Post on 03-Apr-2018
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
1/23
UNIVERSITATEA DE MEDICINA I FARMACIE IULIU
HAIEGANU
REZUMATUL TEZEI DE DOCTORAT
Studiu privind profilaxia i
tratamentul Borreliozei Lyme narealul Transilvaniei
Doctorand Violeta Tincua Briciu
Conductor de doctorat Prof. Dr. Dumitru Crstina
Cluj-Napoca 2013
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
2/23
CUPRINS
INTRODUCERE 11
STADIULACTUALALCUNOATERII 1.EtiopatogeniaiepidemiologiaBorreliozeiLyme 15
1.1.EtiologiaBorreliozeiLyme.ComplexulBorreliaburgdorferisensulato
15
1.1.1.Morfologie,formistructuramembranei 15
1.1.2.Genomuliorganizareagenomului 17
1.1.3.ComplexulBorreliaburgdorferisensulato 18
1.2.EpidemiologiaBorreliozeiLyme 19
1.2.1.Terminologie 19
1.2.2.Vectori 20
1.2.3Gazderezervor 20
1.3.InteraciuneB.burgdorfericugazdaipatogenez 201.3.1.InteraciuneaB.burgdorfericugazda 201.3.2.Patogeneza 21
2.TabloulclinicidiagnosticulBorreliozeiLyme 232.1.Tabloulclinic 23
2.1.1.Afectareacutanat 232.1.2.Afectareasistemuluinervos 242.1.3.Afectareaarticular 25
2.1.4.CarditaLyme 25
2.1.5.Afectareaocular 26
2.1.6.AltepotenialemanifestrialeBorreliozeiLyme 26
2.1.7.BorreliozaLymecronic 262.1.8.BorreliozaLymensarcin 27
2.1.9.BorreliozaLymelapacientulimunocompromis 27
2.2.DiagnosticulBorreliozeiLyme 27
2.2.1.Metodedirectedeevideniereaagentuluipatogen 27
2.2.2.Metodeindirectedediagnostic 28
2.2.3.AlteexaminridelaboratornBL 31
2.2.4.Testenerecomandateaseutilizanscopdiagnostic 32
2.3.Diagnosticdiferenial 32
3.TratamentuliprofilaxiaBorreliozeiLyme 333.1.TratamentulBorreliozeiLyme 33
3.1.1.TerapiarecomandatpacienilorcuBorreliozaLyme 33
3.1.2.Bazelefarmacologiceimicrobiologicealetratamentului 343.1.3.CetrebuiefcutncazulpersisteneisimptomelordupterapiaantibioticadecvatBL? 36
3.1.4.Terapiapacienilorasimptomaticicuserologiepozitiv 37
3.2.ProfilaxiaBorreliozeiLyme 37
3.2.1.Msurideprotecieindividual 37
3.2.2.Managementulmediuluiambientalcpuelor. 39
CONTRIBUIAPERSONAL 1.Ipotezadelucru/obiective 432.Metodologiegeneral 453.Studiul1-EtiologiaBorreliozeiLymenRomnia:IdentificareagenospeciilordeBorreliaburgdorferisensu
47
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
3/23
latoncpuelecolectatedelasubieciiumani3.1.Introducere 47
3.2.Ipotezadelucru/obiective 47
3.3.Materialimetod 47
3.4.Rezultate 50
3.5.Discuii 543.6.Concluzii 56
4.Studiul2-StudiuprospectivprivindrisculdetransmiterealinfecieicuBorreliaburgdorferisensulatodelacpueleIxodeslasubieciiumani
57
4.1.Introducere 57
4.2.Ipotezadelucru/obiective 57
4.3.Materialimetod 57
4.4.Rezultate 58
4.5.Discuii 61
4.6.Concluzii 64
5.Studiul3-Metodedediagnosticpentrudetecia Borrelieiburgdorferisensulatoncpuelecolectatedelasubieciiumani
65
5.1.Introducere 65
5.2.Ipotezadelucru/obiective 65
5.3.Materialimetod 65
5.4.Rezultate 69
5.5.Discuii 72
5.6.Concluzii 76
6.Studiul4.ClinicaBorreliozeiLymenarealul
Transilvaniei:Eritemulmigrator
6.1.Introducere 77
6.2.Ipotezadelucru/obiective 77
6.3.Materialimetod 77
6.4.Rezultate 78
6.5.Discuii 83
6.6.Concluzii 86
7.Studiu5-ClinicaBorreliozeiLymeinarealulTransilvaniei:NeuroborreliozaLyme
87
7.1.Introducere 87
7.2.Ipotezadelucru/obiective 87
7.3.Materialimetod 877.4.Rezultate 89
7.5.Discuii 97
7.6.Concluzii 99
8.Studiul6-AltemanifestrialeBorreliozeiLymenarealulTransilvaniei
101
8.1.Introducere 101
8.2.Ipotezadelucru/obiective 101
8.3.Materialimetod 101
8.4.Rezultate 102
8.5.Discuii 109
8.6.Concluzii 112
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
4/23
9.Discuiigenerale 11310.Concluziigenerale 11511.Originalitateaicontribuiileinovativealetezei 119 REFERINE 121
ANEXA1 141ANEXA2 143
Cuvinte Cheie: Borrelioza Lyme, Ixodes ricinus, Borrelia burgdorferi, Transilvania, Real TimePCR,imunohistochimie,lichidcefalorahidian,serologie,ELISA,WesternBlot
IntroducereBorreliozaLyme (BL), o afeciunemultisistemic, ceamai frecventboal transmisde
cpuenEuropaiAmericadeNord,esteozoonoz,agentuletiologiccirculndntrecpueledincomplexulIxodesricinusiomarevarietatedegazdevertebrate.Animalulrezervorcedeazagentul patogen capuei vector care l transmite fie altor gazde rezervor, fie omului, care
ptrundeaccidentalnfocarulnaturalinchideciclulinfeciei,fradevenilarndulsugazdarezervor.ComplexulBorrelia(B.)burgdorferisensulato,agentuletiologicalbolii,estempritnprezentn19speciigenomicecudistribuiegeograficdiferit.BiroulregionalpentruEuropaalOMS,nraportuldin2006privindBL,menioneaznicioinformaienprivinaRomniei[ 1].
Riscul de infecie cu B. burgdorferi depinde de interaciunea omului cu mediulnconjurtor.StrategiilepentrupreveniaBLse bazeazpe cunoatereadatelorepidemiologice,
ecologiceifiziopatologiatransmiteriiinfecieilaom,implicndmsurideprotecieindividualicolectiv.nRomnianuexistpnnprezentniciunstudiuprivindprevalenainfecieicu B.burgdorferi n cpuele colectate de pe subiecii umani.Deoarece diversele genospecii au unpotenialpatogendiferitiparsfieasociatecutablouriclinicediferite,naceastcercetareamdorit s evalum prevalena i diversitatea B. burgdorferi n cpuele vectori colectate de pe
subieciiumanidinzonaTransilvaniei,utilizndmetodemodernedediagnosticmolecular.BL,boalacu1000de fee,poateafectao largvarietatedeorgane,iartabloulclinici
evoluiasuntfoartediferitedelaunpacientlaaltul.nprezentnuexistunstandarddeaurpentrudiagnosticulBLiarmetodeleexistentetrebuiecombinatelogicpentruaobineceamainalteficien posibil. npracticaclinicserologia reprezintprima ide celemaimulteoriunicametoddelaboratorutilizatndiagnostic.ninterpretareaunuirezultatserologictrebuie
avut ns nvedere faptulc o serologiepozitiv nupoatediferenia ntre o infecie activimarcaimunologicauneiinfeciianteriaresimptomaticesauasimptomatice.Prevalenanaltaanticorpilorspecificinpopulaiageneralnzoneleendemicepuneproblemeprivindrelevanaclinica unei serologii pozitive. nplus polimorfismulmanifestrilor clinice poate ridicarealeproblemedediagnosticdiferenial.
Publicitatea privind BL a crescut adresabilitatea pacienilor spre serviciile de boliinfecioasedupintepturadecapuasaucususpiciuneadeboal.
ContributiapersonalaIpotezadelucru/obiective
AspecteleprivindprofilaxiasautratamentulBLnupotficercetateattatimpctnusecunoscdatelelocaleprivindinfeciavectoruluicuB.burgdorferi.OriceinformaienouprivinddistribuiagenospeciilordeB.burgdorferincpueledetaatedepegazdaumanapoateoferinoiperspectivennelegereaecologieicomplexeaBL.
nlucrareadefaamdoritacercetadiverseaspectealeBLnregiuneanoastr:vectoriii
prevalenainfecieicuB.burgdorferi ,genospeciileexistentelanivellocal,risculdetransmitereainfecieidelacpulaomirolulexaminriicpueidetaate,aantibioticeloriamonitorizrii
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
5/23
serologice n profilaxia bolii. n plus, deoarece n prezent nu exist un standard de aur ndiagnostic,amcomparatdiferitemetodededeteciea B.burgdorferincpue,nscopulstabiliriiroluluilornpracticaclinic.
Un alt obiectiv al studiului a fost reprezentat de descrierea aspectelor epidemiologice,clinicesiserologicealepacienilordiagnosticaicuBLnregiuneanoastr,urmatdeevaluarea
rspunsuluilatratamentiaprognosticuluipacienilor.Datoritpolimorfismuluimanifestrilori absenei unui tablou clinic specific, afectarea neurologic din cadrul BL poate mima alteafeciunineurologice.Ghidurileeuropenedediagnosticrecomandstabilireadiagnosticuluideneuroborrelioza Lyme (NBL) prin analiza lichidului cefalorahidian. Pn n prezent aceast
recomandare nu a fost utilizat n practica noastr clinic, avnd ca i consecin posibilasupradiagnosticareaNBLlapacienicualteafeciunineurologiceilipsaderspunslaterapiaantibiotic.Unobiectivalstudiuluiafostreprezentatdeimplementareanoilorrecomandridediagnosticievalurearspunsuluilatratamentapacienilor.
