Sensitivity, specificity and comparison of three commercially available immunological tests in the diagnosis of Cryptosporidium species in animals

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Veterinary

Microbiology

Sensitivity,

specificity

and

comparison

of

three

commercially

available

immunological

tests

in

the

diagnosis

of

Cryptosporidium

species

in

animals

Olga

Dani ˇsová

_,

_Monika

_Halánová

_,

_Alexandra

_{Valenˇcáková}

_,

_Lenka

_Luptáková

a_{UniversityofVeterinaryMedicineandPharmacy,DepartmentofBiologyandGenetics,Koˇsice,Slovakia} b_{PavolJozef ˇSafárikUniversity,FacultyofMedicine,DepartmentofEpidemiology,Koˇsice,Slovakia}

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Articlehistory:

Received11October2016 Accepted28March2017

Availableonline6September2017 AssociateEditor:RoxanePiazza

Keywords: Cryptosporidiumspp. Immunologytests Comparison PCR Geneticdiversity

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Thestudywasconductedtocomparethespecificityofimmunologicaldiagnostic meth-odsusedforthediagnosisofCryptosporidiumspeciescapableofcausinglife-threatening infectioninbothimmunosuppressedandimmunocompetentpatients.Forthedetection ofCryptosporidiumspeciesin79animalswithdiarrhoea,weusedthreeCopro-antigentests: RIDASCREEN®Cryptosporidiumtest,Cryptosporidium2ndGeneration(ELISA)andRIDA®QUICK Cryptosporidium. Forimmunoassaysweused positiveandnegativesamplesdetectedby meansofpolymerasechainreactionandvalidatedbysequencingandnestedpolymerase chainreaction toconfirm thepresence sixdifferentspeciesof Cryptosporidiumspecies. Prevalence ofcryptosporidiosisin the entire groupdetermined byenzyme immunoas-say,enzymelinkedimmunosorbentassay,immuno-chromatographictestandpolymerase chainreactionwas34.17%,27.84%,6.33%and27.84%,respectively.Sensitivityofanimal sampleswithenzymeimmunoassay,enzymelinkedimmunosorbentassay,and immuno-chromatographic test was 63.6%, 40.9% and 22.7%, resp., when questionable samples were considered positive,whereas specificity of enzymeimmunoassay, enzymelinked immunosorbent assay and immuno-chromatographictest was75.9%, 78.9% and 100%, respectively.Positivepredictivevaluesandnegativepredictivevaluesweredifferentforall thetests.Thesedifferencesresultsarecontroversialandthereforereliabilityand repro-ducibilityof immunoassaysasthe onlydiagnosticmethod isquestionable.The useof variousCryptosporidiumspeciesindiagnosisbasedonimmunologicaltestingand differ-entresultsobtained byindividualtestsindicatepotentialdifferencesinCopro-antigens producedbyindividualCryptosporidiumspecies.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthorat:PavolJozef ˇSafárikUniversityinKoˇsice,FacultyofMedicine,DepartmentofEpidemiology,Koˇsice,Slovakia.}

E-mail:[email protected](M.Halánová).

https://doi.org/10.1016/j.bjm.2017.03.016

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Cryptosporidiaare cosmopolitanwidespread parasite, with broadhostspecificity,primarilyoccurringinyounglivestock. Humans are also susceptible, especially immunodeficient individuals.Inthe recentyears,considerable attention has been paid tocryptosporidiosis caused byzoonotic species, especiallytohostspecificityofthesespecies,andthe asso-ciatedpossibility ofdisease transmissionbetweendifferent hostsintheenvironment.

