Synergistic activity of antibiotics combined with ivermectin to kill body lice

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Synergistic activity of antibiotics combined with

ivermectin to kill body lice

Abdoul Karim Sangare, Jean-Marc Rolain, Jean Gaudart, Pascal Weber,

Didier Raoult

To cite this version:

Abdoul Karim Sangare, Jean-Marc Rolain, Jean Gaudart, Pascal Weber, Didier Raoult. Synergistic

activity of antibiotics combined with ivermectin to kill body lice. International Journal of

Antimicro-bial Agents, Elsevier, 2016, �10.1016/j.ijantimicag.2016.01.001�. �hal-01307125�

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ContentslistsavailableatScienceDirect

International

Journal

of

Antimicrobial

Agents

jou rn a l h om ep a ge : h t t p : / / w w w . e l s e v i e r . c o m / l o c a t e / i j a n t i m i c a g

Synergistic

activity

of

antibiotics

combined

with

ivermectin

to

kill

body

lice

Abdoul

Karim

Sangaré

a

_,

_Jean

_Marc

_Rolain

a

_,

_Jean

_Gaudart

b

_,

_Pascal

_Weber

a

_,

Didier

Raoult

a,∗

a_{Aix-Marseille}_Université,_IHU_{Méditerranée}_Infection,_URMITE,_UM63,_CNRS_7278,_IRD_198,_INSERM_1095,_Faculté_de_Médecine_et_de_Pharmacie,

27Bd.JeanMoulin,13385Marseillecedex5,France

b_{Aix-Marseille}_Université,_UMR912_SESSTIM_{(AMU-Inserm-IRD),}_Department_of_Public_Health_and_Medical_Information,

CentreHospitalierUniversitaireLaTimone,13005Marseille,France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received26October2015 Keywords:

Pediculushumanushumanus Antibiotics

Ivermectin Synergistictreatment

a

b

s

t

r

a

c

t

Ivermectinanddoxycyclinehavebeenfoundtobeindependentlyeffectiveinkillingbodylice.Inthis study,450bodylicewereartiﬁciallyfedonaParaﬁlmTM_membrane_with_human_blood_associated_with

antibiotics(doxycycline,erythromycin,rifampicinandazithromycin)aloneandincombinationwith ivermectin.Fluorescenceinsituhybridisationandspectraldeconvolutionwereperformedtoevaluate bacterialtranscriptionalactivityfollowingantibioticintakebythelice.Intheﬁrstseries,alethaleffectof antibioticsonlicewasobservedcomparedwiththecontrolgroupat18days(log-ranktest,P≤10−3),with asigniﬁcantdifferencebetweengroupsintheproductionofnits(P=0.019,Kruskal–Wallistest).Ahigh lethaleffectofivermectinalone(50ng/mL)wasobservedcomparedwiththecontrolgroup(log-rank test,P≤10−3).Fluorescenceofbacteriocytesinlicetreatedwith20␮g/mLdoxycyclinewaslowerthan inuntreatedlice(P<0.0001,Kruskal–Wallistest).Inthesecondserieswithantibiotic–ivermectin com-binations,asynergisticlethaleffectontreatedlice(log-ranktest,P<10−6₎_was_observed_compared_with

thecontrolgroupat18days,associatedwithasigniﬁcantdecreaseintheproductionofnits(P≤0.001, Kruskal–Wallistest).Additionally,survivalofliceinthecombinationtreatmentgroupscomparedwith ivermectinalonewassigniﬁcant(log-ranktest,P=0.0008).Thesedatademonstratethatthesynergistic effectofcombinationsofantibioticsandivermectincouldbeusedtoachievecompleteeradicationoflice andtoavoidselectionofaresistantlousepopulation.

1. Introduction

ThebodylousePediculushumanushumanusisastrictlyhuman parasite,livingandmultiplyinginclothing;theirinfestationis asso-ciatedwithcoldweatherandalackofhygiene[1].Itsprevalence reﬂectsthesocioeconomiclevelof thesociety[2].In developed countries,infestationismorecommonamongthehomeless pop-ulationand variesfrom 7%to22% [3]. Bodyliceare extremely contagious and posea seriouspublic health problem. Theyare vectorsofthepathogensRickettsiaprowazekii,Bartonellaquintana andBorreliarecurrentis,whichareresponsibleforepidemictyphus, trenchfeverandrelapsingfever,respectively[1,4],andtheymay beconsideredaplague[5].