MetodologiegeneralStudiulaprimitaprobareacomisieideeticaUniversitiideMediciniFarmacieIuliu
Haieganu, Cluj Napoca,Romnia.Fiecare pacientinclus nstudiua primitinformaii privind
scopuliprotocolulstudiuluiii-adatconsimmntulscrisinformat.Douchestionarepentrumonitorizareapacieniloraufostutilizate.Chestionarulpentrumonitorizarea nepturii de cpu a fost completat pentru fiecare subiect la momentulnepturii, urmrind activitile recreaionale i ocupaionale cu risc pentru nepturi de
cpue,numruldecpuedetaate,duratadeataareacpuei,loculatarii,alergiicunoscute,antecedentepatologice,prezenasarcinii.ChestionarulpentrumonitorizareaBLafostaplicatladiferite momente n funcie de design-ul studiilor. S-a notat ocupaia, antecedente personalepatologice,nepturidecpusaueritemmigratornantecedente,prezenaunorsimptomeisemneneurologice,articulare,cutanate,oculare,cardiace.
Testele serologice pentrudeterminareaanticorpilor antiB. burgdorferi s-au efectuat n
diferitemomente,nfunciededesign-ulstudiilor.AufostutilizatetruseleELISA:Borreliaafzelii
IgMELISA,Borrelia afzelii + VlsE IgGELISA (GenzymeVirotech GmbH,Germania)i IgM, IgGanti-Borrelia ELISA (Euroimmun AG, Germania), respectiv trusa Western Blot IgM, IgG Anti-BorreliaEUROLINE-RN-AT(EuroimmunAG,Germania).
Studiu 1. Etiologia Borreliozei Lyme n Romnia: Identificarea genospeciilor de
BorreliaburgdorferisensulatoncpuelecolectatedelasubieciiumaniIntroducereComplexul B. burgdorferi s.l. include 19 genospecii diferite din care 10 raportate n
Europa:B.burgdorferis.s.,B.garinii,B.afzelii ,B.valaisiana,B.lusitaniae,B.bavariense,B.bissettii,B.spielmanii,B.kurtenbachii iB.finlandensis.PrevalenainfecieicuB.burgdorferincpuele
colectatedepesubieciiumanipoatefidiferitdeceancpuelecolectatedepevegetaiedin
aceeaizonageografic.ObiectiveIdentificarea speciilor de cpue care atac omul n regiunea noastra i a prevalenei
infecieicudiferitegenospeciideB.burgdorferinacestecpue.Materialsimetoda
Toatecpuelecolectatedelapacieniicares-auprezentatnClinicaBoliInfecioaseClujNapoca n perioada 01.04.2010-07.09.2010 au fost studiate prospectiv. Cpuele au fostidentificateprivindspecia,stadiuldedezvoltareisexul.IdentificareaspeciilordeB.burgdorferiincapuelecolectateautilizatometodderealtime(RT)PCRavndcaintgenahbb[2].
RezultateAufostcolectateiexaminatemorfologic532cpue,dela386pacieni.Vrfulmaximal
prezentrilordupnepturadecpuafostnlunamai.Majoritateacpuelorauaparinutspeciei Ixodesricinus (97.3%),vectorulBL nEuropa.Altespecii decpueidentificateau fost
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
6/23
Dermacentor marginatus, Haemaphysalis concinna i Haemaphysalis punctata; acestea nuprezint risc de transmiterea a BL, dar sunt vectori pentru alte boli bacteriene, virale sauparazitare.
PrevalenainfecieicuB.burgdorferi sensulatoncpuele Ixodesexaminateafost11,1%.Unprocentmaimarealinfecieiafostdescrisinadulii Ixodes decat innimfe (12,9%versus
10,7%), dar nu semnificativ statistic (p=0,55). Pe baza rezultatelor regiunea noastr poate fincadratnzoneleeuropenecuprevalenjoasainfecieicpuelorcuB.burgdorferi.GenospeciileidentificateaufostreprezentatedeB.afzelii(62,7%),B.garinii (16,2%),B.
burgdorferi sensu stricto (6,9%), B. lusitaniae (2,3%), B. valaisiana (2,3%) i B.
spielmanii/valaisiana (9,3%).HbbRT-PCRnuapututdifereniantreB.spielmanii iB.valaisiana ,fiindnecesaraseefectuatehnicidesecvenierepostPCR.AceastaesteprimadocumentareaB.burgdorferi sensu stricton cpuele colectate din Romnia. Studiul dovedetec principalelegenospeciileumanpatogenesuntprezentencpueledinregiuneanoastr,constituindastfelunriscdeinfeciepentruom.
Studiu 2. Studiu prospectiv privind riscul de transmitere al infeciei cuBorrelia
burgdorferisensulatodelacpueleIxodeslasubieciiumaninRomniaIntroducereRisculdedezvoltareaBLdupnepturadecpuinfectatcu B.burgdorferi ieficiena
chimioprofilaxieipostexpunerenRomniaestenecunoscut. ObiectiveDeoarece diferitele genospecii de B. burgdorferi sunt asociate cu diverse manifestri
clinicei nu toate speciile sunt uman patogene, n acest studiu amdorit sa evalum evoluiaclinicasubiecilorumaninfunciedegenospeciileidentificatencpuadetaatiatitudineaprofilacticurmat.
Materialimetod
Toipacieniinepaidecpuepozitivepentru B.burgdorferi prinhbbRT-PCRiungrup
decontrol(studiucaz-martor)aufostchemailacontroldupunan(mai-august2011).Grupulde controla inclus indivizi comparabili (vrst, sex, reedin) nepai n 2010 de cpueB.burgdorferinegativeiseronegativipentru B.burgdorferi(utilizndtestulELISA)lamomentulnepturii. Chestionar demonitorizare a fost completat pentru fiecare subiect (la momentulinepaturiisilacontrol).
La momentul nepturii de cpu s-a efectuat un test ELISA (pentru a determinaanticorpiIgMsauIgGantiB.burgdorferi datoraiunornepturidecpuanterioare)utilizndkitul Genzyme Virotech. Lacontrol, un test ndou treptea fost efectuat, pentruinvestigareaanticorpilorIgMiIgGanti B.burgdorferiutilizndkitulELISAGenzymeVirotechcascreening,urmatdekitulWesternBlotEuroimmunpentruconfirmare.
Rezultate
SeroprevalenapentruinfeciacuB.burgdorferiafost9,1%la263pacieniexaminailamomentulnepturii,rezultatecareconfirmnivelulrelativnaltalseropozitivitiinpopulaiaasimptomaticdinzoneendemice.20persoanedincele39nepatedecpuepozitivepentru B.burgdorferi prinhbb RT-PCR iungrup de controlformatdin 20pacieni nepai decpuenegativepentruB.burgdorferi ,s-auprezentatlacontrolulcliniciserologic.Doudinpersoanele
investigate au dezvoltat manifestri de BL acut (eritem migrator) i 5 persoane au fcutseroconversiedelamomentulnepturiipnlaevaluareade1an.Nuaufostgsitediferenesemnificativestatistic,nceeacepriveteprezenasimptomelorclinicesauevoluiaserologic,ntregrupulpersoanelornepatedecpue B.burgdorferipozitiveversusgrupuldecontrol,deiambele cazuri de eritem migrator (EM) au aprut n grupul cu cpue pozitive. B. afzelii,principala genospecie patogen n zona noastr geografic, a fost identificat n cpuele
colectatedelapersoanelecuEM.Suntadusedoveziprivindexistenaunorsimptomenespecificenpopulaiageneral,carearputeafiinterpretategreitdrepBLcronic.Deinumrulmical
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
7/23
cazurilornunepermitescalculmeficienasaueeculantibioticelornpreveniaBL,ambelecazurideEMs-audezvoltatnciudachimioprofilaxiei,iarpersoanelefrchimioprofilaxienuauprezentatseroconversiesausimptomeclinicedeBL.Studiulnuadoveditexistenauneilegturi
ntrestatusulinfectantalcpuei,rezultatulmonitorizriiserologiceimanifestrileclinicealepacienilor. Aceste rezultate susin recomandrile conform crora investigarea infeciei cu B.
burgdorferincpuadetaatimonitorizareaserologicnusuntindicatenscopulpredicieiinfecieilasubieciiumani.Studiu3.MetodedediagnosticpentrudeteciaBorrelieiburgdorferisensulaton
cpuelecolectatedelasubieciiumani IntroducereDei nprezent nuexistunstandard deaur ndiagnosticul infecieicu B. burgdorferi,
pacienii se prezint adeseori n serviciul nostru, solicitnd examinarea cpuei n scopulaprecieriirisculuidetransmiterealinfeciei.
Obiective
Scopulstudiuluiafostcomparareaa3metodedediagnosticutilizatepentrudetecia B.
burgdorferincpue,evalundavantajeleidezavantajeleacestormetodeirolullornpracticaclinic.MaterialimetodUnnumrreprezentativdincpuelecolectatedelapacieniicares-auprezentatnClinica
BoliInfecioase,ClujNapoca,nperioada01.04.2010-07.09.2010aufosttestateprintreimetodedediagnostic:ometodimunohistochimici2metodedeRT-PCR( hbbRT-PCRiospART-PCR)[3,2,4].