Clinical manifestation of cryptosporidiosis comprises asymptomaticformsbutalsoseverechronicstatescausing damagetothegastrointestinaltractandaccompaniedby diar-rhoea, anorexia, cachexia, dehydration with dissemination ofparasites tothe surrounding organs withpotential fatal impactonimmunosuppressedsubjects.1,2 _{Cryptosporidiosis}

withclinicalmanifestationaswellasasymptomaticshedding ofoocystsismorefrequentintheyoungthaninadults. Reli-ableandearlydiagnosisisrequirednotonlyofinfectionwith fatalconsequencesbutalsoofasymptomaticinfections.3,4

A direct microscopic diagnosis of Cryptosporidium from stoolsamplesislaboriousand requiresqualified personnel to identify the pathogen. The diagnostic accuracy is sig-nificantly reduced bya low concentrationof oocystsor by mechanically/enzymaticallydamagedoocystsandirregularly excreted oocysts.5,6 _{Worldwide seroprevalence in livestock}

reaches27–30%.7

Intherecentyears,Copro-antigencommercialtests,such asenzymeimmunoassay(EIA),orimmunochromaticdipstick test(ICT)havebeenusedforrapiddiagnosis.Accordingtothe manufacturer,thesetestsarerapidandsensitiveenough,but provideonlyquantitativeresultswhichsufficeonlyfor detec-tionofthepresenceofpathogenintheholdingsbutnotfor individualdiagnosisandidentification.6,8_{Diagnosisbasedon}

Copro-antigenasasingletestfordetectingthe presenceof cryptosporidiaisinadequate,particularlyinriskgroupssuch asimmunodeficientpatientswithlife-threateningdiarrhoea.9

Therefore, molecular methods,including polymerase chain reaction(PCR),becamereferencemethodsforthedetection, identification,differentiationandgenericgenotypingof Cryp-tosporidiumspp.10,11

The aim of our study was to evaluate and compare three commercially available Copro-antigen tests, namely RIDASCREEN® Cryptosporidium test (Enzyme Immunoassay – EIA), Cryptosporidium 2nd Generation (Enzyme Linked Immunosorbent Assay – ELISA), and RIDA®QUICK Cryp-tosporidium (Immuno-chromatographic test – ICT). PCR method,sequencingandphylogeneticanalysiswereusedto confirmthepresenceofcryptosporidiaanddisprovefalse pos-itivityandnegativityofsamples.

Materials

and

methods

Studypopulation–samples

Stoolsampleswere collected from 79animals divided into threegroups(1st–35pigs;2nd–34calves;3rd–10lambs),

withclinicalsymptoms(diarrhoea,abdominalpain,anorexia, weightloss,dehydration).

BymeansofPCRanalysisandsubsequentsequencing,we trulydetectedpositiveandnegativesamplesthatwereused fortheimmunoassay.Weused22positivesamplesofvarying localization.Intestinalspecies:calves–C.parvum(10);C.bovis (2);pigs–C.scrofarum(5),C.suis(2);andgastricspecies:pigs –C.muris(2), C.andersoni(1).Thesensitivities,specificities, positivepredictivevaluesandnegativepredictivevalueswere calculatedaccordingtoLoong.12

Molecularanalysis

DNAisolation

Genomic DNA was extracted from 100mg of stoolsample usingaDNA-Sorb-BNucleicacidExtractionkit(AmpliSence, Russia)accordingtothemanufacturer’sinstructions.Before extraction,wehomogenizedthestoolanddisruptedoocysts at6500rpmfor90swithadditionof0.5-mm-glassbeads, 1.0-mm-zirconbeadsand300␮Llysissolutioninahomogenizer Precellys24(Bertintechnologies).PurifiedDNAwasstoredat −20◦_{CuntiluseinPCR.}

NestedPCR

UsingamodifiedprotocolfornestedPCRweamplified350bp longampliconsspecificfor18SSUrRNAgeneof Cryptosporid-iumspecies.13,14

The volume of the PCR reaction mixtures was, in both cases,50␮L,fromwhichtheDNAsamplewas5␮L.Inthese reactions,weusedprimerswithaconcentrationof0.2␮Mand 5UTaqDNApolymerases(FIREPol).

ThePCRswereruninathermocycler(XPThermalCycler Blocks)withaninitialdenaturationat95◦Cfor5min,followed by35cyclesat95◦Cfor1min,61/57◦Cfor1min,and72◦Cfor 2min.Afinalelongationstepat72◦Cfor7minwasincluded forthecompleteextensionoftheamplifiedproducts.

