ContentslistsavailableatScienceDirect
Acta Tropica
j ou rn a l h o m epa g e :w w w . e l s e v i e r . c o m / l o c a t e / a c t a t r o p i c a
Detection of a broad range of Leishmania species and determination of parasite load of infected mouse by real-time PCR targeting the
arginine permease gene AAP3
Marit Gjerde Tellevik
a,∗, Karl Erik Muller
b, Karen Rebbestad Løkken
b, Audun Helge Nerland
baNationalCentreforTropicalInfectiousDiseases,DepartmentofMedicine,HaukelandUniversityHospital,5021Bergen,Norway
bDepartmentofClinicalScience,UniversityofBergen,5021Bergen,Norway
a r t i c l e i n f o
Articlehistory:
Received19November2013 Receivedinrevisedform13May2014 Accepted14May2014
Availableonline22May2014
Keywords:
Leishmania qPCR
Argininepermease AAP3
Quantification Detection
a b s t r a c t
Leishmaniasisisoneoftheworld’smostneglectedinfectiousdiseases,affectingaround12millionpeople andmorethan350millionatriskofinfection.Theclinicalpicturevariesfromself-healingcutaneous lesionstoseverevisceralinfections,butstillnocommercialvaccinesforhumansareavailableandthe currentlyuseddrugshaveunpleasantsideeffects.Herewereportareal-timePCRassaytargetingthe argininepermeasegeneAAP3thatcanbeappliedforalltheninedifferentspeciesoftheLeishmania genustested;4OldWorldspeciesand5NewWorldspecies,frombothL.(Leishmania)andL.(Viannia) subgenera.Nocross-reactionwasseenwithTrypanosomacruzi,Trypanosomabrucei,humanormouse genomicDNA.Theassayhasahighsensitivity,withalimitofdetectionof10fgDNAforL.(L.)majorand L.(L.)donovani,and100fgDNAforL.(V.)braziliensis,andcanbeusedforbothqualitativeandquantitative purposes.ThisAAP3-Assay,runinduplexwithahostspecificgene-assay,wasalsosuccessfullyusedfor quantificationofparasiteloadoffootpadsfromL.(L.)major-infectedmice.Itcanthereforebeavaluable toolinapplicationslikemonitoringeffectsofdrugs,theselectionofvaccinecandidatesandinscreening patients,includingasymptomaticcarriers.
©2014TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/3.0/).
1. Introduction
Leishmaniasisisavector-bornediseasewithdifferentclinical picturescausedbyprotozoaofthegenusLeishmania.Itisoneofthe world’smostneglectedinfectiousdiseasesandthesecondcauseof parasiterelateddeathsaftermalaria(Mathersetal.,2007).Spread oftheLeishmaniaparasiteiscausedbythebiteofinfectedsand flies. Worldwidemore than350million peoplein 98 countries orterritoriesareatrisk(WorldHealthOrganization,2010).The estimatedincidenceofnewcaseseachyearis0.2–0.4millionfor visceralleishmaniasisand0.7–1.2millionforcutaneousleishman- iasis,causing20000to40000deathsannually,andthesedataare probablyunderestimates(Alvaretal.,2012).Insomeaffectedareas bothdomesticandsylvaticanimalsseemtobeimportantreservoirs oftheparasite,contributingtopromotehumaninfections(Quinnell
∗Correspondingauthor.Tel.:+4755977888.
E-mailaddresses:[email protected](M.G.Tellevik), [email protected](K.E.Muller),[email protected](K.R.Løkken), [email protected](A.H.Nerland).
andCourtenay,2009).Availabledrugsfortreatingthediseasecan becharacterizedaslimited,expensiveandoftenwithunpleasant sideeffects.Moreover,therehasbeenanemergenceofdrugresis- tance(SundarandChakravarty,2013).Nocommercialvaccinesare currentlyavailableforpreventingleishmaniasisinhumans.There- fore,inordertocontrolthedisease,thereisaneedfordevelopment ofnewdrugs,vaccinesandmorespecificandsensitivediagnostic methods.Assaysforquantificationoftheparasiteinthehosttis- suesareessentialfordevelopmentandtestingofprophylacticand therapeuticregimes.
Polymerase chain reaction (PCR) and its variations repre- sent highly sensitive and specific methods for Leishmania DNA detection.PCR hasshowntobesuperior toothermethods like microscopy andvarious immunologictests,reducing time from samplingtotestresult,optimizingsensitivityandspecificityand reducing subjective evaluation (Aviles et al., 1999; Bensoussan etal.,2006;Srivastavaetal.,2011a;Walletal.,2012).Real-time PCRis advantageousoverconventional PCRbecauseit isfaster, lesslabor-intensive,reducesriskofcontamination,andbyusing probesthesensitivityandspecificitycanbeincreased(Dymond, 2013; Mohammadiha et al., 2013; Yang and Rothman, 2004) http://dx.doi.org/10.1016/j.actatropica.2014.05.008
0001-706X/©2014TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Furthermore,byusingstandardcurvesreal-timePCRcanbeused forquantification.ExploringtheLeishmaniaparasitesandtheclin- icalmanifestationstheycause,quantitativereal-timePCR(qPCR) canbeusefulfordetectionandspeciesidentification,butitalsohas awiderpotential,likemonitoringtheeffectofdrugactivityand measuringtheprotectionaspartofvaccinedevelopment.Dueto thehighsensitivity,qPCRcanbeusedforscreeningofpatientsand detectionofasymptomaticcarriers,andtherebyaddressinggaps intheunderstandingofinfectionwithLeishmania(Francinoetal., 2006;Maryetal.,2006;Pourabbasetal.,2013).
