Gavaa, Cristina da Silva Meira-Strejevitcha, Jose Ernesto Vidalb,c,d, Cinara Cássia Brandão de Mattose, Fábio Batista Fredericof, Luiz Carlos De Mattose, Lígia Cosentino Junqueira Franco Spegiorine,g, FAMERP Toxoplasma Research Group, Fernando Henrique An

(1)

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

Molecular

diagnosis

of

symptomatic

toxoplasmosis:

a

9-year

retrospective

and

prospective

study

in

a

referral

laboratory

in

São

Paulo,

Brazil

Lilian

Muniz

Camilo

a

_,

_Vera

_Lucia

_{Pereira-Chioccola}

a,∗

,

Ricardo

Gava

a

,

Cristina

da

Silva

Meira-Strejevitch

a

_,

_Jose

_Ernesto

_Vidal

b,c,d

,

Cinara

Cássia

Brandão

de

Mattos

e

_,

_Fábio

_Batista

_Frederico

f

_,

_Luiz

_Carlos

_De

_Mattos

e

_,

Lígia

Cosentino

Junqueira

Franco

Spegiorin

e,g

,

FAMERP

Toxoplasma

Research

Group,

Fernando

Henrique

Antunes

Murata

h

_,

_Marina

_Neves

_Ferreira

h,m

,

Deusenia

Machado

Ulisses

Barbosa

h

_,

_Fausto

_da

_Silva

_Gonc¸alves

j

_,

Cristiane

Moraes

Dias

j

_,

_Marcia

_Wakai

_Catelan

j

_,

_Rubens

_Camargo

_Siqueira

h

_,

Mariana

Previato

h,k

,

Amanda

Pires

Barbosa

i,l

,

Danilo

Cavallini

i

a_Centro_de_{Parasitologia}_e_Micologia,_Instituto_Adolfo_Lutz,_São_Paulo,_SP,_Brazil b_Instituto_de_Infectologia_Emilio_Ribas,_São_Paulo,_SP,_Brazil

c_Faculdade_de_Medicina,_Hospital_das_Clínicas,_da_Universidade_de_São_Paulo,_São_Paulo,_SP,_Brazil

d_Laboratório_de_{Investigac¸ão}_Médica_(LIM)_49,_Instituto_de_Medicina_Tropical_da_Universidade_de_São_Paulo,_São_Paulo,_SP,_Brazil e_Faculdade_de_Medicina_de_São_José_do_Rio_Preto,_São_José_do_Rio_Preto,_SP,_Brazil

f_Ambulatório_de_{Oftalmologia,}_Fundac¸ão_Faculdade_Regional_de_{Medicina-Hospital}_de_Base,_São_José_do_Rio_Preto,_SP,_Brazil g_Hospital_da_Crianc¸a_e_Maternidade,_São_José_do_Rio_Preto,_SP,_Brazil

h_Faculdade_de_Medicina_de_São_José_do_Rio_Preto,_São_José_do_Rio_Preto,_SP,_Brazil

i_Ambulatório_de_{Oftalmologia,}_Fundac¸ão_Faculdade_Regional_de_{Medicina-Hospital}_de_Base,_São_José_do_Rio_Preto,_SP,_Brazil j_Hospital_da_Crianc¸a_e_Maternidade,_São_José_do_Rio_Preto,_SP,_Brazil

k_Departamento_de_{Oftalmologia,}_Faculdade_de_Medicina_de_Marília,_Marília,_SP,_Brazil l_Departamento_de_{Oftalmologia,}_Universidade_de_São_Paulo,_Ribeirão_Preto,_SP,_Brazil

m_Faculdade_de_Medicina_Veterinária_e_Zootecnia,_Universidade_de_São_Paulo,_São_Paulo,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received17May2017

Accepted23July2017

Availableonline29September2017

a

b

s

t

r

a

c

t

Symptomaticforms oftoxoplasmosisarea seriouspublichealth problemandoccurin

around10–20% of the infected people.Aiming to improve the molecular diagnosis of

symptomatictoxoplasmosisinBrazilianpatients,thisstudyevaluatedtheperformance

ofrealtimePCRtestingtwoprimersets(B1andREP-529)indetectingToxoplasmagondii

DNA.Themethodology wasassayed in807clinicalsamples withknownclinical

diag-nosis,ELISA, andconventional PCR results in a 9-year period. All samples werefrom

∗ _{Corresponding}_author_.

