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RESEARCHOpenAccessAdvantageofusingcolonicwashoutsforBlastocystisdetectionincolorectalcancerpatientsVinothKumarasamy1,AprilCamillaRoslani2,KuppusamyUmahRani3andSureshKumarGovind1*AbstractBackground:TherehavebeenpreviousstudiesassociatingmicroorganismstocancerandwithourrecentfindingsofBlastocytsisantigenhavingahigherinvitroproliferationofcancercellsstrengthensthesuspicion.
Collectingfaecalsamplesalonetoassociatethisparasitewithcancermaynotbeaccurateduetothephenomenonofirregularsheddingandthepossibletreatmentadministratedtothecancerpatients.
Hence,thisbecomethebasistosearchforanalternatemethodofsamplecollection.
ColonicwashoutisanalmostcompletewashedupmaterialfromcolonandrectumwhichincludesvariousmicroorganismssuchasBlastocystisandotherlodgedmaterialwithinthevilli.
ThedetectionofparasiteincolonicwashoutswillgiveabetterreflectionontheassociationbetweenBlastocystisandCRC.
Methods:BlastocytsisdetectionwasmadebyinvitroculturemethodusingJones'medium,formaletherconcentrationtechniqueandconventionalpolymerasechainreaction(PCR)onfaecalsamplesandcolonicwashoutsof204CRCpatientsfromcolonoscopyprocedure.
Faecalsamplesandcolonicwashoutsfrom221normalindividualsservedascontrol.
Results:WeobservedanincreaseddetectionofBlastocystisusingcolonicwashouts(n=53,12.
47%)thanfaecalsamples(n=26,6.
12%).
ElevenfaecalsamplesshowedpositiveresultsforBlastocystiswhichwerealsofoundincolonicwashoutsusingthePCRtechnique.
ThisstudyforthefirsttimeshowedasignificantBlastocystisinfectionamongCRCpatients(n=43,21.
08%)comparedtotheasymptomaticnormalindividuals(n=22,9.
95%).
Blastocystissubtype3infectionwasfoundtobesignificantlymoreprevalent(n=26,12.
75%)comparedtoothersubtypesnamelysubtype1:n=9(4.
41%),subtype2:n=1(0.
49%),subtype5:n=1(0.
49%)andmixedsubtype:n=6(2.
94%)amongtheCRCpatients.
Conclusion:ThestudyshowedthatcolonicwashoutsprovideabetteralternativeforBlastocystisdetectioninCRCpatientscomparedtofaecalsamplesasthispreventstreatmentregimeandthephenomenonofirregularsheddingfrominfluencingthedetectionresultsobtainedfromfaecalsamples.
Keywords:Colorectalcancer,Blastocystis,SubtypesBackgroundBlastocystisisoneofthemostcommonlydetectedmicro-organismsinthehumangut[1].
Itisknowntocausemanynon-specificsymptomssuchasstomachbloatinganddiar-rhea[2].
Amongcolorectalcancer(CRC)patients,theprevalenceofBlastocystisisyettobedeterminedprobablyduetothelackofevidencetoshowitspathogenicrole.
Consideringthefactthathumanintestineisoftenex-posedtovariousmicroorganisms,theputativeroleofinfectiousagentsincausinggastrointestinaldisordersin-cludingCRCisundeniable.
Inaddition,nearly18%ofallcancersworldwidewereassociatedwithinfectiousagents[3].
Humancoloncaneasilyallowthegrowthofover500differentspeciesofbacteriaduetoitsenvironmentwhichisrichinnutrients[4].
BlastocystisinfectionwasalsoreportedtobefrequentincancerandHIV/AIDSpatientswithgastrointestinalsymp-toms[5].
AlthoughtendifferentsubtypesofBlastocystis(Subtype1–Subtype10)havebeenidentifiedthusfar*Correspondence:suresh@um.
edu.
myEqualcontributors1DepartmentofParasitology,FacultyofMedicine,UniversityofMalaya,KualaLumpur50603,MalaysiaFulllistofauthorinformationisavailableattheendofthearticle2014Kumarasamyetal.
;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Kumarasamyetal.
Parasites&Vectors2014,7:162http://www.
parasitesandvectors.
com/content/7/1/162[6-8],epidemiologicalstudiesrelatedtoBlastocystishaveoftenbeenhamperedbythepoorsensitivityofstandardmethodsavailabletodetectBlastocystisgenotypesinfae-calsamples.
Whileapreviousinvitrostudyhavedemon-stratedthatthesolubilizedantigenofBlastocystiscouldproliferatecoloncancercells[3],thepresentstudyat-temptstoassesstheprevalenceofBlastocystisinCRCpa-tients.