TherearemanypublicationsofdifferentvariantsofPCRsfor Leishmania,usingdifferentmoleculartargets,butmostprotocols target a single species (Francinoet al., 2006; Srivastava et al., 2011b), a group of closely related species (Harris et al., 1998;
Odiwuoretal.,2011),ortheyarenotquantitative(Berzunza-Cruz etal.,2009;deAlmeidaetal.,2011;Harrisetal.,1998;Odiwuor etal., 2011; Srivastavaet al., 2011b), and somealsoshow low sensitivity(Wortmannetal.,2005).Thereareonlyfewpublica- tionsof real-timePCR assaysthat targetallornearly allofthe approximately20 differentLeishmaniaspeciesfoundinhumans (Castilho etal.,2008; Tupperwaret al.,2008;Wortmannet al., 2001).Manyprotocolstargetmulticopygenes(Bossolascoetal., 2003;Francinoetal.,2006;Talmi-Franketal.,2010)andsomepro- tocolsformulticopygenesalsouseSYBRGreen(deMonbrisonetal., 2007).Multicopygenesareoftenpreferredtoenhancesensitivity, andthusareadvantageousfordetection,butduetopotentialvaria- tionsandinstabilityincopynumberofthesamegenebothbetween andwithinspecies(Weiratheretal.,2011)theycanbechallenging andconfoundingforquantificationusingstandardcurves.SYBR- Greenhasthedisadvantagesofmoreunspecificbinding,hencea probe,whichismorespecific,istobepreferred.However,itcan bechallengingtodesignaqPCRwithaspecificprobethattargeta DNAsequenceuniversaltoallspeciesoftheLeishmaniagenus.
l-ArginineisanessentialaminoacidforLeishmania,forwhich metabolismdependsonargininesupplyfromexternalsources,as noevidenceforendogenoussynthesishasbeenreported.Theargi- ninetransporterLeishmaniaargininepermeaseAAP3is encoded onchromosome31inLeishmania(L.)majorandotherLeishmania species,andonchromosome30inLeishmania(L.)mexicana(which
duetochromosomefusioneventsistheequivalentofchromosome 31inL.(L.)major(Brittoetal.,1998)).TheAAP3geneisidenti- fiedinseveraldifferentLeishmaniaspecies(Shaked-Mishanetal., 2006).LikelymoreAAP3sequenceswillbepublishedalongwith theincreasingnumberofsequencingprojects.
TheaimofthestudywastodevelopaquantitativePCRmethod thatcouldbeappliedforinfectivitystudiesinmice,focusingonL.
(L.)major,asmurinemodelsarewidelyusedinLeishmaniaresearch, withthebenefit thatitcouldalsobeusedforotherLeishmania species fromboth the Old-and New World groups. We devel- opedaPCR-AssaytargetingtheargininetransportergeneAAP3and includedDNAfromcellpelletsorculturedpromastigotesfromnine differentLeishmaniaspecies,aswellasdifferentnegativecontrols, tovalidatetheassay.ThisAAP3-Assay,runinduplexwithahost specificgene-assay,wasthenusedforquantificationofparasite loadoffootpadsfromL.(L.)major-infectedmice.
2. Materialsandmethods
2.1. Strainsusedinthisstudy
LeishmaniastrainsusedinthisstudyaregiveninTable1.Leish- maniapromastigotesweregrownat26◦CinRPMI1640medium (Sigma–Aldrich,StLouis,MO,USA)supplementedwith10%heat inactivated fetal calf serum (Gibco®, Life Technologies – Invi- trogen, Carlsbad, CA, USA), 100IU/ml penicillin and 100g/ml streptomycin, or in Schneider’s InsectMedium (Sigma–Aldrich) supplementedwith20%heatinactivatedfetalcalfserumand1%
sterile-filteredhumanurine.Parasitesinculturewerewashedand counted by flow cytometry using reference beads (Flow-Count Fluorospheres®, Beckman Coulter, Brea, CA, USA) after stain- ingwithVybrant® DyeCycleTMGreen Stain(Molecularprobes®, Eugene,OR,USA).
L.(L.)mexicanaMHOM/BZ/82/BEL21wasakindgiftfromCen- trodeInvestigacionesRegionales‘Dr.HideyoNoguchi’,Universidad AutónomadeYucatán,Mérida,Yucatán,México.Cellpelletsfrom Leishmania strainsas indicated inTable 1,as wellasfromTry- panosomabruceiandT.cruzi,werekindlydonatedfromDr.Silvia
Table1
Leishmaniastrainsusedinthisstudy,andtheirCq-valuewhen100pgofDNAwasusedastemplateintheAAP3-Assay.
Speciesa Internationalcodeorotherreference Origin Sourceb Cqc
L.(L.)aethiopica MHOM/ET/91/Kassaye Ethiopia SMI 23.4
L.(V.)amazonensis MHOM/BR/73/M2269,LEM0690 Brazil CNRL 23.7
L.(V.)braziliensis MHOM/BR/87/LTB12MAR87,LEM2839 Brazil CNRL 25.0
L.(V.)braziliensis MHOM/BR/75/M2904,LEM2249 Brazil CNRL 25.1
L.(V.)braziliensis SMI2094 Unknown SMI 25.3
L.(L.)donovani MHOM/CY/2006/CH33,LEM5298 Cyprus CNRL 23.8
L.(L.)donovani MHOM/IN/80/DD8,LEM0703 India CNRL 23.7
L.(L.)donovani MHOM/ET/67/HU3 Ethiopia SMI 23.7
L.(V.)guyanensis MHOM/GF/94/22319,LEM2763 FrenchGuiana CNRL 27.6
L.(L.)major MHOM/AF/2006/LEM5344,LEM5344 Afghanistan CNRL 23.9
L.(L.)major MHOM/MA/2004/LEM4905,LEM4905 Morocco CNRL 24.2
L.(L.)major MHOM/TN/2006/LPN296,LEM5373 Tunisia CNRL 23.9
L.(L.)major MHOM/IL/80/FRIEDLIN,LEM3150 Israel CNRL 23.4
L.(L.)major-np MRHO/SU/59/LV39 USSR CNRL 23.6
L.(L.)major-p MRHO/SU/59/LV39 USSR CNRL 24.0
L.(L.)mexicana MHOM/MX/96/NAN01,LEM3816 Mexico CNRL 23.8
L.(L.)mexicana MHOM/MX/93/CRE47,LEM2695 Mexico CNRL 23.5
L.(L.)mexicana-np MHOM/BZ/82/BEL21 Belize CIR 24.3
L.(L.)mexicana-p MHOM/BZ/82/BEL21 Belize CIR 24.4
L.(V.)naiffi MHOM/GF/97/CRE88,LEM3426 FrenchGuiana CNRL 24.0
L.(L.)tropica MHOM/SU/74/K27,LEM0419 USSR CNRL 24.2
L.(L.)tropica MHOM/MA/2000/INHW10,LEM5277 Morocco CNRL 24.1
anp:notbeenpropagatedinmouse;p:propagatedinmouse.
