Comparison of PCR-based methods for the simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical samples

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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

Comparison

of

PCR-based

methods

for

the

simultaneous

detection

of

Neisseria

meningitidis,

Haemophilus

influenzae

,

and

Streptococcus

pneumoniae

in

clinical

samples

Ivano

de

Filippis

∗

_,

_Claudia

_Ferreira

_de

_Andrade,

_Nathalia

_Caldeira,

Aline

Carvalho

de

Azevedo,

Antonio

Eugenio

de

Almeida

Fundac¸ãoOswaldoCruz(FIOCRUZ),InstitutoNacionaldeControledeQualidadeemSaúde(INCQS),RiodeJaneiro,RJ,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15August2015 Accepted14April2016 Availableonline30May2016

Keywords: Diagnostic Neisseriameningitidis Streptococcuspneumoniae Haemophilusinfluenzae

a

b

s

t

r

a

c

t

Background:Severalin-housePCR-basedassayshavebeendescribedforthedetectionof bac-terialmeningitiscausedbyNeisseriameningitidis,Streptococcuspneumoniae,andHaemophilus influenzaefromclinicalsamples.PCR-basedmethodstargetingdifferentbacterialgenesare frequentlyusedbydifferentlaboratoriesworldwide,butnostandardmethodhaseverbeen established.Theaimofourstudywastocomparedifferentin-houseandacommercial PCR-basedtestsforthedetectionofbacterialpathogenscausingmeningitisandinvasivedisease inhumans.

Methods:A totalof110isolatesand134clinicalsamples(99cerebrospinal fluidand35 bloodsamples)collectedfromsuspectedcasesofinvasivediseasewereanalyzed.Specific setsofprimersfrequentlyusedforPCR-diagnosisofthethreepathogenswereusedand comparedwiththeresultsachievedusingthemultiplexapproachdescribedhere.Several differentgenetargetswereusedforeachmicroorganism,namelyctrA,crgAandnspAforN. meningitidis,plyforS.pneumoniae,P6andbexAforH.influenzae.

Results:Allusedmethodswerefast,specificandsensitive,whilesomeofthetargetsusedfor thein-housePCRassaydetectedlowerconcentrationsofgenomicDNAthanthecommercial method.AnadditionalPCRreactionisdescribedforthedifferentiationofcapsulatedand non-capsulatedH.influenzaestrains,thewhilecommercialmethodonlydetectscapsulated strains.

Conclusions: Thein-housePCRmethodsherecomparedshowedtoberapid,sensitive,highly specific,andcheaperthancommercialmethods.Thein-housePCRmethodscouldbeeasily adoptedbypubliclaboratoriesofdevelopingcountriesfordiagnosticpurposes.Thebest resultswereachievedusingprimerstargetingthegenesnspA,ply,andP6whichwereable todetectthelowestDNAconcentrationsforeachspecifictarget.

∗ _{Corresponding}_author.

E-mailaddress:ivano.defilippis@incqs.fiocruz.br(I.deFilippis). http://dx.doi.org/10.1016/j.bjid.2016.04.005

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Introduction

InfectionscausedbyNeisseriameningitidis(Nm),Streptococcus pneumoniae(Sp)andHaemophilusinfluenzae(Hi)are responsi-ble forhigh morbidity and mortalityrates amongchildren andadultsinmanycountrieseachyear.1,2 _Despite_effective

antimicrobialandsupportivetherapy,mortalityratesamong childrenremainhigh,withsignificantlong-termsequelaein survivors.Althoughadequatetreatmentrequiresrapid detec-tionandidentificationofthebacteria,traditionallaboratory diagnostic methods such as culture for the identification of bacterial meningitis pathogens may take 36h or more. Becauseofthesimilarityofclinicalsymptomsamong inva-sive infections causedby Nm,Sp and Hi, physiciansmust frequentlyrequestlaboratoriestotesttheclinicalsamplefor thesethreemicroorganismstodeterminethecausative bacte-riaanddecideonthemanagementofthecase.Rapiddiagnosis oftheetiologicalagentisalsoimportantforhealth surveil-lance,incasesofinvasivedisease,toavoidtransmissionto closecontacts.1–3