Duplex ospA RT-PCR este o metod descris pentru prima dat n cadrul prezenteicercetri.Pelngsistemuldedeteciealgenei ospAaB.burgdorferi[3],unaldoileasistemPCRafostimplementatcaicontrolinternalamplificrii(IAC).Avndnvederecamlucratcupiese
stocatenformolifixatenparafin,acestIACaavutscopuldeaexcludeinhibiiaPCR-ului,care
arputeaducelarezultatefalsnegative.Pentrudeterminareaspecificitiimetodei,22tulpinidereferin ale B. burgdorferi s.l. au fost testate pentru inclusivitate, n timp ce 18 tulpini deLeptospira(incluzndgenospeciileinterrogans,borgpetersenii ,kirschneriibiflexa),precumiotulpin de Borrelia miyamotoi i una de Treponema phagedenis, au fost testate pentruexclusivitate.
RezultateDuplexospART-PCRaprezentatosensibilitatentre1-10copiiADN/PCR,oeficiende
99,85%,exclusivitatede100%iinclusivitatede86,36%(nedetectate B.lusitaniae,tulpinilePotiB2siPotiB3iB.japonica).
Dincele136cpueanalizateprintoatetreimetodeledediagnostic,16(11,8%)aufost
pozitiveprinimunohistochimie(IH),12(8,8%)aufostpozitiveprin hbbRT-PCRi26(9,11%)au
fost pozitive prin ospA RT-PCR. n nici una din probe nu a fost detectata inhibitie prin IAC.Concordana de 86,7%, a celor dou tehnici de RT-PCR din studiul nostru susine existenasensibilitilordiferiteatehnicilorPCRavndcaintgenediferite.Valoarealuip(p
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
8/23
pentruB.burgdorferincpueledetaatedepesubieciiumanipentruprediciainfecieilaomnuserecomand.
AspecteleclinicealeBLaufosturmriten3studiidiferite,efectuateasuprapacienilorcares-auprezentatnperiodaianuarie2011-octombrie2012cudiagnosticuldeEM,suspiciunea
deNBLsaualtemanifestriclinicealeBL.Studiul4.ClinicaBorreliozeiLymenarealulTransilvaniei:Eritemulmigrator Introducere
EritemulmigratoresteceamaifrecventispecificmanifestareclinicaBL.PrognosticulpetermenlungalpacienilortrataicudiagnosticuldeEMesteconsideratbun,deipuinestudiiauevaluatacestaspectnEuropaipnnprezentnuafoststudiatnRomnia.
Obiectiven studiul 1 am dovedit infecia cpuelor I. ricinus colectate de pe subiecii umani cu
principalele genospecii deB. burgdorferi implicate ndezvoltarealeziunilor deEM.Astfel,am
consideratoportunevaluareaprofiluluiepidemiologic,cliniciserologicalpacienilorcuEM
dinarealulnostrugeograficicomparareacurezultatelealtorstudii.Materialimetodanstudiuaufostincluiprospectivpacienicares-auprezentatnClinicaBoliInfecioase,
ClujNapoca,nperioadaiunie2011-august2012cudiagnosticuldeEM.Pacienilorlis-ainstituit
terapieconformrecomandrilorexistente[5,6,7].Evoluiaclinicsiserologicaafostinvestigatacomparnd2 grupuri de pacieni: pacieni cuo perioad scurt demonitorizare de 3 luni sipacientioperioadademonitorizaremailunga,de9luni-1an.AufosturmariteprofilelecineticiianticorpilorspecificidetipIgMsiIgGlainitiereaterapieisilacontrol.
RezultateSuntcomparaterezultateleobinuteprinmonitorizareaa40pacienicuEMcurezultatele
altor studii similare din Europa i America de Nord. Diametrul mediu al leziunii de EM la
prezentareafostmaimiccomparativcualtestudii,sugerndobuninformareapacienilorimedicilordealtespecialitiprivindmonitorizareanepturiidecpu,urmatdeprezentareaprecoceapacienilornserviciuldeBoliInfecioase.
Cincidin34pacienicareaurecunoscutnepturadecpudinstudiulnostru(14,7%)audezvoltatleziuneadeEMnciudachimioprofilaxieipostnepturdecpu,demonstrndc
antibioticeleadministrateprofilacticnuconferprotecie.Dincei40pacieniluainstudiu,15aufostmonitorizaipeoperioadscurt(grupA),iar23pacienipeoperioadmedie(grupB).Doi pacieni nu s-au prezentat la control. Un singur pacient care a urmat terapie incompletpentruEMafostdiagnosticatcaBLtardivcumanifestriarticulare,dar16pacientiauprezentatsimptome nespecifice la reevaluare, in lipsa unor semne obiective, fr a exista diferene
semnificative statistic n ceea ce privete rspunsul la terapie ntre cele 2 grupuri (p=1).
Rezultateleauartatabsentauneilegaturiintreprofilulserologicsievolutiaclinica,susinndrecomandrilerecente,conformcrorarepetareaserologieinuseindicnurmrireapacienilorcuEM.
Studiu5.ClinicaBorreliozeiLymenarealulTransilvaniei:NeuroborreliozaLyme IntroducereNeuroborrelioza Lyme (NBL) poate mima orice afeciune neurologic i vice-versa,
pacienii suferind de alte afeciuni neurologice, precum scleroza multipl, scleroza lateral
amiotrofic(SLA),polineuropatie,parkinsonism,boalaAlzheimer,putndfigreitdiagnosticaica BL. Criteriile europene de diagnostic, spre deosebire de cele americane, includ analizalichiduluicefalorahidianideterminareaproducieiintratecaledeanticorpispecifici [
8].
ObiectiveStudiul a introdus pentru prima dat n practica noastr clinic criteriile europene de
diagnosticaleNBL.
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
9/23
MaterialimetodAu fost inclui n studiu pacieni internai n Clinica Boli Infecioase, Cluj Napoca, n
perioadaianuarie2011-octombrie2012cususpiciuneadeNBLcuafectareaSNC.Criteriide
includere: pacienii prezentaumanifestri deafectare a SNC,Atc antiB. burgdorferinser,auacceptatefectuareapuncieilombare(PL)nscopdediagnosticinuauprimitterapieanterioar
parenteralpentrususpiciuneadeNBL.Toi pacienii inclui n studiu au fost tratai caNBL cu afectare SNC [8], indiferent inecunoscnd rezultatulde laborator al Atc specifici produi intratecal. Simptomele incluse nchestionaruldemonitorizareaufosturmritelanceputul,lasfritulterapieiilacontrolulde3
luniutilizndunscorsimptomatic.S-aurealizatdoumodelederegresiebinomialnegativmixt,cumsurtorirepetate,
avnd ca variabil dependent numrul de simptome, respectiv scorul simptomatic. Timpul(corespunztorobservaiilorfcute),msuratnsptmni,afostluatcavariabilaleatoare.
PentrudeterminareaanticorpilorIgMiIgGBorreliadinLCRiser(ELISA)s-auutilizatnanul 2011 un kit Genzyme Virotech GmbH, Germania iar in 2012 un kit Euroimmun AG,
Germania.Laprezentares-aefectuattestarendoutreptepentruinvestigareaatcIgMiIgGanti
B.burgdorferi,utilizndcascreeningunkitELISA,iarca testdeconfirmareWesternBlotkitulEuroimmun.Lareevaluarede3lunis-arepetattestareaELISA.RezultateConformdefiniieidecazeuropeneamclasificat7cazuricaNBLposibil,33cazuricaNBL
infirmat,iar2cazurinuaupututficlasificatedatoritcantitiiinsuficientedeLCR.NiciuncaznuandeplinitcriteriiledeNBLdefinit.AceststudiuaduceevideneasuprasupradiagnosticuluiNBLnregiuneanoastr.
Dei evoluia pacienilor a fost spre ameliorare, un numr de simptome a persistat.PacieniicuNBLinfirmatauprezentatmaimultesimptomepeparcursulmonitorizriidectceicuNBLposibil statisticsemnificativ(p=0,03)i unscorsimptomaticmaimare,aproapedelimitasemnificaieistatistice(p=0,055).Astfelrezultatelearataoevoluiemaibunsubterapia
antibioticangrupulpacienilorcuNBLposibildectngrupulcelorcuNBLinfirmat.Nus-auidentificatsimptomecaresaparmaifrecvent(semnificativstatistic)ngrupulpacienilorcuNBLposibil.Toipacieniiaufostabordaiinterdisciplinar,astfel22dinpacieniauprezentatundiagnosticneurologic, iarla restul cazurilor nus-au gsitmodificri nurmaconsultuluineurologic,deiexistausimptome.Terapiaspecificindicats-aasociatsauaurmat
terapiei antibiotice. Nu am dovedit existena unei legturi ntre diagnosticul de NBLposibil/infirmatiprezenaunorleziunidedemielinizarepeRMNcraniu(p>0,05).
Afeciunireumatologiceaufostdescrisela9dinpacieni.Monitorizareaserologicaartatovariabilitateaprofilelorcineticiianticorpilor.13pacieni(30,9%)auprezentatreaciiadverselaterapiaantibiotic,incluznduncazdeentrocolitcuClostridiumdifficile.
Discuii
Avnd n vedere prevalena crescut a serologiei pozitive pentru B. burgdorferi npopulaia general, aspect descris n studiul 2 al tezei i n regiunea noastr, apariia unorsimptomeneurologicelaacetipacienipunerealeproblemedediagnosticpozitividiferenial.nstudiulnostru,unpacientdingrupulcuNBLinfirmatafostconfirmatcaSMiali2caSLApeparcursul monitorizrii. Nici unul din aceste cazuri nu a prezentat ameliorare sub terapia
antibiotic. Rezultatele susin afirmaia lui Puchal i Sibilia (2009) [9], conform creiapersistena unor simptome sedatoreazmai degrab unui diagnosticincorect, dect eeculuiterapiei.DateleprezentateconstituieprimadovadnecesarntreruperiiutilizriinejustificateaantibioticelorlapacieniineconfirmaicuNBL.