Electrophoresisandsequencing

A secondaryPCRproductwas evaluatedbyelectrophoresis andvisualizedunderUVlightwith312nmwavelength. Sam-plesthatwerepositiveaftersequencingwereconsequently comparedtosequencesstoredintheGenBankinaccordance withthegeneticmarkerof18SSUrRNAgene.15

PCRproductsweredirectlysequencedinbothdirections. The sequences were aligned and completed using Chro-masProProgrammeand comparedtoknown sequencesin theNationalCentreforBiotechnologyInformationGenBank database.

Phylogeneticanalysis

Thesequenceddatawereprocessedtoformasequence align-ment for identifying similarities using MEGA6 software in subdirectory Align with CLUSTAL W option. Subsequently, thephylogenetictreewasconstructedalsowithMEGA6 soft-ware using a Phylogeny menu and Maximum Likelihood method.

A

C

B

Fig.1–ComparisonofthreecommerciallyavailableCopro-antigentests.(A)RIDASCREEN®Cryptosporidiumtest[Enzyme Immunoassay(EIA)],(B)Cryptosporidium2ndGeneration[EnzymeLinkedImmunosorbentAssay(ELISA)],(C)RIDA®QUICK

Cryptosporidium[Immuno-chromatographictest(ICT)].

Immunologytests

EIA

A commercial Copro-antigen ELISA EIA kit [RIDASCREEN®Cryptosporidium (R-BIOPHARM-AG, Germany)] wasusedaccordingtothemanufacturer’sinstructions(Fig.1). Briefly, 100␮L of the liquid faecal or stool samples were diluted1:10withdilutionbuffer,mixedwellandincubatedat roomtemperaturefor15min.Then,100␮Lofthesupernatant fromeachsamplewasplacedinatestwell.Then,100␮Lof theenzymeconjugatewasaddedandplateswereincubated atroomtemperaturefor60min.Afterwashingfivetimeswith dilutedwashingbuffer,thewellswereincubatedwith100␮L substratesolutionfor15minbefore50␮Lofstopreagentwere added.Opticaldensitiesweredeterminedat450and620nm using an Epoch ELISA reader (Biotek, Bad Friedrichshall). Accordingtothecut-offvalueprovidedbythemanufacturer (extinction for the negative control+0.15), samples were

considered positive if their extinction was morethan 10% abovethecalculatedcut-offvalueandquestionableifitwas betweenthecut-offandthecut-offplus10%.

ELISA

Quantitative detection of Cryptosporidium antigen in stool samples was carried out byin vitro immunoassayusing a commercialkitCryptosporidiumELISA(DiagnosticAutomation, INC,Calabasas,CA),(Fig.1).Inthecaseofapositive immuno-logicalreaction forthepresenceofCryptosporidiumantigen, theabsorbance(450/630nm,ELISAReaderOptysMRThermo Labsystems)wasreadof0.15ODunitsandabove.Absorbance readinglessthan0.15ODunitsindicatedthatthesampledid notcontaindetectablelevelsofCryptosporidiumantigen.

ICT

The commercially available kit RIDA®QUICK Cryptosporid-ium/Giardia Combi (R-BIOPHARM AG) was used according

tothe manufacturer’s instructions(Fig.1).Briefly, a100-␮L aliquotfrom theliquid faecalorstoolsampleswasdiluted 1:10withthedilutionbuffer,vortexedthoroughlyandallowed tosettlefor3min.Fromthesupernatant,500␮Laliquotswere transferredtonewtesttubes.Teststripswereimmersedinthe supernatantfor5–10min.Asamplepositivefor Cryptosporid-iumgivesabluebandwhichappearsalongwithagreencontrol band.16

Ethicalclearance

Thestudywasapprovedbytheinstitutionalethics commit-teeoftheUniversityofVeterinaryMedicineandPharmacyin Koˇsice,Slovakia.

