Low risk of seroconversion or clinical disease in humans after a bite by an Anaplasma phagocytophilum-infected tick

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Low risk of seroconversion or clinical disease in

humans after a bite by an Anaplasma

phagocytophilum-infected tick

Anna J Henningsson, Peter Wilhelmsson, Paula Gyllemark, Monika Kozak Ljunggren,

Andreas Matussek, Dag Nyman, Christina Ekerfelt, Per-Eric Lindgren and Pia Forsberg

Linköping University Post Print

N.B.: When citing this work, cite the original article.

Original Publication:

Anna J Henningsson, Peter Wilhelmsson, Paula Gyllemark, Monika Kozak Ljunggren,

Andreas Matussek, Dag Nyman, Christina Ekerfelt, Per-Eric Lindgren and Pia Forsberg, Low

risk of seroconversion or clinical disease in humans after a bite by an Anaplasma

phagocytophilum-infected tick, 2015, Ticks and Tick-borne Diseases, (6), 6, 787-792.

http://dx.doi.org/10.1016/j.ttbdis.2015.07.005

Copyright: 2015 The Authors. Published by Elsevier GmbH. This is an open access article

under the CC BY-NC-ND license

http://www.elsevier.com/

Postprint available at: Linköping University Electronic Press

http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-122245

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ContentslistsavailableatScienceDirect

Ticks

and

Tick-borne

Diseases

jo u r n al hom ep a g e :ww w . e l s e v i e r . c o m / l o c a t e / t t b d i s

Original

article

Low

risk

of

seroconversion

or

clinical

disease

in

humans

after

a

bite

by

an

Anaplasma

phagocytophilum-infected

tick

Anna

J. Henningsson

a,∗

_,

_Peter

_Wilhelmsson

b

_,

_Paula

_Gyllemark

c

_,

_Monika

_Kozak

b

_,

Andreas

Matussek

a

_,

_Dag

_Nyman

d

_,

_Christina

_Ekerfelt

e

_,

_Per-Eric

_Lindgren

a,b

_,

_Pia

_Forsberg

f

a_Department_of_Clinical_{Microbiology,}_Ryhov_County_Hospital,_S-551₈₅_Jönköping,_Sweden

b_Division_of_Medical_{Microbiology,}_Department_of_Clinical_and_Experimental_Medicine,_Linköping_University,_S-581₈₅_Linköping,_Sweden c_Department_of_Infectious_Diseases,_Ryhov_County_Hospital,_S-551₈₅_Jönköping,_Sweden

d_The_Åland_Group_for_Borrelia_Research,_Bimelix_Biomedical_Laboratory,_Torggatan_10,_AX-22100_Mariehamn,_Åland,_Finland

e_Division_of_Clinical_Immunology,_Department_of_Clinical_and_Experimental_Medicine,_Linköping_University,_S-551₈₅_Jönköping,_Sweden f_Clinic_of_Infectious_Diseases,_Linköping_University_Hospital,_and_Department_of_Clinical_and_Experimental_Medicine,_Linköping_University,_S-551₈₅

Jönköping,Sweden

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received21January2015 Receivedinrevisedform6July2015 Accepted6July2015

Availableonline8July2015 Keywords: Anaplasamaphagocytophilum Anaplasmosis Human Seroconversion Tickbite Ehrlichiosis

a

b

s

t

r

a

c

t

Theriskofcontractinghumangranulocyticanaplasmosis(HGA)afteratickbiteismainlyunknown.In thisstudyweinvestigatedtheclinicalandserologicalresponsein30humansbittenbytickspositivefor Anaplasmaphagocytophilum(GroupA),30humansbittenbyBorreliaburgdorferisensulato(s.l.)-positive ticks(GroupB),and30humansbittenbyticksnegativeforbothA.phagocytophilumandB.burgdorferis.l. (GroupC).Ticks,bloodsamplesandquestionnaireswerecollectedfromtick-bittenhumansat34primary healthcarecentresinSwedenandintheÅlandIslands,Finland,atthetimeofthetickbiteandafterthree months.Atotalof2553ticksdetachedfromhumansin2007–2009wereanalyzedbypolymerasechain reaction,and31(1.2%)werepositiveforA.phagocytophilum,556(21.8%)werepositiveforB.burgdorferi s.l.,andeight(0.3%)wereco-infectedbyA.phagocytophilumandB.burgdorferis.l.Theoverallprevalence ofAnaplasmaIgGantibodiesintheincludedparticipants(n=90)was17%,andtherewasnosigniﬁcant differencebetweenthegroupsA-C.Onlyoneoftheparticipants(inGroupC)showedafour-foldincrease ofIgGantibodiesagainstA.phagocytophilumatthethree-monthfollow-up,butreportednosymptoms. ThefrequencyofreportedsymptomsdidnotdifferbetweengroupsA-C,andwasunrelatedtotheﬁndings ofA.phagocytophilumandB.burgdorferis.l.inthedetachedticks.WeconcludethattheriskforHGAor asymptomaticseroconversionafteratickbiteinSwedenorintheÅlandIslandsislow,evenifthetickis infectedbyA.phagocytophilum.