E-mailaddress:pchioccola@gmail.com(V.L.Pereira-Chioccola).

http://dx.doi.org/10.1016/j.bjid.2017.07.003

(2)

Keywords: Toxoplasmagondii

Symptomatictoxoplasmosis

Diagnosis

Real-timepolymerasechain

reaction

patientswithclinicalsuspicionofseveralfeaturesoftoxoplasmosis.Accordingtothe

min-imumdetectionlimitcurve(inCT),REP-529hadgreatersensitivitytodetectT.gondiiDNA

thanB1.Bothprimersetswereretrospectivelyevaluatedusing515DNAfromdifferent

clin-icalsamples.The122patientswithouttoxoplasmosisprovidedhighspecificity(REP-529,

99.2%andB1,100%).Fromthe393sampleswithpositiveELISA,146hadclinicaldiagnosis

oftoxoplasmosisandpositiveconventionalPCR.REP-529andB1sensitivitieswere95.9%

and83.6%,respectively.ComparisonofREP-529andB1performanceswasfurtheranalyzed

prospectivelyin292samples.Thus,fromatotalof807DNAanalyzed,217(26.89%)had

posi-tivePCRwith,atleastoneprimersetandsymptomatictoxoplasmosisconfirmedbyclinical

diagnosis.REP-529waspositivein97.23%,whereasB1amplifiedonly78.80%.After

com-paringseveralsamplesinaBrazilianreferrallaboratory,thisstudyconcludedthatREP-529

primersethadbetterperformancethanB1one.Theseobservationswerebasedafterusing

caseswithdefinedclinicaldiagnosis,ELISA,andconventionalPCR.

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

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

Introduction

The infection by Toxoplasma gondii in humans is generally

asymptomatic.Duringthechronicphase,theparasites

per-sistencystedinthebrainandmuscleinthemajorityofthe

cases.1–3_However,_symptomatic_forms_may_occur_in_around

10–20%oftheinfectedpeople.Thus,toxoplasmosiscouldbe

aseriouspublichealthproblem,asitmayleadtomoresevere

symptoms.4–7

Infectionsoccurringduringpregnancycanresultinsevere

neonatalimpairmentinthefetus.Intheocularform,lesions

resultfromcongenitalorafterbirth-acquiredinfectionsand

normallyarenecrotic.Theselesionsmaydestroythe

architec-tureoftheneuralretinaandsometimesinvolvethechoroid

(retinochoroiditis).8–13

Thereactivationoflatentinfectionasresultofan

immuno-deficiencysuchasseverelyimmunosuppressedHIV-infected

patients in absence of highly active antiretroviral therapy

andprophylaxisresultsmorefrequentlyincerebral

toxoplas-mosis.Inrarecases,disseminatedtoxoplasmosiscanoccur,

whichinvolvesatleasttwoorgans,infailureofaneffective

Th1immuneresponse.5,6,14–18

The laboratory diagnosis is based on serological and

moleculartests (PCR).Inocularform, thediagnosis is

typ-ically clinical but the laboratory tests normally provide

confirmation.13,19 _Serological _diagnosis _regularly _monitors

seronegativepregnantwomenandseroconversionofchildren

under congenital toxoplasmosis investigation.20 _In _case _of

seroconversion,detection ofT. gondiiDNAbyPCRin

amni-oticfluid(AF) confirmscongenitaltoxoplasmosis.21–24 _After

birth,congenitaldiagnosiscanbemadeincerebrospinalfluid

(CSF)orblood(BL)samplesfromnewborns.Incasesof

suspi-cionofcerebralordisseminatedtoxoplasmosis,thetreatment

isusuallyinitiatedbasedonclinical,radiological,and

sero-logicalfeatures.6,15_PCR_has_been_shown_to_be_an_important

diagnostictoolsinceitisusedinallformsofsymptomatic

toxoplasmosis.25–29_Such_diagnosis_leads_to_rapid_and_specific

treatment,thusavoiding,orreducingseriousdamagecaused

byT.gondii.

Real-timequantitativePCR(qPCR)hasbeenusedfor

molec-ulardiagnosisandstudiesdemonstratedqPCRapplicabilityin

toxoplasmosisdiagnosisusingdifferenttargets.24–26,28,30–34

Aiming toimprove molecular diagnosis ofsymptomatic

toxoplasmosis in Brazilian patients, this study evaluated

the performanceof qPCR in detecting T. gondii DNA using

two primer sets for routine diagnosis. These markers are

used in laboratories in European countries for routine

diagnosis.23,29,35

Material

and

methods

Patientsandclinicalsamples

This retrospective and prospectivestudy evaluated clinical

samplesfrom807patients.InstitutoAdolfoLutz(SãoPaulo,

Brazil) received these samples from January 2008 through

November 2016 for molecular diagnosis of toxoplasmosis.

Clinical samplesoriginated frompatients withknown final

clinical diagnosis were chosen. The clinical samples

ana-lyzedduringthisperiodwereasfollows:173(2008–2010),204

(2011–2013), 138 (2014–2015), and 292 (2016). These clinical

samplessent tothe laboratoryincluded CSF(302), BLwith

EDTA (439), AF (47), and formalin-fixedparaffin-embedded

(FFPE)tissuessectionedin4-␮m-thick(19)(AU).Forserological

tests,BLsampleswerecollectedintubeswithno

anticoag-ulants. Clinical samplesforPCRand serology weresent to

thelaboratorywithin48haftercollection, andimmediately

processed.Intheretrospectivepartofthisstudy,theroutine

diagnosisof515clinicalsamplescollecteduntil2015included

conventionalPCR(cPCR)andenzyme-linkedimmunosorbent

assay(ELISA).Intheprospectivestudy,the292clinicalsamples

senttothelaboratoryin2016wereanalyzedbyqPCRusingthe

primerssetsREP-529andB1.