∗ Correspondingauthor.Tel.:+33491324375;fax:+33491387772. E-mailaddress:didier.raoult@gmail.com(D.Raoult).

Fordecades,severalantibioticshaveprovedtobeeffectiveinthe treatmentoflouse-bornediseases.Asingledoseof200mg doxycy-clinecancurepatientssufferingfromR.prowazekii-inducedtyphus

[6],and500mgoftetracyclineorerythromycinisoptimal ther-apyforthetreatmentofB.recurrentisinfections[7].Azithromycin hasbeenrecommendedforthetreatmentofB.quintanainfections

[8].Inaddition,rifampicinwasshowntobeeffectiveagainstsome intracellular bacteria[9].These resultsindicatethepotentialof antibioticsforthemanagementoflouse-bornediseases.Recently, wedemonstratedtheinvitroefﬁcacyofdoxycyclineatdifferent dosesonbodyliceandfoundthatdoxycyclinehasadirecteffecton endosymbiontsoflicethroughthemycetomeswitch[10].

Severalstrategiesforeradicationoftheseparasiteshavebeen usedtoavoidthespreadand/oroutbreakofthesediseases. How-ever,someproductshavealreadyshowntheirlimits.Theresistance oflicetolindaneinEurope,AfricaandAsia[11]andtoDDTinKorea andJapan[12,13]hasbeenreported.Pyrethroidshavebeenwidely used,howeverresistancetothesecompoundshasemergedand

http://dx.doi.org/10.1016/j.ijantimicag.2016.01.001

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218 A.K.Sangaréetal./InternationalJournalofAntimicrobialAgents47(2016)217–223

spreadoverthelastdecade.Intheheadlouse,resistanceto per-methrinwasﬁrstreportedinFrancein1994andthroughoutthe worldthereafter[14].Ivermectinisasyntheticderivativeofthe antiparasiticclassofcompoundsknownasavermectins[15]. Resis-tancetoivermectinandrelateddrugsisanincreasingproblemfor parasitecontrol[16].PreviousstudieshaveassociatedATP-binding cassette (ABC) transporters with drug resistancein nematodes

[17]andarthropods[18].However,resistancetoivermectinhas alsobeendocumentedininsectsandmitesofplants[19]andin nematodes[16].Resistancetoivermectinhasbeendocumentedin Rhipicephalus(Boophilus)microplusticksinMexico[20].The occur-renceofresistancetomacrocycliclactoneshasbeenreportedin otherarthropods[21].Usedinscabies,clinicalandinvitro resis-tancehavebeendocumentedintwopatientswhoreceived30and 58dosesofivermectin,respectively[22].

Today,post-therapeuticre-infestationofthevectorofdiseases (lice) is commonand still remains a realchallenge, thus there is a needto develop newtherapeutic strategies toavoid these re-infestations.ItwasobservedthatavermectinB1acouldact syn-ergisticallywiththeantibioticmeticillintokillmeticillin-resistant Staphylococcusaureus(MRSA),renderingthebacteriaonceagain vulnerable tometicillin [23]. The hypothesis that the bacterial symbiontCandidatus Riesiapediculicola is a possible target for the development of louse control strategies [24] was recently conﬁrmedinourpreviouswork[10].Inthisstudyweaimedto demonstratetheinvitrocombinationofivermectinwithseveral antibioticsasasynergisticassociationtoachievecomplete eradi-cationofliceandtoavoidtheselectionofaresistantpopulationof lice.

2. Materialsandmethods

2.1. Lousefeedingsysteminvitro

A long-established inbred line of the humanbody louse (P. h.corporis, Orlando strain), which had been bred onrabbits in alaboratorypetshopbyourURMITEteaminMarseille,France, wasused as a model in the experiments in this study[25]. A HemotekTM_feeding_system_(Discovery_Workshops,_Accrington,_UK) withaParafilmTM_membrane_(Chicago,_IL),_a_PS5A/220_Power_Unit (HemotekTM_5W1_System;_Discovery_Workshops)_and_six_heating unitswasused(Fig.1).Asquareofsyntheticfeedingmembrane measuringca.3cm×3cm wasstretchedaroundtheapertureof thebloodmealreservoirandwassecuredwithanO-ring(Fig.1a). Thefeedingareawassufficientfor100lice(Fig.1b).The blood mealreservoirwasremovableforeasycleaningandautoclaving ifnecessary.Thesystemdoesnotuseanimalhostsandenables modificationofthemealbyintroductionofchemicalsorinfectious agentsandaccurateadjustmentofthetemperaturethrough heat-ingunits(Fig.1c)connecteddirectlytothepowerunit(Fig.1d). Licewerefedeverydayat37◦Cfor1h(Fig.1e).

