Assessment of conventional PCR and real-time PCR compared to the gold standard method for screening Streptococcus agalactiae in pregnant women

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Assessment

of

conventional

PCR

and

real-time

PCR

compared

to

the

gold

standard

method

for

screening

Streptococcus

agalactiae

in

pregnant

women

Michele

Berger

Ferreira

_,

_Fernanda

_de-Paris

_,

_Rodrigo

_Minuto

_Paiva

_,

Luciana

de

Souza

Nunes

a_{UniversidadedeSantaCruzdoSul(UNISC),EscoladeFarmácia,SantaCruzdoSul,RS,Brazil}

b_{HospitaldeClínicasdePortoAlegre(HCPA),Servic¸oClínicodePatologia,DepartamentodediagnósticoPersonalizado,PortoAlegre,RS,} Brazil

c_{UniversidadeFederaldoPampa(UNIPAMPA),EscoladeMedicina,Uruguaiana,RS,Brazil}

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Articlehistory:

Received14June2017 Accepted13September2018 Availableonline22November2018

Keywords: Streptococcusagalactiae Pregnantwomen Screening ConventionalPCR Real-timePCR

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GroupBStreptococcusisacausativeagentofinvasiveneonatalinfections.Maternal colo-nizationbyStreptococcusagalactiaeisanecessaryconditionforverticaltransmission,with efficientscreeningofpregnantwomenplayinganessentialroleinthepreventionofneonatal infections.Inthisstudy,weaimedtocomparetheperformanceofconventionalpolymerase chainreactionandreal-timePCRassaysasscreeningmethodsforS.agalactiaeinpregnant womenagainstthemicrobiologicalculturemethodconsideredasthegold-standard.Atotal of130samplesfrompregnantwomenwereanalyzedforsensitivity,specificity,positive pre-dictivevalue,andnegativepredictivevalue.Statisticalanalysiswasperformedusingthe SPSSsoftware,version20.0.Theverifiedcolonizationratewas3.8%withthegold-standard, 17.7%withconventionalPCRassay,and29.2%withthereal-timePCRtest.Thetrialswith conventionalPCRandreal-timePCRhadasensitivityof100%andaspecificityof85.6% and73.6%,respectively.Thereal-timePCRassayhadabetterperformancecomparedto thegold-standardandagreaterdetectionrateofcolonizationbyS.agalactiaecompared toconventionalPCRassay.Withitsquickresults,itwouldbesuitableforusingin rou-tinescreenings,contributingtotheoptimizationofpreventiveapproachestoneonatalS. agalactiaeinfection.

©2018PublishedbyElsevierEspa ˜na,S.L.U.onbehalfofSociedadeBrasileirade Infectologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mailaddress:[email protected](L.S.Nunes). https://doi.org/10.1016/j.bjid.2018.09.005

1413-8670/©2018PublishedbyElsevierEspa ˜na,S.L.U.onbehalfofSociedadeBrasileiradeInfectologia.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Streptococcus agalactiae or group B streptococcus (GBS) is a commensalmicroorganismthatcolonizesthelower gastroin-testinalandgenitourinarytracts.However,itisalsoamajor causativeagentofinvasiveinfectionsinnewborns,pregnant women,andelderlyandimmunocompromisedpatients.1–3

GBSistheleading infectiouscause ofneonataldiseases suchaspneumonia,meningitis,andsepsis,withhigh morbid-ityandmortalityratesintheUnitedStates.4–6_Theincidence

ofneonatalGBSinfectionsrangesfrom0.80to3.06per1000 livebirthsindevelopingcountries.7 _{InBrazil,somestudies}

haveindicatedanincidencebetween0.39and 1.0per 1000 livebirths.8–11

MaternalcolonizationwithGBSisanecessarycondition forverticaltransmissionatdelivery,aswellasforthe occur-renceofearly-onsetneonatalinfection.In1996,theCenters forDiseaseControlandPrevention(CDC)releasedguidelines forthepreventionofneonatalGBSinfections.Theguidelines were updatedin2002and 2010.Current guidelines recom-menduniversalscreeningofpregnantwomenbetween35and 37weeksofgestationusingcombinedvaginalandanalclinical specimens.Inpositivecases,themothershouldreceive intra-partumantibioticprophylaxis.12InBrazil,however,thereis currentlynospecificpreventionstrategiesforneonatal infec-tions by GBS, and universal screening routine inpregnant womenisnotstandardizedfortheprenatalperiod.13

Microbiological culture is considered the gold-standard methodforGBSscreening,buttheturnaroundtimeforresults is between48 and 72h. Furthermore,it has limitations in detectinglownumber ofbacteria, which maylead tofalse negativeresults.14,15

Thestudyofsensitive,specific,andrapidtechniquesfor detectingGBSinpregnantwomenisextremelyimportantto optimizeapreventiveapproachforneonatalinfections.The aimofthisstudywastoassesstheperformanceofstandard polymerasechainreaction(PCR)andreal-timePCR(qPCR)as screeningmethodsforGBS inpregnantwomenwhen com-paredtothegold-standard.