The samples evaluated in this study were from

symp-tomatic patients with the following clinical diagnosis of

toxoplasmosis: cerebral(404HIV-patients),ocular(193with

ocular alterations), congenital infection by transplacental

(3)

women),anddisseminated(19brainautopsiesfromdeceased HIV-patients).

HIV-infected patients with suspicion of cerebral

toxo-plasmosis presented progressive neurological deficits and

contrast-enhancing mass lesion(s) on computed

tomogra-phy or magnetic resonance imaging. These cases were

includedin“definitionforcerebraltoxoplasmosis”previously

described.6,15_Those_who_died_from_severe_disseminated

toxo-plasmosishadpositiveserologyandimmunohistochemistry

fortoxoplasmosisofatleasttwoorgans.Bothgroupofpatients

were admitted and treated atthe Instituto de Infectologia

EmilioRibasbyDrJoseE.Vidal’sgroup.Patientswithsuspicion

ofoculartoxoplasmosishadocularalterations,suchas

tox-oplasmicretinochoroiditisscarsorretinalexudativelesions.

They were admitted and treated at Ambulatorio de

Oftal-mologia,byDrFábio B.Frederico’sgroup.Pregnant women

withsuspicionofacutetoxoplasmosisinfectionwere

admit-tedand treated atthe High RiskAntenatal Careand Fetal

Medicine Service. After enrollingin a high-risk pregnancy

clinic,pregnantwomenwereroutinelyscreenedforTORSCH

(Toxoplasmosis,Rubella, Syphilis,Cytomegalovirus,

Hepati-tisandHIV).36,37_Those_clinically,_{epidemiologically}_suspected

oftoxoplasmosisandhavingpositiveIgMorlowIgGavidity

foranti-T.gondiiantibodiesunderwentamniocentesisforPCR

testinginamnioticfluid.Beforecollectionpregnantwomen

wereinformedoftheprocedureandrequiredtosigninformed

consent forms. Thenewborns ofthese mothers with

sus-pectedorconfirmedfetalinfectionwereadmittedandtreated

attheHighRiskPediatricServiceandFetalMedicineService.

MothersandnewbornsweretreatedbyDrLígiaCJFSpegiorin’s

group.

T.gondiiantigenandserologicaldiagnosis

T.gondiilysateantigenandELISAwereperformedexactlyas

describedbefore.19,38,39

DNApurification

BLsamples(5mL)werepreparedasdescribedbefore.28_Plasma

sampleswereseparatedbycentrifugationandusedin

sero-logical diagnosis. Pellets were mixed with three times the

volumeofabuffercontaining150-mMammoniumchlorate,

1-mM potassiumbicarbonate, 0.1-mM EDTA, pH 7.3,

incu-batedfor 15min atroom temperature undermild shaking

andcentrifuged(2500g/10min).AFsamples(10mL)were

cen-trifuged(2500g/10min)andthe supernatantwasdiscarded.

Blood pellets, containingonlynuclei cells, AFpackedcells

and crushed tachyzoites (for control and standard curves)

weredigestedfor10minat56◦_C_with_proteinase_K₍₂₀_␮_g)_in

200␮LofALbuffer(Qiagen).AUfragments(5sections of

4-␮m-thickFFPE)weredissolvedinxylene(1mL),incubatedfor

30sec,centrifugedtwice(8000g/1min)andsupernatantswere

removed.Pelletsweremixedwith1mLethanol,centrifuged

(8000g/1min)andincubatedfor10min,atroomtemperature

forcompletexyleneevaporation.40

DNAmoleculeswereextractedfromBL,AF,andtachyzoites

byQIAampDNAMiniKit(Qiagen);andfromAU,byQIAamp

DNA FFPETissue kit (Qiagen).Theprotocols were followed

according to the manufacturer’s instructions in a Robotic

workstationforautomatedpurificationofDNA(QIAcube,

Qia-gen).DNAmoleculesfromCSFsamples(3mL)wereextracted

asdescribed before.41 _DNA _{concentrations}_and _purity_were

determinedbytheratioofO.D.at260and280nminNanoDrop

ND1000.ForuseinPCR,sampleswithhighDNA

concentra-tions(assomeBLandAU)weredilutedwithultra-purewater

untilconcentration100ng/␮L.

cPCRtargetfortoxoplasmosisandinternalcontrol

T. gondiiwas identifiedbycPCR usingthe primer pair

B22-B23,31_designed_to_amplify_a_115-bp_amplicon_from_the_B1_gene

astarget.Thereactionswererunfollowingthesame

condi-tions describedelsewhere.42 _Absence_of_PCR_inhibitors_was

verified usingahousekeepinggene thatamplifieda140bp

fragment of the human ␤-globulin gene.43 _Each

amplifica-tion run containedtwo negativecontrols(ultra-pure water

andnegativeDNAfortoxoplasmosis)andonepositive(DNA

extractedfromtachyzoites).Afterthermalcycles,PCR

prod-uctswereelectrophoresedin2%agarosegelandstainedwith

ethidiumbromideandvisualizedunderUVillumination.