2.2. Preparationofbloodmealsupplementedwithantibioticsand ivermectin

HumanbloodgroupArhesuspositive(A+₎_[code₇₄₈₈₃_from theFrenchBloodService(EFS)]wasused.Ethicalapprovalforthe useofhumanblood invitro wasobtainedfromtheLaboratory ResearchEthicsBoardofMolecularHematology,EFS.Bloodwas distributedinto5mLtubesandwaskeptat−20◦_C_before_use._The bloodwaswarmedinawaterbathat37◦Cfor30minbeforeuse. Drugsusedwereininjectableandoralformasfollows:doxycycline 20mg/mL (Vibraveineuse® batch A262408; Laboratoires SERB, Paris,France);erythromycin1g(Erythrocine®batch24370TB22; AmdipharmLtd.,Dublin, Ireland);rifampicin 600mg (Rifadine®

batchA3468;Sanofi-Aventis,Paris,France);azithromycin600mg (Azadose®batch490004;PfizerHolding,Paris,France);and iver-mectin10mg/mL(Ivomec®batchBM295/12;Merial,Lyon,France). For each group of lice,15␮L of uncombined antibioticdiluted atconcentrations(doxycycline20␮g/mL,erythromycin4␮g/mL, rifampicin10␮g/mLandazithromycin8␮g/mL)previously estab-lishedforthefirstseriesofexperimentswasaddedtoavolumeof 1.485mLofhaemolysedblood.Inthesecondseriesforeachgroup oflice,15_␮Lofantibiotic(doxycycline20_␮g/mL, erythromycin 4␮g/mL, rifampicin 10␮g/mL and azithromycin 8␮g/mL) plus 15␮Lofivermectindilutedataconcentration(50ng/mL) previ-ouslyestablishedwasaddedtoavolumeof1.470mLofhaemolysed blood.Thefinalvolumeof1.5mLobtainedforeachconcentration wasintroducedintodifferentreservoirs.Thereservoir wasthen attachedtothefeederbyscrewingitontoanylonstudontheheat transferplateatthebottomofthefeeder.

2.3. Monitoringofliceandnits

Intotal,450adultlice[excluding10licesacrificedaslive con-trolsinfluorescenceinsituhybridisation(FISH)]wereusedinthe experiments.Liceweredividedintogroupsof45,wereplacedon apieceofblackclothandwerekeptinjarsinanovenat29◦Cand 90%humidityasbreedinglice.Thefirstseriesofexperimentswas performedwithoutcombinationofdrugsandthesecondwasbased ontheantibiotic–ivermectincombinations.Agroupoflicetreated withivermectinalone wasmonitoredinparallelwiththe com-binationseries.Thecontrolgroupwasfedexclusivelywithblood withoutdrugsthroughouttheexperiments.Licewereplacedonthe membranedailyfor1hofbloodmealfeeding.Everyday,survival wasdeterminedbyassessingthenumberofnitsineachgroupand wasrecordedonadatacollectionsheetbeforethenextmeal (Sup-plementaryTablesS1andS2).Alloftheexperimentswererepeated threetimesforreproducibility.

Supplementary Tables S1 and S2 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.

ijantimicag.2016.01.001.

2.4. Fluorescenceinsituhybridisation(FISH)

FortheFISHtechnique,theaimwastohighlighttheeffectof theantibiotic(especiallydoxycycline)onthelouseendosymbiont aspreviouslydemonstrated[10].Thus,tenspecimensweretested ineachuntreated group(livingcontrolsanddeadcontrols)and inthedoxycycline20g/mLgroup(Table1).Followingﬁxation, samplesweredecolourisedin6%H2O2inethanolfor2handwere thenplacedinhybridisationbuffer[20mMTris–HCl(pH8.0),0.9M NaCl,0.01%sodiumdodecylsulphate and30%formamide] con-taining10pmolofﬂuorescentprobe/mLbasedontheCandidatus Riesiapediculicola 16SrRNA sequence:Cy3-Lice1255R (5 -Cy3-TTGGCTCGCTCTTACGAGT-3)[10].