Studiu6.AltemanifestrialeBorreliozeiLymenarealulTransilvaniei
Introducere
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
10/23
Investigarea,tratamentulimonitorizareaunuipacientcareseprezintcususpiciuneadeBL,reprezintoprovocarepentrumediculinfecionist.nprezentnuexistunstandarddeaurpentrudiagnosticulBL.CuexcepiaEM,diagnosticatclinicpebazaleziuniicaracteristiceiaNBL,
cndseindicevaluareaLCR,multiplealtemanifestripotfintlnitentabloulclinicalBL,maimult sau mai puin specifice. Un rol important n diagnosticul acestor pacieni l reprezint
cunoatere aspectelor epidemiologice (nepturi de cpu, EM n antecedente), asociatmanifestrilorclinice i serologiei pozitive pentruB. burgdorferi. Serologia, principalametodutilizat n diagnosticul de laborator, trebuie interpretat cu pruden datorit limitelorcunoscute,nprincipalreaciifalspozitive.nevaluareaacestorpacieni,unrolimportantrevine
diagnosticuluidiferenial.MaterialimetodAufostincluinstudiupacienicares-auprezentatnClinicaBoliInfecioasenperioada
ianuarie2011-octombrie2012cususpiciuneadeBL.Criteriideincludere:pacieniiprezentaudiversemanifestrideorgandintabloulclinicalBLiserologiepozitivpentru B.burgdorferi.Criterii de excludere: pacienii cu EM i cei cu suspiciunea clinic de NBL care au acceptat
efectuareapuncieilombare,incluinstudiileanterioare,sauceipierduidinurmrire.
ChestionaruldemonitorizareaBLafostaplicatlamomentulincluderii,lasfritulterapieiilareevaluarede3luni.Pacieniiaufostmpriingrupuri,nfunciedeprobabilitateadeaavea BL dup un scor dejapublicat de Clarissou et al. [10]. La prima prezentare s-a efectuattestarendoutrepte,utilizndcascreeningnanul2011kitulELISAVirotech,pentrucananul
2012 s se utilizeze kitul Euroimmun; ca test de confirmare Western Blot s-a utilizat kitulEuroimmun.Lareevaluarede3lunis-arepetattestareaELISA.Terapiaantibioticarespectatindicaiileghidurilornfunciedeafectareadeorgan,vrst,comorbiditi[ 5,6,7].
Rezultatenstudiuaufostincluiprospectiv64pacieni.57dinpacieniiinclui(89%)auprezentat
testulndoutrepte(TTT)pozitiv,restulde7pacieniprezentndtestulELISAsauWesternBlot
pozitiv.
BLafostdescrismaifrecventlapacieniproveninddinmediulurban,lasexulfemininin grupa de vrst 50-64 ani. 58 pacieni (90,6%) au prezentat afectri multiple de organ.Utilizndscoruldeprobabilitate,44pacieniaufostclasificaicaBLfoarteprobabil,18caBLprobabili2pacienicaBLpuinprobabil.Nuaufostdescrisediferenesemnificativestatisticaleevoluieintimpanumruluidesimptomesauscoruluisimptomaticintrecele3grupuride
pacieni.Porninddelapremisaclipsaderspunslatratamentsepoatedatoraunuidiagnosticgreit, rezultatele obinute sugereaz c utilizarea scorului de probabilitate prezentat deClarissouetal.[10]nuaducebeneficiinstabilireadiagnosticuluideboal.
RspunsullaterapiaantibioticafostmaibunngrupulpacienilorcareauprezentatTTTpozitiv, comparativcu grupulcelorcareau prezentatTTTnegativ. Utilizarea testuluiWestern
Blot ca screening,sau testarea sa lapacienii cu testulELISAnegativscadevaloarea clinic a
rezultatuluiipoateducelaundiagnosticgreit,urmatdelipsaderspunslaterapiaantibiotic.Acesterezultatesusinindicaiadetestareaanticorpiloranti B.burgdorferiprinmetodaWesternBlotdoarlapacieniicutestELISApozitiv.
EvaluareapacientilorcuBLnstudiiterapeuticeestedificil,deoareceintervinfactorideeroareprecumexistenaunorcomorbiditi.Dincei64pacieniinvestigai,24auprezentatun
diagnosticsecundarneurologic,33undiagnosticsecundarreumatologic,8auprezentatafeciunitiroidiene,10afeciunipsihice,3afeciunicutanate,2serologiepozitivapentruinfectiacuvirusulhepatiticC,unulcuvirusulhepatiticB,2serologiepozitivapentruMycoplasmapneumoniae.
Monitorizarea serologic la trei luni dup terapie a artat o variabilitate a profilelorcineticiianticorpilor,subliniindlipsautilitiiacesteianpracticaclinic.
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
11/23
DiscuiigeneraleDin perspectiva BL, medicii pot fi supui la dou greeli: subdiagnosticul i
supradiagnosticulBL.Este o greeal snunegndimla aceast afeciune,atta timp ctne
aflmntr-ozonendemic,avndcpueinfectatecuprincipalelespeciipatogeneumanedeB.burgdorferinimediatavecintate,daresteogreealisignormalteafeciuni,launpacient
cuserologiepozitivpentruB.burgdorferi.PolimorfismulmanifestrilorclinicealeBLestentr-adevr un aspect cunoscut, dar ndeprtarea de orice alt diagnostic, atta timp ct serologiapersistpozitiv,poateprivapacientuldeterapiespecificpentrualtboaltratabil.Astfel,lapacieniicusimptomecronice,lipsaderspunslaterapiaantibioticsugereazposibilitateaunui
diagnostic iniial greit i nevoia unei reevaluri clinice dect a unei noi cure antibiotice. ncontextulinformaiiloreronatesaucontradictoriipecareleprimescprinmass-media,pacieniisolicitadeseorirepetareacurelorantibioticedatoritpersisteneiserologieipozitivei/sauaunoracuzesimptomatice.Pacientultrebuieinformatpebazaunuidialogdeschisderisculuneiterapiiantibioticeprelungite,freficienadovediticuposibilereaciiadverse.PrezentareapublicaunorpacienicuafeciunineurologicedegenerativeprecumSLAsauSMcafiindBL,nu
facedectscreascconfuziaitemerilepacienilorcareniseadreseaz.Deoareceanticorpii
anti B. burgdorferi pot persista 10-20 ani dup primoinfecie, iar seroprevalena n cadrulpopulaiei generale este crescut, n evaluarea acestor pacieni trebuie s inem cont dembtrnireafiziologic,stres,publicitateaprivindBL,darinprimulrndcomorbiditi.
Concluziigenerale1. CercetareadefaaprezintprimulstudiuefectuatinRomniaasupracapuelor
colectatedelasubieciiumani.2. Majoritateacpuelorexaminate(97.3%)aparingenului Ixodes,vectorulBL.3. UtilizndinscopdiagnosticometoddeRT-PCRavandca intgenahbbaB.
burgdorferi,prevalenainfecieicuB.burgdorferis.l.incapueleIxodesexaminateafost11,1%.
4. Pe baza rezultatelor obtinute, regiunea noastr poate fi incadrat in zonele
europenecuprevalenajoasainfecieicapuelorcuB.burgdorferi.5. Studiul dovedete caprincipalele genospeciiuman patogene sunt prezente incapueledinRomnia,constituindunriscdeinfeciepentruom.
6. Seroprevalena pentru infecia cu B. burgdorferi a fost 9,1% la 263 pacieniexaminailamomentulnepaturii,rezultatecareconfirmnivelulrelativinaltalseropozitivitii
inpopulaiaasimpomaticdinzoneendemice.7. Studiul nua doveditexistenaunei legturi intrestatusul infectantal capuei,
rezultatulmonitorizriiserologiceimanifestrileclinicealepacienilor.8. Acesterezultatesusinrecomandrileconformcrorainvestigareainfecieicu
B.burgdorferiincapuadetaatsimonitorizareaserologicanusuntindicateinscopulprediciei
infecieilasubieciiumani.
9.
Beneficiilechimioprofilaxieipostnepturdecpunuaupututfidovedite.Eritemulmigrators-adezvoltatnciudachimioprofilaxiei,iarpacieniifrchimioprofilaxienuaudezvoltatmanifestrideboalsauseroconversie.
10. Duplex ospA RT-PCR, o metod de RT-PCR descris pentru prima dat nprezentulstudiu,s-adoveditafiometodeficientdescreeningalcomplexuluiB.burgdorferis.l.,
utilpentrustudiipeunnumrmaredeprobe.11. VariaiarezultatelorobinuteprinimunohistochimiesiceledouametodedeRT-
PCRsubliniazexistentarezultatelordiscordanteadiferitelormetodeutilizatepentrudetecia B.burgdorferiincapue.
12. Atttimpctnuexistunstandarddeaurndiagnostic,beneficiilelaniveldeindivid,printestareacpueidetaate,suntlimitatederezultatelefalspozitiveifalsnegative.
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
12/23
13. Un singurpacientcu terapie incompletpentruEM a fost diagnosticat caBLtardivcumanifestriarticulare,sugerndunprognosticbunalpacienilorcuEMinregiuneanoastr.
14. NiciunpacientdinstudiunuaindeplinitcriteriiledeNBLdefinitsidoar16,6%dincazuriaufostclasificatecaNBLposibila.
15. Acest studiu aduce evidente asupra supradiagnosticarii NBL in regiuneanoastra.16. Datele prezentate constituie prima dovada necesara intreruperii utilizarii
nejustificateaantibioticelorlapacientiineconfirmaticuNBL.