To address this problem, nonculture methods like PCR havebecomeavailableduringthe lasttwodecades, provid-ingearlyandaccuratediagnosisofbacterialmeningitis.1,4–10

Because of its rapid, sensitive and specific results, PCR is an important tool to improve the diagnostic of such microorganisms. AnadditionaladvantageofPCRover con-ventional laboratory methods is the possibility to detect genomic DNA from clinical samples without the need of previous culture. Recently, the use of a quantitative PCR methodcalledRealTime-PCR,haveimprovedthediagnosis ofinfectiousagentsbasedonmolecularmethods.11–17

How-ever, the higher cost of reagents and equipment could be apossibledrawbacktothe implementationofthis method in public laboratories of developing countries. The use of a basic PCR method, can be still more appropriate for diagnostic purposes in low-budget laboratories. A number of different target genes have been used in several PCR-based assays to specifically detect the genomic DNA of theseagents. For N.meningitidisnspA, ctrA andcrgA, forH. influenzae bexA and P6, and forS. pneumoniae ply and lytA

geneshave beenused astargets.4–10,18,19 _In _order_to

deter-mine the best combination ofprimers, wehave developed a simple multiplex PCR for the simultaneous detection of the three main agents of meningitis worldwide, Nm, Sp, and Hi. The multiplex PCR method here described allows thedetection ofthe threemicroorganismsina single-tube reaction, saving time and reagents. Target species specific genes used for Nm, Hi, and Sp detection were nspA, P6,

andply respectively.Primersamplifyinghigherlength frag-mentswerechosenforidentificationbecausethevisualization ofbands below200bpthrough electrophoresismay not be cleardepending ofthe agarosegel typeand concentration used. Furthermore, we have compared the specificity and sensitivityofourmethodtootherin-houseandcommercial PCR-based methods previously described forsimultaneous or single detection of Nm, Hi, and Sp from clinical samples.4,5,7–10,18,19

Materials

and

methods

Bacterialisolates,clinicalsamples,andcultureconditions

Wehaveanalyzedatotalof110strainsisolatedfromclinical samples(N.meningitidisn=31,H.influenzaen=29,S. pneumo-niaen=50)whicharepartoftheBacterialCultureCollection oftheINCQS/FIOCRUZinstitute.Thestrainswereisolated dur-ingthelastfiveyears,fromclinicalsamplesofcerebrospinal fluid(CSF)andblood.Thesesampleshavebeencollectedfrom culture-confirmedcasesofpatientswithsuspected meningi-tisinBrazilianpublichospitals.Theisolateswererecovered from clinical samples after inoculation in chocolate agar (bloodagarbasewith5%steriledefribinatedrabbitbloodat 56◦_C)_and_incubated_at₃₇◦_C_in_a_5%_CO₂_environment._The

isolated strains were identifiedasNm, Hi, orSpbycolony morphology, latexagglutination ofspecific antisera(Slidex Meningitis–Biomerieux),Gramstainingandoptochin suscep-tibility,thelatteronlyforSpdetermination.Eachspecieswas confirmedbyspecificPCRtests8,18,19_freeze-dried_and

incor-poratedintotheculturecollection.

Wehavealsoanalyzed134clinical samples(99CSFand 35blood)collectedfromsuspectedcasesofinvasivedisease. Thirty four (25.3%)ofthesesampleswere considered posi-tiveforoneofthethreepathogenstargetedinthisstudyby conventionallaboratorytests(culture,microscopy,andlatex agglutination)and100sampleswereconsiderednegativeafter conventionallaboratorytests,butcollectedfrompatientswith suspectedsymptomsorepidemiologicallinktoinvasive dis-easecausedbyoneofthethreeagentsheredescribed.The studyhasbeenapprovedbythelocalresearchethics commit-tee.