However,detectionofBlastocystisbecomesharderinthiscohortasthesepatientsarelikelytoundergochemotherapyandotherdrugtreatmentsthatprobablycouldhavekilledtheorganism.
Theirregularsheddingre-portedpreviously[9]raisesthepossibilitythatBlastocystisdetectioninfaecalsamplescouldbemissed.
ThereforeinthepresentstudyweattemptedtocomparetheprevalenceofBlastocystisusingcolonicwashoutsandfaecalsamplestoidentifytheBlastocystisgenotypepresent.
ItishighlyprobablethatabetterBlastocystisoccurrenceinpatientswithCRCcanbeobtainedwhencolonicwashoutsareusedassamplematerial.
Patientsresorttocolonoscopyonlyasalastresorttofindingthecausewhentheinitialscreeningandtreatmentdonotrelievesymptoms.
Thepossibilityofre-coveringparasitesfromcolonicwashoutishigherasitcontainsanalmostcompletewashedupma-terialfromcolonandrectumwhichincludesvariousmicroorganismssuchasBlastocystisandotherlodgedma-terialwithinvilli.
MethodsPatientsThisisahospitalized-basedcross-sectionalstudyof425patientswhounderwentdiagnosticcolonoscopyinUniver-sityofMalayaMedicalCentre(UMMC).
Thesampleswerecollectedovera2-yearperiod,between2010and2012.
Colonoscopyisusuallyrecommendedforscreeningandpreventionofcolorectalcancer(CRC).
Over751potentialparticipantswerebriefedaboutthisstudybutonly425pa-tientswereinterested.
Theywereprovidedwithaconsentforminperson.
Theybelongedtotwocohorts-agroupof221patientswhocamefornormalscreeningand204pa-tientspresentingcolorectalmalignancies.
ThisstudywasapprovedbytheMedicalEthicsCommitteeoftheUMMCinaccordancewiththedeclarationofHelsinki.
SpecimencollectionandscreeningColonicwashoutswereobtainedatthetimeof(orimme-diatelypriorto)thediagnosisofCRC.
Whereas,faecalsampleswereobtainedviathestandardclinicalprocedureswherebypatientswillbegivenadurationof1–2weekstodelivertheirfaecalsamples.
Colonicwashoutswerecol-lectedincleandisposablebowls.
Eachsamplewascentri-fugedat1,400*gandthepelletsobtainedwereculturedinJones'medium[10]supplementedwith10%horseserum[11]andincubatedat37°Cfor24handthenscreenedforBlastocystis.
DNAwasextractedfromtheremainingfreshcolonicwashoutforgenotypingpurposes.
Apeasizeofthefaecalsamplescollectedfromthesamein-dividualsweredirectlyculturedinJones'mediumandotherprocedurescarriedoutweresimilartothatofcolonicwash-out.
PredominantBlastocystissubtypewasidentifiedusingpolymerasechainreaction(PCR)technique.
FormaletherconcentrationtechniqueFreshcolonicwashoutandfaecalsampleswereroutinelyprocessedbytheformaletherconcentrationtechnique(FECT)toobtainstoolconcentrate.
Ficoll-PaquedensitygradientcentrifugationmethodwascarriedouttoisolateBlastocystiscystfromtheconcentrate[11].
Briefly,thestoolconcentratere-suspendedinPBSwaslayeredon5mlofFicoll-Paqueandcentrifugedat1,600*gfor20minutes.
BlastocystiscystlayerwhichwasformedaftercentrifugationwasremovedintoanotherFalcontubeandre-suspendedin1mlPBSandobservedundermicroscopeforthedetectionofBlastocystiscyst.
DNAextractionandgenotypingPCRtechniquewasusedtodetectBlastocystisinadditiontostandardstoolculturetechniqueinbothcolonicwash-outsandfaecalsamples.
DNAextractionwasconductedusingQIAampDNAStoolMiniKit(QIAGEN).
Briefly,200mlofthesedimentsofcolonicwashoutsamplesorfaecalsampleswereusedtoextractDNA.
PCRwascarriedoutwithspecificsequence-taggedsite(STS)primers(Table1).
SevendifferentSTSprimerswereusedforgeno-typingasdescribedpreviously[6,7,12-18].
TheSTSprimersusedwereSB83(351bp),SB340(704bp),SB227(526bp),SB337(487bp),SB336(317bp),SB332(338bp)andSB155(650bp)forsubtype1,2,3,4,5,6and7re-spectivelybasedonarecentclassificationterminology[19].