1. Introduction

Tickborne fever is a well-recognisedproblem in veterinary medicineinEuropeandinScandinavia(Egenvalletal.,1997;Stuen etal.,2002;Stuen,2007).Thecausativeagent,Anaplasma phagocy-tophilum,istransmittedbyIxodesticks,andknowntooccasionally alsocause humandisease(Chenet al.,1994).The ﬁrstveriﬁed casesofhumangranulocyticanaplasmosis(HGA)inEuropewere reportedfromSloveniain1997,followedbycasesfromSweden, NorwayandtheNetherlands(Petrovecetal.,1997;Lotric-Furlan et al., 1998; Bjoersdorff et al., 1999a; vanDobbenburgh et al., 1999; Karlsson et al., 2001). HGA is typically a febrile illness

∗ Correspondingauthor.

E-mailaddress:anna.jonsson.henningsson@rjl.se(A.J.Henningsson).

withheadache,myalgiaandmalaiseaccompaniedbyleukopenia, thrombocytopeniaandelevatedhepaticenzymes.Thesymptoms aregenerallymildtomoderate,andsubclinicalinfectionsoccur. However, the disease course can be severe and even fatal in immunocompromisedindividuals(Bakkenetal.,1996b;Dahlgren etal.,2011;Jerebetal.,2012;Schotthoeferetal.,2014).SinceA. phagocytophilumsharesvectorswithBorreliaburgdorferisensulato (s.l.),co-infectionsoccur(Bakkenetal.,1996a;Nadelmanetal., 1997;Bjoersdorffetal.,1999b).Datafromanimalstudiessuggest thatsuchco-infectionsaremoresevere,possiblydueto immuno-suppressioninducedbyA.phagocytophilum(Bakkenetal.,1996b; Thomasetal.,2001).

TheprevalenceofA.phagocytophiluminﬁeld-collectedticksin Swedenwasrecentlyestimatedtobe1.3–15%(Severinssonetal., 2010).ThehumanseroprevalenceinSwedenhaspreviouslybeen reportedtobe8–21%(Dumleretal.,1997;Bjoersdorffetal.,1999b;

http://dx.doi.org/10.1016/j.ttbdis.2015.07.005

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788 A.J.Henningssonetal./TicksandTick-borneDiseases6(2015)787–792

Wittesjöetal.,2001),andtheseroconversionrates3.9–11.1%per yearinahighlyendemicareainthesouth-eastofSweden(Wittesjö etal.,2001).However,theimportanceofA.phagocytophilumasa humanpathogeninSwedenisstilluncertain,andlittleisknown aboutco-infectionswithB.burgdorferis.l.as wellastherisk of contractingHGAafterasingletickbite.

ThisstudywaspartoftheScandinavianTick-BorneDiseases (TBD)STINGstudydescribedbelow,andtheaimswereto inves-tigatetheprevalenceofA.phagocyophiluminticksthathavebitten humans,andtoevaluatetheriskforhumansofdeveloping clini-calHGAorsubclinicalseroconversionagainstA.phagocytophilum afteratickbiteinSwedenandontheÅlandIslands,Finland, includ-ingtheriskof diseaseafterabite bya tickco-infectedwithA. phagocytophilumandB.burgdorferis.l.