Twentyninereferencestrainswereusedaspositive con-trolsasfollows:N.meningitidisserogroupA(ATCC13077),N. meningitidisserogroupB(ATCC13090),N.meningitidisserogroup C(ATCC13102),N.meningitidisserogroupW135(ATCC35559),

H.influenzae(ATCC33391),H.influenzaeaegyptius(ATCC11116),

H.influenzaeNT(ATCC49247),H.influenzaeserotypea(ATCC 9006), H. influenzae serotype b (ATCC 33533) H. influenzae

serotypec(ATCC9007),H.influenzaeserotyped(ATCC9008),

H. influenzaeserotype e(ATCC 8142), H. influenzae serotype f (ATCC 9833), S. pneumoniae (ATCC 33400), S. pneumoniae

serotype 14 (ATCC 6314), S. pneumoniae serotype 3 (ATCC 6303),S.pneumoniae serotype33 (ATCC8333),S.pneumoniae

serotype 41 (ATCC 10341), S.pneumoniaeserotype 51 (ATCC 10351), S.pneumoniaeserotype19F(ATCC49619),S. pneumo-niae serotype 61 (ATCC 10361), S. pneumoniae serotype 19A (ATCC 700673),S. pneumoniaeserotype 14 (ATCC 700672),S. pneumoniaeserotype9V(ATCC700671),S.pneumoniaeserotype 6B(ATCC700670),S.pneumoniaeserotype4(ATCCBAA-334),

S. pneumoniae serotype 6A (ATCC BAA-659), S. pneumoniae

serotype 5 (ATCCBAA-341). Other 9reference strains were alsousedasnegativecontrolsastheycanalsocause bacte-rial meningitisinhumans:Neisserialactamica (ATCC23970),

Neisseria subflava (ATCC 11076), Moraxella catarrhalis (ATCC 25238),Streptococcusagalactiae(ATCC13813),Streptococcus pyo-genes(ATCC19615),Klebsiellapneumoniae(ATCC13883),Listeria

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monocytogenes (ATCC 15313), Acinetobacter sp. (ATCC 14293),

Escherichiacoli(ATCC11775).

ExtractionandpurificationofbacterialDNA

GenomicDNAextractionandpurificationfromisolatedstrains and negative clinical samples was carried out using the DNeasyBlood and Tissue extraction kit (Qiagen) according to manufacturer instructions. Briefly, 200␮l of the clinical samplewere processedusingthe above mentioned extrac-tionkit,whichallowstotalpurificationofbacterialgenomic DNA free of contaminants and enzyme inhibitors. Extrac-tion and purification ofDNA was achievedusing different washing buffers and separation on Anion-Exchange Resin

columnsbybindingofnucleicacidsandfurtherelutionfor finaluse.PurifiedDNAwerekeptat−20◦_C_for_repeated

exper-iments.

PrimerdesignandPCRconditions

SpecificsetsofprimersfrequentlyusedforPCR-diagnosisof thethreeabove-mentionedagentswereusedandcompared withtheresultsachievedusingthemultiplexapproachhere described(Table1).For thedetectionofNm byPCR, target-genesctrA, crgA andnspA were used,for Spdetectiontwo differentregions ofthe target-gene ply were used. Primers

ply-894andply-974weredescribedbyCorlessetal.,11

target-ingaspecificregionofthepneumococcuspneumolysingene (ply)amplifyinga80bpamplicon.Anewpairofprimerswas designedtoamplifyalargersegmentoftheplygene.Weused theS.pneumoniaepneumolysingeneGenBankaccess#M17717 sequenceastemplateforprimersdesign.Theregion ampli-fiedwaslocatedbetweenpositions555and1554oftheply

geneM17717.ForHidetection,targetgenesP6andbexAwere

used(Table1).PCRwascarriedoutusingthePCR-MasterMix (Promega) DNAamplificationkitaccordingtomanufacturer specificationswithanequimolarmixtureofthefourprimer pairsat50pmol/␮leachforthemultiplexapproach.PCRwas runonanEppendorfMastercyclerEPthermocyclerwith35 cyclesof95◦_C_for₃₀_s,₅₀◦_C_for₁_min,₇₂◦_C_for₁_min_and₃₀_s

