepartamentodeAçõesProgramáticasEstratégicas.Atençãoà saúdedorecém-nascido:guiaparaosprofissionaisde saúde: intervençõescomuns,icteríciaeinfecções.2ed.Brasília: Min-istériodaSaúde;2013.p.109---22.

(8)

12.CamargoME.Improvedtechnique ofindirect immunofluores-cenceforserologicaldiagnosisoftoxoplasmosis.RevInstMed TropSaoPaulo.1964;6:117---8.

13.LundénA.Immuneresponsesinsheepafterimmunizationwith Toxoplasmagondiiantigensincorporatedintoiscoms.Vet Para-sitol.1995;56:23---5.

14.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265---75.

15.ChumpitaziBF,BoussaidA,PellouxH,RacinetC,BostM,Fleuret AG. Diagnosis of congenital toxoplasmosis by immunoblot-ting and relationship with other methods. J Clin Microbiol. 1995;33:1479---85.

16.GrossU,Lüder CG,HendgenV,HeegC,SauerI,WeidnerA, etal.ComparativeimmunoglobulinGantibodyprofilesbetween motherandchild(CGMCTest)forearlydiagnosisofcongenital toxoplasmosis.JClinMicrobiol.2000;38:3619---22.

17.RillingV,DietzK,KrczalD,KnotekF,EndersG.Evaluationof acommercial IgG/IgM westernblotassay forearly postnatal diagnosisofcongenitaltoxoplasmosis.EurJClinMicrobiolInfect Dis.2003;22:174---80.

18.MachadoAS,AndradeGM,JanuárioJN,FernandesMD,Carneiro AC, Carneiro M, et al. IgG and IgM western blot assay for diagnosisofcongenitaltoxoplasmosis.MemInstOswaldoCruz. 2010;105:757---61.

19.HomanWL,VercammenM, DeBraekeleer J,Verschueren H. Identificationofa200-to300-foldrepetitive529bpDNA frag-ment in Toxoplasma gondii, and its use for diagnostic and quantitativePCR.IntJParasitol.2000;30:69---75.

20.SensiniA.Toxoplasmagondiiinfectioninpregnancy: opportu-nitiesandpitfallsofserologicaldiagnosis.ClinMicrobiolInfect. 2006;12:504---12.

21.LagoEG,OliveiraAP,BenderAL.Presenceanddurationof anti-ToxoplasmagondiiimmunoglobulinMininfantswithcongenital toxoplasmosis.JPediatr(RioJ).2014;90:363---9.

22.MagiB,MiglioriniL.Westernblottingforthediagnosisof con-genitaltoxoplasmosis.NewMicrobiol.2011;34:93---5.

23.L’OllivierC,WallonM,FaucherB,PiarrouxR,PeyronF,Franck J.Comparisonofmotherandchildantibodiesthattarget high-molecular-massToxoplasmagondiiantigensbyimmunoblotting improvesneonataldiagnosisofcongenitaltoxoplasmosis.Clin VaccineImmunol.2012;19:1326---8.

24.GamgneuxFR,CommereV,SchaeferCT,CametJD.Performance ofaWesternBlotassaytocomparemotherandnewborn anti-Toxoplasmagondiiantibodiesfortheearlyneonataldiagnosis of congenital toxoplasmosis.Eur JClin Microbiol Infect Dis. 1999;18:648---54.

25.SunX,LuH,JiaB,ChangZ,PengS,YinJ,etal.A compara-tivestudyofToxoplasmagondiiseroprevalenceinthreehealthy Chinese populations detected using native and recombinant antigens.ParasitVectors.2013;6:241---7.