h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /
Veterinary
Microbiology
Development
and
validation
of
a
modified
TaqMan
based
real-time
PCR
assay
targeting
the
lipl32
gene
for
detection
of
pathogenic
Leptospira
in
canine
urine
samples
Bruno
Alonso
Miotto
a,∗,
Aline
Santana
da
Hora
b,
Sueli
Akemi
Taniwaki
b,
Paulo
Eduardo
Brandão
b,
Marcos
Bryan
Heinemann
b,
Mitika
Kuribayashi
Hagiwara
aaDepartamentodeClínicaMédica,FaculdadedeMedicinaVeterináriaeZootecnia,UniversidadedeSãoPaulo,SãoPaulo,SP,Brasil bDepartamentodeMedicinaVeterináriaPreventivaeSaúdeAnimal,FaculdadedeMedicinaVeterináriaeZootecnia,UniversidadedeSão
Paulo,SãoPaulo,SP,Brasil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received27April2017 Accepted12September2017 Availableonline2December2017 AssociateEditor:AgnesFigueiredo
Keywords: Leptospirosis Dog Urine qPCR lipl32
a
b
s
t
r
a
c
t
AmodifiedTaqManreal-timepolymerasechainreactiontargetinga138bpfragmentwithin thelipl32genewasdevelopedtoidentifyexclusivelypathogenicLeptospiraspp.indogurine samples.Thirty-fivesamplesfromdogswithsuspectedclinicalleptospirosisand116 sam-plesfromapparentlyhealthydogsweretestedforpresenceofleptospiralDNAusingthe TaqMan-basedassay.Theresultswerecomparedwiththosefromawell-established con-ventionalPCRtargetingthe16SRNAencodinggeneassociatedwithnucleotidesequencing analysis.Theoverallagreementbetweentheassayswas94.8%(confidenceinterval[CI]95% 88–100%).Thenewlydevelopedassaypresented91.6%(CI95%71.5–98.5%)relativesensitivity (22[+]lipl32PCR/24[+]16SRNAandsequencing),100%(CI95%96.3–100%)relativespecificity and98.7%accuracy(CI95%94.8–100%).Thelipl32assaywasabletodetectandquantifyat least10genomeequivalents/reaction.DNAextractedfrom17pathogenicLeptospiraspp., 8intermediate/saprophyticstrainsand21differentpathogenicmicroorganismswerealso testedusingthelipl32assay,resultinginamplificationexclusivelyforpathogenicleptospiral strains.Theresultsalsodemonstratedhighintraandinter-assayreproducibility(coefficient ofvariation1.50and1.12,respectively),therebyqualifyingthenewlydevelopedassayasa highlysensitive,specificandreliablediagnostictoolforleptospiralinfectionindogsusing urinespecimens.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).
∗ Correspondingauthorat:LaboratóriodeMicrobiologiaeImunodiagnóstico,DepartamentodeClínicaMédica,FaculdadedeMedicina
VeterináriaeZootecnia,UniversidadedeSãoPaulo,RuaProfessorOrlandoMarquesPaiva87,Butantã,zipcode05508-270,SãoPaulo,SP, Brazil.
E-mail:[email protected](B.A.Miotto).
https://doi.org/10.1016/j.bjm.2017.09.004
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Leptospirosis is a reemerging bacterial disease of global importancecausedbypathogenic spirochetes ofthegenus
Leptospira.1PathogenicLeptospiraiscurrentlyclassifiedinto10 distinctgenomospeciesandmorethan260serovars,2which canaffect awide varietyofmammalianspecies, including dogsandhumans.Leptospiraltransmissionoccursthrough directcontactwithcontaminatedbiologicalmaterialor indi-rectly through contact with contaminated environmental waterresources.3 Thus,identificationofinfectedanimalsis akeystrategyforpreventingthedisease.