and afinalextensionof72◦_C_for₁₀_min._A₁₀_␮l_sample_of

thecompletedreactionmixturewasrunina1%agarosegel stainedwithethidiumbromide.Amplifiedproductswere visu-alizedandphotographedunderUVlight.Wehavealsouseda commercialkitforthedetectionofthethreeagentstargeted inthis study,namedSPEED-OLIGOBACTERIALMENINGITIS fromVircellwhichisaPCR-basedmethodcoupledtoa dip-stick device that enablesspecificdetection ofH. influenzae, N.meningitides,andS.pneumoniaeincerebrospinalfluid sam-ples.Thereactionswerecarriedoutaccordingtotheproducer instructions.

PurifiedDNAofthreereferencestrains:Nm ATCC13090, HiATCC33533,49247,andSpATCC33400weredilutedtoan undetectablelevelforsensitivitydetermination.Tenfoldserial dilutionsrangingfrom10␮gto10fgDNAper␮lwereusedin singleandmultiplexPCRgivingarangeofdifferentsensitivity accordingtothespecificsetofprimersused(Table2). Consid-eringagenomesizeofroughly2MbforNm,Sp,andHiand since1kbofdsDNAhasamolecularmassof660kDa,10fgof DNArepresentfivegenomes.

Thedifferentiationofcapsulatedand non-capsulatedH. influenzae strains was carried out bythe inclusion ofbexA

and P6primer sets ina second multiplexassay. The pres-enceoftwobandsat343bpand273bpindicatedacapsulated Histrain. WiththisresultanadditionalPCRisrequiredfor serotype determination. The presence of a single band at 273bpindicates a non-capsulatedHistrain and nofurther serotypedeterminationwasnecessary.

Table1–Primerssequencesusedinthisstudy.

Microrganismsandprimers Ampliconlength(bp) Reference

Streptococcuspneumoniae

ply-894:5′_{-TGCAGAGCGTCCTTTGGTCTAT-3}′

ply-974:5′_{-CTCTTACTCGTGGTTTCCAACTTGA-3}′ ₈₀ _Corless_et_al.₍₂₀₀₁₎11

Multiplex

ply-1:5′_{-GACCCCAGCAATTCAATTCAAGTGT-3}′

ply-2:5′_{-TACGCACTAGTGGCAAATCG-3}′ ₁₀₀₀ _This_study

Neisseriameningitidis

ctrA-617:5′_{-GCTGCGGTAGGTGGTTCAA-3}′

ctrA-727:5′_{-TTGTCGCGGATTTCGAACTA-3}′ ₁₁₁ _Corless_et_al.₍₂₀₀₁₎11

crgA-F:5′_{-GCTGGCGCCGCTGGCAACAAAATTC-3}′

crgA-R:5′_{-CTTCTGCAGATTGCGGCGTGCCGT-3}′ ₂₃₀ _Taha₍₂₀₀₀₎5

Multiplex

nspA1:5′_{-AGCACTTGCCACACTGATTG-3}′

nspA2:5′_{-GGAACGGACGTTTTTGACAG-3}′ ₄₈₁ _de_Filippis_et_al.₍₂₀₀₅₎8

Haemophilusinfluenzae Multiplex

HiP6-F5′_{-ACTTTTGGCGGTTACTCTGT-3}′ ₂₇₃ _van_Ketel_et_al.₍₁₉₉₀₎18

HiP6-R5′_{-TGTGCCTAATTTACCAGCAT-3}′ bexA-142:5′_{-GGCGAAATGGTGCTGGTAA-3}′

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Table2–Sensitivityofin-house-PCRandcommercialPCR-baseddetectionkit.