2. Materialandmethods

2.1. Studydesign

This study was part of the on-going TBD STING study (Wilhelmssonetal.,2010,2013;Frylandetal.,2011;Lindblometal., 2014),inwhichtick-bittenindividualsareaskedtodonatethetick alongwithabloodsampleattheirlocalprimaryhealthcare cen-tre(PHC).Threemonthslatertheparticipantsareaskedtocome backtothePHCandgiveasecondbloodsample.Atbothvisits, theparticipantsarealsoaskedtoﬁllinquestionnairesconcerning localityofthetickbite,symptoms,etc.Iftheparticipantsshould observeadditionaltickbitesduringthethreemonthsstudyperiod, theyareaskedtocollectthoseticksaswellanddonatethemtothe study(thesetickswerenotanalyzedinthepresentstudy).Medical recordsarescrutinizediftheparticipantsseekmedicalcare dur-ingthestudyperiod.Onlytick-bittenindividuals≥18yearsofage andwithnoimmunosuppressivediseaseortreatmentareincluded inthestudy.Noantibioticprophylaxisisgivenafterthetickbite butthestudyparticipantsareorderedtoseekmedicalcareincase theygetsymptoms.Theticksandbloodsamplesfromindividuals includedinthisparticularpartoftheTBDSTINGstudywere col-lectedduring2007–2009at34PHCsinSwedenandontheÅland Islands,Finland(Fig.1).

2.2. Ticks

The ticks that participants provided were transported and handledaspreviously described (Wilhelmsson et al., 2013).All ticks were examined under a USB microscope (Dino-Lite Long AM4013TL,AnMoElectronicsCorporation,Hsinchu,Taiwan)and photographed.Speciesandlifestageoftheticksweredetermined, aswellasthecoxalandscutalindices inorder toestimatethe feedingdurationaccordingtoGrayetal.(2005).

2.3. Tickhomogenization,totalnucleicacidextractionand reversetranscriptionofnucleicacid

Tickpreparation,homogenization,processoftotalnucleicacid (NA)extractionandreversetranscribedNA(RTNA)synthesishave beendescribedelsewhere;fortickscollectedin2007(Wilhelmsson etal.,2010)andfor tickscollectedin2008–2009(Wilhelmsson et al., 2013). Brieﬂy, total NA was extracted using a BioRobot M48workstation(Qiagen,Hilden,Germany)andMagAttract®_RNA

TissueMiniM48Kit(Qiagen).Theextractionswereperformed fol-lowingtheprotocolfromthemanufacturer,exceptfornotadding DNasetotheRDDbuffer,thusobtainingtotalNAincludingDNA. FortheRTNAsynthesis,20␮LoftheextractedtotalNAwasused togetherwiththeIllustraTM _Ready-to-Go_RT-PCR _Beads _kit _(GE

Healhcare,AmershamPlace,UK)accordingtothemanufacturer’s instructions.

Fig.1.Mapshowingthefourregionswherethe34primaryhealthcarecentres (PHCs)arelocated.(A)SouthernmostSweden(12PHCs).(B)South-centralSweden (18PHCs).(C)NorthernSweden(3PHCs).(D)TheÅlandIslands(1PHC).SE,Sweden; NO,Norway;FI,Finland;DK,Denmark;EE,Estonia;LV,Latvia;LT,Lithuania. ReprintedfromLindblometal.(2014)withpermissionfromElsevier.

2.4. Real-timePCRandconventionalPCRassays

Detection of A. phagocytophilum DNA was based on a real-time PCR method targeting the 16S rRNA gene. Each reaction consisted of 12.5␮L Maxima Probe qPCR Mastermix 2X (Fer-mentas St. Leon-Rot, Germany), 0.6␮L (10␮M)of each primer (AphGERfn 5-TAGATCCTTCTTAACGGAAGGGCG-3 and AphGERr 5-AAGTGCCCGGCTTAACCCGCTGGC-3) (Sigma–Aldrich Sweden AB, Stockholm, Sweden) (Goodman et al., 1996; Severinsson et al., 2010), 0.25␮L (10␮M) of the probe (AphGERp 5 -[6FAM]-CTGTCGTCAGCTCGTGTCGTGAGATGTTG-[BHQ-1]-3) (Sigma–AldrichSwedenAB),6.05␮LRNasefreewaterand5.0␮L cDNA. The reaction was performed according to the following thermalcycleconditions:95◦Cfor10min,45cyclesof95◦Cfor 15s,60◦Cfor30sand72◦Cfor30s.