17. PacientulcareseprezintcususpiciuneadeBLpoateprezentauntabloucliniccuafectrimultipledeorgansinumeroasecomorbiditai.
18. Rspunsul pacienilor la terapia antibiotic depinde de corectitudineadiagnosticuluiirespectareaindicaieidetestareserologicndoutrepte(screeningELISAiconfirmareWesternBlot).
19. Nus-adoveditexistenauneilegturintrerspunsullaterapiealpacienilori
ncadrareaacestorandiversescorurideprobabilitate.
20. Dateleobinutesusindiscordanantrerezultateleserologicesiclinice,carenupermitapreciereaeficieneiterapieiprintestareapostterapeuticaanticorpilorspecifici.21. Datoritvariabilitaiicineticiianticorpilorsipersisteneiacestoradupaterapie
la un numar important din pacieni, monitorizarea posterapeutic a pacienilor cu BL se
recomanadafiomonitorizareclinic.Originalitateaicontribuiileinovativealetezei Originalitatea tezei const n abordarea multidisciplinar a unei afectiuni complexe,
BorreliozaLyme,dinperspectivaentomologului,epidemiologului,clinicianuluiimediculuidelaborator.Esteprezentatprimulstudiuefectuatasupracpuelorcolectatedelasubieciiumani
din Romnia, evalund riscul de transmitere al infeciei cu B. burgdorferi la om. Pe baza
rezultatelorobinutes-aefectuatunprotocollocalprivindatitudineapostnepturdecpu.Examinareastatusuluiinfectantal cpuei,monitorizareaserologici chimioprofilaxianu suntrecomandate,monitorizareapacienilordupnepturadecpufiindunaclinic.
O dat cu proiectul acestei teze, au fost introduse n practica noastr clinic criteriileeuropenedediagnosticale NBL. Pe lng dateletiinifice furnizate,acest studiuare caprim
beneficiucreareaunuiprotocoldeabordareapacienilorcareseadreseazserviciuluinostrucususpiciuneadeBL,urmndoconduitdiagnosticiterapeuticconformindicaiilorghidurilor,cuajutoruluneiechipemultidisciplinare.
ToipacieniiincluinstudiuaufostinternaisauconsultaiambulatornSpitalulClinicde Boli Infecioase, Cluj Napoca. Analiza morfologic a cpuelor colectate a fost realizat n
cadrulDepartamentuluideParazitologieiBoliParazitarealFacultiideMedicinVeterinar
USAMV,ClujNapoca.Diagnosticulimunohistochimicalinfecieicu B.burgdorferincpues-aefectuatncadrulLaboratoruluideAnatomopatologiealSpitaluluiClinicdeBoliInfecioase,ClujNapoca. Studiile de diagnosticmolecular au fost realizate n cadrulmobilitii transnaionaleefectuatelaCentrulNaionaldeReferinpentruBorrelia,InstitutulBavarezpentruSntateiSigurana Alimentului, Oberschleissheim, Germania. Diagnosticul serologic al infeciei cu B.
burgdorferi s-a efectuat n cadrul Laboratorului Clinic al Spitalului de Boli Infecioase, ClujNapoca.
Toate aceste realizri au fost posibile prin colaborarea realizat cu Departamentul deParazitologieiBoliParazitarealFacultiideMedicinVeterinar,USAMVClujNapocancadrulproiectuluiIDEI-PCCECNCSIS84,7/2010Studiifundamentalei aplicatedeecoepidemiologie,biologieigeneticamolecularalevectorilorboliiLymeiprinproiectulUMFIuliuHaieganu
ClujNapocaco-finanatE.S.FPOSDRU88/1.5/S/56949.
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
13/23
IULIU HAIEGANU UNIVERSITY OF MEDICINE AND
PHARMACY, CLUJ-NAPOCA
Study regarding prophylaxis and
treatment of Lyme Borreliosis in
Transylvania
ABSTRACT OF THE DOCTORAL THESIS
Doctoral Candidate Violeta Tincua BriciuScientific CoordinatorProf. Dr. Dumitru Crstina
Cluj-Napoca 2013
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
14/23
TABLEOFCONTENTSINTRODUCTION 11CURRENTSTAGEOFKNOWLEDGE 1.Etiology,pathogenesisandepidemiologyofLymeborreliosis
15
1.1.EtiologyofLymeborreliosis.Borreliaburgdorferisensulatocomplex
15
1.1.1.Morphology,ShapeandMembraneStructure 15
1.1.2.GenomeandGenomeOrganization 17
1.1.3.Borreliaburgdorferisensulatocomplex 18
1.2.EpidemiologyofLymeborreliosis 19
1.2.1.Terminology 19
1.2.2.Vectors 20
1.2.3Reservoirhosts 20
1.3.B.burgdorferi hostinteractionandpathogenesis 201.3.1.B.burgdorferi-hostinteraction 20
1.3.2.Pathogenesis 212.ClinicalmanifestationsanddiagnosisofLymeborreliosis 23
2.1.Clinicalmanifestations 23
2.1.1.SkinInvolvement 232.1.2.NervousSystemInvolvement 242.1.3.JointInvolvement 25
2.1.4.CardiacInvolvement 25
2.1.5.EyeInvolvement 26
2.1.6.OtherPotentialManifestationsofLymeborreliosis 26
2.1.7.ChronicLymeborreliosis 26
2.1.8.Lymeborreliosisinpregnancy 272.1.9.Lymeborreliosisinimmunocompromisedhost 27
2.2.Lymeborreliosisdiagnosis 27
2.2.1.DirectDetectionofB.burgdorferi 272.2.2.ImmunologicDiagnosisofB.burgdorferiInfection 28
2.2.3.OtherlaboratoryassaysinLB 31
2.2.4.Testsnorrecommendedtobeusedfordiagnosis 32
2.3.Differentialdiagnosis 32
3.TreatmentandprophylaxisofLymeborreliosis 333.1.TreatmentofLymeborreliosis 33
3.1.1.TreatmentrecommendedforLymeborreliosis 33
3.1.2.Pharmacologicandmicrobiologicbasesofthetreatment 343.1.3.WhatshouldbedoneincaseofpersistentsymptomsafteradequateantibiotictreatmentforLymeborreliosis?
36
3.1.4.Therapyinasymptomaticpatientswithpositiveserology 37
3.2.ProphylaxisofLymeborreliosis 37
3.2.1.Personalprotection 37
3.2.2.Integratedtickmanagement 39
PERSONALCONTRIBUTION 1.Hypothesis/objectives 432.Methods 453.Study1-LymeBorreliosisetiologyinRomania: Borrelia
burgdorferisensulatospeciesintickscollectedfromhumans
47
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
15/23
3.1.Introduction 47
3.2.Hypothesis/objectives 473.3.Materialandmethods 47
3.4.Results 50
3.5.Discussion 54
3.6.Conclusions 564.Study2-ProspectivestudyonthetransmissionriskofBorreliaburgdorferisensulatofromIxodesricinus tickstohumans
57
4.1.Introduction 57
4.2.Hypothesis/objectives 57
4.3.Materialandmethods 57
4.4.Results 58
4.5.Discussion 61
4.6.Conclusions 64
5.Study3-Diagnosticassaysusedfor Borreliaburgdorferisensulatodetectionintickscollectedfromhumans 65
5.1.Introduction 65
5.2.Hypothesis/objectives 65
5.3.Materialandmethods 65
5.4.Results 69
5.5.Discussion 72
5.6.Conclusions 76
6.Study4-ClinicalmanifestationsofLymeBorreliosisinTransylvania:Erythemamigrans
6.1.Introduction 77
4.2.Hypothesis/objectives 776.3.Materialandmethods 77
6.4.Results 78
6.5.Discussion 83
6.6.Conclusions 86
7.Study5-ClinicalmanifestationsofLymeBorreliosisinTransylvania:LymeNeuroborreliosis
87
7.1.Introduction 87
7.2.Hypothesis/objectives 877.3.Materialandmethods 87
7.4.Results 89
7.5.Discussion 977.6.Conclusions 99
8.Study6OtherclinicalmanifestationsofLymeBorreliosisinTransylvania
101
8.1.Introduction 101
8.2.Hypothesis/objectives 101
8.3.Materialandmethods 101
8.4.Results 102
8.5.Discussion 109
8.6.Conclusions 112
9.Generaldiscussion 11310.Generalconclusions 115
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
16/23
11.Theoriginalityandinnovativecontributionsofthethesis
119
REFERENCES 121ANNEX1 141
ANNEX2 143Keywords:LymeBorreliosis,Ixodesricinus ,Borreliaburgdorferi ,Transylvania,RealTimePCR,imunohistochemistry,cerebrospinalfluid,serology,ELISA,WesternBlot.
IntroductionLymeBorreliosis(LB),amultisystemicdisorder,themostfrequenttickbornediseasein
EuropeandNorthAmerica,isazoonosis,theetiologicalagentbeingtransmittedbetweenalargevarietyofreservoirhostsbythehardticksbelongingtothe Ixodesricinus complex.Thereservoirhostpassesthepathogentothevectortickthattransmitsittoanotherreservoirhostortothehuman,thatincidentallyentersthecycleofevolutionandclosestheinfectiouscycle. Borrelia(B.)burgdorferi sensu lato complex, the etiologicalagentof thedisease,is currentlydivided in19genospecieswithdifferentgeograficaldistribution.WHOregionalofficeforEuropementionesin2006nodataforRomaniaregardingLB[1].
TheriskofinfectionwithB.burgdorferidependsonthehumaninteractionwithnature.LBprevention strategies are based on epidemiological, ecological and phisiopathological data,involvingpersonalandgeneralprotectivemeasures.Tillnow,nostudyisavailableinRomaniaon the infection rate of ticks collected from human patients. As different B. burgdorferigenospeciesappeartobeassociatedwithdifferentclinicalpictures,ouraimwastoevaluatetheprevalenceanddiversityoftheLymediseasespirochetesinvectortickscollectedfromhumanpatientsincentralTransylvania,usingmodernmoleculardiagnostictools.