b SMI:PublicHealthAgencyofSweden,Sweden;CNRL:CentreNationaldeRéfèrencedesLeishmanioses,Montpellier,France;CIR:CentrodeInvestigacionesRegionales, UniversidadAutónomadeYucatán,Mérida,Yucatán.
c Cq-valuefromtheAAP3-assayusing100pgDNAastemplate.
Botero-KleivenandDr.LeighDavidssonatthePublicHealthAgency ofSweden,Sweden,andwerekeptinethanolduringtransportto ourlaboratoryanduntilDNAextraction.
2.2. DetectionoftheAAP3geneinLeishmaniaspecies 2.2.1. DNAextraction
Culturedpromastigotesfromthestationaryphaseandcellpel- letsinethanolwerewashedwithDulbecco’sphosphate-buffered saline(10mMPhosphate,137mMSodiumchloride,2.7mMPotas- siumchloride,pH7.4)(DPBS)beforesubjectedtoDNAextraction using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, except that Pro- teinaseKwasusedinsteadofprotease.DNAwaselutedin100lof BufferAE(Qiagen).InallDNAextractionsanegativecontrolofDPBS wasincludedtomonitorfor contamination.DNAconcentration andqualitywasdeterminedbyaNanoDrop®ND-1000Spectropho- tometer(ThermoScientific,Wilmington,DE,USA).AllDNAsamples werestoredat−20◦C.
2.2.2. Real-timePCR
Primersandhydrolysisprobe(TaqMan®MGBprobe)targeting a74bpregionoftheL.(L.)majorgeneencodingtheargininetrans- porterAAP3weredesignedbytheCustomTaqMan®AssayDesign ToolfromApplied Biosystems(AppliedBiosystems,Warrington, UK).Thesequencesoftheprimersand probewere5-GGCGGC- GGTATTATCTCGAT-3 (Forward), 5-ACCACGAGGTAGATGACAGA- CA-3(Reverse)andFAM5-ATGTCGGGCATCATC-3 NFQ(probe).
In silico analysis of specificity of the assay was performed using the Vector NTI software (Life Technologies). A global BLAST search was undertaken by the 74bp region wherein the primers and probe bind. In addition, complete genome sequences of some relevant bacteria (like Mycobacteria, Pseu- domonas, Streptococcus, Staphylococcus) were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and investi- gatedfortheextentofbinding(BLAST)oftheprimersandprobe.
EachPCRtest wasperformedintriplicatein a20lreaction mixture.Thereactionmixtureincluded:1×TaqManUniversalMas- terMixIIwithUNG(AppliedBiosystems),1×CustomTaqMan® GeneExpressionAssayMixwithprimersandprobetargetingthe LeishmaniaAAP3gene,1×TaqMan®CopyNumberReferenceAssay, Mouse,Tfrc,waterand100pgofDNAsample.ThePCRwasrunasa duplexassayaftercomparingtheresultsofsingleplexAAP3-Assay andtheduplexwiththeTfrc-Assay(resultsnotshown).
TheqPCRwasperformedwithanAppliedBiosystems7900HT FastReal-TimePCRSystem(AppliedBiosystems),withcyclingcon- ditionsasfollows:50◦Cfor2min,95◦Cfor10min,followedby40 cyclesat95◦Cfor15sand60◦Cfor1mineach.Allsampleswere runonMicroAmp®Optical96-wellReactionPlates(AppliedBiosys- tems) sealed with MicroAmp® Optical Adhesive Film (Applied Biosystems). Each run included multiple no-template controls.
HumanDNA,T.bruceiandT.cruziwasusedasnegativecontrolsto checkforcross-reactivity.ThehumanDNAforcontrolwasprovided byChristelG.Haanshuus,NationalCentreforTropicalInfectious Diseases,DepartmentofMedicine,HaukelandUniversityHospital, Bergen,Norway.
Tenfold dilution series of L. (L.) major DNA (range 12ng–1.2×10−7ng)werepreparedforcreatingstandardcurves and estimating assay performance for theAAP3-Assay. Besides quantification,dilutionseriesandstandardcurveswereusedfor estimation of PCR efficiency, limit of quantification (LOQ) and limitofdetection(LOD).LOQandLODwerealsoestimatedforL.
(L.) donovaniand L. (V.)braziliensis. The dilutioncorresponding totheLOQwasthehighestdilutionusedforthestandardcurve.
For estimating repeatability and reproducibility,replicates of a DNAsamplefromthefootpadsofBALB/cmiceinfectedwithL.(L.)
majorparasiteswereused.DNAwasquantifiedusingtheAbsolute QuantificationAssay.Thresholdsweresetautomatically.Ampli- conswererunona2%agarosegel(SeaKemTM,Lonza,Rockland, ME,USA)with1XGelRedTM(Biotium,Hayward,CA,USA)tocheck forthecorrectsize.Replicateswithquantificationcycle(Cq)-value differingbymorethan0.3wereomitted.