Materials

and

methods

Clinicalspecimens

One hundred and thirty combined rectal/vaginal speci-mens were collected to conduct this study, as per CDC recommendations.12_{Clinicalspecimenswerecollectedfrom}

pregnantwomenat35weeksormoreofgestationwhohad receivedcareattheHospitaldeClínicasdePortoAlegre(HCPA) betweenAugust2014andNovember2014.

Microbiologytests

The swabs were inoculated onto a Todd Hewitt selective medium (Himedia Laboratories, India) supplemented with gentamicin (8␮g/mL) and nalidixic acid (15␮g/mL). The selectivemediumwasincubatedat36◦ C,5%CO2for18h.It

wasthensubculturedontobloodagarplates (BioMerieux®,

Marcy-l’toile, France),whichwere incubatedat35–37◦C,5% CO2for18–24h.Afterincubation,theplateswereinspected

for ␤-hemolytic colonies. If no ␤-hemolytic colonies were observed after 24h, plates were reincubated for another 24hand inspectedagain. The ␤-hemolytic colonieswhose morphologywas consistentwithgroupBStreptococcuswere subcultured inbrothand submitted tothe CAMP(Christie, Atkins, Munch, Petersen) test. The colonies that tested positivewerepresumptivelyconsideredGBS.

Molecular

analysis

BacterialisolatespreparationandDNAextraction

Theswabswereincubatedfor15to18hontoaToddHewitt selectivemedium.Aftercentrifugatingthebroth,the precipi-tatewaswashedwitha1×PBSsolutionandresuspendeditin TEbuffer(10mMTris–HCl,0.1mMEDTA,pH7.5).Thissolution wassubmittedtothermallysis.Thermallysiswasperformed usingtheTEsolutionfor15minat100◦Ctolysebacterialcell walls.TheconcentrationoftheextractedDNAwasassessed underaspectrophotometer.AllDNAsweredilutedto5ng/␮L andstoredat−20◦_{CuntiluseinPCRassays.}

Conventionalpolymerasechainreaction(PCR)

For theconventionalPCRreaction,weusedprimers5-CAA CGATTCTCTCAGCTTTGTTAA-3and5-TAAGAAATCTCT TGTGCGGATTTC-3,producinga779-bpfragmentthatis spe-cifictotheatrgene.16_{Thistargetgeneencodesanaminoacid}

transporterprotein,gs0538,whichisspecifictoGBS.Apositive controlconfirmedbyDNAsequencingwasusedintheassay (http://www.ncbi.nlm.nih.gov/BLAST).

ApositivecontrolconfirmedbyDNAsequencingwasused intheassay.

Amplification conditions were described in a previous study.17_{Theampliconsweredetectedbyelectrophoresisusing}

10␮Loftheamplifiedreactionmixtureina2%agarosegel con-tainingSYBR® SafeDNAgelstain(Invitrogen®,Calbad,USA) asdye.A100-bpmolecularweightmarker(Invitrogen®, Cal-bad,USA)andapositivecontrolforS.agalactiaewereusedto evaluatethePCRproducts.Thefragmentspresentinga779-bp ampliconwereconsideredpositiveforGBS.

Real-timePCR(qPCR)

The real-time PCR (qPCR) reaction targeted the cfb gene, which codifies a diffusible extracellular protein pro-duced by GBS (CAMP factor). We used primers Sag59 5-TTTCACCAGCTGTATTAGAAGTA-3 and Sag190 5 -GTTCCCTGAACATTATCTTTGAT-3.AsdescribedbyKeetal., theseprimerswerespecifictoS.agalactiaeandtestedagainst manyotherspecieswhichalsohavegenessimilartocfb.18_For

theinternalcontrol(IC)reaction,weusedasyntheticIC,with primersTBIC90F5-ATCGCTGATCCGGCCACA-3 andTBIC90R 5-TCGGTGACAAAGGCCACGTA-3 fordetection.Thepositive controlwasthesameasinconventionalPCR.