LB, the disease with 1000 faces, mayaffecta large variety of organs and theclinicalmanifestationsandevolutionmaybedifferentfromonepatienttoanother.Nowadaysthereisnogolden standard for diagnostic and the assays available have to be combined to obtain the
highest sensitivity and specificity. A positive serology cannot differentiatebetween anactiveinfectionandtheimmunologicalhallmarkofaprevioussymptomaticorasymptomaticinfection.The high prevalence of positive serology in the general population in endemic areas raisesproblems in interpreting the clinical relevance of a positive serology while the polymorphicclinicalmanifestationscomplicatethedifferentialdiagnosis.
DuetopublicityandthegeneralinformationonLB,thenumberofpatientsthatpresenttotheinfectiousdiseasesdepartmentaftertickbitesorwiththesuspicionofLBincreasedinthelastyears.
PersonalcontributionHypothesis/objectivesProphylaxisortreatmentofLBcannotbeevaluatedaslongaswehavenodataregarding
the vector infectionwith B. burgdorferi.Any newdata regarding genospecies distribution in
questingticksorinticksremovedfromhumansmayprovidenewinsightsinthecomplexecologyofLB.
The objectives of the present thesis were to investigate different aspects of LB in ourregion:thevectorsandtheprevalenceofinfectionwithB.burgdorferi,thelocalgenospecies,theriskofB.burgdorferitransmissionfromtickstohumanandtheimportanceoftestinginfectioninthedetachedtick,antibioticsuseandserologicalfollowupinLBprophylaxis.
Anotherobjectiveofthethesiswastodescribetheepidemiological,clinicalandserologicaldataofthepatientsdiagnosedwithLB,evaluatingtheresponsetotreatmentandtheprognosis.Due to the various clinical manifestations and the absence of specific symptomatology, theneurological manifestations in LB may mimic other neurological diseases. The EuropeanguidelinesrecommendthediagnosisofLymeneuroborreliosis(LNB)basedonthecerebrospinalfluid(CSF)analysis.Untilthepresentstudytheformerrecommendationhasnotbeenfollowedin
ourclinicalpractice,causingoverdiagnosisofLNBinpatientswithotherneurologicaldiseasesfollowedbylackofresponsetoantibiotictherapy.Theaimofourstudywastointroducethenewdiagnosticrecommendationsandtoevaluatetheresponsetotreatmentofthepatients.
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
17/23
MethodsThe study was approved by the Ethics Committee of the University of Medicine and
PharmacyIuliuHatieganuCluj-Napoca,Romania.Eachpatientwasincludedinthestudyafteraninformedconsentandreceivedinformationontheaimsandprotocolofthestudy.
Two questionnaires were used for patients monitoring.The questionnaire for tick bitemonitoringwascompletedforeachpatientat themomentof tickbite, regardingoccupational
and recreational risk for tick bites, site of tick attachment, numberof ticks detached, knownallergiestoantibiotics,pathologicalantecedents,pregnancy(ifpresent).ThequestionnaireforLBmonitoring was completed at different moments, depending on the design of the study,regardingprofession,personalantecedents,knowntickbitesorerythemamigrans,cutaneous,musculoskeletal,neurological,cardiac,ocularsymptomsandsigns.
The serologicaltests for antiB. burgdorferi antibodiesdetectionwere used atdifferentmoments,dependingonthedesignofthestudy.TheELISAtestsusedwere:BorreliaafzeliiIgMELISA,Borreliaafzelii+VlsEIgGELISA(GenzymeVirotechGmbH,Germany)andIgM,IgGanti-BorreliaELISA(EuroimmunAG,Germany)andtheWesternBlottestsusedwere:IgM,IgGAnti-BorreliaEUROLINE-RN-AT(EuroimmunAG,Germany).
Study 1 - Lyme Borreliosis etiology in Romania:Borrelia burgdorferi sensu lato
speciesintickscollectedfromhumans.IntroductionTheB.burgdorferi sensulato(s.l.)complexcurrentlyincludes19differentgenospecies,10
ofwhichhavebeenreportedfromEurope:B.burgdorferi s.s.,B.garinii,B.afzelii,B.valaisiana,B.lusitaniae ,B.bavariense,B.bissettii,B.spielmanii,B.kurtenbachiiandB.finlandensis.Prevalenceof infection with B. burgdorferi s.l. in ticks collected from humans may be different than inquestingticksfromcloselyrelatedgeographicalareas.
ObjectivesTheaimsofthestudyweretodeterminewhichtickspeciesbitehumansinRomaniaand
theprevalenceofB.burgdorferigenospeciesintheseticks.MaterialandmethodsAlltickscollectedfrompatientswho presentedto theClinicofInfectious Diseases,Cluj
Napoca, between 01.04.2010-07.09.2010 were prospectively studied. Ticks were identified
regarding species, sex and developmental stage. B. burgdorferi genospecies identification incollectedticksusedareal-timePCRassaytargetingthehbbgene[2].ResultsA total number of 532 ticks were collected from 386 human patients. The maximum
numberofpresentationswasinthemonthofMay.Themajority(97.3%)oftheticksexaminedbelongtothespecies Ixodesricinus,thevectorofLBinEurope.OthertickspeciesidentifiedwereDermacentormarginatus,HaemaphysalisconcinnaandHaemaphysalispunctata;thesespeciesdonottransmitLB,butarevectorsforotherbacterial,viralorparasiticdiseases.
In Ixodes ticks theprevalenceof infectionwith B. burgdorferi s.l. was 11.1%. A higherprevalenceofinfectionwasfoundinadult Ixodes(12.9%)thaninIxodesnymphs(10.7%),butnotstatistically significant (p=0,55). Based on our results, our region might be included in theEuropeanlowinfectionratesregions.
Species identification revealedmainlyB. afzelii(62.7%), but also B. garinii (16.2%), B.
burgdorferi sensu stricto (6.9%), B. valaisiana (2,3%), B. spielmanii/valaisiana (9,3%) and B.lusitaniae(2.3%).Hbbreal-timePCRwasnotabletodistinguishB.spielmaniifromB.valaisiana,needing post PCRassays for differentiation. This was the first reportofB. burgdorferi sensustricto in ticks collected from Romania. All relevant human pathogenic genospecies of B.burgdorferiarepresentinticksfromourregion,thusposingariskofinfectionforhuman.
Study2 -Prospectivestudyon thetransmissionriskofBorreliaburgdorferisensu
latofromIxodesricinustickstohumansIntroductionThe risk of developing LB after B. burgdorferi infected tick bite and the benefit of
chemoprophylaxisinRomaniaisunknown.ObjectivesAs different B. burgdorferi genospecies are associated with different clinical
manifestations andnotallthegenospecies arehumanpathogenic,theaimofthestudywasto
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
18/23
evaluate the clinical andserological outcomeof patients bittenbyBorreliainfectedtickswithregardtodifferentgenospeciesandtheprophylaxisfollowed.
MaterialandMethodsAllpatientsbittenbyB.burgdorferipositiveticksinhbbRT-PCRandacontrolgroup(case-
controlstudy)were asked for followup oneyearlater (May-August2011).Thecontrolgroupincluded age, sex and residence (urban/rural) matched individuals bitten by B. burgdorferi
negativeticksandseronegativeforB.burgdorferi(usinganELISAtest) atthemomentoftickbite.Thequestionnairewascompletedforeachindividualatthemomentoftickbiteandatthefollowup.
Atthemomentof tickbiteanELISAassaywasperformed(todeterminepresentIgMorIgGB.burgdorferiantibodiesduetoprevioustickbites)usingtheGenzymeVirotechkit.Atthefollowup a two-tieredtest (TTT)wasperformedto determineIgM and IgGantibodies antiB.burgdorferiusingtheVirotechELISAkitasscreeningfollowedbyEuroimmunWesternBlotkitasconfirmation.
ResultsTheseroprevalencefor B. burgdorferiinfectionwas9.1%in263personstestedatthe
moment of tick bite, results that confirm the high level of seropositivity in asymptomaticpopulationinendemicarea.20personsoutofthe39personsbittenbythe B.burgdorferipositiveticksand20matchingpersonsbittenbynegativetickspresentedthemselvesforthefollowup.Two of the investigated persons developed acute LB (erythema migrans) and 5 personsseroconvertedattheoneyearfollowup.Nodifferencewasfoundregardingpresenceofclinicalsymptomsorseroconversionbetweenthegroupbittenby B.burgdorferipositiveticksversusthegroup bitten by B. burgdorferi negative ticks, though both cases of erythema migrans (EM)occurredinthegroupbittenbypositiveticks.B.afzelii,themainpathogenicgenospeciesinourregion,wasidentifiedintickscollectedfrompersonswithEM.Thestudyprovidesevidenceforunexplained symptoms in the general population, that could be interpreted as chronic LB.ThoughthesmallnumberofpatientsinvestigateddidnotenableustoassesstheefficacyorthefailureofantimicrobialprophylaxisforthepreventionofLB,bothcasesofEMdevelopedinspiteof chemoprophylaxis and patients without chemoprophylaxis did norseroconvertor developsymptoms of LB. Our study did not show a statistical significant relationship between theinfectivestatusofthetick,theserologicalfollowupandtheclinicalfollowupofthepatients.The
results underline the lack of benefit for testingB. burgdorferi in detached tick or serologicalfollowupforpredictinginfectioninhumans.Study3.DiagnosticassaysusedforBorreliaburgdorferisensulatodetectioninticks
collectedfromhumansIntroductionAlthoughthereisnogoldenstandardfor B.burgdorferi detectioninticks,patientspresent
inourdepartmentaskingfortheinvestigationofthedetachedtick,topredictthetransmissionriskofinfection.