2.5. Lightandconfocalmicroscopy

Thespectraldeconvolutiontechniquewasusedtodifferentiate autofluorescencecausedbychitinfromcleanfluorescence.First, thelousewasmarkedandmountedbetweenslideandcoverslip andimagingwasperformedasfollowsusingaLeicaSP5AOBS® con-focalresonantscanner(LeicaMicrosystems,Mannheim,Germany) inLambdamode.Thesamplewasscannedonatotalbandof160nm andaslitwidthof5nmtakingcaretoneversaturatethesignal. Fluorescencemeasurementwasdefinedastheextractionofcurves (oneforfluorescenceandoneforautofluorescence).Aftercreation ofthereferencecurves,allacquisitionsweremadewithexactlythe samesettingsforgain,offset,laserpowerandobjective.Foreach acquisition,32pictureswerecollected.Oncethereferenceswere

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Fig.1.TheHemotekTM_5W1_membrane_feeding_system_for_lice:_(a)_the_device’s_meal_reservoir_(a1,_blood_meal_reservoir;_a2,_plastic_ring;_and_a3,_ParaﬁlmTM_);_(b)_assembling

thedevice;(c)theheatingunits;(d)thePS5powerunit;and(e)bloodmealfeedingofliceonartiﬁcialmembrane.

Table1

Measurementofﬂuorescenceintensityinthreepopulationsoflice.

Group Day Totalarea Surfacemarked Intensityratio Doxycycline 20␮g/mL 1 16,217 1205 7.4 2 4808 325 6.7 3 29,970 1385 5.9 4 32,994 2179 6.6 5 19,730 1190 6 6 29,211 1537 5.2 7 51,047 2468 4.8 8 20,479 1216 4.6 9 37,185 1231 3.3 10 16,231 395 2.4 Dead con-trols 1 8977 1462 16.2 2 9523 1275 13.3 3 9488 1358 14.3 4 13,279 1755 13.2 5 12,391 1518 12.2 6 9873 1280 12.9 7 9173 1051 11.4 8 10,506 1163 11 9 9923 986 9.9 10 12,675 1269 10 Living con-trols 1 12,861 1943 15.1 2 12,496 1605 12.8 3 11,058 1467 13.2 4 7558 1249 16.5 5 10,913 1134 10.3 6 7945 1156 14.5 7 8962 1580 17.6 8 12,381 1397 11.2 9 9914 1290 13 10 13,091 1898 14.4

determinedandassigned,thesampleswerespectrallydeconvolved andthesignalswereseparatedbyassigningthemadistinctcolour. 2.6. Calculationofintensityratios

MetaMorphv.4.6(MolecularDevices,Sunnyvale,CA)anda gen-eratedExceltable(MicrosoftCorp.,Redmond,WA)wereusedto calculatetheintensityratios.

2.7. Statisticalanalysis

Data werestored in Excel.Analyses wereperformed withR softwarev.3.1.1(RFoundationforStatisticalComputing,Vienna, Austria).Kaplan–Meierestimationsandlog-ranktest wereused for survivalanalysis.AnalysisofKruskal–Wallisnon-parametric testwasusedformeancomparisons.Bonferronicorrectionwas appliedforsubgrouptests.Thestatisticaltestsrepresentbilateral situations,andP-valuesof≤0.05wereconsideredsigniﬁcant.

3. Results

Feedingliceonartiﬁcialmembranesenabledthemtobetreated withantibioticsandivermectin.Thebloodfeedingratewas>95% inalltreatmentarms(Fig.1e).