qPCRtargetfortoxoplasmosisandinternalcontrol

The performance of two different primer sets for qPCR

was analyzed. The first (B1) amplified a 71-bp

frag-ment of the B1 gene as template,29,35 _which _has ₃₅

copies in the genome and is conserved in different

parasite strains.31 _The _design _included _the _forward

(5′_{-GAAAGCCATGAGGCACTCCA-3}′₎ _and _reverse ₍₅′

-TTCACCCGGACCGTTTAGC-3′₎ _primers; _and _a _{hybridization}

probe (5′_{-CGGGCGAGTAGCACCTGAGGAGATACA-3}′₎ _labeled

with FAM (6-carboxyfluorescein) and BHQ1 (Black Hole

Quencher1)atthe5′_and₃′_ends,_{respectively.}

The second (REP-529) amplified a 112-bp of the highly

repetitivesequenceREP-529(GenbankAF487550),23,29 _which

has200–300copiesinT.gondiigenome.30_The_design_included

theforward(5′_{-AGAGACACCGGAATGCGATCT-3}′₎_and_reverse

(5′_{-TTCGTCCAAGCCTCCGACT-3}′₎_primers;_and_the

hybridiza-tionprobe(5′_{-TCGTGGTGATGGCGGAGAGAATTGA-3}′₎_labeled

with FAMand BHQ1.The designand location in the gene

of each primer pair is described in Fig. 1. Both molecular

markersequenceswerepreviouslystandardizedinour

labora-toryusingserialdilutionsofDNAfromtachyzoites(RHstrain)

astemplate.ToconfirmtheabsenceofPCRinhibitors,aprimer

settoaEukaryotic18SrRNAgene(GenBankaccessioncode

X03205.1),purchasedfromAppliedBiosystemswasusedas

housekeepinggene.Thereactionswereperformedinfinal

vol-umeof20␮L.DNAsamples(3␮LofDNAuntil100ng/␮L),DNA

control(5ng/␮L)orDNAforstandardcurve(3␮Lforeachpoint)

wereaddedtoreactionmixturecontaining10␮Lof2XTaqMan

UniversalPCRMasterMix.Next,1.25␮Lof“AssayMix”(18␮M

offorward and reverse primersand 5␮M ofthe hydrolysis

probe).AmplificationswereperformedinanApplied

Biosys-tems7500Real-timePCRSystemusingthefollowingthermal

profile:2min,50◦_C,_and₉₅◦_C_for₁₀_min._Next,₄₀_cycles_were

(4)

Gene B1 AF179871 ( 1 to2214 bp) 1

2214 1920

1740

F (B22 cPCR) 1793-1817 (Length:25)

R (B23 cPCR) 1882-1907 (Length:26)

F (B1 qPCR)

1774-1793 (Length:20) 1795-1821 (Length:27)1826-1844 (Length:19) R (B1 qPCR) PROBE B(1 qPCR)

Product length 115

Product length 71

REP 529 AF487550 ( 1 to404 bp)

1 270 390 404

Productlength112

F (REP-529 qPCR) PROBE (REP-529 qPCR) R (REP-529 qPCR) 270-290 (Length:21) 310-334 (Length:25) 363-381 (Length:19)

A

C

B

Fig.1–Designandgeneslocationofprimersetsusedinthisstudy.(A)cPCR(B22-B23),(B)qPCRB1,and(C)qPCRREP-529. Theanalysesweredoneinhttps://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi?ctgtime=1486921082&jobkey=

1N4LxE9DQutl1VjQVbB84i-rbdACuHbNAw&CheckStatus=CheckforB22/B23;

https://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi?ctgtime=1486924834&jobkey=l51I5ugp5YHCu3W-eN5RjALFQL4v1lujLgfor(B1),andhttps://www.ncbi.

nlm.nih.gov/tools/primer-blast/primertool.cgi?ctgtime=1487009304&jobkey=ycMWuJUKmKKmAidBf0sr3mPZ1S9SaAUw&

CheckStatus=CheckforREP-529.TheschemeswerebasedonLinetal.46

Standardcurveanalysis

Two T.gondiistandardcurves usingREP-529 and B1primer

setswereconstructedinnineconcentrationsofDNAobtained

from 1×107 _tachyzoites_(in _triplicate)._Initial_{concentration}

(1×107_tachyzoites)_was₃₅_ng/_␮_L_and_serial_dilutions_ranged

upto10−1_tachyzoites_(3.5_fg)._The_cycle_threshold_values₍_C_T₎

wereplottedasmean(triplicate)againstthestandardcurve

valuestodeterminethedetectionlimitofbothprimersets.

Parasite concentrationswere determined afterthe

calcula-tionofthelinearregressionequation(y=ax+b),wherey=CT;

a=curveslope(slope);x=parasitenumber;andb=wherethe

curveintersectsy-axis(yintercept).44

Qualityassurance

IneachPCRrun,ablankcontrolwasusedconsistingof

DNA-freewaterplusthePCRmix.Separateroomswereusedfor

(i)DNAextraction;(ii)PCRmixandprimerpreparation;(iii)

addingDNAfromclinicalsamplesandpositivecontrol;and

(iv)post-PCRagarosegelelectrophoresisanalysisincaseof

thecPCR.DNAsampleswereassayedintriplicateand,atleast

twicetodeterminereproducibility.ThequalityofDNA

sam-plesstockedfornine yearsand retrospectivelyre-analyzed

with qPCR was confirmed by the positivity using the 18S

primerset.