The qPCR reaction was based on the SYBR® Detection System (InvitrogenTM_{, USA). The reaction was carried out}

using the Platinum SYBR® Green amplification kit – qPCR SuperMix-UDG,with10␮Mofthereverseandforwardprimers andaDNAconcentrationadjustedto5ng/␮L.Amplification wasperformedinthe7500Real-TimePCRSystem(Applied Biosystems,FosterCity,CA)underthefollowingconditionsof temperatureandcycling:oneinitialcycleat50◦Cfor2min; onecycleat95◦Cfor10min;40cyclesat95◦Cfor15sandat 60◦Cfor1mintoamplifyDNA;and2cyclesat95◦Cfor15s andat60◦Cfor15stomeasuremeltingtemperature(Tm)and

detectfluorescence.

Theresultswere analyzedusing thecyclethreshold(Ct)

andTm.PositiveresultsfortheGBStargetareaTmbetween

76and78◦C,andfortheIC,82–84◦C.

Limitofdetection(LoD)

TheLoDoftheqPCRassaywasdeterminedusingfourserial dilutions(5,10,50and100copies/␮L)ofacommercialstrain ofGBS(AmpliRun,Vircell,Granada,Spain).Thedilutionswere submitted to the qPCR protocol in 46 trials. The LoD was definedasthelowestdilutiontestedwithpositiveresults(DNA amplificationproduct)inatleast95%ofthereplicates.

Statisticalanalysis

Sensitivity, specificity, positive predictive value (PPV), and negativepredictive value(NPV)forthePCRtechniquewere calculatedusingcultureasthegoldstandard.Concordance betweenassayswasdeterminedusingthekappacoefficient. StatisticalanalysiswasperformedusingtheSPSS® software, version20.0(IBM,Inc.,Chicago,USA).

Results

Amongthe130clinicalspecimensusedinthestudy,allfive (3.8%)samplespositiveintheCAMPtestwerepositiveby con-ventionalPCRfortheatrgene.Incomparison,23(17.7%)ofthe clinicalspecimenstestedpositiveforGBScolonizationwith conventionalPCR,and38(29.2%)testedpositivewithqPCR. Tables1and2showtheresultsforconventionalPCRandqPCR assays,respectively,comparedtothemicrobiologicalculture. Alloftheculturepositiveclinicalsamplestestedpositive withbothPCRtechniques,indicatingasensitivityof100%of theassays. Amongthe 125clinical specimenswithculture negativeresults,18 turnedout positiveintheconventional PCRassay,and107werenegativeinboth,pointingtoa speci-ficityof85%forthetest.TheqPCRassaypresentedaspecificity of73.6%whencomparedtothe,giventhat92clinical speci-menswerenegativeinbothmethods.

BothconventionalPCRandqPCRtechniquespresenteda negativepredictive valueof100%and a positivepredictive valueof21.7%and13.1%,respectively.Concordancebetween microbiologicalcultureresultsandbothconventionalPCRand qPCRwereweak(kappa=0.314and0.177,respectively).

TheLoDfoundintheqPCR assaywas10copies/␮LofS. agalactiae.

Discussion

MaternalrectovaginalcolonizationwithSGBisthemainrisk factorforverticaltransmissionduringlaborandforthe occur-renceofearly-onsetneonatalinfection.TheCDCrecommends screeningprogramsforpregnantwomenbetween35and37 weeksofgestationtodetectS.agalactiaecolonization,asan intrapartum antimicrobialprophylaxis isrequired for colo-nizedwomen.12_{SincetheintroductionofSGBscreeningsin}

theUSA,theearlyneonatalinfectionratebySGBdecreased from1.8casesper1000livebirthsto0.26casesper1000live births.19_{However,inBraziltherearenopublichealthpolicies}

inplaceforthestandardizationofuniversalroutine screen-ingsofpregnantwomenduringtheprenatalperiod.13

In our study, the maternal colonization rate was 3.8% with microbiological culture,17.7% with conventional PCR, and 29.2% with qPCR. Other Brazilian studies using both microbiologicalandPCRmethodsforSGBdetectionfounda colonizationratebetween14.6%and35.9%.20–23_The

coloniza-tionratesinpregnantwomencanvarywidelyduetofactors relatedtothecharacteristicsofthestudiedpopulationandthe detectionmethodsused.24,25