Canine leptospirosisis frequently reported worldwide1,4 and infected dogs manifest a broad spectrum of clinical symptoms, ranging from hepatic and renal failure, often accompaniedbyhemorrhagic and pulmonarydisorders, to mild, self-limiting febrile illness.3 Although susceptible to many serovars, dogs can act as maintenance hosts for different pathogenic Leptospira spp., notably L. interrogans
sv. Canicola, thus contributing to the spread of lep-tospiresintotheenvironmentwithoutovertclinicalsignsof infection.1
Thediagnostic approachforcanine leptospirosisis pre-dominantlybasedonserologicalevidenceofinfection,usually obtainedthroughthemicroscopicagglutinationtest(MAT).5 However,useofMATpresentsseverallimitationsandthe sero-logical resultsshould be interpreted with parsimony: MAT presents lowsensitivityduring theearly stagesofthe dis-ease, and multiple samples from both the acute and the convalescentphasesareusuallyrequiredforconfirmingthe infection.6,7 Additionally, serum titers may therefore rep-resent recent exposure to the pathogen instead of active infection,6,7 and the interpretation of serological data can beparticularlyconfusinginrelationtorecentlyimmunized dogsorthosewithunknownimmunizationstatus.The iden-tificationofchronicallyinfectedindividuals usingMATcan also bechallenging, given that serum titersmay not nec-essarilybeassociatedwith asymptomaticrenal carriage of leptospires.3
Althoughculturingleptospiresisstillconsideredthe eli-gible test to unmistakably confirm leptospiral infection, recoveryofviableleptospiresfromculturemediafaces crit-icaldrawbacks,suchas frequentcontamination, fastidious growthofthepathogenandlowdiagnosticsensitivity,1which undermineitsuseinclinicalandepidemiologicalstudies.On theother hand,PCRhasbeen successfullyusedtoconfirm leptospiral infection andenables earlydiagnosis ofclinical leptospirosis,8,9alongwithidentificationofdogswith asymp-tomaticurinarysheddingofleptospires.10–13
AgreatnumberofPCRassaystargetingdifferent protein-encodinggeneshavebeendevelopedfordiagnosinghuman and animal leptospirosis.5 Different quantitative PCR pro-tocols targeting the lipl32 gene have previously been described11,14–16;however, fewstudies havevalidatedthese assays for detecting leptospires in canine specimens.11,14
LipL32 is an abundant outer membrane protein that
occurs exclusively in pathogenic Leptospira and presents highly pairwise DNA sequence identity among pathogenic species.17
Thecurrent report describes thedevelopment and vali-dationofamodifiedquantitativePCRassaytargetingthelipl32
geneforidentifyingpathogenicLeptospiraincanineurine sam-ples.
Material
and
methods
Samplesandisolates
Urinesamplesfrom35dogswithsuspectedclinical leptospiro-sisthatwereattendedattheUniversityofSãoPauloVeterinary Hospital Service(Hovet FMVZ-USP)between2013and 2015 were used toevaluatethe diagnostic sensitivity, specificity andaccuracyoftheTaqManPCRassay.Thedogsincludedin thisstudywereconsideredsuspectedforacuteleptospirosis when presenting high blood urea nitrogen (BUN) and cre-atininelevels(above60mg/dLand 1.4g/dL,respectively),in associationwithtwoormoretypicalclinicalmanifestations ofleptospirosis(hemorrhagicdisorders,fever,jaundice, pros-trationormuscleweakness).Anadditional116urinesamples fromapparentlyhealthydogsthatwerebeingkeptintwo dif-ferentlocalpublicshelters,whichwerecollectedduring2013, werealsotested.
TheEthicsCommitteeoftheSchoolofVeterinaryMedicine and Animal Science, University of São Paulo (protocols 2706/2012and2406140614)approvedallproceduresinvolving animalmanipulationinthecurrentstudy.
Theleptospiralstrainsusedinthisstudywereprovidedby theBacterialZoonosisLaboratory,DepartmentofPreventive VeterinaryMedicineandAnimalHealth,SchoolofVeterinary MedicineandAnimalScience,UniversityofSãoPaulo.The iso-lates(Table1)weremaintainedinsemisolidFletchermedium (BDDiagnostics,Sparks,MD,USA)containing1.5%agar(BD Diagnostics)at28◦Cunderaerobicconditions,andwere col-lectedatmid-logphase.
SampleprocessingandDNAextraction
Urinesampleswerecollected viaurinarycatheterization or cystocentesis and were centrifuged within2h after collec-tion(6500×gfor25minat25◦C)aspreviouslydescribed.16,18 Thepelletswereresuspendedin2mLofsterile phosphate-buffered saline (PBS; 0.01M PO4; 0.1M NaCl; pH 7.2) and
all DNA extractions were processed not more than 48h after collection. In order toaccess the performanceof the DNA extractionprocedures and verify the presenceofPCR inhibitors,allclinicalsamplesweresubjectedtoquantitative amplificationtargetingthemelanocortin1receptor-encoding gene (MC1R),anuclear genethat encodesforaseven-pass transmembrane Gprotein-coupled receptorprotein that is involved in hair and fur coloringin mammals.19 Genomic DNAfrom clinicalsampleswasextractedusingNucliSens® miniMAGTM (BioMérieux Inc.,Durham, NC, USA), in
accor-dance with the manufacturer’s instructions, with slight modifications:1mLofresuspendedsolutionwasusedinthe initiallysisstepandthefinalelutionstepwasperformedwith 40L.
Additionally, pure cultures of leptospires and non-spirochetal pathogens used to determine the analytical
Table1–Pathogenic,intermediateandnonpathogenicLeptospirastrainsusedforsensitivityandspecificitytestsand resultsfromTaqManreal-timePCRand16SPCRassays.