Micro-organism Genetarget DNAdetectionlimitsa

In-housePCR

Neisseriameningitidis ATCC13090

nspA(deFilippisetal.,20058₎ _36.8_fg/␮L(U/M)

ctrA(Corlessetal.,200111₎ ₅₂_pg/␮L(U)

crgA(Taha,20005₎ ₅₂_pg/␮L(U)

Streptococcuspneunoniae ATCC33400

ply(Corlessetal.,200111₎ _21.4_pg/␮L(U)

Streptococcuspneunoniae ATCC33400

ply(thisstudy) 80fg/␮L(U/M)

H.influenzae ATCC49247

P6(vanKeteletal.,199018₎ ₁₀₀_fg/␮L(U/M)

Speed-oligo/MEDIVAX Neisseriameningitidis ATCC13090 ctrA 520fg/␮L Streptococcuspneunoniae ATCC33400 lytA 214pg/␮L Haemophilusinfluenzae ATCC33533 bexA 206pg/␮L a _U,_uniplex-PCR;_M,_{multiplex-PCR.}

Results

NovelmultiplexPCR

The multiplex PCR approach here described is capable to simultaneouslydetectgenomicDNAofthethreeagentsabove described.Thethreeprimerpairsusedforspecifically indi-catetheetiologicalagentarenspA(Nm),P6(Hi),andply(Sp) givingbandswithdifferentsizes:481bp,273bp,and1000bp, respectively (Fig. 1).When apositive resultisachieved for Hi,anadditionalPCRiscarriedoutinordertodetermineif thestrainiscapsulated.ForthissecondPCRround,specific primersfortargetingthebexAgeneareused. Amplification ofa343bpDNAfragment,confirmsthepresenceofa capsu-latedstrainwhichshouldbefurtheranalyzedtodetermine theserotype. Noamplificationafterthesecond PCRround, indicatesanon-typeableHi(HiNT)(Fig.2).

Specificity

Thespecificityoftheassaywasassessedbytestingapanel ofnon-Nm,Sp,orHistrains,whichincludedDNAsfromnine pathogenicbacteriawhichmaycauseinvasivediseasewith symptomssimilartothosecausedbythethreetarget orga-nisms.NoamplificationwasobservedafterPCRwithanyof themicroorganismstested.

Sensitivity

UniplexandmultiplexPCRusingtheprimersetsandthe com-mercialkitheredescribed,showedpositiveamplificationfor atleastoneofthethreetargetswithallthe110clinicalisolates ofNm,Sp,andHi.ForCSFandbloodsamples,thesensitivity ofPCRfordetectingbacterialmeningitisor septicemiawas

able todetect bacterialgenomicDNA ofall the34samples previouslyscoredaspositiveand foradditional12samples whichwerescoredasnegativeafterconventionallaboratory testsgivingatotalof46(34.3%)PCRpositivesamples(Table3).

Fig.1–AgarosegelofamultiplexPCRshowingthe detectionofthethreepathogensfromthreedifferent samples.Lane1:100bpDNAladder;lane2:Neisseria meningitidis(nspAgenewith481bp);lane3:Haemophilus influenzae(P6genewith273bp);lane4:Streptococcus pneumoniae(plygenewith1000bp);lane5:negativecontrol.

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Table3–Sensitivityandspecificityofprimersets.

PCR primers Positive samples by conventional tests/by PCR

Nm Sp Hi PPV/NPV (%) nspA - 1/2 15/20 a --- --- 100/100 Neisseria ctrA - 617/727 15/20 --- --- 100/100 meningitidis crgA- F/R 15/20 --- --- 100/100 Streptococcus ply -894/974 --- 12/16 --- 100/100 pneumoniae ply - 1/2 --- 12/16 --- 100/100 Haemophilus bexA - 142/241 --- --- 7/10 100/100 influenzae P6- F/R --- --- 7/10 100/100 Speed - oligo Nm 15/20 --- --- 100/100 Sp --- 12/16 --- 100/100 Hi --- --- 7/10 100/100 HiNT --- --- 7/10 100/100

PPV, Positive Predictive Value; NPV, Negative Predictive Value; Nm, Neisseria meningitides; Sp, Streptococcus pneumoniae; Hi,Haemophilus influenza. (a) total number of clinical samples tested for each agent: Nm n=20, Sp n=16, Hi n=10.