Withthegoldstandardmethod,thepercentageofpregnant womenwhotestedpositiveinourstudywaslowin compari-sontothePCRassays.Similarly,Castellano-Filhoetal.26_have

described a colonizationrateof32.6% detectedby conven-tionalPCRcomparedto9.5%withthegoldstandard.These dataareconsistentwithotherstudiesassessingqPCRassays, whichshowedconsiderableincreaseintheidentificationof pregnantwomencolonizedbySGB.27–29

Inourstudy,thesensitivityofbothPCRmethodswas100%. De-Parisetal.17_{alsoevaluatedtheuseofconventionalPCRas}

amethodforSGBdetectioninpregnantwomenandfound ahigh sensitivityrate(100%) forthetest. Instudieswhere theqPCRtechniquewasused,wefoundvariableandlower sensitivity values, between89.1% and 95.4%.28,30,31 _Despite

presentinghighsensitivity(100%),theqPCRassayassessed inthepresentstudyalsodetectedahighernumberofpositive clinicalspecimenscomparedtotheconventionalPCR.

TheuseofaselectivemediumbeforePCRisrecommended bytheCDCandisessentialtoensurehighsensitivityinthe screeningtest,suchastheoneobservedinourstudy.12

Sub-mitting clinical specimenswithout previous enrichmentto thePCRassayreducesthetimeuntilresults,butaffects per-formance. Mashouf et al.32 _{performed a directassay using}

conventional PCRwiththe 16S rRNAgene asatarget.The testsensitivitywas88.23%comparedtothemicrobiological culturemethod.Thedirectuseofclinical specimensinthe qPCR also shows lower sensitivity (<75%)compared to the gold-standard.33

The discrepancy between the rates of positive results identified in the culture (3.8%) and those obtained with conventionalPCR(17.7%)andreal-timePCR(29.2%)mayedue tonon-viablebacteriaoralowbacterialloadinthecollected clinical specimens. In these situations, the gold-standard method is usually insufficient for detection.34 _{For that}

reason, although microbiological culture is considered the gold-standardforSGBdetection,itisimportanttohighlight

Table1–ComparisonbetweentheconventionalPCRtechniqueandthemicrobiologicalculture(goldstandard)in detectionofS.agalactiae.

Techniques MicrobiologicalCulture(goldstandard) Total Kappa

Positive Negative

ConventionalPCR

Positive 5(100%) 18(14.4%) 23(17.7%) 0.314

Negative 0(0%) 107(85.6%) 107(82.3%)

Total 5(100%) 125(100%) 130(100%)

Table2–ComparisonbetweentheqPCRtechniqueandthemicrobiologicalculture(goldstandard)indetectionofS. agalactiae.

Techniques MicrobiologicalCulture(goldstandard) Total Kappa

Positive Negative

qPCR

Positive 5(100%) 33(26.4%) 38(29.2%) 0.177

Negative 0(0%) 92(73.6%) 92(70.8%)

Total 5(100%) 125(100%) 130(100%)

situationsinwhichterminfantswithneonatalinfectionsare borntowomenwhotestednegativeforcolonization.35,36

Itisthereforerelevantthatthe chosenmethodology for routinescreeningprogramshasahighanalyticalsensitivity, suchastheoneverifiedintheqPCRassayinthisstudy,which wascapableofdetectingaslowas10copies/␮LofS.agalactiae.

TheLoDindicatedinourassayissuperiortotheonedescribed byElAilaetal.26_{whoperformedaqPCRassaywiththesame}

targetgene(cfb),butusingaprobesystem.Theyfounda min-imumLoDof20copies/␮L.

ThePPVoftheconventionalPCRandqPCRtechniqueswere 21.7%and13.1%,respectively.Thepredictivevaluesrelateto thespecificityandprevalenceofcolonization,whichmeans thatthe lowPPV foundin theconventional PCRand qPCR assaysmustbeanalyzedcomparativelytothegold-standard, inwhichtherewerefive(3.8%)positiveclinicalsamples.37

Inourstudy,thespecificityoftheconventionalPCR tech-niquewas 85.6%, a slight increase comparedto the 82.6% foundbyMunarietal.22_{fortheatrgene.TheqPCRreaction}

showedaspecificityof73.6%whencomparedtotheculture. ThisresultisconsistentwiththatreportedbyYeungetal.38

(73.1%), who targetedthe cpsGgene locus ofthe SGB cap-sularpolysaccharide.Both PCRassays presentedanNPVof 100%.Thispercentageisclinicallyimportantasitensuresthat negativecasesareindeedtrue,thuspreventingunnecessary exposure ofpregnant women to antimicrobialprophylaxis duringlabor.