Dataanalysis

ThediscordantresultsinqPCR werere-analyzed,usingthe

sameprotocols,atleasttwice.Sensitivitiesandspecificitiesof

bothprimersetsinclinicalsamplesweredetermined

consid-ering the true diagnosis (clinicaland laboratory diagnoses)

andwerecalculatedas:(i)percentofsensitivity=ratiooftrue

positives/truepositives+falsenegatives×100;(ii)percentof

specificity=ratiooftruenegatives/(truenegatives+false

pos-itives)×100. Reproducibilitycalculationswere done exactly

asdescribedbefore.45_The_percentages_of_concordant_indices

werecalculatedasthefollowingratio:(numberofconcordant

results)/(number total of samples)×100. Linear regressions

wereconstructedfromthestandardcurvesforREP-529and

B1andCTmeancomparisonofbothprimerpairs.Bothcurves

werestatisticallyanalyzedusinganunequal-variancet-test

basedonacriticalvalueofp≤0.05.Allcalculationswererun

inGraphPadPrism6.0software.

Results

REP-529andB1testedinT.gondiitachyzoites

The first step was to evaluate the reportable range of the

reactionthatwascalculatedusingDNAextractfrom

(5)

A

40 40

30 30

20

C

T

C

T

20

10 10

Y= -3.069 . X + 43.26 Y= -3.611. X + 40.11

R2= 0.9985 R2= 0.9923

10

Number of tachyzoites (in log₁₀)

102 103 104 105 106 107 102 103 104 105 106 107 1

B

Fig.2–StandardcurveofT.gondii,tachyzoitesusingREP-529(A)andB1(B)primersets,respectivelyusingthehydrolysis probeFAMdye-labeled.Resultsareshownasmeancyclethreshold(CT)obtainedfromtriplicateofeachDNAconcentration.

Standardcurveanalysiswasdonein10-foldserialdilutionsofDNAextractedfromtachyzoites,atinitialconcentrationof 35ng/␮L(1×107tachyzoites).ForREP-529,R2=0.9923andB1,R2=0.9985.

Table1–SummaryofretrospectiveofqPCRanalysisemployingREP-529andB1primersetsin515DNAsampleswith resultsinclinicaldiagnosis,ELISA,andcPCR(B22-B23).

Clinicalsamples Results

Serologicaldiagnosis(ELISA) n cPCR(B22-B23) qPCR(REP-529) qPCR(B1)

Negative Positive Negative Positive % Negative Positive %

Negative 122 122 0 121 1 99.2a ₁₂₂ ₀ ₁₀₀a

Positive 393 247 146b ₂₅₃ ₁₄₀ _95.9b ₂₈₁ ₁₂₂ _83.6

Total 515 369 146 374 141 – 403 122 –

a _Percent_of_specificity_was_calculated_as_ratio_of_true_{negatives/true}_negatives₊_false_positives_×₁₀₀_(clinical_diagnosis_and_ELISA_results_were

consideredgoldstandardmethod− 122patientswithouttoxoplasmosis).

b _Percent_of_sensitivity_was_calculated_as_ratio_of_true_{positives/true}_positives₊_false_negatives_×₁₀₀_(clinical_diagnosis_and_cPCR_were_considered

goldstandardmethod).

Theresulting standardcurveshowedthe detectionlimitof

eachcurve.R2₌_0.9923_for_REP-529_and_0.9985_for_B1_showed

highlinearityamongthevariables.46_The_minimum_detection

limit(inCT)was39.28(correspondingtooneparasite)for

REP-529, higherthan the 37.18(corresponding to100 parasites)

forB1.

REP-529andB1qPCRtestedinBrazilianclinicalsamples

Thefirst515DNAsamples(collecteduntil2015)hadresultsof

clinicaldiagnosis,ELISAandcPCR(B22-B23).TheseDNA

sam-pleswereusedtotestREP-529andB1inqPCR,andresultsare

showninTable1.DNAsamplesof122patientshadnegative

serological,molecular,andclinicaldiagnosis (without

toxo-plasmosis).REP-529andB1providedspecificitiesof99.2%and

100%,respectively.Allsampleswerenegativewhen

consider-ingresultsofatleastoneprimerset.

Table1alsoshowsPCRresultsof393clinicalsampleswith

positiveELISA.PositiveIgG antibodies anti-T.gondiiare not

necessarilyindicative ofasymptomaticinfection, whereas

detectionoftachyzoiteDNA inbiologicalfluidsascerebral,

ocular, congenital or disseminating forms can

character-izeasymptomaticinfection.19,40,42,47,48_Thus,_{sensitivities}_of

REP-529 and B1 were 95.9% and 83.6%, respectively were

calculatedconsideringcPCRandclinicaldiagnosis(146

sam-ples,whichincluded94patientswithcerebraltoxoplasmosis,

10newbornswithcongenitalinfection,21patientswith

ocu-lartoxoplasmosis,sixpregnantwomenwithtoxoplasmosis,

and15postmortempatientswithseveredisseminated

toxo-plasmosis)(Table2).REP-529amplified140/146sampleswith

averageCTof31.71±0.36.B1amplified122/146,withaverage

CTof32.12±0.38.

