Rev. Bras. Reumatol. vol.57 número6

w w w . r e u m a t o l o g i a . c o m . b r

REVISTA

BRASILEIRA

DE

REUMATOLOGIA

Brief

communication

Molecular

evidence

of

Borrelia

burgdorferi

sensu

lato

in

patients

in

Brazilian

central-western

region

Fernando

Aguilar

Lopes

,

Jania

de

Rezende

,

Danielly

Beraldo

dos

Santos

Silva

,

Fernanda

de

Cássia

Gonc¸alves

Alves

_,

_Carina

_Elisei

_de

_Oliveira

_,

Izaías

Pereira

da

Costa

a_{UniversidadeFederaldeMatoGrossodoSul(UFMS),FaculdadedeMedicina(Famed),ProgramadePós-Graduac¸ãoemSaúdee}

DesenvolvimentonaRegiãoCentro-Oeste,CampoGrande,MS,Brazil

b_{UniversidadeFederaldeMatoGrossodoSul(UFMS),HospitalUniversitárioMariaAparecidaPedrossian(Humap),CampoGrande,MS,}

Brazil

c_{UniversidadeCatólicaDomBosco(UCDB),ProgramadePós-Graduac¸ãoemBiotecnologia,CampoGrande,MS,Brazil}

d_{UniversidadeEstadualPaulista(Unesp),FaculdadedeCiênciasAgráriaseVeterinárias(FCAV),ProgramadePós-Graduac¸ãoem}

GenéticaeMelhoramentoAnimal,Jaboticabal,SP,Brazil

e_{UniversidadeCatólicaDomBosco(UCDB),ProgramaInstitucionaldeBolsasdeIniciac¸ãoCientífica(PIBIC),CampoGrande,MS,Brazil}

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Articlehistory:

Received22November2016 Accepted14April2017 Availableonline2June2017

Keywords: Borreliaburgdorferi Lymedisease flgE

Baggio-Yoshinarisyndrome Brazil

a

b

s

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a

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t

WeaimedtodetectDNAofBorreliaburgdorferiinwholebloodandserumsamplesofpatients withclinicalsymptomsandepidemiologycompatiblewithBrazilianLyme-likedisease.Four patientswithpositiveepidemiologicalhistorieswererecruitedforthestudy.Bloodsamples werecollected,screenedbyserologictestingbyELISAandWesternblottingandmolecular identificationofB.burgdorferibyamplifyingafragmentoftheconservedgenethat synthe-sizesthehookflagellarflgE.Theresultsshowedpositiveserologyandforthefirsttime,the presenceofB.burgdorferisensulatoinhumansintheMidwestregionofBrazil.Theresulting sequencesweresimilartoGenBankcorrespondingsequencesofB.burgdorferiflgEgene.By neighbor-joiningthephylogeneticanalysis,theflgEsequenceoftheBrazilianstrain clus-teredinamonophyleticgroupwiththesequenceofB.burgdorferisensulatounder100% bootstrapsupport.Thisstudyopensuppromisingperspectivesandreinforcestheneedfor additionalstudiestodeterminetheepidemiologicalcharacteristicsofthedisease,aswell astheimpactoftheprevalenceofBrazilianborreliosisinMatoGrossodoSulState,Brazil.

©2017ElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:fernando.lopes@ufms.br(F.A.Lopes). http://dx.doi.org/10.1016/j.rbre.2017.05.001

Evidência

molecular

de

Borrelia

burgdorferi

sensu

lato

em

pacientes

no

centro-oeste

brasileiro

Palavras-chave: Borreliaburgdorferi Doenc¸adeLyme flgE

SíndromedeBaggio-Yoshinari Brasil

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e

s

u

m

o

Esteestudopromoveuadetecc¸ãodeDNAdeBorreliaburgdorferisensulatoemamostrasde sangueesorodepacientescommanifestac¸õesclínicaseepidemiologiacompatíveiscom adoenc¸adeLyme-símilebrasileiraousíndromedeBaggio-Yoshinari.Paratanto,foifeita triagemsorológicapelosmétodosdeElisaeWesternblottingeaidentificac¸ãomolecularde B.burgdorferipormeiodaamplificac¸ãodeumfragmentodogeneconservadoquesintetiza oganchoflagelar(flgE).Osresultadosdemonstraramsorologiapositivae,pelaprimeiravez, apresenc¸adeDNAdeBorreliaburgdorferisensulatoemhumanosnaRegiãoCentro-Oestedo Brasil.Aanálisegenéticadassequênciasdosisoladosmostrousimilaridadeàssequências disponíveisnoGenBank.Pelaanálisefilogenéticainferidapelasequênciaparcialdogene flgE,acepabrasileiraagrupou-secomasequênciadeB.burgdorferisensulato.Esteestudo abreperspectivaspromissorasereforc¸aanecessidadedeestudosadicionaisafimde deter-minarascaracterísticasepidemiológicasdadoenc¸a,bemcomooimpactodaprevalência daborreliosebrasileiranoEstadodeMatoGrossodoSul,Brasil.