The real-time PCR-positive samples were further analyzed with a nested conventional PCR assay to amplify a longer sequence of the 16S rRNA gene for species identification. The first reaction consisted of 5␮L Phusion HF buffer and 0.75␮L PhusionHFpolymerase(2U/␮L) (Thermo FisherScientificInc.), 2_␮L(10_␮M)ofeach primer(HGE15 -TCCTGGCTCAGAACGAAC-3andAph16S5nR5-TAGTCTTGCGACCGTAGTC-3)(Sigma–Aldrich SwedenAB,Stockholm,Sweden),35.25␮lRNasefreewaterand

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Table1

Numberofcollectedticks,theirlifestageandestimatedfeedingtime.

Total Larvae Nymphs Adults N.D. Medianfeeding-time(h)

Allcollectedticks 2553 89 1806 612 46 31b_(n₌₁₈₉₅₎

TickspositiveforA.p. 31 0 23 8 0 29(n=20)

TickspositiveforB.b.s.l. 30a ₀ ₂₁ ₉ ₀ ₂₆_(n₌₂₇₎

TicksnegativeforA.p.andB.b.s.l. 30a ₀ ₂₄ ₅ ₁ ₃₇_(n₌₂₈₎

N.D.,lifestagenotdetermined;A.p.,Anaplasmaphagocytophilum;B.b.s.l.,Borreliaburgdorferisensulato.

a_Randomly_selected_from_the_collected_ticks.

b_Median_duration_of_tick_feeding_is_based_on_nymphs_(n₌₁₄₅₁₎_and_adult_female_ticks_(n₌_444).

5␮lcDNA.Thereactionwasperformedaccordingtothefollowing thermalcycleconditions:98◦Cfor10min,35cyclesof94◦Cfor 30s,60◦Cfor45s,and72◦Cfor30s.Thiswasfollowedby72◦C for7min.Analiquot(5␮L)of thePCR productobtainedin this assaywasaddedtothesecondPCRmixture,whichwasprepared usingthesamevolumes,concentrations,andampliﬁcation pro-grammeasthoseusedfortheﬁrstmixture,exceptwithadifferent primer pair (Aph16SnF 5-CAAGCTTAACACATGCAAGTCG-3 and Aph16S6nR 5-GAGTGCTTAACGCGTTAGCTAC-3) (Sigma–Aldrich SwedenAB,Stockholm,Sweden),and thenumberofcycleswas increasedto42.TheobtainedPCRproductsweresentto Macro-genInc.(Seoul,SouthKorea),fornucleotidesequencingbasedon BigDyechemistry.Sequencechromatogramswereinitiallyedited usingBioEditSoftware(V7.0)(TomHall,IbisTherapeutics, Carls-bad,CA)followedbyastandardBLASTsearchagainsttheGenBank database(http://blast.ncbi.nlm.nih.gov).

Theextractioncontrolconsistedofareal-timePCRassay tar-getingtheIxodes16SmitochondrialDNA,previouslydescribedby

Schwaiger and Cassinotti (2003). Detectionof B.burgdorferi s.l. wasperformedbyreal-timePCRtargetingthe16SrRNAgeneas describedpreviously(Wilhelmssonetal.,2013).

2.5. Serumsamples

Theacuteandconvalescentserumsamples(collectedat inclu-sioninthestudyand afterthreemonths) correspondingtothe tickspositiveforA.phagocytophilum(GroupA)wereanalyzedfor thepresence ofA.phagocytophilumIgG antibodiesusinga com-merciallyavailableindirectimmunoﬂuorescentassay(IFA);(Focus Diagnostics,Cypress,CA,USA).Acuteandconvalescentserafrom individualsbittenbyB.burgdorferis.l.positiveticks(GroupB),and serafromindividualsbittenbyticksnegativeforbothA. phagocy-tophilumandB.burgdorferis.l.(GroupC)wererandomlyselected foranalysisregardingIgGantibodiesagainstA.phagocytophilum. Allserumsampleshadbeenstoredat−20◦_C_before_analysis_and