Comparisonofdetectionlimitsofsingle,multiplex,and

commercialPCRmethods

Wehavecompareddifferentprimersetsforthedetectionof the three microorganismson single and multiplexed reac-tions.PCRwascarriedoutasasingleormultiplexedreaction

Fig.2–Agarosegelshowingthedifferentiationof capsulatedandnon-capsulatedHaemophilusinfluenzae

strains.Lane1:100bpDNAladder;lane2:capsulatedH. influenzae(bexAandP6geneswith343bpand273bp respectively);lane3:non-capsulatedH.influenzae(HiNT), (bexAgenewith343bp);lane4negativecontrol.

performedwiththesetsofprimersdescribedinTable1.For thecommercialkit,thereactionswere performedonlyasa singletest(uniplex)foreachpathogen asrecommendedby theproducer.AsitisshowninTable2,conventionalPCRtest usingprimerstargetinggenesnspA,8_ply_(this_study)_and_P618

wereabletodetectthelowestDNAdilutionsofeachspecific target(36.8fg/␮L,80fg/␮Land100fg/␮L,respectively)bothin theuniplexormultiplexapproach.ThecommercialtestSPEED OLIGOtargetinggenesctrA,lytA,andbexAshowedtobeless sensitivethantheconventionalPCRtest(Table2).

Discussion

ConventionalPCRisstillwidelyusedasastraightforward lab-oratorytesttodetectspecificDNAofdifferentpathogenssuch asvirus,bacteria,andprotozoafromhumanoranimal clini-calsamples.SeveralconventionalPCR-basedtestshavebeen described forsingleor simultaneousdetection ofthemost frequentbacterialagentscausinginvasivediseaseand menin-gitis inhumans, namelyN. meningitidis,S. pneumoniae, and

H. influenzae.4–10 _Gene _targets _vary _according _to_the

micro-bial agentshowingsimilarsensitivityandspecificity. There arefewcommercialtestsbasedonmolecularstrategies avail-ableforthedetectionofthesepathogens.Oneofthesetests istheOligoSpeedherecomparedtoconventionalPCR-based methodstargetingdifferentgenesforeachmicrobialagent.A greatconcernonthismatterlatelyraisedistheintra-strain genepolymorphism foundwithinmeningococcalstrainsas describedonastudy.20_{False-negative}_results_by_real-time_PCR

weredescribedforthectrAgeneusedasadiagnostictarget. Ourresultsshowthatthecombinationofspecificprimers tar-getinggenesnspA,ply,andP6onamultiplexapproachwas abletodetectatleast10genecopiesofeachbacterialagent.

TwovariantsofH.influenzaecausingeitherinvasiveor non-invasivediseasehavebeendescribed.Themostcommonis thecapsulatedHivariantwhosecapsuleisclassified accord-ingtoitspolysaccharidestructureintosixdifferentserotypes (a,b,c,d,eandf)whichcanbedetectedusingPCR.19_However,

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thisapproachfailstoidentifynon-capsulatedstrainssinceit isbasedontheamplificationofageneticcapsulestructure. ThemostcommonvirulencefactorofHistrainsisthecapsule whichhelpsthebacteriatoevadethehostimmunesystem. Nevertheless,anincreasingnumberofnon-typeablestrains hasbeenreportedcausingdifferentformsofdisease21–23_and

the occurrence ofthese strains associated toinvasive and non-invasivediseasehasbeenreportedasaconcernfornew vaccines design.24,25 _van _Ketel _et _al.18 _described _an _outer

membraneprotein(P6)which isexclusivelyfoundinallH. influenzaestrainsanditisnotrelatedtothepresenceofthe capsule,being suitableto discriminatebetween capsulated andnon-capsulatedstrainsthroughasimplePCR.Failureto amplifythebexAgeneandamplificationoftheP6gene indi-catesanon-capsulatedHistrain.Thesingleuseofprimersfor thedetectionoftheP6genewouldbesufficientforthe detec-tionofHistrains,butthisapproachalonewouldnotindicate ifthestrainisnon-capsulated,animportantinformationfor epidemiologicsurveillanceaftertheintroductionofthe con-jugatedHibvaccine.AnadditionalPCRreactionisrequired targetingthebexAgenetodetermineifthestrainiscapsulated andinthiscaseaserotype-specificPCRcanbeperformedfor serotypedeterminationasdescribedbyFallaetal.19_The

inclu-sionofanadditionalPCRwithprimerstargetingtheHi capsu-largenebexAisimportant,sinceitcandifferentiatecapsulated tonon-capsulatedHistrains(NTHi)whichhasbeen consid-eredasapotentialemergingpathogenincountrieswherethe conjugatedvaccineagainstHibhasbeenintroduced.21,24–29