Thespecificity of PCRassays must beanalyzed against theresultsofthemicrobiologicalculture.Althoughtheuse ofa selectivebrothand chromogenic mediafacilitatesthe ␤-hemolytic identification of S. agalactiae, there is a non-hemolyticfractionofSGBstrainsthathinderdetectionbythe traditionalmethod.12_{Inpractice,thismeansthattheresults}

considered as false-positives inthe PCRtechniques are in facttruepositives,bearinginmindthegreatersensitivityof themolecular method.Indeed,inthe Microbiology Labora-torytheyinvestigateonly␤-hemolyticcoloniesintheCAMP test, sincethis characteristicappears inmostS. agalactiae.

Thelowpositivityrateinthecultureisbasicallyduetothe

growthoftheanal-vaginalmicrobiota,whichhindersthe iso-lationofS.agalactiae,andnotbecausewedonotinvestigate thenon-hemolyticcoloniesofS.agalactiae,whichhavealow percentageinthespecies.

InastudyperformedbyFeuerschuetteetal.,27_thesamples

withdiscrepantresults,negativebythecultureandpositive byreal-timePCR,weresubmittedtoconventionalPCR,which providedmainlypositive results.Theresultsobtainedwith qPCRwerethusconsideredtruepositives,whilethenegative resultswithculturewereconsideredfalsenegative.

Inthecaseofourstudy,thelowspecificityofthePCR tech-niquesmayresultfromitshighperformancewhendetecting SGBinsampleswheretheculturemethodfails.

Overthepastfewyears,manycommercialkitstodetect SGBbyqPCRwerereleased.AmongthemaretheIDI-StrepBkit (IDI,Sainte-Foy,QC,Canada)andtheGeneXpertkit(Cepheid, Sunnyvale,CA,EUA),bothofwhichtargetthecfbgene. Stud-ieshaveassessedtheperformanceofthesetestsasanoption todetect maternalintrapartumcolonization, with sensitiv-ityresultsbetween85%-98.5%andspecificityresultsbetween 97.6%and99.6%.39–42_{However,boththeoperatingplatforms}

and thekits haveanelevated cost,makingthem economi-callyunfeasibleasascreeningstrategyinthepreventionof neonataldiseasesbySGBindevelopingcountrieslikeBrazil.

Following the CDCrecommendations iseffectivein pre-ventingneonatalinfectionsbySGB,reducingaround80%of theirincidence.18_{However,toachievesuchrates,itisessential}

tousesensitive,specificandfastscreeningmethodsthatallow forthecorrectdetectionandadequateapproachtocolonized pregnantwomen.

The high sensitivity ofa testis an essential parameter whenscreeningisthefocus,alsoconsideringthatthe fail-ureinidentificationofindividualpositiveresultsisariskfor thedevelopmentofdiseases.37_{ThePCRtechniquesassessed}

inourstudycouldbesuitableforscreeningroutinesdueto theirhighersensitivityandfasterturnaroundtimeofresults comparedtotraditionalcultures.TheNPV(100%)ofthetests isalsorelevantasit indicatesreliabilityincorrectly identi-fyingnegativeclinicalsamplesandensurestherationaluse

ofantimicrobialprophylaxis.TheqPCRtechniquehada bet-terperformanceinidentifyingpositiveSGBclinicalspecimens comparedtoconventionalPCR.Theseresultsmayberelated toabettersensitivityofthereal-timemodality,whichuses afluorophore and allows fora more specificamplification system.Besides,theamplificationcanbemonitoredandthe numberofmoleculesineachcyclecanbequantifiedasthe reactionoccurs,providing resultsinaconsiderably shorter timecomparedtothegold-standard.Ourstudyconfirmsthe highsensitivityoftheqPCRmethodanditspotentialforusein screeningroutines,providingreliableandsaferesultsforthe rationaluseofantimicrobialprophylaxisinthepreventionof SGBverticaltransmissionsandintheoccurrenceofneonatal infections.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

FinancialsupportwasprovidedbyFIPE-HCPA(Researchand EventsSupportFundatHospitaldeClínicasdePortoAlegre).

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