Species Serogroup Serovar Strain TaqManlipl32 PCR16S
Pathogenic
L.interrogans Pomona Pomona pomona + +
Canicola Canicola HondUtrechtIV + + Icterohaemorrhagiae Icterohaemorrhagiae rga + + Icterohaemorrhagiae Copenhageni m20 + + Sejroe Hardjo(Hardjoprajitno) hardjoprajitno + +
L.kirschneri Autumnalis Butembo butembo + +
Cynopteri Cynopteri 3522c + +
Pomona Mozdok m36/05 + +
L.santarosai Shermani Shermani 1342k + +
Grippothyphosa Bananal 2a/cap + +
Sejroe Guaricura guaicurus + +
L.borgpetersenii Ballum Castellonis castellon3 + +
Celledoni Whitcombi whitcomb + + Javanica Javanica veldratbatavia46 + +
Mini Mini sari + +
L.noguchii Panama Panama cz214 + +
Panama u73 + +
Intermediate/nonpathogenic
L.inadai Lyme m34/99 − +
L.meyeri 19cap − +
L.biflexa Doberdo Rufino rpe − +
Seramanga Patoc patoc − +
Nazaré Nazaré nazaré − +
Andamana Bovedo bovedo − +
BuenosAires buenosaires − +
Garcia Garcia garcia − +
specificityofthelipl32TaqManreal-timePCRweresubjectedto
twowashingsteps(6500×g,25minat25◦C)usingsterilePBS
priortoDNAextraction;thepelletswereresuspendedin2mL
ofsterilephosphate-bufferedsalineandgenomic DNAwas
extractedusingQIAampDNAMiniKit®(QiagenInc.,Valencia,
CA),inaccordancewiththemanufacturer’sinstructions.
Primerandprobedesign
Primers and probe targeting the lipl32 gene were selected
based on a previously reported real-time PCR reaction
described byRojas etal.11 Atotal of113 sequences
encod-ing the LipL32 proteinthat were available in the National Center for Biotechnology Information Nucleotide Database (NCBI;http://www.ncbi.nlm.nih.gov/)werealignedusingthe Mega5.10software20inordertoevaluatetheinsilico speci-ficity of the previously described primers. The selected sequences included all availableL. interrogans sv. Canicola sequences,andsequencesfromL.interrogansserovarsthatare commonlyattributedtocanineleptospirosis(serovars Ictero-haemorrhagiae, Pomona and Grippotyphosa), along with other representative serovars of the species L. interrogans, L.kirschneri,L.borgpetersenii,L.noguchii, L.weiliiand L. san-tarosai (Supplementary Material 1). Two of the sequences thus selected (AY461924 and DQ286416) presented one nucleotidemismatch withthe degeneratedforwardprimer previouslydescribed.11Oligonucleotidestructuralanalysisof the described primers was performed using OligoAnalyser 3.1 (http://www.idtdna.com/), which showed the possibil-ity of self-dimer and hairpin formation. To accommodate
all available sequences tested and to overcome possible structural primer instability, a newly non-degenerated for-wardprimerwasdesigned(5-TAAAGCCAGGACAAGCGCC-3), resultinga138bpampliconwhencombinedwiththereverse primerpreviouslydescribed,whichwasnotmodified.The flu-orescentprobesequencedescribedbyRojasetal.11wasalso notmodified,exceptforuseofminorgroovebinder(MGB)as aquencheratthe3-endinsteadofnonfluorescentquencher (NFQ).
TaqManreal-timePCRassays
Optimal concentrations of primers (100, 300, 500, 600, 700 and 900nM) andprobe (200, 250and 300nM)targeting the
lipl32geneweretestedinaccordancewiththemanufacturer’s recommendations(AppliedBiosystems,ThermoFisher Scien-tific Inc.,Carlsbad,CA,USA).Thisresultedinanoptimized reaction withafinal volumeof25Lusing600nM ofeach primer, 250nM of the probe, 1× TaqMan® Universal Mas-terMixII(ThermoFisher,ScientificInc,Carlsbad,CA,USA), DNase-free waterand 5L of extracted DNA.The amplifi-cation protocol consistedof2minat 50◦C,10minat95◦C and 45 cycles of amplification (95◦C for 15s and 60◦C for 60s).
TheMC1Rassaywascarriedoutusingprimersandprobe previouslydescribed byKanthaswamyet al.21 Thereaction consisted of1× TaqMan® UniversalMaster MixII(Thermo Fisher, Scientific Inc, Carlsbad, CA, USA), 900nM of each primer, 250nM ofthe probe, sterile DNase-free water and 5Lofextracted DNA,resultingin afinalvolume reaction
of25L.Thecyclingconditionsconsistedofaninitialstep of50◦Cfor2min,followedby10minat95◦Cand40cycles ofamplification(95◦C for15sand 60◦Cfor60s).All MC1R
runswere performed concomitantlywith analysison DNA extractedfrompureculturesofcaninefibroblasts,aspositive controls.