Aunidirectionalworkflowpre-topost-qPCRwasenforced,and preparationofqPCRreactionmixture,DNApreparationsandqPCR werecarriedoutinfacilitiesphysicallyseparatefromeachother.
2.3. DetectionoftheAAP3geneinL.(L.)major-infectedmice 2.3.1. Mousetissuesamples
The left footpads of female BALB/c mice were inoculated with10lof106ml–1L.(L.)majorparasitesinstationaryphase.
After swelling and lesions had developed, 56 days post inocu- lation, mice were euthanized after first using Isoba vet. 100%
(Intervet/Schering-PloughAnimalHealth,IntervetDenmarkA/S, Denmark)foranesthesia.Footpadsofcontrolmice(2animals)and infectedmice(8animals),andliverforprovidingcontrolDNA,were harvestedandstoredat−80◦C.
2.3.2. Ethicalclearance
TheanimalexperimentswereapprovedbytheNationalAnimal ResearchAuthorityinNorwayandcarriedoutattheLaboratory AnimalFacility(AAALAC-accredited)attheUniversityofBergen, Bergen,Norway.
2.3.3. DNAextractionfrommicetissue
Mouse footpads were subjected for DNA extraction by a phenol-chloroform based protocol from Instituto Oswaldo Cruz, Rio de Janeiro, Brazil, available at http://clioc.fiocruz.br/
documents/mmp.pdf(LeishmaniasisEpidemiologyNetworkSouth America, 2009). DNA pellet was dissolved in 200l TE-buffer (10mMTris–HCl,1mMEDTA,pH8.0)andincubatedatroomtem- peratureovernight.Mouseliverswereincubatedovernightat56◦C inBufferATL(Qiagen)with2mg/mlProteinaseK(Qiagen),before subjectedtoDNAextractionusingQIAampDNAMiniKit(Qiagen) accordingtothemanufacturer’sinstructions.DNAwaselutedin 100lofBufferAE(Qiagen).
InallDNAextractionsanegativecontrolofDPBSwasincluded to monitor for contamination. DNA concentration and quality was determined by a NanoDrop® ND-1000 Spectrophotometer (ThermoScientific,Wilmington,DE,USA).AllDNAsampleswere storedat−20◦C.
2.3.4. Quantitativereal-timePCR
Inordertonormalizetheparasiteloadforamountofmousetis- sueDNA,andhenceovercomethepossiblequantificationerrors duetodifferentcuttingpointswhenharvestingthefootpads,the qPCR was run asa duplex-assay withthe AAP3-Assay and the TaqMan® CopyNumber ReferenceAssay, Mouse, Tfrc (Applied Biosystems,FosterCity,CA,USA),asareferenceassay.Thispre- madereactionmixtureconsistsofprimersandaVIC®dye-labeled TAMRATMprobewhichdetectsthesingle-copytransferrinreceptor gene(Tfrc)inthemousegenome.
EachqPCRtestwasperformedintriplicateina20lreaction mixture.ThereactionmixturewasasinSection2.2.2,exceptthat 100ngofDNAsamplewasusedastemplate.Initialdilutionexper- imentsshowedthatwith100ngofDNAthebackgroundlevel,or inhibition,wasnegligible.
Fivefold dilution series of BALB/c mouse DNA (range 1050ng–0.33ng) were prepared for creating standard curve and estimating assay performance for theTfrc-Assay. For esti- mating repeatability and reproducibility, replicates of a DNA sample from the footpads of BALB/c mice infected withL. (L.)
majorparasiteswereused.DNAwasquantifiedusingtheAbsolute Quantification Assay. Thresholds were set automatically. The parasiteloadin tissuesamples wasgivenas theratiobetween Leishmania DNA and genomic mouse DNA in the 100ng DNA appliedtoeachPCRreaction.SincetheamountofDNAcannotbe reliablyestimatedoutsidethelinearareaofthestandardcurve, theDNAquantityforsampleswithLeishmaniaDNAlessthanthe LOQwassettobeequaltoorlessthantheLOQ.Parallelswith Cq-valuedifferingbymorethan0.3wereomitted.
3. Results
3.1. SpecificityoftheAAP3-AssayandtheTfrc-Assay
TheprimersandprobefortheAAP3-Assayweredesignedfor L.(L.)major.We alsoexperimentallytested theability ofthese oligonucleotidestodetectotherdifferentLeishmaniaspecies,rep- resentingninedifferentspecies,eitherisolatedfromcellpelletsor culturedpromastigotes.L.(L.)aethiopica,L.(V.)amazonensis,L.(V.) braziliensis,L.(L.)donovani,L.(V.)guyanensis,L.(L.)major,L.(L.) mexicana,L.(V.)naiffiandL.(L.)tropicacouldallbeamplifiedwith theAAP3-Assay,asseenfromTable1.ThePCRresultedinampli- consofthecorrectsize,74bp,asvisualizedbygelelectrophoresis (resultsnotshown).NeithertheAAP3-Assay northeTfrc-Assay couldamplifyT.bruceiandT.cruzi,orgenomicDNAfromhumans ormice(AAP3-Assayonly).
TheglobalBLAST searchwiththe74bpregion(whereinthe primersandtheprobesbind)gaveonlyhitsindicatinggeneration ofpositivesignalsof theassaywhentargetingDNA fromLeish- maniaspecies. Likewise,investigation of the bacterial genomic sequencesdidnotrevealstrongbindingofanycombinationsof appliedprimersinawaythatwouldgenerateamplificationofany segmenttowhichtheprobewouldbind,andtherebygivingriseto apositivesignal.