Intheprospectivestudy(2016),atotalof292clinical

sam-pleswereanalyzedbyREP-529andB1.AsshowninTable2,

among807clinical samples,217(26.89%) had positivePCR,

atleast oneprimer set anda confirmedclinical diagnosis.

These included: (i) 30.94% (125/404) patients with cerebral

toxoplasmosis; (ii) 12.80% (16/125) newbornswith

congeni-tal alterations; (iii) 18.18%(12/66)ofpregnant womenwith

acutetoxoplasmosiswithT.gondiiDNAinAForBL;(iv)23.83%

(46/193)patientswithocularalterationsandT.gondiiDNAin

BL;and (v)94.74%(18/19)postmortempatientswithsevere

disseminatedtoxoplasmosis.

Table3describestheperformanceofprimersetsand

clin-icalcharacteristicsof217positivesamples.REP-529amplified

T.gondiiDNAin97.23%(211/217)ofpositivesamples,andB1

(6)

Table2–ClinicalandPCRresultsofthe807clinicalsamplesanalyzed.

Toxoplasmosis(clinical form)

Totalnumberof clinicalsamples

analyzed

Positivesamplesclinical,molecular,and serologicaldiagnosis

%Positivity (bothstudies)

Retrospectivestudy (total–515samples)

Prospectivestudy (total–292)

Bothstudies(total– 807samples)

Cerebral 404 94 31 125 30.94

Congenital 125 10 6 16 12.80

Gestational 66 6 6 12 18.18

Ocular 193 21 25 46 23.83

Disseminated (postmortem)

19 15 3 18 94.74

Total 807 146 71 217 26.89

Table3–Characteristicsofclinicalsamples,sensitivitiesofREP-529andB1consideringthe217positivesamples.

Clinicalform(n) Sample Totala _REP-529 _B1

Positive % Positive %

Cerebral(125) CSF 49 49 100 41 83.67

BL 76 72 94.74 62 81.58

Congenital(16) CSF 3 3 100 2 66.67

BL 13 13 100 9 69.23

Gestational(12) AF 8 8 100 5 62.50

BL 4 4 100 3 75.00

Ocular(46) BL 46 44 95.65 36 78.26

Disseminated(18) AU 18 18 100 13 72.22

Total 217 211 97.23 171 78.80

a _At_least_one_primer_set_positive.

Table4–ComparisonbetweenmeanCTvaluesobtainedwithREP-529andB1qPCRconsideringthe217positivesamples.

Clinicalform(n) Sample Totala _Mean_±_SEM_(n)_CT _p_valueb

REP-52 B1 CT ttest

Cerebral (125)

CSF 49 32.86± 0.43(49) 33.71± 0.40(41) 0.85 0.2420(ns)

BL 76 31.08± 0.54(72) 31.92± 0.51(21) 0.84 0.2830(ns)

Congenital (16)

CSF 3 35.61± 0.22(3) 36.37± 1.67(2) 1.68 0.2628(ns)c

BL 13 31.06± 1.06(13) 32.99± 1.13(9) 1.93 0.6757(ns)

Gestational (12)

AF 8 34.69± 0.63(8) 36.13± 0.56(3) 1.44 0.0554(ns)

BL 4 35.02± 0.58(4) 36.36± 0.21(2) 1.34 0.0192(ns)

Ocular(46) BL 46 32.07± 0.62(44) 33.22± 0.66(24) 1.15 0.0225(ns)

Disseminated(18) AU 18 30.92± 0.96(18) 31.27± 1.48(10) 0.35 0.2172(ns)

a _SEM_(standard_error_of_the_mean).

b _Significant_value_at_a_p_<_0.01.

c _{Non-significant.}

AlthoughaverageCT ofREP-529islowerthanforB1,the

CT withboth primersets were notstatisticallydifferent in

positivesamples(Table4).

Theconcordanceindexamongtheprimer setswas

cal-culated basedon the positiveresults. Thetotal number of

sampleswasbasedontheresultsof515clinicalsampleswith

positive(146)andnegative(369)resultsincPCR(B22-B23)until

2015,and807clinicalsampleswithpositive(217)andnegative

(590)resultsinqPCR(REP529andB1)(2008–2016).Asshownin

Table5,concordanceofresultsamongthethreeprimersets

was83.56%.ConcordancebetweenB22-B23andREP-529was

95.89%; betweenB22-B23and B1 was 83.56%;and between

REP-529andB1,83.87%.

Discussion

Until 2015,molecular diagnosis in our laboratory included

cPCR using primer set B22-B23, which amplified a

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Table5–ConcordanceindexbetweenthreemarkersfordetectingT.gondiiinclinicalsamples.