AllTaqManreal-timePCRrunswereperformedinthesame machine(AppliedBiosystems® 7500Real-TimePCRSystem, ThermoFisherScientificInc,Carlsbad,CA,USA).
Analyticalsensitivityofthelipl32assay
Theanalyticalsensitivityofthelipl32assaywasdetermined through amplification of serial dilutions of genomic DNA extractedfromapurecultureofL.interrogansserovarCanicola strainHondUtrechtIV.DNAwasextractedfroma2mL sam-pleandquantifiedinduplicateusingaQubit® 2fluorimeter (Invitrogen,ThermoFisherScientificInc,Carlsbad,CA,USA) withaQuant-iTTMDNAAssayKit(Invitrogen,ThermoFisher
ScientificInc, Carlsbad,CA, USA).Thenumber ofGenomic Equivalents(GEs),assumingonegenomecopyperleptospire, wasestimatedbasedonagenomicsizeof4.659Mb.Toprepare standardcurves,theextractedDNAwasstandardizedtoan initialconcentration of1×106GE/reaction, in orderto
per-formserial10-folddilutionsuntilreaching1×101GE/reaction.
All standard-curve dilutions were tested in triplicate, and eachrunincludedasinglenegativecontrolcontaining ster-ilenuclease-freewater.Thelower limitofdetection(LLOD) wasdeterminedastheconcentrationatwhich95%ormore of the replicates from eight runs (performed on different occasions)presentedamplification. Thecycle threshold(Ct)
cutoffvaluewasdeterminedbycalculatingtheaverageCt
val-ues ofLLOD replicatesfrom differentstandard-curve runs. Coefficientsofvariation(CV)andaverageCtvalueswere
cal-culatedtodefineinterandintra-assayvariationsandestimate theassayreliability.TheCVvalueswerecalculatedusingthe standarddeviation(SD)andthemeanCt usingtheformula
CV=(SD×100%)/meanCt.
Analyticalspecificityofthelipl32assay
The specificity of the new set of primers targeting the
lipl32 gene was initially confirmed by BLAST analysis (http://blast.ncbi.nlm.nih.gov/), followed by performing the assay using extracted DNA from several spirochetes as a template,including pathogenic strains, saprophytic strains andstrainswithintermediatepathogenicity.DNAextracted from pureculturesofother non-leptospiralpathogens was also tested, including Brucella canis (strain RM6/66 – ATCC 23365),Enterococcusfaecalis(fieldstrain),Escherichiacoli(strain DH5␣),Salmonellaentericasv.Typhi(ATCC14028),Yersinia ente-rocolitica(field strain),Proteus mirabilis(field strain), Candida albicans(ATCC90112),Staphylococcusepidermidis(fieldstrain),
Staphylococcus aureus (ATCC 25923), Streptococcus pyogenes
(fieldstrain),Klebsiellapneumoniae(fieldstrain), Pseudomonas aeruginosa (ATCC 27853), Serratia marcescens (field strain),
Mycobacteriumtuberculosis(strainH37Rv),Mycobacteriumbovis
(strainAN5),Rickettsiarickettsii(strainTAIAC¸U),Rickettsia bel-lii(strainMOGI),Rickettsiaparkeri(strainAT24),Ehrlichiacanis
(strainSÃOPAULO),Leishmaniainfantumchagasi(strainCBT56
RIO GRANDE DO NORTE) and Toxoplasma gondii (strain
RH).
Diagnosticsensitivity,specificityandaccuracyofthe lipl32assay
Quantitative amplification of DNA extracted from clinical samplesthatwereobtainedfromsymptomaticand asymp-tomaticdogswasperformedintriplicate.Thesampleswere consideredpositivewhenatleasttwothirdsofthereplicates presentedCtvalueslowerthanthecutoffvaluethathadbeen
established.Forcomparativeanalysis,theperformanceofthe modified lipl32TaqMan real-time PCRassay wascompared with resultsobtainedfrom awell-established conventional PCRinwhichaLeptospira genus-specificsetofprimers tar-geting a331bpfragmentofthe 16SrRNAgene(Lep1Fand Lep2R)wasused.22ConventionalPCRamplificationwas car-riedoutasfollows:94◦Cfor5min,40cyclesat94◦Cfor30s, 60◦Cfor30s,72◦Cfor30sandafinalextensionat72◦Cfor 5min.PureL.interrogansserovarCanicola(strainHondUtrecht IV)genomicDNAwasusedasapositivecontroland DNase-freewaterasanegativecontrolinallPCRruns.Theamplified productswereseparatedbyelectrophoresisona2%agarose gelstainedwithSYBRSafeDNAgelstain(Invitrogen,Thermo FisherScientificInc, Carlsbad,CA,USA)and wereanalyzed underUVtransillumination.Thenucleotidesequencesofthe amplifiedfragmentsweresequencedusingtheBigDye Termi-nator3.1cyclesequencingkit(ThermoFisherScientificInc, Carlsbad,CA,USA)onanABI7500GeneticAnalyzer(Thermo Fisher Scientific Inc, Carlsbad, CA, USA), in accordance with the manufacturer’s recommendations.The sequences obtainedwerecomparedwiththoseintheGenBankdatabase (http://www.ncbi.nlm.nih.gov/BLAST), to determine species identification.Agreementbetweentheassayswasassessed usingthe kappatest,witha95%confidenceinterval,along withcalculationofrelativesensitivity,specificity and accu-racy.The95%confidenceintervals(CI)forthesensitivityand specificityvalueswerecalculatedasproportionCI.The95% confidence intervals for proportions (sensitivity, specificity andaccuracy)werecalculated,asalltheotherstatistics,using thesoftwareRStudio(Version1.0.136–©2009–2016RStudio,
Inc).