3.2. SensitivityoftheAAP3-Assay
LODandLOQwereestimatedforL.(V.)braziliensis,L.(L.)dono- vaniandL.(L.)majorusingserialdilutionsofDNApurifiedfrom invitro cultivated parasites. Withtheappliedmethodused for DNAextraction,100fgcorrespondedto2parasites(p).ForL.(V.) braziliensis,L.(L.)donovaniandL.(L.)majortheLODs,giveninfg withnumberofparasiteswithourextractionmethodinbrackets, were:≥100fg(2p),≥10fg(0.2p)and≥10fg(0.2p),respectively.
TheLOQsforL.(V.)braziliensis,L.(L.)donovaniandL.(L.)majorwere
≥1000fg(20p),≥10fg(0.2p)and≥100fg(2p),respectively.
3.3. Assayperformance
From the DNA dilution series parameters of assay perfor- manceotherthansensitivitywascalculated.Fromrepeatedruns themeanslopesoftheAAP3-AssayandTfrc-Assaywere−3.205 (range−2.994to−3.305)and -3.193(range−3.083to−3.313), respectively.Efficiency,asdeterminedfromtheslopeandusing the formula E=10−1/slope−1, was 105.1% for the AAP3-Assay and 105.7% for the Tfrc-Assay. The correlation coefficient, R2, was 0.999 (range 0.996–0.999) for the AAP3-Assay and 0.997 (range0.996–0.999)fortheTfrc-Assay,andtheY-interceptwas 19.48(range18.26–20.85) fortheAAP3-Assayand31.83(range 31.43–32.11)fortheTfrc-Assay.ADNAsamplefromthefootpads ofBALB/cmiceinfectedwithL.(L.)majorparasiteswasusedfor estimatingrepeatabilityandreproducibility.Theintra-assaycoef- ficientofvariation(CV)fortheAAP3-Assay,calculatedfromDNA quantityofreplicates,wasforeachofthreeseparateruns0.0908, 0.0901and0.0882,respectively.FortheTfrc-Assaytheintra-assay CVwasforeachoftwoseparateruns0.0179and0.021.Inter-assay
Fig.1. AmplificationplotfortheAAP3-AssayandtheTfrc-Assay.Amplification curvesforDNAisolatedfromfootpadofuninfectedControlmouse(Control)and mouseinfectedwithL.(L.)major.
CV,calculatedfromDNAquantityofseparateruns,was0.052for theAAP3-Assayand0.059fortheTfrc-Assay.Theratioin100ng DNAbetweenquantityofL.(L.)majorDNA,asmeasuredbythe AAP3-Assay,andmouseDNA,asmeasuredbytheTfrc-Assay,was 3.22×10−4and3.25×10−4forthesamesampleontwoseparate runs.
No amplification of any of the no-template controls was detected.
3.4. EstimationofL.(L.)majorDNAinmicefootpads
Foranalyzing andquantification ofparasiteloadoffootpads fromL.(L.)major-infectedmice,theAAP3-Assaywasruninduplex with a host specific gene-assay; the Tfrc-Assay, which detects mouseDNA.LeishmaniaDNAwassuccessfullyamplifiedfromall theinfected mice,but not from thecontrolmice. Fig.1 shows amplificationplotsfortheAAP3-AssayandtheTfrc-Assaywhen analyzinga sample ofLeishmania-infectedtissue,together with plotsfortheTfrc-Assaywhenanalyzingacontrolsampleofunin- fectedtissue.AsthemiceDNAconstitutethemajorityoftheDNA intheinfectedtissue,allplotsfromtheTfrc-Assaymakeupacon- currentcurve.ValuesfortheAAP3-Assaytriplicates andforthe triplicatesoftheTfrc-AssayaregiveninTable2.Inaddition,DNA isolatedfromthefootpadofoneoftheotherL.(L.)major-infected micewasanalyzed,wherewemadetwodifferentdilutionsofthe DNAtoconfirmthatthesameratiobetweenLeishmaniaDNAand mouseDNAwouldstillbeobtained.For148.5ngsampletheratio was1.99×10−4andusing74.3ngofthesamesampletheratiowas 1.93×10−4.
4. Discussion
Microscopy has traditionally been the cheapest and easiest methodfor detectingand counting,Leishmania parasites. How- ever,real-time PCR issuperior regarding sensitivity,specificity, capacityandhasinadditionshorttimeofanalysisandminimized subjectivityoflaboratorystaff.WehavedevelopedaqPCRassay abletodetectaslittleas10fgofDNA,andtoquantifydownto thelimit of 10fg(L.(L.)donovani).The AAP3-Assayis effective in detecting several different species, and possibly the species rangeforourassayisbroaderthanwehavetestedfor.Regarding specificity,bothexperimentalandbioinformaticanalyzesshowno homologyofthetargetsequencetonon-Leishmaniasequences.
Table2
QuantificationofL.(L.)majorDNAinfootpadfromexperimentalinfectedmouse.
Samplea CqAAP3-Assayb,c QtyLeishDNAd CqTfrc-Assayb QtymouseDNAe QtyLeishDNA/QtymouseDNA
L.(L.)major-1 28.08 2.3×10−3 25.55 90.03 2.55×10−5
L.(L.)major-2 27.96 2.5×10−3 25.46 96.18 2.60×10−5
L.(L.)major-3 28.03 2.4×10−3 25.46 95.99 2.50×10−5
Control1-1 Undet. – 25.46 95.79 –
Control1-2 Undet. – 25.51 92.54 –
Control1-3 Undet. – 25.49 93.84 –
aControl1,uninfectedmice;resultsareshownforalltriplicatesforeachsample.
bCq,quantificationcycle.
c Undet.,Undetermined.
d QtyLeishDNA,QuantityLeishmaniaDNAinngdetectedbytheAAP3-Assayper100ngDNA.
eQtymouseDNA,QuantitymouseDNAinngdetectedbytheTfrc-Assayper100ngDNA.