processedaccordingtothemanufacturer’sinstructions,exceptthat dilutions1:80,1:160,1:320,etc.wereused.Endpointtitrewas recordedasthereciprocalofthelastserialdilutionatwhich spe-ciﬁcapple-greenﬂuorescenceofAnaplasmainclusionbodieswere focallylocatedin the cytoplasmof theinfected cells. IFA titres ≥1:80wereinterpretedaspositive.Thiscut-offvaluewasbased onlocalSwedishserumsamplesusedwhenvalidatingthemethod (Bjoersdorffetal.,1999b).Forthediagnosisofon-goingorrecent A. phagocytophiluminfection,at leasta four-foldriseof theIFA titrewasrequiredwhenacuteandconvalescentserumsamples weretestedsimultaneously.Allserumsampleshadbeenanalyzed previouslyforthepresenceofantibodiesagainstB.burgdorferis.l. (Frylandetal.,2011).

2.6. Questionnaires

Thequestionnaireswerescrutinizedforsymptomssuggestive ofHGA;fever,headache,myalgia,arthralgiaandmalaise(Fryland etal.,2011).

2.7. Statistics

Comparison of clinical and serological databetween groups wereperformedusingthechi2_-test,_and_the_{Kruskal–Wallis}_test

wasappliedforcomparisonoffeeding-time(SPSSforWindows, version15.0).p-Values<0.05wereconsideredassigniﬁcant. 2.8. Ethics

ThestudywasapprovedbytheRegionalEthicsReviewBoardin Linköping(M132-06),andbythelocalEthicsCommitteeofÅland Healthcare,2008-05-23.Written consentwasobtainedfrom all participants.

3. Results

3.1. Ticks

Intotal,2553ticksthathadbittenhumanswerecollectedduring theyears2007–2009.AllthecollectedticksbelongedtotheIxodes ricinusspecies;89(3.5%)larvae,1806(70.7%)nymphs,612(24.0%) adults.Forty-six(1.8%)oftheticksweresodamagedthatlifestage couldnotbedetermined(Table1).

3.2. PrevalenceofA.phagocytophilumandB.burgdorferis.l.in theticks

Thirty-oneticks(1.2%)werepositiveforA.phagocytophilum,and eightofthemwereco-infectedwithB.burgdorferis.l.(0.3%).Five hundred-and-ﬁfty-sixticks(21.8%)werepositiveforB.burgdorferi s.l.alone(Wilhelmssonet al.,2013).OftheA. phagocytophilum-positiveticks,23werenymphsandeightwereadults.Feeding-time couldbeestimatedin20oftheA.phagocytophilum-positiveticks; median29h,range<15hto>69h.Therewerenosigniﬁcant differ-encesregardingestimatedtickfeeding-timebetweenthegroups A-C(pp=0.47)(Table1).

3.3. Seroprevalence,seroconversionandreportedsymptomsof thetick-bittensubjects

Acuteandconvalescentserumsamplesfromthethirty individ-ualsbittenbytickspositiveforA.phagocytophilumwereobtained (oneparticipantthathad beenbittenbyanA. phagocytophilum-and B.burgdorferis.l.-positiveadult tickwaslostto follow-up). ThepairedserafromtheindividualsbittenbyA. phagocytophilum-positivetickswereanalyzedforthepresenceofA.phagocytophilum IgG antibodies(Group A),aswellas serafromthirty randomly chosen individuals bitten bytickspositive for B. burgdorferis.l. (GroupB),andthirtyindividualsbittenbyticksnegativeforboth A. phagocytophilumandB.burgdorferi s.l.(Group C).Theoverall seroprevalenceforA.phagocytophiluminallthreegroupswas17% (15/90)withnosigniﬁcantdifferencebetweenthegroups(Group A:13%,GroupB:23%,GroupC:13%,p=0.45).Noneofthe partici-pantsreportedhavinghadconﬁrmedHGApreviously.Thedetected antibodytitreswere1:80(n=8)and1:160(n=7).Sixindividuals