Results
Themodifiedlipl32TaqManreal-timePCRassaywasableto detect1×106to1×101GE/reactionoftheextracted
leptospi-ralDNAinalinearmanner,withacoefficientofcorrelation (of 0.998 and efficiencyof 98.96%.The analytical dataalso showedthattheassaywasabletodetect10copiesper reac-tion(LLOD):from42replicatescontaining10copies/reaction fromeightdifferentruns,41replicatespresented amplifica-tion.
ThereactionpresentedaCt cutoffvalueof37.5arbitrary
units (SD± 0.324)and wasdetermined based onthe aver-ageCtvalueof17replicatesamplifying10copiesperreaction
fromfivedifferentstandard-curveruns.Thestandardcurves were selectedbasedon adequateefficiencyvalues(ranging
from 92.74%to98.96%) andhigh Ct values(>38) were not
consideredinthe calculationtoavoidunspecific amplifica-tions.Allselectedrunswereperformedonseparatedaysand thehighestintra-assayandinter-assayCVvaluesconsidering allstandard-curvedilutionswere1.50and1.12,respectively (SupplementaryMaterial2).
Thelipl32TaqManreal-timePCRassaywasabletodetect DNAfromallpathogenicLeptospiraspp.tested,whilenoneof theintermediateorsaprophyticLeptospirastrainspresented amplification(Table1).Allstrainstestedwerealsosubjected to16SrRNAamplificationtoconfirmtheappropriate extrac-tionprocedure,andallsamplesyieldedstrongpositivebands whenanalyzedonagarosegel(Table1).Moreover,the new setofprimersuseddidnotshow anyunspecific hybridiza-tion in the in silico evaluation and no amplification was observedwhenDNAfrom other non-spirochetalpathogens wastested.
Whenconsideringbothsymptomaticandasymptomatic dogs,thelipl32assaypresentedhighoverallsensitivity(91.6%; CI 71.5–98.5%), specificity (100%; CI 96.3–100%) and accu-racy (98.7%; CI 94.8–100%), also revealing high agreement withthe conventional PCRassay associatedwith sequenc-ing analysis (kappa=0.948; CI 0.88–1). All the parameters used to compare the lipl32 TaqMan real-time PCR assay andthe16SPCRassay(withandwithoutDNAsequencing), accordingtotheclinicalstatusofthedogsarepresentedin
Table2.
Out of the 35 urine samples collected from dogs pre-senting suspected acute leptospirosis, 11 (31.42%) showed positive results in the lipl32 TaqMan real-time PCR assay. All lipl32-positive samples also tested positive in the 16S rRNA conventional PCR; DNA from all samples could be sequenced and BLAST analysis revealed high similarity (>99%)topathogenicLeptospiraspp.However,fiveadditional samplestestedpositivesolelythroughthe16SrRNA ampli-fication. Threeofthem presentedhighsequence similarity to non-leptospiral organisms (two in relation to uncul-turedbacteriaand oneinrelationto Canislupusfamiliaris),
and two PCR-positive samples presented high sequence similarity topathogenicLeptospira spp.,thus revealing sen-sitivity differences between the lipl32 TaqMan real-time PCR assay and the 16S assay performed with or without the association withsequencing analysis (Table2). Among the 116 samples collected from apparently healthy dogs, 12 (10.34%) tested positive in the lipl32 TaqMan real-time PCR assay; all lipl32-positive samples also tested positive in the conventional PCR and were successfully identified as pathogenic Leptospira species through further sequenc-ing analysis (>99% similarity). Nevertheless, an additional 15 urine samplespresented positiveresults solelythrough the 16S rRNA PCR (Table 2); for three samples, no read-able sequences were retrieved, and sequence analyses on the other 12 samples revealed unspecific amplifica-tions,withhighsequencesimilarity toCollinsellaintestinalis
(n=2), Corynebacterium sp. (n=2), Pasteurellaceae bacterium
(n=1),unculturedorganisms(n=6)andCanislupusfamiliaris
(n=1).