3.1. Effectofantibioticsonthesurvivalofliceandonnit production

To determine the effect of antibiotics, in the ﬁrst series of experiments each group of lice treated with either doxycy-cline 20␮g/mL, erythromycin 4␮g/mL, rifampicin 10␮g/mL or azithromycin8_␮g/mLaswellasthecontrolswerefolloweduntil theend.Survivalofliceinthecontrolgroupwassigniﬁcantly dif-ferentfromsurvivalinthetreatedgroups(log-ranktest,P≤10−3)

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Fig.2.Evolutiontrendsintheantibiotictreatmentgroups.(a)Kaplan–Meiercurvesillustratingsurvivaloflicetreatedwithantibioticsincomparisonwiththecontrolgroup. (b)Mean(±95%CI)numberofeggslaidperdayinthedoxycycline-,erythromycin-,rifampicin-andazithromycin-treatedgroupsandthecontrolgroup.CI,conﬁdence interval;Doxy,doxycycline;Ery,erythromycin;Rifam,rifampicin;Azith/Azithro,azithromycin.

(Fig. 2a). Moreover, a decrease in the production of nits was observedinthetreatedgroupscomparedwiththecontrolgroup (Fig.2b),withmean[95%conﬁdenceinterval(CI)]numberofeggs laidper dayof 7.5 (2.5–12.5), 9.8(4.3–15.3), 10 (4.6–15.6), 7.7 (2.2–13.2)and17.8(13.3–22.3)inthedoxycycline-, erythromycin-, rifampicin- and azithromycin-treated groups and the control group,respectively. Thedifferencein mean nitproduction was signiﬁcantbetweenthegroups(P=0.019,Kruskal–Wallistest). 3.2. Directeffectofdoxycyclineonthelicemycetomeanalysed throughFISH

Toshowthatdoxycyclineaffectsthelouse’sendosymbionts,we focusedonanewanalysisbasedonspectraldeconvolutionto cal-culatetheratiosbetweenthetwosignals(Table1).Byusingthe functionextractioncurvecomputation ofreplyinemission and positioningthereferenceareasondistinctivesignals,twocurves were extracted:one for specific fluorescence of the bacterium andoneforautofluorescenceofthelouse.Thus,theresultsshow thatbacterialfluorescenceinthegrouptreatedwithdoxycycline 20_␮g/mL(Fig.3a)waslowerthanintheuntreatedgroups(living anddeadcontrols)(Fig.3bandc)(P<0.0001,Kruskal–Wallistest). 3.3. Effectofivermectinaloneonthesurvivalofliceand

productionofnits

Todeterminetheantiparasiticactionofivermectinalone,lice treatedwith50ng/mLivermectinwerefolloweduntiltheendof theexperiment.Ahighmortalityratewasfoundcomparedwith thecontrolgroup(log-ranktest,P_≤10−3)at18days(Fig.4a).A signiﬁcantdifferencebetweengroupswasobservedinmeannit production (P=0.001,Kruskal–Wallis test),with mean(95% CI) numberofeggslaidperdayof5.2(0.6–9)and17.8(13.3–22.3), respectively(Fig.4b).

3.4. Synergisticeffectofantibioticsandivermectinonsurvivalof liceandproductionofnits

Todeterminetheantiparasiticsynergisticactionofivermectin combined with antibiotics, in a second series of experiments ﬁve groups of lice were tested for their survival kinetics. In

the combination groups (doxycycline 20␮g/mL+ivermectin 50ng/mL, erythromycin 4␮g/mL+ivermectin 50ng/mL, rifampicin 10␮g/mL+ivermectin 50ng/mL or azithromycin 8␮g/mL+ivermectin 50ng/mL), lice were more rapidly killed compared withthecontrol group,exhibiting potentsynergistic action (log-rank test,P<10−6) (Fig. 5a).Moreover, a significant decreaseintheproductionofnitswasobservedinthecombination groupscomparedwiththecontrolgroup(Fig.5b),withmean(95% CI)eggsperdayof2.4(−0.9to5.8),4.4(−0.1to9.0),4.0(−0.6 to8.6),3.6(−0.5to7.7)and17.8(13.3–22.3),respectively.Thus, thedifferencein mean production ofnits between groups was significant(P≤0.001,Kruskal–Wallistest).Moreover,overall sur-vivalofliceinthecombinationtreatmentgroupscomparedwith ivermectin alone wasalso significant(log-rank test, P=0.0008) (Fig.6a).Therefore,thedifferencewassignificantwhencomparing thecombinationgroupswiththeivermectinandcontrolgroups (Fig.6b).