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790 A.J.Henningssonetal./TicksandTick-borneDiseases6(2015)787–792 inGroupAreportedsymptoms(e.g.headache,neckpain,fatigue,

vertigo,myalgia,arthralgia),butnoneofthemdisplayedsigniﬁcant increaseinAnaplasmaantibodytitreorseroconversion.Twoofthe sevenindividualsbittenbyticksco-infectedbyA.phagocytophilum andB.burgdorferis.l.reportedsymptomsbutshowednoantibody responseagainstanyofthebacteria.SixindividualsinGroupBand ﬁveindividualsinGroupCreportedsymptoms(e.g.headache,neck pain,fatigue,vertigo,myalgia,arthralgia,paresthesia)butshowed noseroconversionorincreasedantibodytitresagainstA. phagocy-tophilumorB.burgdorferis.l.,exceptforonepatientinGroupCthat wasdiagnosed withLymeneuroborreliosis.Onlyoneindividual inthestudy(inGroupC)showedseroconversionforA. phagocy-tophilumatthethree-monthfollow-up,butreportednosymptoms. FourparticipantsshowedseroconversionforB.burgdorferis.l. dur-ingthestudyperiod(oneinGroupA,twoinGroupBandonein GroupC),butreportednosymptoms.

4. Discussion

Althoughincreasinglydetected,HGAisstillaratherunknown illnessinEurope(BlancoandOteo,2002;Strle,2004),asopposed tothesituationintheUSAwhereHGAisanotiﬁablediseasewith increasingincidence(CentersforDiseaseControlandPrevention, 2011).Underreporting,differencesinhostrangeorreduced vir-ulencein humans of the circulating A. phagocytophilumstrains inEuropemaybepossibleexplanations, butpreciseknowledge isstilllacking.Thereisa largeamountofgenetically diverseA. phagocytophilumlineagesinEurope(Tveten,2014),e.g.the mouse-associatedlineagescomprisingvariantsthatalsohavebeenisolated fromhumancases,andthedeer-associatedlineagesthatdonot appeartobeinfectiousfor humans(Massunget al.,2002).This impliesthatthereispossiblyaEuropeanriskscenariosimilarto theoneintheUSA.

Inthisstudy,wefoundanA.phagocytophilumprevalenceof1.2% inticksthathavebittenhumansinSwedenorontheÅlandIslands, whichiswithinthesamerangeastheprevalencefoundin ﬁeld-collectedticksfromSweden(Severinssonetal.,2010).Only0.3%of theanalyzedtickswereco-infectedwithA.phagocytophilumand B.burgdorferis.l.,similartotheco-infectionratesfoundin previ-ousstudiesonﬁeld-collectedticksandtickscollectedfrombirdsin Europe(Schichtetal.,2011;Coipanetal.,2013;SolengandKjelland, 2013;Capliginaetal.,2014).Toourknowledge,nopreviousdata onprevalenceofA.phagocytophilumandco-infectionswithother pathogensinticksdetachedfromhumanshasbeenreportedfrom northernEurope.

Theseroprevalenceof17%thatwefoundamongtheTBDSTING studyparticipantsisinaccordancewithpreviousstudiesonother tick-exposedpopulationsinSweden,i.e.patientswithLyme bor-reliosisorpeoplelivingintick-endemicareas(Dumleretal.,1997; Bjoersdorff et al.,1999b; Wittesjö et al., 2001). The seropreva-lence among healthy blood donors (probablya population less exposedtotickbitesthantheparticipantsintheTBDSTINGstudy) fromJönköpingcounty,usingthesameIFAandthesamecut-off levelasinthepresentstudy,hasbeenfoundtobe8.3% (unpub-lisheddata).Noneofthesubjectsinthisstudyhadbeendiagnosed withHGApreviously,whichmayindicatethatthediseaseis gen-erallymildoreven asymptomatic,but itmayalsoindicatethat physiciansrarelysuspectHGAintheirpatients.Inthisstudy, sub-jectsreceivingimmunosuppressivetreatmentswerenotincluded, anditisplausiblethatthecourseofHGAismoresevereinthe immunocompromised,ashasbeenshownpreviously(Bakkenetal., 1996b;Dahlgrenetal.,2011;Jerebetal.,2012;Schotthoeferetal., 2014).ThishasalsobeenshownforinfectionscausedbyCandidatus Neoehrlichiamikurensis,anothermemberofthefamily Anaplas-mataceae(Welinder-Olssonetal.,2010;Grankvistetal.,2014).