All lipl32-negative samples tested positivefor the MC1R
gene,thusconfirmingthattheDNAextractionand amplifi-cationprocedureswereappropriate.
Discussion
ThemodifiedqPCRassaydescribed inthisstudypresented high analytical sensitivity, thus enabling consistent detec-tionoflowconcentrationsofleptospiralDNAextractedfrom canineurinespecimens(atleast10copiesperreaction).These resultswerecompatible withthe sensitivitylevelsof previ-ousstudiesthatusedthelipl32geneasatargetformolecular detectionofleptospiresinclinicalsamples.11,14,16Thereaction presentedlow analytical variationwithinor betweenruns, thusconfirmingthehighanalyticalreliabilityoftheassay.
The newly designedforward primer showed consensus areas in relation to all sequences retrieved from Gen-Bank,including sequences fromthe twoleptospiral strains belonging to L. santarosai and L. borgpetersenii that had presented nucleotidemismatches withthe forward primer originallydescribed.11Althoughunusual,L.santarosaiandL.
borgpetersenii infections were recentlyreported indogs.23,24 Consequently,developmentofhighlysensitiveassays capa-bleofdetectingamorediverserangeofpathogenicLeptospira
sp.couldbecrucialforwiderapplicationofmolecularassays. Thelipl32TaqManreal-timePCRassaywasabletodetectDNA fromallthepathogenicLeptospiraspp.tested,whilenoneof theintermediateandsaprophyticstrainspresented amplifi-cation.Moreover,noinsilicounspecificprimerhybridization wasdetectedandnoamplificationwasobservedwhenDNA fromnon-leptospiralpathogenswastested,thusconfirming thehighspecificityoftheassay.
All the positive TaqMan real-time PCR results obtained from clinical suspected cases were confirmed through the conventional16SPCRwhencombinedwithfurther sequenc-ing analysis. Despite the high overall agreement between theassays(kappa=0.874),twosamplestestedpositivesolely throughthe16SPCR.Thenucleotidesequencesobtainedfrom bothofthesesamplespresentedhighsimilaritytothespecies
L.interrogans,thusconfirmingleptospiralinfection.The per-formance of the TaqMan real-time PCR assay was tested severalmonthsafterrunningthe16SPCR,andthereforethe discrepancybetweentheseresultsmightbeexplainedby pos-siblelossofDNAintegrityduringsamplestorage,aspreviously described.25 Moreover,conventionalPCRprotocolsaremore susceptibletocontaminationduringlaboratorialprocedures than are real-time assays,26 and the differences observed mightalsobeattributabletocontaminationwhilehandling the clinical material. In relationtothe lipl32 TaqMan real-timePCRassayalone,24ofthe35dogs(68.6%)withsuspected clinicalleptospirosisdidnotpresentamplificationof leptospi-ralDNAinurinesamples.Theseresultscanbeattributedto severalcauses,including:(I)intermittenturinarysheddingof leptospiresviaurine;(II)hyperacutemanifestationofthe dis-ease,thusprecedingmigrationofthepathogentotherenal tubules;(III)clinicalmanifestationsattributedtoothercauses notrelatedtoleptospiralinfection;and(IV)sheddingofvery lowquantitiesofleptospiresintheurine,whichwouldlimit detectionofthepathogenthroughtheassay.
The molecular evaluationon urine samples taken from asymptomatic dogs showed 100%agreementbetween Taq-Man real-time PCR and conventional PCR in association withnucleotidesequence analysis(kappa=1).Nonetheless,
Table2–Kappatestandaccuracycalculationandrelativesensitivity/specificityvaluesofthelipl32TaqManreal-timePCR assayusingthe16SPCRassayand16SPCRassayassociatedwithLeptospiraidentificationbyDNAsequencingas referencetests.