4. Discussion

Fordecades,completeeradicationofhumanlicehasremained challenging.Inthefirstinvitroseriesofexperiments,thelethal effectoftheantibioticsdoxycycline,erythromycin,rifampicinand azithromycin on lice wasobserved compared withthe control groupat18 days, associatedwitha decreasein theproduction ofnits.Ofthefourantibioticstested,doxycyclineappearedtobe moreeffectivethan rifampicin,azithromycinand erythromycin, withavariablesurvivaltimeaccordingtotreatmentgroup(Fig.2a; SupplementaryTableS1).Recently,theinvitroefficacyof doxy-cyclineatdifferentdosesonbodylicewasdemonstrated[10].In lightoftheseresults,we cansuggest thattheuseofantibiotics (doxycycline,rifampicin,azithromycinanderythromycin)aloneor incombinationcouldbeanefficientwaytodevelopnewdrugsand toovercomedrugresistanceinlice.

Severaldecadesagoitwasdemonstratedthatbodylicedepend on obligate endosymbionts to supplement their nutritionally-deﬁcientblood diet[26].However,theymaintainorganscalled mycetomes that house the primary endosymbiont [27]. Thus, throughFISHanalysiswewereabletodemonstratethatdamageto thesymbiontbacteriacausedthedeathoftheirhostasconﬁrmed

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Fig.3. Imagesoflicebyconfocalmicroscopythroughthetechniqueofspectraldeconvolution:(a)lousetreatedwithdoxycycline20␮g/mLtakenatDay10showinglower bacterialfluorescence;(b)livingcontrolshowinghigherbacterialfluorescence;(c)deadcontroltakenatDay10showinglowerbacterialfluorescence;and(d)lousetested withnegativeprobeshowingnobacterialfluorescence.

Fig.4.Evolutiontrendsintheivermectinalonetreatmentgroup.(a)Kaplan–Meiercurvesillustratingsurvivaloflicetreatedwithivermectin(Iv)incomparisonwiththe controlgroup.(b)Mean(±95%CI)numberofeggslaidperdayintheivermectin(Iver)-treatedgroupandthecontrolgroup.CI,conﬁdenceinterval.

byadecreaseinbacterialﬂuorescence(Fig.3a).Theseresults cor-roborateourpreviousworkshowingthatdoxycyclinemayaffect theendosymbiontsoflicethroughthemycetomeswitch,thus caus-ingdeathofthelouse[10].

Despitetheintroductionofsomeproductssuchas organophos-phates,pyrethrinsandpyrethroids,insecticideresistanceremains anincreasingprobleminmanyinsectvectorsofdisease[14], espe-ciallyamonglice.ExpressionlevelsofABCtransportershavealso beenassociated withdrugresistance in arthropods and nema-todes.OverexpressionoftheseABCtransportersisassociatedwith ivermectinresistance inHaemonchus contortus[16], Caenorhab-ditis elegans[17],P.h. humanus[28],Sarcoptes scabiei [29] and

R.microplus[18].Inacohort ofhomelesssubjectsinMarseilles (France),aprevalenceofbodyliceof85%wasobserved[30].This infestationwasreducedtemporarilyto19%withthreedosesoforal ivermectinadministeredat7-dayintervals.However,treatment failure or early recrudescence is commonfollowing ivermectin alone,andmultipledosesareusuallyrequiredtoachieveacure

[31].Moreover,intheirrandomisedtrialChosidowetal.suggest thativermectincouldbeanalternativetreatment[32],butits tran-sienteffectwasalsodemonstratedinsomestudies[3].Theﬁndings ofthecurrentstudyshowthetransienteffectofivermectinalone onlicewithamonitoringtimeof10days,associatedwithaslow decreaseintheproductionofnits(Fig.4;SupplementaryTableS2).

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Fig.5. Evolutiontrendsintheantibiotic–ivermectincombinationtreatmentgroups.(a)Kaplan–Meiercurvesillustratingsurvivaloflicetreatedwiththeantibiotic–ivermectin combinationsincomparisonwiththecontrolgroup.(b)Mean(±95%CI)numberofeggslaidperdayinthecombinationtreatmentgroupsandthecontrolgroup.CI,conﬁdence interval;Iv/Iver,ivermectin;Doxy,doxycycline;Ery,erythromycin;Rifam,rifampicin;Azith/Azithro,azithromycin.