An additional advantage of the multiplex system here describedisthatthereisnopossibilityofmisinterpretation ofthespecificbandswithprimerexcess,whichisfrequently foundbelow100bp,sincethelengthoftheshortestbandis 273bpforthebexAgenefragmentandotherbandsarelocated at 343bp (P6), 481bp (nspA) and 1000bp (ply). Other PCR-basedmethodsusereal-timePCRprimerswhichmaygenerate short-lengthampliconswithdifficultpreviewbyagarosegel electrophoresis.7,10

Sensitivitywasassessedusingclinicalsamples(CSFand whole blood) from culture-confirmed cases for one of the three organisms. Sensitivity for Nm,Sp, and Hi was 100% sinceallpositivecasesconfirmedbyculturegavepositivePCR result.Furthermore,12samplesassociatedtosuspectedcases werealsoscoredpositivebythemultiplexPCRapproachhere described.Itislikelythatthesesampleswereconsidered neg-ativeduetopreviousantibiotictreatment.Theuseofthenovel multiplexPCRgaveatotalof46(34.3%)PCRpositivesamples (Table3).Theseresultsshowedhowsensitiveisthetestwhen otherphenotypicorepidemiologicalresultsindicatethatthe agentcausingdisease couldbeoneofthosethreebacterial pathogens.Problemsrelatedtocultureandisolationofthese bacteriaarefrequentlyreportedbyseverallaboratories,where aspecificandsensitivemoleculartestiscrucialfortherapid andaccurateidentificationofthediseasecausingagent.

Real-time PCR has been pointed as advantageous over conventional PCR due to its higher sensitivity and faster resolutionsincetheresultcanbemonitoredduringtherun. However,real-timePCRdoesnotappeartoofferadvantages whenusedbypubliclaboratoriesforbroad-rangePCRtesting alargenumberofsamples.Infact,theremaybe disadvan-tages, such as need of trained technical staff, controlled

environmentforassaysetups,andhighercostofequipment andreagents.Forpublicorprivatelaboratoriesthatstilluse conventional PCR todetect such pathogens,the choicefor thebestmultiplexsystemiscrucialtoachieveaccurateand reliableresults.Inthepresentstudy,wewereabletocompare several different primers combinations and gene targets recentlydescribed bydifferent authors,witha commercial PCR-based test for the detection of the three agents. Our datashowthatthemostsensitiveandreliablecombination of primers for a multiplexapproach include the following targets: nspA gene for N. meningitidis generating a 481bp amplicon,8 _ply _gene _for _S._pneumoniae _generating_a ₁₀₀₀_bp

amplicon (sequence described in this study), and P6 gene forH.influenzaegeneratinga273bpamplicon.18_For_the

dis-criminationbetweencapsulatedandnon-capsulatedstrains ofH.influenzae,anadditionalPCRisrequired,targetingthe

bexA genegeneratinga343bpamplicon,19 _when_a_positive

amplificationoftheP6geneisobserved.

Webelievethatthestandardizationofconventional PCR-basedtestsforthedetectionoftheinvasivebacterialagents heredescribedcouldimprove,interoperability,reproducibility, andqualityamongdifferentworldwidelaboratoriesinvolved inroutineclinicalanalysis.

Funding

FinancialsupportwasprovidedbyINCQS/FIOCRUZandFAPERJ Proc.N.E26/110.761/2010.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsaregratefultotheSequencingcore“Plataforma GenomicadeSequenciamentodeDNA/PDTIS-FIOCRUZ”.The studywasapprovedbythelocalEthicsCommittee.

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