WiththeappliedmethodusedforDNAextraction,100fgcor- respondedto2 parasites. Assuming 80fgof Leishmania DNA is equivalenttooneparasite,then100fgcorrespondsto1.2parasites, indicatingthatthesensitivityoftheAAP3-Assayactuallyisbetter thanexperienced,andcanbefurtherimprovedbyoptimizingDNA extraction.
RegardingsensitivityintermsofDNAquantity,theLODofour assayequalsthatofsomeofthepublicationstargetingbothrRNA- andkDNA minicirclegenes (Berzunza-Cruz etal.,2009; Nicolas etal.,2002; Prinaetal.,2007; Gomeset al.,2012;Talmi-Frank etal.,2010;Wortmannetal.,2001), whicharegeneswithhigh copynumbers,buttherearealsopublicationstargetingmulticopy genesshowinghighersensitivity(Francinoetal.,2006).However, whenusingmulticopygenesforquantificationtheremightbea needforaspeciesspecificstandardcurveasnumberofgenecopies varybetweenspecies(Weiratheretal.,2011),thusmakingthose assayslittlesuitableforauniversalquantitativeLeishmania-assay.
LiketheAAP3-Assay,theassayof(Wortmannetal.,2001),targeting theLeishmania16SrRNA,wasalsoabletoamplifyawiderangeof Leishmaniastrainsatthegenuslevel.However,thesensitivitywas onlydeterminedforL.(L.)mexicana,theCq-valuesbothwithinand betweenspecieshadagreatrange,thoughtheynotclearlyspec- ifyifthesameamountofDNAwasused,andparametersforassay performance,likeefficiency,LOQ,reproducibilityandrepeatability wasnotestimated,oratleastnotgiveninthetext.
ThechromosomethathoststheAAP3geneissupernumerary;
tetrasomicforsomespecies,likeL.(L.)major,L.(L.)infantumand L.(L.)donovani,andhexasomicforL.(V.)braziliensis(Rogersetal., 2011).InL.(L.)major,L.(L.)infantum,L.(V.)amazonensisandL.(L.) donovanitherearetwoidenticalcopiesofAAP3(Castilho-Martins etal.,2011;Shaked-Mishanetal.,2006),whereasinL.(L.)mex- icanathereisnoreportedequivalentgeneduplication.Wehave included22Leishmaniastrains, representing9differentspecies.
TheCq-valuesforthesestrainsusing100pgDNAastemplatein theAAP3-Assayareshownin Table1.Thereisvery littleintra- species variation in Cq-value, but some inter-species variation, mainlybetweenL.(V.)braziliensisandL.(V.)guyanensisagainstthe othersspeciesincludedinourstudy.Thismightindicatethatploidy betweenstrainsandspeciesdoesnotlargelyaffecttheresultsof theassay,butthisshouldbeinvestigatedfurther.Tupperwaretal.
(2008)describesaPCRassaytargetingtheLeishmaniaGP63able toamplifyseveraldifferentspecies, butcomparedtotheAAP3- AssaythereisamarkedlydifferenceintheCq-valuablebetween specieswhenthesameamountof templatewasused.Whether thereisaneedforaspeciesspecific,orevenstrainspecific,standard curvefortheAAP3-assaydependsonthespecificapplicationand therequiredaccuracy,andhasyettobemorethoroughlyevalu- ated.IncontrasttoothermulticopygeneslikethekDNAminicircle andITS1,withcopynumbersaround10000and200respectively, andasseenfromTable1,itislesslikely,andforseveralspecies probablyspeciesgroup-specificstandardcurvewillbesufficient.
Awayofavoiding aspeciesspecificstandard curvecouldbeto usetheDNAconcentrationsforthedilutionsofthestandardcurve insteadofthenumberofparasites,andthenusingspeciesrelated conversionfactors.
InthisstudywedevelopedaqPCRassaythatcanbeappliedon abroadrangeofspeciesoftheLeishmaniagenus,fromboththe Old-andNewWorldgroups,includingboth L.(Leishmania)spp.
andL.(Viannia)spp.,andwhichinadditioncanbeusedtoanalyze DNAfrombothcellculturesandmousetissue,andmostlikelyDNA fromseveralotherkindsofsamplematerials.Byusingastandard curveonecandeterminetheamountoftargetDNAinasample, and fromthatalsocalculatethenumber ofparasites. However, whenisolatingDNAfrominfectedtissuestheamountofDNAwill bedependentonthesizeofthetissuesampleandtherecovery ofDNAatthedifferentstepsoftheisolationprotocol.Theuseof hostreferencegenecancircumventthisproblem.Whenapplied toLeishmaniainfectedmouse,theAAP3-andTfrc-Assaysgivea ratiobetweenLeishmaniaDNAandmouseDNA.Thisratio,which isindependentoftheamountoftotalDNAappliedfortheanaly- sis,isreflectingparasiteloadinthetissueandhencedifferences canbedetected.InastudybyTupperwaretal.(2008),theynor- malizedtheparasitenumbertothetotalDNAisolatedfromthe micetissuesamples,andfinallyreportedtheparasitenumberper milligramoforiginaltissue,notusingahostreferencegene.This reportedresultcouldthenbepronetodifferencesinrecoveryof DNAduringtheextractionprocess.Nicolasetal.(2002)describe a real-time PCR assay which successfully detects four different Leishmaniaspeciesfromawiderangeofmouseinfectedtissues.
However,thestudydoesnotincludeamethod,likeusingahost referencegene,forestimatingandnormalizationoftheparasite burdenofthetissue.Theyalsotargetthehighcopynumbermini- circlekDNA,whichmighthavedrawbacks,asmentioned above.
Alimitationofourmethodisthatitnotnecessarilydiscriminates betweenliveanddeadparasites.However,thelevelsofLeishmania DNAdetectedwillstillindicatetheparasiteloadatthetimeof sampling.