©2017ElsevierEditoraLtda.Este ´eumartigoOpenAccesssobumalicenc¸aCC BY-NC-ND(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Lyme disease (LD) is an emerging multisystemic

zoono-sis, caused by spirochetes of the Borrelia burgdorferi sensu lato group and transmitted by ticks of the Ixodes ricinus complex.1

Thisisadiseasewithwidegeographicaldistribution,and itsclinicalmanifestationsvaryaccordingtothespeciesofB. burgdorferisensulato complexfoundinacertain geographi-callocation.2,3_{Theetiologicalandantigenicdiversityexplains} the organotropism andthe appearance ofdifferentclinical and laboratory pictures indifferent regions, presenting an

increasing challenge to the management of this emerging

zoonosis.1

InBrazil,theleadingcasesweredescribedinthestateof São Paulo in1992,4 _{and sincethen other caseshave been} described with the use ofserological and molecular tech-niques in several Brazilian states: Mato Grosso do Sul,5–7 Amazonas,8_Tocantins,9_andParaná.10

Differencesobservedinepidemiological,clinicaland lab-oratory characteristics in relation to LD described in the

northern hemisphere allowed characterizing the Brazilian

Lyme Disease-like Syndrome (BLDS) or Baggio-Yoshinari

Syndrome11,12_{which,despitetheclassicmigratoryerythema}

and the usual systemic complications found in LD,

fre-quently occurs with a high frequency of recurrences and

the production of autoantibodies over its lengthy clinical evolution.13

Studies using molecular methodsfrom human samples

withBLDS-compatiblesymptomatologyhavereinforcedthe

existenceofcasesofborreliosisinBrazil10,14_{Thus,thisstudy} aimed toinvestigate the presenceof B. burgdorferi DNA in samplesfrompatientswithaclinicalandserological diagno-sisofborreliosisinMatoGrossodoSul,Brazil.

Patients

and

methods

Fourfemalepatientswithameanageof33.3(±11.9)yearsold andpresentingclinicalmanifestationsandBLDS-compatible epidemiologytreatedattheRheumatologyDepartment, Hos-pitalUniversitarioMariaAparecidaPedrossian,Universidade FederaldeMatoGrossodoSul(HUMAP-UFMS),wereselected. These patients had a history oftick bites and ofvisits to high-risk areas insoutheastern and midwesternregionsof Brazil;theymettheBraziliancriteriaforthediagnosisof bor-reliosis, as adopted bythe Laboratóriode Investigac¸ão em

Reumatologia (Rheumatologic ResearchLaboratory),

Hospi-taldasClínicas,FaculdadedeMedicina,UniversidadedeSão Paulo(LIM-17),areferencecenterinBrazil.12,15

One of the patients was in an acute phase (diagnosed

withinthreemonthsofonsetofdisease)andtheotherthree wereinalatephase(diagnosedmorethan3monthsafterthe onsetofthedisease)ofborreliosis.Thepatientwhohadearly Lymepresentedflu-likesymptoms,includingfever,headache, myalgia,andarthralgia.Theotherthreepatients,whohadlate Lymedisease,developedarthritisandalsocognitivedisorders,

manifestingwithnonspecificsymptoms,including memory

loss,sleepdisturbancesandmoodchanges,adepressivemood withsocialindifferenceandlossofappetite,andrecurrenceof flu-likesymptomsandchronicfatigue.Forallpatientsstudied, serologicaltestsforB.burgdorferiG39/40,ofNorthAmerican origin,were positive, usingELISA and WB16 _{methodologies} according tothestandardizationand interpretation recom-mendedbyLIM-17,anationalreferencelaboratory.12,15

DNAwasextractedusingQIAampDNAKit(QIAGEN®)from samplesof100␮Lofperipheralbloodandofserum, accord-ingtothemanufacturer’sinstructions.Purity(260nm/280nm)

and total DNA concentration (ng/␮L) were determined by

optical density spectrophotometry (BioDrop® Touch Duo

SpectrophotometerbyBioDropEngland).