Clinicalstatusofthedogs 16SPCRassay 16SPCR+Leptospirasp. identificationbysequencing Negative Positive Total Negative Positive Total Symptomatic TaqManreal-time
assay Negative 19 5 24 22 2 24 Positive 0 11 11 0 11 11 Total 19 16 35 22 13 35 Sp=100%(79.1–100%) Se=68.8%(41.5–87.9%) Sp=100%(81.5–100%) Se=84.6%(53.7–97.3%) kappa=0.705(0.48–0.93); accuracy=85.7%(68.9–94.6%) kappa=0.874(0.70–1); accuracy=94.3%(79.5–99.0%) Negative Positive Total Negative Positive Total Asymptomatic TaqManreal-time
assay Negative 89 15 104 104 0 104 Positive 0 12 12 0 12 12 Total 89 27 116 104 12 116 Sp=100%(94.8–100%) Se=44.4%(26.0–64.4%) Sp=100%(95.6–100%) Se=100%(69.9–100%) kappa=0.551(0.36–0.74); accuracy=87.1%(79.2–92.3%) kappa=1.0(1.0–1.0); accuracy=100%(96.0–100%) Negative Positive Total Negative Positive Total Total TaqManreal-time
assay Negative 108 20 128 127 2 129 Positive 0 23 23 0 22 22 Total 108 43 151 127 24 151 Sp=100%(95.7–100%) Se=53.4%(37.8–68.5%) Sp=100%(96.3–100%) Se=91.6%(71.5–98.5%) kappa=0.593(0.48–0.76) accuracy=86.7%(80.0–91.5%) kappa=0.948(0.88–1.0) accuracy=98.7%(94.8–100) Sp:relativespecificity;Se:relativesensitivity.
15samples testedpositivesolely throughthe conventional
PCR assay, and sequencing analysis provided evidence of
unspecific amplification in most of these samples (n=12).
Despiteseveralattempts,itwasnotpossibletorecover
read-able sequences or reproduce amplification from the other
threesamples, which were consequentlyconsidered
nega-tive.16SribosomalRNAgenesequencingisthemostusual
approachformolecularcharacterizationofspirochetesandit
isconsideredtobeawell-establishedPCR-basedtoolfor
dis-tinguishingleptospiral species.27 However,previous reports
haveshownthatthereare considerablerisksofamplifying DNAfromcertaincommensalandenvironmentalbacteria,14 which undermines its use for confirmation of leptospiral infectioninclinicalsamples.Althoughthe16SPCRwasshown toberemarkablyunspecificinthepresentstudy,theresults alsoshowedthatuseofprimerstargetingthe16SrRNAgene can provide reliable results if associated with sequencing methods. Furthermore,all unspecificamplifications in 16S PCRtestednegativeinthequantitativePCR,whichconfirmed thehighspecificityofthenewlydevelopedqPCRassay.
TheapplicationoftheTaqManreal-timeassayusing com-bined samples from both symptomaticand asymptomatic dogsalsorevealedhigh sensitivity,specificityand accuracy standards, withhighoverall agreementbetween thenewly developedTaqManassayandtheconventionalPCRassociated withsequencingmethods,asourresultshaveshown.
In conclusion, the present study described the devel-opment and validation of a reliable and highly sensitive
quantitativeassaytargetingexclusivelypathogenicLeptospira
species. These results may contribute to a more precise diagnostic approach for identification of symptomatic and asymptomaticdogsinfectedbypathogenicleptospires.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
WewouldliketothankMarciaReginaFranzolinofthe Butan-tan Institute forprovidingbacterial strains. Thiswork was supportedbytheResearchSupportFoundationoftheStateof SãoPaulo(Fundac¸ãodeAmparoàPesquisadoEstadodeSão Paulo,FAPESP;no.2012/14681-7);B.A.M.holdsaFAPESPgrant (no.2012/13022-0)andaPhDfellowship(no.164284/2014)from theNationalCouncilforScientificandTechnological Devel-opment(ConselhoNacionaldeDesenvolvimentoCientíficoe Tecnológico,CNPq).
Appendix
A.
Supplementary
data
Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2017.09.004.
r
e
f
e
r
e
n
c
e
s
1. SchullerS,FranceyT,HartmannK,etal.European consensusstatementonleptospirosisindogsandcats.J SmallAnimPract.2015;56:159–179.
2. BourhyP,ColletL,BrisseS,PicardeauM.Leptospira mayottensissp.nov.,apathogenicspeciesofthegenus Leptospiraisolatedfromhumans.IntJSystEvolMicrobiol. 2014;64:4061–4067.
3. Andre-FontaineG.Canineleptospirosis–dowehavea problem?VetMicrobiol.2006;117:19–24.
4. Azocar-AedoL,MontiG.Meta-analysesoffactorsassociated withleptospirosisindomesticdogs.ZoonosesPublicHealth. 2016;63:328–336.
5. PicardeauM,BertheratE,JancloesM,SkouloudisAN,Durski K,HartskeerlRA.Rapidtestsfordiagnosisofleptospirosis: currenttoolsandemergingtechnologies.DiagMicrobiolInfec Dis.2014;78:1–8.
6. MillerMD,AnnisK,LappinMR,LunnKF.Variabilityinresults ofthemicroscopicagglutinationtestindogswithclinical leptospirosisanddogsvaccinatedagainstleptospirosis.JVet IntMed.2011;25:426–432.