Fig.6.Evolutiontrendsintheantibiotic–ivermectincombinationtreatmentgroupsandtheivermectinalonetreatmentgroup.(a)Kaplan–Meiercurvesillustratingsurvival oflicetreatedwithantibiotic–ivermectincombinationsandwithivermectinalone.(b)Kaplan–Meiercurvesillustratingthecomparisonofoverallsurvivalofliceinthe antibiotic–ivermectincombinationgroups,theivermectinalonegroupandthecontrolgroup.Iv/Iverm,ivermectin;Doxy,doxycycline;Ery,erythromycin;Rifam,rifampicin; Azith,azithromycin.

Thus,inlightofthesevarioustherapeuticfailures,wemay con-cludethativermectincouldbeusefulwithotherdrugsofdifferent classesespeciallyantibiotics,inthecontrolofbodyliceinfestation. The efﬁcacyof drugcombination withivermectin hasbeen demonstratedinnematodes[33]andectoparasites[34].For iver-mectin,itwassuggestedthatheadlicemaybemoresusceptible thanbodylice;thiscouldbejustiﬁedbythemodelofMumcuoglu etal.,whoobservedhighmortalityofnymphsandfemalebodylice fedonrabbitstreatedwith200␮g/kgivermectinfor3days, fol-lowedbyasharpdeclinereachingthelevelofthecontrolsonDay6

[35].Inlightofthevarioustherapeuticfailuresdescribedinlice,we decidedtestantibioticcombinationswithivermectin.Inthe sec-ondseriesofinvitroexperiments,wewereabletodemonstrate theantiparasitic synergisticactivityofa combinationofseveral drugs.Themonitoringtimerangedfrom5daysto7daysinthe

combinationgroup,whilstthatofivermectinalonewas10days (SupplementaryTableS2).Interestingly,thedifferenceinoverall survivalofliceinthecombinationtreatmentgroupscomparedwith ivermectinalonewasalsosigniﬁcant.Thus,theseresultshighlight theefﬁcacyofdrugcombinationscomparedwithivermectinalone. Intheliterature,compoundsincludingantibioticshavebeenshown to increase intracellular concentrations of macrocyclic lactones

[36].Inaddition,itwasshownthatdrugcombinationsincluding ivermectinprovideantiﬁlarialactivitywithancillarybeneﬁtson intestinalhelminthsand ectoparasitessuchaschiggersandlice

[34].Whendoxycyclinewascombinedwithmacrocycliclactones inadultworms,ivermectinefﬁcacywasca.80%compared with 9%whentreatmentwasperformedwithdoxycyclinealone[37]. Hoeraufetal.recommendconcurrentadministrationofivermectin withdoxycyclineinthetreatmentofOnchocercawormscarrying

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Wolbachia endobacteria[38], and later Coulibalyet al. found a significantreduction of circulatingantigen levelsof Wuchereria bancroftiingroupstreatedwithacombinationofdoxycyclineplus ivermectinfor12months[39].Theeffectofthiscombinationhas also been confirmed in dogs naturally infected with Dirofilaria immitis[40].Thus,thecurrentresultsareinagreementwiththe literature,especially that of Guoet al., which identifies forthe firsttimethatavermectin(macrocycliclactonederivativeincluding ivermectin)could act synergisticallywith meticillin(an antibi-otic)tokillbacteria(MRSA)[23].Inaddition,thecurrentstudy demonstratesthefirstsynergisticassociationofthesedrugsinvitro againstbodylice.Webelievethatthesynergisticeffectof com-binationsofantibioticsplusivermectincouldbeusedtoachieve completeeradicationofliceandtoavoidtheselectionofaresistant populationoflice.

5. Conclusion

Thepresentstudydemonstratesasynergisticeffectof antibi-oticscombinedwithivermectintoevenmoreeffectivelycontrol lice.Thus,itwouldbeinterestingtodevelopthismodelinvivoin thecontrolofliceinfestationinhumans.

Acknowledgement

TheauthorsaregratefultoJean-MichelBerengerfortechnical support.

Funding:Thisworkwassupportedby‘IHUMéditerranéeInfection’ (Marseille,France).

Competinginterests:Nonedeclared.

Ethicalapproval:Ethicalapprovalfortheuseofhumanbloodinvitro wasobtainedfromtheLaboratoryResearchEthicsBoardof Molec-ularHematology,FrenchBloodService(EFS).

Patent:Title:‘Composedandcombinationofcompoundsforthe treatmentoflice’.Yearofsubmission:2014.

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