Periodofthestudy Primersets Clinicalsampleswith symptomatic toxoplasmosis

PCRresults

Concordanta _Discordantb _Concordance

indexc_(%)

B22-B23/REP-529/B1 146 122 24 (83.56)

2008–2015 B22-B23/REP-529 146 140 06 (95.89)

B22-B23/B1 146 122 24 (83.56)

2008–2016 REP-529/B1 217 182 35 (83.87)

a _Positive_in_all_markers.

b _Negative_at_least_one_marker.

c _Percents_of_concordant_index_were_calculated_as_the_ratio:_(number_of_concordant_{results)/(number}_total_of_samples)_×_100.

Numbertotalofsampleswasbasedonresultsof515clinicalsampleswithpositive(146)andnegative(369)resultsincPCR(B22-B23)until

2015;and807clinicalsampleswithpositive(217)andnegative(590)resultsinqPCR(REP529andB1)(2008–2016).

anddetectsDNAofasingleorganismdirectlyfromacrude

celllysateor10parasitesinthepresenceof100,000human

leukocytes.31 _High_sensitivity_and _specificity _of_this_primer

setwerepreviouslyreportedinCSFandBLsamplescollected

fromAIDSpatientsandfromthosewithoculartoxoplasmosis

in our setting.19,41,42 _Thus, _to _include _qPCR _in _molecular

diagnosis,wepreviouslyevaluatedtheperformanceofqPCR

todetectT.gondiiDNAtestingtwoprimersets.23,29,35

Thefirstexperimentswereaimedtodeterminethe

mini-mumdetectionlimitcurve(inCT)ofeachprimerset.REP-529

wasmore sensitivetodetect T. gondiiDNAthan B1,which

were39.28(correspondingtooneparasite)and37.18

(corre-spondingto100parasites),respectively.Similarresultswere

showninotherstudy33 _and_they_could_be_attributed_at_the

number of repeated copies in T. gondiigenome (B1gene –

35copiesand529bpsequence200–300copies).33_In_fact,_the

REP-529sequenceismoreabundantinnumberofrepetitions

thantheB1.30 _Despite_these_differences_of_sensitivity,_good

reproducibility(100%)wasshownwithbothprimersets,since

similarresultswere presentedinthereplicas(in triplicate)

andindifferentreactions(samesampleanalyzedondifferent

days).

Next,REP-529andB1wereretrospectivelyevaluatedusing

515DNAextractedfrom differentclinicalsamplescollected

fromBrazilianpatientswithknownclinicalandlaboratorial

diagnoses.Thesensitivityandspecificityofdiagnostictests

were assessed on optimal working conditions. To improve

the PCR sensitivity, the clinical samples were processed

rapidlywithin48h ofcollectionto preventTaqpolymerase

inhibition.42,49_Considering_the_variation_of_the_parasite_DNA

concentration, allsamples usedinthis study were

quanti-fiedafterextraction(sinceconcentrationsupto100ng/␮Lcan

inhibitthereaction)andwerecheckedfortheDNAinhibitors

byamplifyingahumanhousekeepinggene.Thismainquality

controlpreventednegativeresultsduetolowDNAquality.50

ThequalityofDNAsamplesstockedforuptonineyearsand

retrospectivelyre-analyzedwithqPCRwasconfirmedby

posi-tivityusingthe18Sprimerset.Theseresultswereessentialto

verifyiflong-termstorageat−20◦_C_after_initial_diagnosis_had

notalteredDNA.Inaddition,alldiscordantresultsbetween

thethreemarkerswererepeated(intriplicate)atleasttwice.

Onlyone DNA (0.8%)sample had positiveqPCR in

REP-529from the122patientswithouttoxoplasmosis(theyhad

negativeELISA,cPCR,andclinicaldiagnosis).Similarresults

havebeenshowninotherstudies.28,51_Thus,_both_primer_sets

providedhighspecificities(99.2%and100%).

Fromthe393clinicalsampleswithpositiveELISA,146had

positivecPCRandthesepatientshadsymptomaticinfection,

according to the clinical diagnosis. Considering both cPCR

andclinicaldiagnosisREP-529andB1sensitivitieswere95.9%

and83.6%,respectively.ThemeansofCTforREP-529andB1

were31.71±0.36and32.12±0.38,respectively.AlthoughDNA

samples stockedup tonine years were checkedusing two

humanhousekeepinggenes(␤-globulinand18Sprimersets),

six(2.8%)werefalsenegativesamplesdeterminedbyREP-529

and24(16.4%)byB1inthefirstpartofthestudy(retrospective).

Theseresultscouldbeattributedto:(i)degradationofT.gondii

DNAbythelong-termstorageat−20◦_C;_(ii)_absence_of_such

sequencesinsomeofthebiologicalsamplesasmentionedby

others51_;_and_(iii)_human_DNA_could_inhibit_PCR_when

target-ingtheB1gene,whichoccursin35copiesinT.gondiigenomeor

in529bpsequence,whichoccursinmorecopies(200–300).30,33

Despitethat,theseresultsconfirmthatREP-529was

consider-ablybetterthanB1indetectingT.gondiiDNA.Thesedatahave

beenconfirmedbyotherstudies.29,32,33,52

When only the qPCR for both primer sets were tested

inmoleculardiagnosis,the reactionsweredoneusingDNA

extractedrecently.ComparisonofREP-529 andB1

perform-anceswasalsoprospectivelyanalyzedin292clinicalsamples

collected in2016 (Table 2). Thus, atotal of 807DNA

sam-pleswereanalyzedretrospectivelyandprospectively.Ofthem,

217 (26.9%) had positive PCR, at least one primer set and

clinicaldiagnosisconfirmedassymptomatictoxoplasmosis.