OnemicroliterofDNApreparationwasusedtoamplifythegenomicsequencesina20μlPCRcocktailcontaining0.
2mMofthefourdNTPs,25pmolofeachprimer,1*PCRbuffer(75mMTris–HCl,pH8.
8,20mM(NH4)2SO4,and0.
01%Tween20),2.
5mMMgCl,and1UTaqDNApolymerase(recombinant)(Fermentas).
PCRwascarriedoutwithonecycledenaturingat95°Cfor5min,42cyclesincludingannealingat56.
3°Cfor90s,ex-tendingatfor60s,andadditionalcyclewitha10minchainelongationat72°C(Thermalcycler,BIO-RAD,USA).
ThePCRproductsandasizemarkerofa100-bpladderwereelectrophoresedin1.
5%agarosegels(Promega,USA)whichwerestainedwithethidiumbromideandphoto-graphedusinganultravioletgeldocumentationsystem(Uvitec,UK).
PCRamplificationforeachprimerpairwasrepeatedthriceforeachpositivesample.
StatisticalanalysisDatawereanalyzedusingStatisticalPackageforSocialSciencesforWindowsSPSS(Version17.
0).
TheChiKumarasamyetal.
Parasites&Vectors2014,7:162Page2of5http://www.
parasitesandvectors.
com/content/7/1/162squaredtestwasusedtodeterminesignificanceofdiffer-encesinprevalenceofBlastocystisbetweenthehealthyindividualsandCRCpatients.
Fisher'sExacttestwasusedtodeterminethepre-dominantBlastocystissubtypeinthecolonicwashoutsofnormalandCRCpatients.
Inalltheanalyses,aprobabilitylevelofp<0.
05wasusedtoindicatestatisticalsignificance.
ResultsBlastocystisdetectioninthefaecalsamplesandcolonicwashoutsTheoverallprevalenceofBlastocystisinfectionobtainedfromthethreemethodsusedwas15.
29%(65/425).
Therewere65/425,4/425,and4/425samples(includingfaecalsamplesandcolonicwashouts)detectedpositiveforBlasto-cystisviaPCR,invitrocultivationandformaletherconcen-trationtechniquerespectively.
Overlappingpositiveresultswereoftenobservedinfaecalsamplesandcolonicwashoutsaswellasamongthedifferenttechniquesused.
Colonicwashoutsandfaecalsamplesshowed12.
24%(n=52)and5.
65%(n=24)ofBlastocystisinfectionrespectivelyviatheconventionalPCRtechnique.
Forty-onecolonicwashoutswerepositiveforBlastocystis,despitethefaecalsamplesfromthesamepatientsbeingnegativeforBlastocystis.
Whereas,11faecalsamplesidentifiedpositiveforBlastocys-tisalsoshowedpositiveforcolonicwashoutsobtainedfromthesamepatientsusingPCRtechnique.
Averysmallper-centage(0.
96%,n=4)offaecalsampleswerefoundpositiveviainvitrocultivationoffaecalsamplesbutnonefromco-lonicwashouts.
AlthoughotherparasitessuchasAscarislumbricoidesandhookwormweredetectedviaformaletherconcentrationtechniquebutthefrequencywasnegli-gibleandstatisticallynon-significant.
BlastocystisinfectionandsubtypeanalysisinCRCpatientsAtotalof43(21.
08%)sampleswerepositiveforBlastocys-tisinfectioninCRCpatientsandwassignificantlyhighercomparedtonormalindividuals(n=22,9.
95%,p<0.
01)(Figure1).
WeconductedconventionalPCR[20]toclassifyBlastocystisintocertainsubtype.
Inthecurrentstudy,fourdifferentBlastocystisgenotypeswereidentifiedamongallthesubjectsnamelysubtype1(ST1),subtype2(ST2),sub-type3(ST3),subtype5(ST5)andmixedsubtypes.
Subtype6(ST6)andsubtype7(ST7)werenotdetectedinallcases.
ST3waspresentathigherlevelscomparedtoothersub-typesdetectedinbothgroupsasshowninTable2.
Overall,ST3wasthemostprevalentsubtype(n=30,14.
71%),whereasST1,ST2,andST5wereseenin5.
39%(n=11),3.
43%(n=7)and0.
49%(n=1)oftheCRCpatients,re-spectively.
Besidesthat,mixedsubtypeinfectionsweredetectedinsixsampleswhichwere0.
98%(n=2,ST1andST2)and1.
96%(n=4,ST2andST3)(Table2).
ST3infectionwasalsofoundtobestatisticallysignificantinCRCpatientsascomparedwiththecontrolgroup(Table2).