ObjectivesThe objective of this studywas to compare 3 different assays used fordetection ofB.
burgdorferi in ticks, evaluating their advantages or disadvantages and their role in clinicalpractice.
MaterialandmethodsArepresentativenumberoftickscollected frompatientswhopresented intheClinicof
Infectious Diseases, Cluj Napoca, between 01.04.2010-07.09.2010 have been tested by 3diagnosticassays:immunohistochemistryandtwoRT-PCRassays( hbbRT-PCRandospART-PCR)[3,2,4].
DuplexospART-PCRisanassayfirstdescribedinourstudy.Aswehaveworkedwithticksfixed in formalin and embedded in paraffin, besides the already described system for thedetection of the ospA gene [3], the internal amplification control (IAC) used excluded theinhibitionofthePCR,whichcouldleadtofalsenegativeresults.Fordeterminingthespecificityofthe duplex ospA RT-PCR, 22 B. burgdorferi s.l. strains were tested for inclusivity, while 18Leptospirastrains(includingthegenospeciesinterrogans,borgpetersenii ,kirschneriandbiflexa),Borreliamiyamotoi andTreponemaphagedenisweretestedforexclusivity.
Results
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
19/23
Sensitivity of the duplex ospA RT-PCR was 1-10 copies/PCR, efficiency 99.85%, theexclusivitywas100%andtheinclusivitywas86.36%(notdetectedB.lusitaniaestrainsPotiB2,PotiB3andB.japonica).
Outof136tickstestedbyallthethreeassays,16 (11.8%)werefoundtobepositivebyimmunohistochemistry(IH),12(8.8%)byhbbRT-PCR,and26(19.1%)byduplexospART-PCR.InallsamplesnoinhibitionwasdetectedbytheIACsystem.The86.7%concordanceofthetwo
RT-PCRassaysunderlinesthedifferentsensitivitiesofPCRtechniquestargetingdifferentgenes.The p-value below the statistical significant value (p
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
20/23
sclerosis(ALS),polyneuropathy,Parkinsonism,AlzheimersdiseasemaybemisdiagnosedasLB.TheEuropeandiagnosticcriteria,unliketheAmericanones,recommendthecerebrospinalfluid(CSF)analysisandintrathecalantibodyproductiondetection[ 8].
ObjectivesThe present study introduced for the first time in our clinical practice the European
diagnosticrecommendationsforLNB.
MaterialandmethodsPatientshospitalisedintheClinicofInfectiousDiseasesbetweenJanuary2011-October
2012withthesuspicionofLNBwithcentralnervoussystemmanifestationswereincludedinthestudy. Inclusion criteriawere: centralnervous systemmanifestations, positive serology forB.burgdorferi,lumbarpuncture,andnopreviousparenteralantibiotictreatment.
PatientsweretreatedasLNBwithCNSmanifestations[ 8],indifferentoftheCSFanalysisresults.Thesymptomsincludedinthequestionnairewerefollowedatadmission,attheendoftreatment and 3 month later, using a scoring scale. Two negative binominal mixt effectsregressionmodels were used. The dependent variablewas the numberof symptoms and thesymptomaticscorerespectively.Thetime(correspondingtotheobservationsmade),measuredinweeks,wasusedasanrandomvariable.
Theintrathecalantibodyindex(IAI)wascalculated,usingaGenzymeVirotechkitin2011and an Euroimmun kit in 2012. At admission the TTT used for screeningGenzyme VirotechELISAkitin2011andEuroimmunELISAkitin2012,followedbyanEuroimmunWesternBlotkit.Clinicalandserologicalevaluation(ELISA)wasrepeated3monthaftertreatment.
ResultsAccording toEuropeanguidelinesno case fulfilled the criteria for definiteLNB, 7cases
werepossibleLNB and in 33 cases LNBwas infirmed.2 cases could not be classified due toinsufficientamountofCSF.ThisstudybringsevidencethatLNBisoverdiagnosedinourpatients.
Though the clinical symptomatology improved, a number of symptoms persisted. Thepatients with infirmed LNBpresentedmore symptoms during the followup then thosewithpossibleLNB,statisticallysignificant(p=0,03),andahighersymptomaticscore,closetothelimitof statistically significance (p=0,055). These results show a better evolution under antibiotictherapyinthegroupofpatientswithpossibleLNBthaninthegroupofpatientswithinfirmedLNB. No symptoms were described more frequent (statistically significant) in the group of
patients with possible LNB. All the patients were evaluated in a multidisciplinary team. 22patients presenteda neurologicaldiagnosiswhile the rest ofthe patients did notpresent anychangesattheneurologicalassessment,thoughpresentedsymptomatology.Thespecifictherapywasaddedor followed theantibiotic treatment.No associationhas been provedbetween thediagnosisofpossible/infirmedLNBanddemyelinatinglesionsonthebrainMRI(p>0,05).
Rheumatologicalmanifestationsweredescribedin9patients.Theserologicalfollowupshowed a very diverse profile of antibodies kinetics. Adverse reactions to medication werepresentin13patients(30.9%),includingoneClostridiumdifficile associateddisease.
DiscussionDue to the high prevalence of positive serology for B. burgdorferi in the general
population,anaspectalreadydescribedinourregion(study2),theoccurrenceofneurologicalsymptomsinthesepatientscomplicatesthepositiveanddifferentialdiagnosis.Inourstudy,apatientfromthegroupwithinfirmedLNBwasconfirmedasMSandtwopatientsasALSduring
the 3 months follow up. None of these patients presented any improvement after antibiotictherapy.TheresultsconfirmthestatementofPuchalandSibilia(2009)[9],thatpersistenceofsymptomatology may be due to an incorrect diagnosis than to treatment failure. The studyrepresentsthefirstproof tostoptheantibioticsoveruse innotconfirmedLNBpatientsinourregion.
Study6OtherclinicalmanifestationsofLymeBorreliosisinTransylvania.IntroductionThe investigation,treatmentand followupof thepatientswith the suspicionof LBmay
poseachallengetotheinfectiousdiseasesspecialist.NogoldenstandardforLBdiagnosisexists.ExceptEM, diagnosedon clinical data, and LNBthathas the recommendation ofCSF analysis,other manifestations may be described, more or less specific. An important aspect in thediagnosisisrepresentedbyepidemiologicaldata(knowntickbitesorEM),associatedtoclinicalmanifestationsandpositiveserologyforB.burgdorferi.Theserologyhastobeinterpretedwith
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
21/23
cautionduetoknownlimitsasfalsepositivity.Animportantaspectintheassessmentofthesepatientsisrepresentedbythedifferentialdiagnosis.
MaterialandmethodsPatientsthatpresentedintheClinicofInfectiousDiseasesbetweenJanuary2011-October
2012 with the suspicion of LB were included in the study. Inclusion criteria were: clinicalmanifestationsofLBandpositiveserologyfor B.burgdorferi.Exclusioncriteria:patientswithEM
andpatientswithclinicalsuspicionofLNBthatacceptedthelumbarpunctureincludedinthepreviousstudies-,orthosethatdidnotreturnforthefollowup.
Thequestionnairewascompletedatinclusion,aftertherapyand3monthslater.PatientsweredividedingroupsontheprobabilityofhavingLBusingascorealreadypublished[ 10].AtinclusiontheTTTusedforscreeningin2011theVirotechELISAkitandin2012theEuroimmunELISAkit;asconfirmationtheEuroimmunWesternBlotkitwasused.Atthe3monthsfollowuptheELISAtestwasrepeated.Theantibiotictherapyfollowedtheguidelinesrecommendationsdependingonclinicalmanifestations,ageandcomorbidities.
Results64patientswereincludedinthestudy.Outofthem57patients(89%)presentedtheTTT
positive,therestofthe7patientspresentingELISAorWesternBlottestpositive.LBwasdescribedmorefrequentinpatientsfromurbanarea,femalegenderandtheage
group50-64yearsold.58patients(90,6%)presentedmultipleclinicalmanifestationsinvolvingdifferentorgansystems.Usingtheprobabilityscore,44patientswereclassifiedasveryprobableLB,18asprobableLBand2patientsaslittleprobableLB.Comparingthese3groupsofpatients,no significant statistical difference was obtained between the number of symptoms/symptomaticscoreevolutionundertreatment.Acceptingthatthatlackofresponsetotreatmentmay be caused by an incorrect diagnosis, these results suggest that the probability scorepresentedbyClarissouetal.[10]doesnotbringanybenefitforLBdiagnosis.
The response to treatmentwasbetter in thegroupofpatients that presented the TTTpositive,comparedtothegroupofpatientsthathadanegativeone.UsingtheWesternBlottestasscreening,orusingitinpatientswithanegativeELISAscreeningtest,decreasestheclinicalvalueoftheresultandmayleadtoafalsepositivediagnosis,followedbythelackofresponsetoantibiotic therapy. Our results sustain the recommendation of testing for B. burgdorferiantibodiesbyWesternBlotonlyinpatientswithapositivescreeningELISAtest.
Following LB patients in therapeutic studies is difficult, as there may limits due tocomorbidities.Outofthe64patientsinvestigated,24presentedaneurologicaldiagnosis,33arheumatologicdiagnosis, 8 presentedwith thyroid disorders,10 with psychiatric disorders,3with cutaneous disorders, 2 with positive serology for hepatitis C virus, one with positiveserologyforhepatitisBvirus,2withpositiveserologyforMycoplasmapneumoniae.
The serological follow up 3 months after therapy showed a very diverse profile ofantibodieskinetics,underliningthelackofbenefitforantibodiesfollowupinclinicalpractice.