TheprimersusedweredesignedbyRezendeetal. (Insti-tutoNacionaldaPropriedadeIndustrial[NationalInstituteof IndustrialProperty]–INPI–ProcessBR1020160215226)for amplificationofa262-bpfragmentfromthecodingregionof theconservedgenethatsynthesizestheflagellarhook(flgE) ofB.burgdorferidescribedbySaletal.17_{Thefollowingprimers} wereused:flgE262FW(5′_{-TCCTCCGGGATTCATACAAG-3}′_)and flgE262Rev(5′_{-TGGGTGCAAATGTAGGTGAA-3}′_).

Amplificationwasperformedin25␮Lofreactionfinal vol-umecontaining17.55␮LofDNAse/RNAse-freeultrapureH2O,

2.50␮Lof10×PCRBuffer,0.75␮LofMgCl2(1.5mM),0.50␮LOf

dNTPmix(10mM),0.25␮Lofeachprimer(10pmoles),0.2␮L ofPlatinumTM_{TaqDNAPolymeraseand3␮LofDNAextracted}

fromthesample.

PCRcycleconditionsconsistedofaninitialdenaturation at95◦_{Cfor3min,followedby45repetitivecyclesconsisting} ofdenaturationat95◦_{Cfor20s,annealingat62}◦_Cfor25s, extension at 72◦_{C for 25s, followed by a final extension} at72◦_{C for5min.Good laboratory practiceswere followed}

to avoid contamination and, in each reaction, a negative

control (water) was included to rule out the possibility of contamination.Borreliaanserinawasusedasapositivecontrol inallreactions.PCRproductswereanalyzedon1.5%agarose gelelectrophoresis,stainedwithSYBRGold(Invitrogen)and analyzedbyUVtransillumination.

PositivesampleswerepurifiedwithQIAEX® IIGel

Extrac-tion Kit (Qiagen GmbH) and sequenced in ABI Prism

3130 automatic DNA sequencer (Applied Biosystems®)

in both directions using BigDye Ready Reaction mix (ABI

Corporation®).Alignment wasperformed onClustalW2®

software using the sequences obtained in this study and

the sequence available on GenBank (Accession Number:

CP009656.1).Phylogeneticanalysiswasinferred fortheflgE

geneusingtheMEGA7.0® programand adendrogramwas

constructed by the Neighbor-Joining method.18 _Confidence valuesfortheindividualbranchesoftheresultingtreewere determinedbybootstrapanalysiswith100replicates.

ThestudywasapprovedbytheResearchEthicsCommittee (REC)ofUniversidadeFederaldeMatoGrossodoSul(UFMS)as indicatedintheresearchprotocolnumber1065681ofMay15, 2015–CAAE42325815.1.0000.0021.Allcontrolsandpatients signedaconsentform.

Results

Thepresenceofanti-Borreliaantibodiesandamplificationof B.burgdorferisensulatoDNAfortheflgEgeneweredetectedin allsamplesanalyzed(Table1).

ForPCRreaction,eightsamples(fourwholebloodandfour sera)and100%(4/4)ofthewholebloodsampleswere submit-tedand25%(1/4)ofthehumanserumsampleswerepositive (Fig.1).

Table1–ResultsofserologyforBorreliaburgdorferiby ELISAandWesternblottingandofPCRfortheflgEgene inpatientssamples.

Patient ELISA Westernblotting PCR

IgM IgG IgM IgG flgE

1 Negative Positive Negative Positive Positive 2 Positive Positive Positive Positive Positive 3 Negative Positive Positive Positive Positive 4 Negative Positive Negative Positive Positive

Thesequencesobtainedinthisstudyshowedhomology

withsequencesofB.burgdorferisensulatodepositedin

Gen-Bank,allowingidentificationatthespecieslevel.Accordingto

theGenBanksequences,thesequencedandassessedextent

oftheflgEgenewas228bp,whichencode76aminoacids.

Thesequencesobtainedinthis studywere depositedin

GenBank under accession numbers KU712208, KU712209,

KU712210, KY073265 and KY073266. The sequences were

groupedintoamonophyleticgroupbyphylogeneticanalysis

bytheNeighbor-Joiningmethod,with100%bootstrapsupport

(Fig.2).

Discussion

Our resultsprovide evidence ofB. burgdorferisensu lato in

whole blood samples and the unprecedented detection of

amplifiedspirocheteDNAfortheflgEgeneinahumanserum sampleinourregion.

The gene that synthesizes the flagellar hook (flgE) is a uniquechromosomalgene,approximately1kb,thatencodes theflagellinprotein(41kDa).Becauseitishighlyconserved,its diversityisvaluableindistinguishingBorreliaspecies.19,20 Phy-logeneticanalysisusingtheflagellingenesequenceconfirmed homologyamongtheisolates.