7. LevettPN.Leptospirosis.ClinMicrobiolRev.2001;14:296–326.
8. HarkinKR,RoshtoYM,SullivanJT.Clinicalapplicationofa polymerasechainreactionassayfordiagnosisof
leptospirosisindogs.JAmVetMedAssoc.2003;222:1224–1229.
9. HarkinKR,RoshtoYM,SullivanJT,PurvisTJ,ChengappaMM. Comparisonofpolymerasechainreactionassay,
bacteriologicculture,andserologictestinginassessmentof prevalenceofurinarysheddingofleptospiresindogs.JAm VetMedAssoc.2003;222:1230–1233.
10.GayN,Soupé-GilbertME,GoarantC.Thoughnotreservoirs, dogsmighttransmitLeptospirainNewCaledonia.IntJEnviron ResPublicHealth.2014;11:4316–4325.
11.RojasP,MonahanAM,SchullerS,MillerIS,MarkeyBK,Nally JE.Detectionandquantificationofleptospiresinurineof dogs:amaintenancehostforthezoonoticdisease leptospirosis.EurJClinMicrobiolInfectDis.2010;29: 1305–1309.
12.SamirA,SolimanR,El-HaririM,Abdel-MoeinK,HatemME. LeptospirosisinanimalsandhumancontactsinEgypt: broadrangesurveillance.RevSocBrasMedTrop.2015;48: 272–277.
13.ZakeriS,KhoramiN,GanjiZF,etal.Leptospirawolffii,a potentialnewpathogenicLeptospiraspeciesdetectedin human,sheepanddog.InfecGenEvol.2010;10:273–277.
14.FinkJM,MooreGE,LandauR,VemulapalliR.Evaluationof three5exonuclease-basedreal-timepolymerasechain reactionassaysfordetectionofpathogenicLeptospiraspecies incanineurine.JVetDiagnInvest.2015;27:159–166.
15.VillumsenS,PedersenR,BorreMB,AhrensP,JensenJS, KrogfeltKA.NovelTaqMan®PCRfordetectionofLeptospira speciesinurineandblood:pit-fallsofinsilicovalidation.J MicrobiolMethods.2012;91:184–190.
16.StoddardRA,GeeJE,WilkinsPP,McCaustlandK,Hoffmaster AR.DetectionofpathogenicLeptospiraspp.throughTaqMan polymerasechainreactiontargetingtheLipL32gene.Diag MicrobiolInfecDis.2009;64:247–255.
17.PinneM,HaakeDA.LipL32isasubsurfacelipoproteinof Leptospirainterrogans:presentationofnewdataand reevaluationofpreviousstudies.PLOSONE.2013;8:e51025.
18.LevettPN,MoreyRE,GallowayRL,TurnerDE,SteigerwaltAG, MayerLW.Detectionofpathogenicleptospiresbyreal-time quantitativePCR.JMedMicrobiol.2005;54:45–49.
19.EvansJJ,WictumEJ,PenedoMCT,KanthaswamyS.Real-time polymerasechainreactionquantificationofcanineDNA.J ForensicSci.2007;52:93–96.
20.TamuraK,StecherG,PetersonD,FilipskiA,KumarS.MEGA6: Molecularevolutionarygeneticsanalysisversion6.0.MolBiol Evol.2013;30:2725–2729.
21.KanthaswamyS,PremasuthanA.Quadriplexreal-timePCR (qPCR)assayforhuman–canine–felinespeciesidentification andnuclearDNAquantification.ForensicSciIntGen. 2012;6:e97–e98.
22.MérienF,AmouriauxP,PerolatP,BarantonG,SaintGironsI. PolymerasechainreactionfordetectionofLeptospiraspp.in clinicalsamples.JClinMicrobiol.1992;30:2219–2224.
23.MiottoBA,MorenoLZ,GuillouxAG,etal.Molecularand serologicalcharacterizationofthefirstLeptospirasantarosai strainisolatedfromadog.ActaTrop.2016;162:1–4.
24.LlewellynJR,Krupka-DyachenkoI,RettingerAL,etal. UrinarysheddingofleptospiresandpresenceofLeptospira antibodiesinhealthydogsfromUpperBavaria.BerlMunch TierarztlWochenschr.2016;129:251–257.
25.CarlsenKH,JensenJS.MycoplasmagenitaliumPCR:does freezingofspecimensaffectsensitivity?JClinMicrobiol. 2010;48:3624–3627.
26.MackayIM.Real-timePCRinthemicrobiologylaboratory. ClinMicrobiolInfect.2004;10:190–212.
27.PosticD,Riquelme-SertourN,MerienF,PerolatP,BarantonG. Interestofpartial16SrDNAgenesequencestoresolve heterogeneitiesbetweenLeptospiracollections:applicationto L.meyeri.ResMicrobiol.2000;151:333–341.