GeneraldiscussionTwo errors may be encountered by the medical doctors with respect to LB:
underdiagnosisandoverdiagnosis.Itisanerrornottothinkaboutthisdisease,aslongaswelivein an endemic area, with ticks infected with the main pathogenic genospecies in ourneighbourhood,aswellasitisanerrortoignoreotherdiseasesinpatientswith B.burgdorferi
positive serology. The polymorphic clinical manifestations of LB is indeed well known, butignoringanyotherdifferentialdiagnosis,aslongastheserologyremainspositive,maydeprivethe patient of a specific therapy for another curable disease. Thus, in patients with chronicobjective complaints,a lack ofresponse to therapy suggests aboveallthepossibility ofinitialmisdiagnosisandtheneedforpromptreassessmentratherthanretreatment.Patients,wrongorcontradictory informed by mass-media, ask very often for antibiotic retreatment, due topersistenceofpositiveserologyand/orsymptomatology.Thepatienthastobeinformedinanopendialogueoftherisksofprolongedantibiotictherapy,notprovedtobeefficientbutpossiblywithsideeffects.ThepublicpresentationofpatientswithneurologicaldiseaseslikeALSorMSasbeingLBaugmentedtheconfusionandfearofpatientsthataddresstoourmedicaldepartment.AsantibodiesantiB.burgdorferimaypersist10-20yearsafterinfectionandtheseroprevalencein general population is high, when evaluating these patients the medical doctors have toconsiderphysiologicalaging,stress,publicityregardingLB,butmostimportantcomorbidities.
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
22/23
Generalconclusions1. ThethesispresentsthefirststudyontickscollectedfromhumansinRomania.2. Themajorityofexaminedticks(97.3%)belongto Ixodesgenus,thevectorofLB.3. Using for diagnosis a RT-PCR targeting the hbb gene of B. burgdorferi, the
prevalenceofinfectionswithB.burgdorferiinexaminedIxodestickswas11.1%.
4. Basedonthethesisresults,ourregionmaybeincludedintheEuropeanregionswithalowprevalenceofinfectionwithB.burgdorferiinticks.
5. The study provides evidence that the most relevant human pathogenicgenospeciesofB.burgdorferis.l.arepresentinticksfromRomania,thusposingariskofinfectiontohumans.
6. TheseroprevalenceforB.burgdorferi infectionwas9,1%in263patientstestedat the moment of tick bite, results that confirm the high prevalence of seropositivity inasymptomaticpatientsinendemicareas.
7. Our study did not show a statistical significant relationship between theinfectivestatusofthetick,theserologicalfollowupandtheclinicalfollowupofthepatients.
8. TheseresultssustainthelackofbenefitfortestingB. burgdorferiindetachedtickorserologicalfollowupforpredictinginfectioninhumans.
9. Benefitsofchemoprophylaxisaftertickbitecouldnotbeproven.EMdevelopedin spite of chemoprophylaxis andpatients without chemoprophylaxis did nor seroconvertordevelopsymptomsofLB.
10. DuplexospART-PCR,aRT-PCRassaydescribedforthefirsttimeinthepresentstudy,provedtobeanefficientmethodforscreeningB.burgdorferis.l.,usefulforstudiesonlargenumberofsamples.
11. The discordant results of immunohistochemistry and the twoRT-PCRassaysunderlinethedifferentresultsofdifferentassaysusedforB.burgdorferidetectioninticks.
12. Aslongasnogoldenstandardexists,thebenefitsfortheindividualintestingthedetachedtickarelimitedbyfalsepositiveandfalsenegativeresult.
13. Asinglepatient,withincompletetherapyforEM,wasdiagnosedwithlateLBwith musculoskeletalmanifestations, suggesting a good prognosis ofpatients with EM in ourregion.
14.
NoneoftheinvestigatedpatientsfulfilledthecriteriafordefiniteLNBandonly16.6%ofthecaseswereclassifiedaspossibleLNB.15. ThisstudybringsevidencethatLNBisoverdiagnosedinourpatients.16. The results represent the first proof to stop the antibiotics overuse in not
confirmedLNBpatientsinourregion.17. Thepatientpresentingwith thesuspicion ofLBmay presentmultipleclinical
manifestationsanddiversecomorbidities.18. Theresponsetotreatmentdependsonacorrectdiagnosisbasedonapositive
two-tieredtest.19. Noassociationwasproved betweenthepatients response to treatementand
thedifferentprobabilityscores.20. Ourresultsshowthattheserologicalprofileisunpredictableanduncorrelated
withtheclinicalcourse,andsustaintherecentrecommendationofavoidingrepeatedserologic
testingforassessingtherapyefficacyinLBpatients.21. Duetothevariabilityofantibodieskineticsandtheirpersistenceaftertherapy,
theassessmentofpatientswithLBinthefollow-uprestsprimarilyontheclinicalpicture.
TheoriginalityandinnovativecontributionsofthethesisThe original contribution of the thesis consists in themultidisciplinary approach of a
complex disease, Lyme borreliosis, from the entomological, epidemiological, clinical andlaboratoryperspective.ThefirstsurveyontickscollectedfromhumansinRomaniaispresented,evaluatingthetransmissionriskof B.burgdorferiinfectiontohumans.Basedonourresults,alocalguidelinewascreatedregardingtickbites.Testingfor B.burgdorferiinthedetachedtick,serologicalfollowupandantibioticprophylaxisarenotrecommended.Thepatientsfollowupisaclinicalone.
Along with the project of the thesis, the European diagnostic criteria for LNB wereimplemented in the clinical practice. Apart from the scientific data, the study has created aprotocol for evaluating patients that present in our department with the suspicion of LB,
7/28/2019 Yh3i4_Briciu Violeta Tincuta Rezumat
23/23
following the diagnostic and therapeutic recommendations of the guidelines, in amultidisciplinaryteam.
Allthepatientsincludedinthestudyhavebeenhospitalisedorinvestigatedasoutpatientsin the Clinic of Infectious Diseases, Cluj Napoca. The morphological characterisation of thecollectedtickshasbeenperformedintheDepartmentofParasitologyandParasiticDiseasesoftheFacultyofVeterinaryMedicine,UniversityofAgriculturalSciencesandVeterinaryMedicine,
Cluj Napoca. The immunohistological diagnosis of B. burgdorferi infection in ticks has beenperformed in the Pathology Laboratory of theClinic of Infectious Diseases, Cluj Napoca. ThemoleculardiagnosticassayshavebeenperformedinthetransnationalmobilityattheNationalReference Centre for Borrelia, Bavarian Health and Food Safety Authority, Oberschleissheim,Germany.TheserologicaldiagnosticofB.burgdorferiinfectionhasbeenperformedintheClinicalLaboratoryoftheClinicofInfectiousDiseases,ClujNapoca.
ThestudyhasbeensupportedbytheprojectIDEI-PCCECNCSIS84,7/2010Fundamentaland applied studies of eco-epidemiology,biologyandmolecular genetics of the Lyme diseasevectorperformedincollaborationwiththeDepartmentofParasitologyandParasiticDiseasesoftheFacultyofVeterinaryMedicine,UniversityofAgriculturalSciencesandVeterinaryMedicine,Cluj Napoca andby the IuliuHaieganu University ofMedicine and PharmacyCluj Napocaprojectco-financedbyE.S.FPOSDRU88/1.5/S/56949.
References
1 Lindgren E, Jaenson T. Lyme borreliosis in Europe: influences of climate and climate change,epidemiology,ecologyandadaptationmeasures.Copenhagen.(Denmark):WHORegionalOfficeforEurope.2006.35p,ReportNo.:EUR/04/5046250.
2Ferdin,J.,Cerar,T.,Strle,F.,Ruzi-Sablji,E.,2010.Evaluationofreal-timePCRtargetinghbbgeneforBorreliaspeciesidentification.J.Microbiol.Methods.82(2),115-9.
3EisendleK,GrabnerT,ZelgerB.Focusfloatingmicroscopy:"goldstandard"forcutaneous
borreliosis?AmJClinPathol.2007;127(2):213-22.4IvacicL,ReedK,MitchellP,GhebraniousN.ALightCyclerTaqManassayfordetectionofBorrelia
burgdorferisensulatoinclinicalsamples.DiagnMicrobiolInfectDis.2007;57(2):137-43.5
WormserGP,DattwylerRJ,ShapiroED,HalperinJJ,SteereAC,KlempnerMS,KrausePJ,BakkenJS,StrleF, Stanek G,BockenstedtL, Fish D,Dumler S,NadelmanR: Theclinicalassessment,treatment, andpreventionofLymedisease,humangranulocyticanaplasmosis,andbabesiosis:clinicalpracticeguidelines
bytheInfectiousDiseasesSocietyofAmerica.ClinInfectDis2006;43:10891134.6StanekG,WormserGP,GrayJ,StrleF.Lymeborreliosis.Lancet.2012;379(9814):461-737HansmannY.TreatmentandpreventionofLymedisease.CurrProblDermatol2009;37:111-298MyglandA,LjstadU,FingerleV,RupprechtT,SchmutzhardE,SteinerI.EFNSguidelinesonthe
diagnosisandmanagementofEuropeanLymeneuroborreliosis.EurJNeurol.2010;17(1):8-169PuchalX,SibiliaJ.Whatshouldbedoneincaseofpersistentsymptomsafteradequateantibiotic
treatmentforLymedisease?CurrProblDermatol.2009;37:191-9.10ClarissouJ,SongA,BernedeC,GuillemotD,DinhA,AderF,etal.Efficacyofalong-termantibiotic
treatment in patients with a chronic Tick Associated Poly-organic Syndrome (TAPOS). MedMal Infect.
2009;39(2):108-15.