Insummary,wehave developedaverysensitivemethodfor detectionofLeishmaniathatcanbeappliedforseveraldifferent speciesoftheparasite.Incombinationwithanassayforquantifica- tionofhostDNA,itispossibletomeasuretheloadofLeishmaniain infectedtissues.Withitshighsensitivitythemethodhaspotential asatoolfordiagnosticpurposes,includingdetectingasymptomatic infections.Duetothequantificationpossibility,themethodcanbe usedtomonitortheprogressofinfection,whichwillbeavaluable toolintestingnewdrugsandinvaccinedevelopment.
Acknowledgements
WearegratefultoSteinarSørnesattheDepartmentofClin- icalScience,University of Bergen,Bergen,Norway,for valuable technical assistance. The study was supported by the National
CentreforTropicalInfectiousDiseases,DepartmentofMedicine, HaukelandUniversityHospital,Bergen,Norway.
References
Alvar,J.,Velez,I.D.,Bern,C.,Herrero,M.,Desjeux,P.,Cano,J.,Jannin,J.,denBoer,M., 2012.WHOLeishmaniasisControlTeam,Leishmaniasisworldwideandglobal estimatesofitsincidence.PLoSOne7,e35671.
Aviles,H.,Belli,A.,Armijos,R.,Monroy,F.P.,Harris,E.,1999.PCRdetectionandiden- tificationofLeishmaniaparasitesinclinicalspecimensinEcuador:acomparison withclassicaldiagnosticmethods.J.Parasitol.85,181–187.
Bensoussan,E.,Nasereddin,A.,Jonas,F.,Schnur,L.F.,Jaffe,C.L.,2006.Comparison ofPCRassaysfordiagnosisofcutaneousleishmaniasis.J.Clin.Microbiol.44, 1435–1439.
Berzunza-Cruz,M.,Bricaire,G.,SalaizaSuazo,N.,Perez-Montfort,R.,Becker,I.,2009.
PCRforidentificationofspeciescausingAmericancutaneousleishmaniasis.
Parasitol.Res.104,691–699.
Bossolasco,S.,Gaiera,G.,Olchini,D.,Gulletta,M.,Martello,L.,Bestetti,A.,Bossi, L.,Germagnoli,L.,Lazzarin,A.,Uberti-Foppa,C.,Cinque,P.,2003.Real-time PCRassayforclinicalmanagementofhumanimmunodeficiencyvirus-infected patientswithvisceralleishmaniasis.J.Clin.Microbiol.41,5080–5084.
Britto,C.,Ravel,C.,Bastien,P.,Blaineau,C.,Pages,M.,Dedet,J.P.,Wincker,P.,1998.
Conservedlinkagegroupsassociatedwithlarge-scalechromosomalrearrange- mentsbetweenOldWorldandNewWorldLeishmaniagenomes.Gene222, 107–117.
Castilho-Martins,E.A.,LaranjeiradaSilva,M.F.,dosSantos,M.G.,Muxel,S.M.,Floeter- Winter,L.M.,2011.AxenicLeishmaniaamazonensispromastigotessenseboththe externalandinternalargininepooldistinctlyregulatingthetwotransporter- codinggenes.PLoSOne6,e27818.
Castilho,T.M.,Camargo,L.M.,McMahon-Pratt,D.,Shaw,J.J.,Floeter-Winter,L.M., 2008.Areal-timepolymerasechainreactionassayfortheidentificationand quantification ofAmerican Leishmania species onthe basis of glucose-6- phosphatedehydrogenase.Am.J.Trop.Med.Hyg.78,122–132.
deAlmeida,M.E.,Steurer,F.J.,Koru,O.,Herwaldt,B.L.,Pieniazek,N.J.,daSilva,A.J., 2011.IdentificationofLeishmaniaspp.bymolecularamplificationandDNA sequencinganalysisofafragmentofrRNAinternaltranscribedspacer2.J.Clin.
Microbiol.49,3143–3149.
deMonbrison,F.,Mihoubi,I.,Picot,S.,2007.Real-timePCRassayfortheidentification ofcutaneousLeishmaniaparasitespeciesinConstantineregionofAlgeria.Acta Trop.102,79–83.
Dymond,J.S.,2013.Explanatorychapter:quantitativePCR.MethodsEnzymol.529, 279–289.
Francino,O.,Altet,L.,Sanchez-Robert,E.,Rodriguez,A.,Solano-Gallego,L.,Alberola, J.,Ferrer,L.,Sanchez,A.,Roura,X.,2006.Advantagesofreal-timePCRassayfor diagnosisandmonitoringofcanineleishmaniosis.Vet.Parasitol.137,214–221.
Gomes,L.I.,Gonzaga,F.M.,deMorais-Teixeira,E.,deSouza-Lima,B.S.,Freire,V.V., Rabello,A.,2012.Validationofquantitativereal-timePCRfortheinvitroassess- mentofantileishmanialdrugactivity.Exp.Parasitol.131,175–179.
Harris,E.,Kropp,G.,Belli,A.,Rodriguez,B.,Agabian,N.,1998.Single-stepmulti- plexPCRassayforcharacterizationofNewWorldLeishmaniacomplexes.J.Clin.
Microbiol.36,1989–1995.
LeishmaniasisEpidemiologyNetworkSouthAmerica,2009.Manual–Molecular Procedures.InstitutoOswaldoCruz,RiodeJaneiro,Brazil.
Mary,C.,Faraut,F.,Drogoul,M.P.,Xeridat,B.,Schleinitz,N.,Cuisenier,B.,Dumon,H., 2006.ReferencevaluesforLeishmaniainfantumparasitemiaindifferentclinical presentations:quantitativepolymerasechainreactionfortherapeuticmonitor- ingandpatientfollow-up.Am.J.Trop.Med.Hyg.75,858–863.
Mathers,C.D.,Ezzati,M.,Lopez,A.D.,2007.Measuringtheburdenofneglectedtrop- icaldiseases:theglobalburdenofdiseaseframework.PLoSNeglect.Trop.D1, e114.