Results

Inourstudy,byanalysing79samplesofanimalfaecesusing PCRandsequencing,wedetectedtruepositivityin22cases (27.84%),inwhichwedetected6species–C.parvum,C.bovis, C.scrofarum,C.suis,C.muris,C.andersoni(Table1).

Resultsofthethreeimmunoassaywereasfollows:using EIA, ELISA and ICT to examine 35 samples from pigs (1st group),positivewere14(40.0%),7(20.0%)and0(0.0%) sam-ples,respectively.Incalves(2ndgroup),therewerepositive13 (38.23%),13(38.23%)and5(14.7%)samples,andinthelambs (3rdgroup)0(0.0%), 2(20.0%) and0(0.0%) samplesbyEIA, ELISAandICT,respectively.Theprevalenceof cryptosporidio-sisinallfaecalsampleswas27(34.17%),22 (27.84%)and 5 (6.33%)byEIA,ELISAandICT,respectively(Table1).The preva-lencedetectedbyEIA(34.17%) andELISA (27.84%) methods wasaboutthesame,butitwasnotaconfirmationofaproven positivityinthesamesamplebyseveralmethods.UsingPCR as a reference method, animal samples sensitivity (SE) of theEIA,ELISAandICTwas63.6%,40.9%and22.7%, respec-tively,whenquestionablesampleswereconsideredpositive, whereasspecificity(SP)ofEIA,ELISA,ICTwas75.9%,78.9%, 100%,respectively.Positivepredictivevalues(PPV)and nega-tivepredictivevalues(NPV)weredifferentforallthreetests

(Table2).

Phylogenictreeforpartialfragmentofthe18SrRNAgene

Forcreatingaphylogenetictree,21sequenceswereidentified, beforeusedforimmunologytests,byBLASTas Cryptosporid-iumspp.and17referencesamplesfromNCBI,wereusedasthe bestmatchingspecies(Fig.2).Asanout-groupweused refer-encesequences(NCBI)Eimeriasp.(JQ993661.1),Eimeriatenella (JX093900.1) and Eimeria zuernii (AY876923.1). Evolutionary branchingofsequencesinthephylogenetictreealsoshowed relationshipbetweensequencesidentifiedasC.parvumandC. bovis,C.scrofarum,C.suis,C.muris,C.andersoni.

Discussion

In the recent years, the number of commercial diag-nostic immunoassays for the presence of the antigen to Cryptosporidium spp. antibodies increased rapidly, focusing especiallyonspeed,easeandsufficientsensitivityoftesting.

However,thesetestsareusedasscreeningtests,provideonly quantitative resultsand are suitableonlyfor detectingthe presence/absenceofinfectioninlargegroupsofanimalsor humans.6,8_{Weitzeletal.,}17_Helmyetal.,16_{andUppaletal.,}18

intheirstudiesshowedtheinaccuracyofimmunoassaysand ahigherdetectionrateofthepathogenbyPCRmethods.In our study, we confirmed the inaccuracy of immunoassays not onlywithregard tothe percentagedifference between the tests, butalso thediversityofindividual positive sam-plesanalyzedbyeachtest.WhenusingthePCRmethodand sequencing,wedetectedinfectionwithCryptosporidiumspp.in 28.57%ofsamplesnotonlyinsamplespositiveinatleastone immunoassaybutalsoinfivesampleswherethepresenceof infectionwasnotconfirmedbyanyofthethreeCopro-antigen immunoassays.TheprevalencedetectedbyEIA(34.17%)and ELISA(27.84%)methodswasnearlythesame,butitwasnota confirmationofaprovenpositivityinthesamesampleby sev-eralmethods(Table1andFig.1).Thesensitivity(SE)ofanimal samplesdetectedwithEIA,ELISAandICTwas63.6%,40.9% and22.7%,respectively,whenquestionablesampleswere con-sidered positive, whereas specificity (SP) of EIA,ELISA and ICT was 75.9%, 78.9%,100%, respectively (Table 2). Despite thefactthattheproducersofthementionedimmunoassays declare100%specificityand100%sensitivity,thedifferences observedinourstudynotonlybetweenindividualassaysbut alsobetweendifferentgroupsindicatecontroversialreliability andreproducibilityoftests.Therefore,theresultsof serolog-icalteststhatareusedtodetectantigensofCryptosporidium spp.inthefaeces,eitherpositiveornegative,obtainedbya sin-glediagnosticmethodarenotreliableandappearinsufficient, particularlywithrespecttopatientsfromlife-threateningrisk groups(immunosuppressedpatients).