Weconcludefromourdatathattheriskof contractingHGA afteratickbiteislowevenifthetickisinfectedbyA. phagocy-tophilumandhasbeenfeedingfor≥29h.Thiscorroboratesprevious findingsfromcentralEurope(Tijsse-Klasenetal.,2011).Thereis possiblyadelayinthetransmissionofA.phagocytophilumfrom theticktothehost,butsincefeeding-timecouldbeestimatedin only20oftheinfectedticksandnoneofthestudyparticipants seroconverted,nofirmconclusionscouldbedrawnfromourdata regardingthis.DelayedtransmissionhasbeenshownforB. burgdor-feris.l.(Piesmanetal.,1987),andattachmentandpartialfeeding of aB. burgdorferis.l.-infected tickdoesnot necessarily leadto infectionordisease(Frylandetal.,2011)whichalsoseemstobe thecaseforA. phagocytophilumaccordingtoexperimental stud-iesonmicebyKatavolosetal.(1998).Theoverallseroprevalence of17%inthistick-bittenpopulationthat,however,didnotreport previouslydiagnosedHGA,mayalsoindicatealowriskfor clini-caldiseaseafterexposuretoA.phagocytophilum.Therehavebeen previousreportsofverifiedsymptomaticHGAcasesfromSweden, howevertheincidenceislow(Bjoersdorffetal.,1999a,2002).In EuropeancountriesthenumberofclinicallydiagnosedcasesofHGA islow,whiletheseroprevalenceofantibodiesagainstA. phagocy-tophilumis2–28%(Strle,2004).Suchhighlevelsofseropositivity inregionswithlownumbersofclinicalcasesmaybeexplainedby thecirculationofstrainsthatarenon-pathogenictohumansorby serologiccross-reactivitywithotherbacteria(RarandGolovljova, 2001).Evidencefromcross-infectionexperimentshaveindicated thatA.phagocytophilumisolatesofdistincthostoriginarenot uni-formlyinfectiousforotherheterologoushosts(RarandGolovljova, 2001;Jinetal.,2012).TheproportionofA.phagocytophilumstrains pathogenictohumansthatoccurinI.ricinusticksinEuropeisstill uncertain,andresultsfromdifferentmolecularstrain characteriza-tionstudiesarenoteasilycomparable,sincedifferentgeneregions andfragmentlengthshavebeeninvestigated.Arecentanalysisof thepopulationstructureofA.phagocytophilumbasedon multilo-cussequencetypingrevealedthatallstrainsisolatedfromhumans inEuropebelongedtothesamedominatingclonalcomplex(Huhn etal.,2014).

Thereportedsymptomsinanyofthestudygroupsare proba-blynotrelatedtoHGAorLymeborreliosis,sincenocorresponding antibodyresponsescouldbedetectedin thereportingsubjects. Only one participant showed seroconversion against A. phago-cytophilum atfollow-up, but reported nosubjective symptoms. ThisparticipanthaddonatedanA. phagocytophilum-,aswellas B.burgdorferis.l.,-negativetick,buthadprobably,inconjunction withthestudyperiod,hadadditionaltickbitesthatpassed unno-ticed.NoincreasedriskforHGAorLymeborreliosisafterabite byaco-infectedtickcouldbedetectedinthisstudy,howeverthe numberofindividualsbittenbyaco-infectedtickwaslow(n=7). The number of individuals that reported symptoms duringthe studyperioddidnotdifferbetweenthegroupsA-C,andseemed tobeunrelatedtowhetherthebitingtickcontainedA. phagocy-tophilum,B.burgdorferi s.l.,both or noneofthesetwo bacteria. Therefore,analysisofthetickafteratickbiteseemstobeoflittle ornovalueforpredictingdiseaseormakingtreatmentdecisionsin humans.

Weused acut-off intheIFA of1:80,as1:64and 1:80have beenwidelyusedforepidemiologicalpurposesbefore.However, ithaspreviouslybeendiscussedbyotherswhetherthesecut-offs maybesettoolow(Aguero-Rosenfeldetal.,2002;Walderetal., 2003),sinceasigniﬁcantproportionofthepopulationwithout clin-icalevidenceofHGAwillthentestpositiveforA.phagocytophilum antibodies.Furthermore,serologicalcross-reactionsmay compli-catetheinterpretationofIFAresults.Falsepositiveresultsmaybe duetoe.g.Epstein–Barrvirusinfection,Lymeborreliosisor autoim-munedisorders(Dumleretal.,2007),andthis,togetherwiththe uncertaintyofthepropercut-off,shouldbekeptinmindwhen