REP-529amplifiedT.gondiiDNAin211(97.23%)clinical

sam-ples, whereas B1 amplified only 78.80% of them. Positive

resultsdeterminedbyREP-529in18AUbrainsampleswere

also confirmed by parasite visualization by

immunohisto-chemistryinbraintissues.However,fiveofthemhadnegative

resultswhentheB1wasused.Althoughtheseanalyseswere

done inBrazilianclinicalsamples,theyconfirmfindingsof

otherstudiesconductedinEuropeanclinicalsamplesaiming

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thatthosefromrepeatregionsweremoresensitivethanthose

fromtheB1gene.26,29,33,52

Theconcordanceindex betweenthreeprimersets

(B22-B23/REP-529/B1)variedfrom83.56%to95.89%.Althoughthe

B22-B23and REP-529genesamplifydifferentT.gondiigenic

regions,bothhadalmostthesamepowerindetectingT.gondii.

Ontheotherhand,theconcordanceindicesbetweenB1and

REP-529 or B22-B23 were 83.87 and 83.56respectively. The

combinationofB1withotherprimersetsdidnotincreasePCR

sensitivity,sincenosamplewerepositiveforB1andnegative

forREP-529.Similartheresultshavebeenpreviousreported.52

Despite B1 and B22-B23 amplify the same B1 gene region

(Fig. 1), they presented different sensitivities. Although T.

gondiigenotypingwasnotdeterminedinthe217patientswith

positive PCR, probably, different Brazilian genotypes could

becirculating amongthesepatients, asalready showed by

others.53,54_One_small_confirmation_of_these_data_was_verified

bythegenotypingof15ofthe18AUbrainsamplesusedhere.

Amongthem,sixgenotypesweredeterminedinourprevious

study.40_One_of_them_was_Toxo_DB_#11,_previously_identified_in

differentBrazilianinfectednewbornsanddomesticanimals.54

TheotherfivegenotypesidentifiedwereTgHuDis1,TgHuDis3

andTgHuDis5,TgHuDis2andTgHuDis4.40_Although_REP₅₂₉

hasbeenregardedwitharegionwithalargevariationinits

copynumberindifferentT.gondiistrains,52_these_results_can

suggestthatindependentoftheinfectingstrain,REP-529can

besatisfactorilyusedformoleculardiagnosisofsymptomatic

toxoplasmosis.

Inconclusion,thiscomparativestudy,whichevaluated

sev-eralsamplesinaBrazilianreferrallaboratoryconcludedthat

REP-529hadbetterperformancethanB1one.These

observa-tionswereconcludedafterusingcaseswithdefinedclinical

diagnosis,ELISA,andcPCR.

Ethics

approval

and

consent

to

participate

TheEthicsCommitteeofallinvolvedInstitutions approved

theentirestudy,whichwasperformedfollowingthe

recom-mendationsofthesameCommittee(CONEP-IAL/SESnumber:

815489).

Consent

to

publish

Allauthors revisedthe manuscript,approvedthefinal

ver-sionsubmitted,published,andagreedtobeaccountableforall

aspectsoftheworkinensuringthatquestionsrelatedtothe

accuracyorintegrityofanypartoftheworkareappropriately

investigatedandresolved.

Funding

ThisstudywasfundedbygrantsfromFAPESP(Fundac¸ãode

AmparoàPesquisadoEstadodeSaoPaulo,Brazil)

2014/09496-1 to VLP-C, 2013/10050-5 to MP, 2013/15879-8 to FHAM,

2013/25650-8to LCM, 2014/01706-7to MNF;by CNPq

(Con-selhoNacionaldeDesenvolvimentoCientíficoeTecnológico)

toVLP-C301369/2015-1.

Authors’

contributions

VL Pereira-Chioccola, LM Camilo and CS Meira-Strejevitch

designed the study and experiments; performed the data

analysis,interpretedthedata.VLPereira-Chioccolawrotethe

manuscript.LMCamiloandRGavaperformedthelaboratorial

experiments(DNAisolation,cPCR,qPCR,ELISAanddata

anal-ysis).JEVidalcriticallyrevisedthemanuscriptanddetermined

theclinicaldiagnosisforpatientswithcerebraland

dissem-inatedtoxoplasmosis.CCBrandãodeMattos,LCMattos,FB

Frederico,RCSiqueira,MPreviato,APBarbosa,FHAMurata,

MNFerreira,LCJFSpegiorin,DMUBarbosa,FSGonc¸alves,CM

Dias, MW Catelan D. Cavallini performed the inclusion of

patients withocular, congenital, and gestational

toxoplas-mosis,samplecollection,developtheclinicaldiagnosis and

evaluationinFAMERP.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

JimHessonofAcademicEnglishSolutionsproofreadthepaper

(http://academicenglishsolutions.com/AES/home.html).

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