Possible explanation of controversial results of Copro-antigenimmunologicaltestsforCryptosporidiumspp.detection isthatnotallcommerciallyproducedantibodiesareableto recognizeallCryptosporidiumspp.oocystsantigensof individ-ual species,althoughallsolubleandinsolubleantigensare detectedby EIAtests. Antibodiesin therespective kits are notrespondingorrespondonlyweaklytoantigensofsome species thatare geneticallydistant from speciesC. parvum andC.hominis,whichserveasabasisforproductionofthese tests.19,20 _{Ourphylogenetic analysisconfirmedgenetic}

vari-abilityofindividualspeciesandbythatalsoprobabilitythat eachCryptosporidiumspp.canproducevariousCopro-antigens whicharenotinevitablydetectablebyimmunologicaltests, asthesetestsare commerciallyproducedusingantigensof speciesC.parvumandC.hominis.19,20 _{SpeciesC.parvumand}

C. hominis are also detectable bymeans ofcommon genes specificforthem(e.g.GP60),whichcomparedtoother Cryp-tosporidiumspp.enablenotonlytheiridentificationbutalso genotypingandthusindicatetheirgeneticvariabilitywithin thespecies.TheimmunologicaltestsuseCryptosporidiumspp. thatoccurmostfrequentlyinindividualanimals,thus exhibit-ingaprobablehostspecificityforthegivenanimalspecies.In ourstudy,positivitywasdetectedbyallthreeimmunological testsonlyforonespecies,namelyC.parvum,butonlyinthree samples,althoughwetested10positivesamplesoffaecesfor thepresenceofC.parvum(Table1).Theprimaryidentification ofnon-specificspeciesC.murisandC.andersoniinpigs, differ-entlocalizationonphylogenetictreeandinabilityofdetection

Table1–ComparisonofpositiveresultsofdifferentdiagnostictestsforthedetectionofCryptosporidiuminfaecal samples.

Species EIA ELISA ICT PCR Identifiedspecies Pigs (n=35) − − − + C.muris + − − + C.muris + − − + C.scrofarum − − − + C.scrofarum + − − − + + − − + + − − + − − − + − − − − − − + C.suis + − − + C.suis − + − + C.scrofarum + − − + C.scrofarum + − − − − + − − + + − − + − − − − + − + C.scrofarum + + − − + − − + C.andersoni

No.ofpositivesamples 14 7 0 10 Calves (n=34) + − − + C.bovis − − − + C.bovis − + − − − − − + C.parvum + + + + C.parvum + + + + C.parvum + + − + C.parvum − + − − + + − + C.parvum + + + + C.parvum + + + + C.parvum + + − − + + − − + + − − + − − − − − − + C.parvum − + − − + − − + C.parvum

No.ofpositivesamples 13 13 5 12 Lambs

(n=10)

− + − −

− + − −

No.ofpositivesamples 0 2 0 0



Positiveanimals/% 27/34.17 22/27.84 5/6.33 22/27.84

byimmunologicaltestsalsoindicatethepotentialofC.muris

andC.andersonitoproducedifferentCopro-antigens. Anotherpossibilityistheoccurrenceofantigensubtypes andnonexistenceofsurfaceantigens,wherespecific glyco-proteinsnecessary fordetection are notexpressed but are boundtounspecificgroups(e.g.hydroxylgroups,basic sug-ars)duetoreorganizationandmodificationofgeneregions, butnotmutations.BabesiaandPlasmodiumhavemanyantigen subtypes,wheremodificationofgeneregionisevolutionary duetoprotectionofthegivenpathogen.Antigenvariations form when pathogen withstands host immunity through sequence modification of antigenslocated on erythrocytes

surface.21 _{Blake et al.}22 _{pointed at antigen variability and}

genome diversity of pathogens from phylum Apicomplexa intheirstudy,whichexamined similaritiesbetweengenera Plasmodium,ToxoplasmaandEimeria,butalsoantigen variabil-ityofindividualspeciesfromthesamegenus.