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interpretingtheresultsandmaybeanexplanationtothe discrep-ancybetweentheratherhighseroprevalencefoundinourstudy populationandthelowprevalenceofA.phagocytophilumfoundin theanalyzedticks.However,Dumleretal.(1997)founda sero-prevalenceof11.4%amonginhabitantsontheKosterislandoffthe westerncostofSweden(cut-off1:80),Wittesjöetal.(2001)founda seroprevalenceof28%ininhabitantsontheAspöislandlocatedoff thesoutheastcoastofSweden(cut-off1:80),andSkarphedinsson etal.(2001)foundaseroprevalenceof21%amongDanishpatients withconﬁrmedorsuspectedLymeborreliosis(cut-off1:64),sothe seroprevalenceof17%foundinourstudyseemsreasonable.As dis-cussedabove,itmayrepresentanover-estimate,buttheselected cut-offallowsforthecomparison.

Another possible limitationto the study is that the second serumsampleswerecollectedafterthreemonths,anditcannot becompletelyruledoutthata fewofthesubjectsbittenbyan A.phagocytophilum-infectedtickactuallydidseroconvertbutthe antibodytitrehaddroppedagainatfollow-up.However,thisseems ratherunlikelysinceantibodiesusuallyremainforseveralmonths orlonger(Bakkenetal.,2002;Aguero-Rosenfeld,2003).

Inconclusion,theprevalenceofA.phagocytophiluminticksthat havebitten humansdonot differfromtheprevalencefoundin ﬁeld-collectedticksinSwedenandontheÅlandIslands.The sero-prevalenceof17%amongthestudyparticipantsindicatethatthe populationinthestudyareaisexposedtoA.phagocytophilum,but theinfectionisseeminglyoftenunnoticedoratleastundiagnosed sincenoneoftheseropositiveindividualsreportedapreviousHGA infection.TheriskofcontractingHGAafterasingletick-biteis how-everlow,evenifthetickisinfectedwithA.phagocytophilumand hasbeenfeedingforseveraldays.Thus,analysisofthetickaftera tickbiteisnotclinicallyusefulandshouldbeavoidedsinceitmay leadtounnecessaryanxietyforthetick-bittenindividualaswellas unfoundeduseofantibiotics.Anyﬁrmconclusionswhethertherisk ofdiseaseisincreasedornotafterabitebyatickco-infectedwithA. phagocytophilumandB.burgdorferis.l.couldnotbedrawnfromthis studyduetothelownumberofco-infectedticks.Lymeborreliosis andHGAco-infectionsneedtobefurtherevaluated,sinceprevious dataisambiguous(Belongiaetal.,1999;Krauseetal.,2002;Steere etal.,2003;Horowitzetal.,2013),andanimalstudiessuggestan aggravatedcourseofsuchinfections(Bakkenetal.,1996b;Thomas etal.,2001).ThecontinuingTBDSTINGstudyprovidespossibilities tobringnewinsightsintotheinterplaybetweenticks,tick-borne pathogensandhumans,andpermitsimprovedriskassessments forhumandiseasefollowingatickbitewhichisimportantfroma publichealthperspective.

Acknowledgements

Theauthorswouldliketothankallparticipantsofthestudy aswellasthestaff atthePHCs participatingintheTBD STING study.We wouldalsoliketothanktheTBDSTINGstudygroup, consisting of Clas Ahlm, Johan Berglund, Sten-Anders Carlsson, LindaFryland,MatsHaglund,PontusLindblom,PeterNolskogand KatharinaOrnsteinfor allthevaluable workandadvice onthe studydesign.AspecialthankstoLiselottLindvall,Susanne Olaus-son,Mari-AnneÅkesonandHeleneJardeforsfortheircontinuous workwiththestudyandcollectionlogistics,toAnnikaLindkvist forexcellentworkwiththeIFA,toJanErnerudhforadviseonthe studydesign,andtoDagHvidstenforkindlyprovidingblooddonor seraforvalidationoftheIFAmethod.Thisstudywassupported byTheSwedishResearchCouncil,TheMedicalResearchCouncil ofSoutheast Sweden (FORSS),FuturumAcademy ofHealthcare, Jönköping County Council, the Interreg IV A Programme Scan-dTick,andtheDivisionofMedicalServices,RyhovCountyHospital, Jönköping.

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