Mohammadiha,A.,Mohebali,M.,Haghighi,A.,Mahdian,R.,Abadi,A.R.,Zarei,Z., Yeganeh,F.,Kazemi,B.,Taghipour,N.,Akhoundi,B.,2013.Comparisonofreal- timePCRandconventionalPCRwithtwoDNAtargetsfordetectionofLeishmania (Leishmania)infantuminfectioninhumananddogbloodsamples.Exp.Parasitol.
133,89–94.
Nicolas,L.,Prina,E.,Lang,T.,Milon,G.,2002.Real-timePCRfordetectionandquan- titationofLeishmaniainmousetissues.J.Clin.Microbiol.40,1666–1669.
Odiwuor,S.O.,Saad,A.A.,DeDoncker,S.,Maes,I.,Laurent,T.,ElSafi,S.,Mbuchi,M., Buscher,P.,Dujardin,J.C.,VanderAuwera,G.,2011.UniversalPCRassaysforthe differentialdetectionofallOldWorldLeishmaniaspecies.Eur.J.Clin.Microbiol., 209–218.
Pourabbas,B.,GhadimiMoghadam,A.,Pouladfar,G.,Rezaee,Z.,Alborzi,A.,2013.
Quantification ofLeishmaniainfantum kinetoplastDNA formonitoringthe responsetomeglumineantimoniatetherapyinvisceralleishmaniasis.Am.J.
Trop.Med.Hyg.88,868–871.
Prina,E.,Roux,E.,Mattei,D.,Milon,G.,2007.LeishmaniaDNAisrapidlydegraded followingparasitedeath:ananalysisbymicroscopyandreal-timePCR.Microbes Infect.9,1307–1315.
Quinnell,R.J.,Courtenay,O.,2009.Transmission,reservoirhostsandcontrolof zoonoticvisceralleishmaniasis.Parasitology136,1915–1934.
Rogers,M.B.,Hilley,J.D.,Dickens,N.J.,Wilkes,J.,Bates,P.A.,Depledge,D.P.,Harris, D.,Her,Y.,Herzyk,P.,Imamura,H.,Otto,T.D.,Sanders,M.,Seeger,K.,Dujardin, J.C.,Berriman,M.,Smith,D.F.,Hertz-Fowler,C.,Mottram,J.C.,2011.Chromosome andgenecopynumbervariationallowmajorstructuralchangebetweenspecies andstrainsofLeishmania.GenomeRes.21,2129–2142.
Shaked-Mishan,P.,Suter-Grotemeyer,M.,Yoel-Almagor,T.,Holland,N.,Zilberstein, D.,Rentsch,D.,2006.Anovelhigh-affinityargininetransporterfromthehuman parasiticprotozoanLeishmaniadonovani.Mol.Microbiol.60,30–38.
Srivastava,P.,Dayama,A.,Mehrotra,S.,Sundar,S.,2011a.Diagnosisofvisceralleish- maniasis.T.Roy.Soc.Trop.Med.H.105,1–6.
Srivastava,P.,Mehrotra,S.,Tiwary,P.,Chakravarty,J.,Sundar,S.,2011b.Diagnosis ofIndianvisceralleishmaniasisbynucleicaciddetectionusingPCR.PLoSOne6, e19304.
Sundar,S.,Chakravarty,J.,2013.Leishmaniasis:anupdateofcurrentpharmacothe- rapy.ExpertOpin.Pharmacol.14,53–63.
Talmi-Frank,D.,Nasereddin,A.,Schnur,L.F.,Schonian,G.,Toz,S.O.,Jaffe,C.L.,Baneth, G.,2010.DetectionandidentificationofoldworldLeishmaniabyhighresolution meltanalysis.PLoSNeglect.Trop.D4,e581.
Tupperwar,N.,Vineeth,V.,Rath,S.,Vaidya,T.,2008.Developmentofareal-time polymerasechainreactionassayforthequantificationofLeishmaniaspecies andthemonitoringofsystemicdistributionofthepathogen.Diagn.Micr.Infec.
Dis.61,23–30.
Wall,E.C.,Watson,J.,Armstrong,M.,Chiodini,P.L.,Lockwood,D.N.,2012.Epidemi- ologyofimportedcutaneousleishmaniasisattheHospitalforTropicalDiseases, London,UnitedKingdom:useofpolymerasechainreactiontoidentifythe species.Am.J.Trop.Med.Hyg.86,115–118.
Weirather,J.L.,Jeronimo,S.M.,Gautam,S.,Sundar,S.,Kang,M.,Kurtz,M.A.,Haque,R., Schriefer,A.,Talhari,S.,Carvalho,E.M.,Donelson,J.E.,Wilson,M.E.,2011.Serial quantitativePCRassayfordetection,speciesdiscrimination,andquantification ofLeishmaniaspp.inhumansamples.J.Clin.Microbiol.49,3892–3904.
WorldHealthOrganization,2010.Controloftheleishmaniases,WHOTechnical reportseries;no,949,Switzerland.
Wortmann,G.,Sweeney,C.,Houng,H.S.,Aronson,N.,Stiteler,J.,Jackson,J.,Ock- enhouse,C.,2001.Rapiddiagnosisofleishmaniasisbyfluorogenicpolymerase chainreaction.Am.J.Trop.Med.Hyg.65,583–587.
Wortmann,G.,Hochberg,L.,Houng,H.H.,Sweeney,C.,Zapor,M.,Aronson,N.,Weina, P.,Ockenhouse,C.F.,2005.RapididentificationofLeishmaniacomplexesbya real-timePCRassay.Am.J.Trop.Med.Hyg.73,999–1004.
Yang,S.,Rothman,R.E.,2004.PCR-baseddiagnosticsforinfectiousdiseases:uses, limitations,andfutureapplicationsinacute-caresettings.LancetInfect.Dis.4, 337–348.