Serologicaltestsareanappropriatediagnosticmethodin numerous clinical groups(e.g. animal herd)for determina-tion of the presence of a pathogen, because ofits speed, sufficientsensitivityand affordability.However,oneshould keep in mind that immunological tests are not capableof detectingeveryCopro-antigenofeachofmorethan30species and many genotypes identified up to 2016. Thespecies C.

Table2–Sensitivity(SE/%),specificity(SP/%),positivepredictivevalues(PPV/%),andnegativepredictivevalues(NPV/%)of testsfordetectionCryptosporidiumspp.instoolsamples.

EIA ELISA ICT

Pigs SE 35.7 20.0 0 SP 64.0 80.0 100.0 PPV 35.7 28.5 0 NPV 76.2 71.4 71.4 Calves SE 75.0 58.3 41.6 SP 81.8 72.7 100.0 PPV 69.2 53.8 100.0 NPV 85.7 76.2 75.8 Lambs SE 0 0 0 SP 100.0 80.0 100.0 PPV 0 0 0 NPV 100.0 100.0 100.0



scrofarum, C. suis and C. andersoni identified in our study have been identified alsoin humans.23,24 _Thelow

reliabil-ity of immunological tests, particularly when identifying infection in one patient, necessitates to use for accurate diagnosis atleast two, optimally threedifferent diagnostic methods(microscopy,immunologyandmolecularmethods). Molecularmethods,includingthepolymerasechainreaction

(PCR)areconsideredreferencemethodsnotonlyfordetection ofcryptosporidiosisinfection,butalsoforidentification indi-vidualCryptosporidiumspp.andgenotyping.Anditisexactly the identification and genotyping of individual species by molecularmethodsthatconfirmsvariabilityamong individ-ualspeciesandthusalsodifferentgenotypeandphenotype manifestations.21,22 C.muris (isolate 2) C.parvum (isolate 51) C.parvum HQ 259571.1 C.parvum FJ 379574.1 C.parvum KT151554.1 C.parvum (isolate 47) C.parvum (isolate 65) C.parvum (isolate 58) C.scrofarum (isolate 32) C.scrofarum KJ 790202.1 C.scrofarum KF 975530.1 C.scrofarum KC 481231.1 C.scrofarum JX 424840.1 C.suis (isolate 18) Eimeria sp.JQ 993661.1 Eimeria zuernii AY 876932.1 Eimeria tenella JX 093 C.suis (isolate 19) C.suis AB 449870.1 C.bovis (isolate 38) C.bovis (isolate 39) C.bovis KJ 70243.1 C.bovis KF 128749.1 C.bovis JX 515546.1 C.bovis HQ 179571.1 C.scrofarum (isolate 4) C.scrofarum (isolate 5) C.scrofarum (isolate 21) C.scrofarum (isolate 20) C.parvum (isolate 60) C.parvum (isolate 52) C.parvum (isolate 48) C.parvum (isolate 53) C.parvum (isolate 46) C.muris (isolate 3) C.muris KJ 162056.1 C.andersoni (isolate 33) C.andersoni AY 954886.1 C.andersoni KP 704554.1) C.muris AJ 307669.1 99 92 84 94 89 92 41 72 68 44 87 80 81 59 89 0.04 72 82 74 78 74 66 89 91 94 41 100 68 89 100 54 56 77 74 57 81 49 38 100 90

Fig.2–EvolutionaryrelationshipsamongspeciesC.parvumandC.muris,C.andersoni,C.suis,C.scrofarum,C.bovisinferred fromapartialfragmentofthe18SrRNAgene.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

Thisstudy was supported bythe Grant APVV-15-0134 and VEGA1/0061/16.

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