Allanalyzedwholebloodsamplesshowedpositivityand

only asingle serum sample was positive in the PCR

reac-tion.Coburn et al.21 _{demonstratedthe bindingabilityofB.}

burgdorferitohostplateletsandGoodmanetal.22_statethat perhaps this is the reasonfor the higher concentrationof plasmaversusserumspirochetesandsuggestthatplasmais thepreferredsampleforresearch.

PM C- 1 2 3 4 5 6 7 8 C+

262bp 300bp

200bp

Fig.1–PCRofflgEgeneanalyzedon1.5%agarosegel

stainedwithethidiumbromide:MW,molecularweight

(100pb);C−,negativecontrol;1,2,3and4,serumsamples

frompatientswithBrazilianborreliosis;5,6,7and8,whole

bloodsamplesfrompatientswithBrazilianborreliosis;C+,

72

LS1 human blood (KU712208)

LS2 human blood (KU712209)

LS2 human serum (KU712210)

B31 (CP009656.1)

LS3 human blood (KY073265)

LS4 human blood (KY073266)

Fig.2–PhylogenetictreebasedonthecomparisonofthecodingregionoftheflgEgeneofBorreliaburgdorferi.

InBrazil,patientsdiagnosedwithcharacteristics sugges-tiveofborreliosis present a history ofexposure toticks, a suggestiveclinicalpicturewithfrequentrecurrences,a posi-tiveserologybutwithlow,littlepersistenttiters,includingthe visualizationofstructuressuggestiveofspirochetesinblood samplesofpatientswithacompatibleclinicalprofile.12,13

Thepresenceofanti-BorreliaantibodiesdetectedbyELISA

andWBtechniqueshasbeendemonstratedinsymptomatic

and asymptomatic patients, withpositive epidemiology of

contactwithticksinseveralregionsofBrazil.5,6,8–10

However,theobservationofaserologicalreactivitypattern differentfromLDwithlowsensitivityandvariabletitersled tothedefinitionofnationalcriteriafortheinterpretationof serologicaltests.LIM-17,areferencecenterinBrazil,adopts aWBevaluationcriterionthatisbasednotonthepresence ofspecificbands,butonthequantificationofbands:atestis consideredpositivewiththepresenceof2bands forIgM,4 bandsforIgGoryetacombinedpatternof1IgMbandand2 IgGbands.12,15

ThedescriptionofspirochetesintheirL-form,knownas cell wall-deficientbacteria and whichmay alter their mor-phologywhensurvivalconditionsarenotfavorable,12_andthe subsequent detectionof B. burgdorferisensustricto by Man-tovani et al.,14 _{have aided in the understanding of most}

controversies on BLDS: a protracted permanenceof

spiro-chetesinthe hostdueto theevasionmechanisms ofhost

defenses,23 _{difficulties tocultivate these organisms in}

tra-ditional mediasuchasBSK, althoughsuchmedia describe

the visualization of structuressuggestive of spirochetes in humanbloodsamplesofpatientswithacompatibleclinical profile,12 _{lowimmunological responseagainstthe bacteria,} causinglow,littlepersistent,titersofantibodies,development ofautoimmunity12_{andresistancetotheireliminationfrom} thehost,withclinicalandserologicalrelapses.13,23–25

Theseresultsconfirmtheexistenceofborreliosiscaused byB.burgdorferisensulatointhestateofMatoGrossodoSul, Brazil,withsymptomssimilartoclassicLDandsuggestthe needtocontinueepidemiological,serologicalandmolecular studiesinordertobettercharacterizethisemergingzoonosis intheregion,whichisrarelyfatalbutofgreatmorbiditywhen notadequatelydiagnosedandtreatedduetorecurrencesand progressiveclinicalcomplications.

Funding

ThisstudywasfundedbytheConselhoNacionalde

Desen-volvimento Científico e Tecnológico (National Council for

Scientific and Technological Development) (CNPq) and the

Fundac¸ãode ApoioaoDesenvolvimento doEnsino,Ciência

eTecnologia(FUNDECT)(FoundationforSupporttothe Devel-opmentofEducation,Science,andTechnology)oftheStateof MatoGrossodoSul.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsthanktheLIM-17teamfortheirtrainingand coop-erationduringthis studyandProf.Dr.SusanaElisaMoreno (UCDB),Dr.AnaRitaCoimbraMottadeCastro(UFMS)andDr. CristianoMarceloEspinolaCarvalho(UCDB)fortheir invalu-ablecollaborationinthisstudy.

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