DiscussionFaecalsamplecollectionofmorethanthreetimesfromthesamepersonhavebeenshowntosignificantlyraisethepossibilityofdetectingparasites[9].
Howeverthiswouldbetootediousandtroublesometoexecute.
ThefaecalsamplecollectionwaspurposelycarriedoutashowitwouldbeforroutinescreeningpurposeaftertheCRCdiagnosisregardlessofpreorpost-treatmentregimewhichcouldnotbeavoided.
Therefore,thechancesofre-coveringparasiteswhichcouldhavebeeninfluencedbythetreatmentregimecannotbeoverruled.
TheirregularTable1PrimersequencesusedforBlastocystisgenotypingSubtypesSTSprimersetsProductsize(bp)Sequencesofforward(F)andreverse(R)primers(5′-3′)SourceofprimerGenBankaccessionno.
ReferenceCladeintheSSUrRNAphylogenya1SB83351FGAAGGACTCTCTGACGATGANandIIAF166086Yoshikawaetal.
[6]IRGTCCAAATGAAAGGCAGC2SB155650FATCAGCCTACAATCTCCTCBAF166087VIIRATCGCCACTTCTCCAAT3SB227526FTAGGATTTGGTGTTTGGAGARHV93–13AF166088Yoshikawaetal.
[7]IIITTAGAAGTGAAGGAGATGGAAG4SB332338FGCATCCAGACTACTATCAACATTHJ96AS-1AF166091VIRCCATTTTCAGACAACCACTTA5SB340704FTGTTCTTGTGTCTTCTCAGCTCHJ96–1AY048752Yoshikawaetal.
[18]IIRTTCTTTCACACTCCCGTCAT6SB336317FGTGGGTAGAGGAAGGAAAACASY94–3AY048751VRAGAACAAGTCGATGAAGTGAGAT7SB337487FGTCTTTCCCTGTCTATTCTGCARN94–9AY048750IVRAATTCGGTCTGCTTCTTCTGKumarasamyetal.
Parasites&Vectors2014,7:162Page3of5http://www.
parasitesandvectors.
com/content/7/1/162sheddingofBlastocystisinfaecalsamplefurthercom-poundthechallengefordetectingtheseparasitesdes-pitetheuseofthestandardfaecalculturetechnique[9].
Inaddition,detectionusingmicroscopeisagreaterchallengewhenparasitespresentinverysmallnumbersinthefaecalsamples.
Assuch,theusageofcolonicwashoutprobablyfarmoreeffectiveasitcanbecol-lectedalmostimmediatelyduringdiagnosisandthede-tectionwithouttheinfluenceofanyprevioustreatmentregime.
Thediagnosticmethodtodeterminethepres-enceofBlastocystisvarywidelyinsensitivity.
PossibilityofBlastocystisbeingeliminatedbytreatmentusuallysoughtbyCRCpatientsfortheirinitialsymptomscouldhaveresultedinzerodetectioninthepresentstudyusingbothformal-etherconcentrationtechniqueandthegoldstandardinvitrocultivationmethod.
Therefore,PCRtech-niquewasemployedforthescreeningofBlastocystisinbothfaecalsamplesandcolonicwashouts.
BlastocystiswasdetectedincolonicwashoutsamplesfrompatientswhoweretestednegativeforBlastocystisbyusingfaecalsample.
ThisisthefirststudythatdetectsBlastocystisincolonicwashoutsamplesfromCRCpatientsviatheconventionalPCRmethod.
Thesesampleswerecollectedattheinitialstageofcancerdetectionwhichexcludedthepossibilityofthepatientsundergoingmedicaltreatmentssuchaschemo-therapywhichcouldhavekilledtheparasites.
AlthoughpathogenicityofBlastocystisiscontroversial,BlastocystisscreeninginCRCpatientsiscrucialconsideringtherecentinvitrostudiesprovidingevidencesoftheparasite'sexacer-batingpotentialinproliferatingcancercells[21-23].
ThismethodprobablycanbeoneofthemoreeffectivewaystoscreenforBlastocystisforhighriskindividualsandthosewhoaresuspectedofinfectionbutfoundnegativebyotherlesssensitivemethods.
Thecollectionofco-lonicwashoutsiseasierasitisawasteproductpro-ducedfromcolonoscopyprocedure.
Furthermore,thechancesforcross-contaminationcanbepreventedasitcanbecollecteddirectlyandalmostimmediatelyfrompatientswhoareundergoingcolonoscopy.
ThismethodmaypreferablybeusedforindividualsthatgothroughcolonoscopyprocedureforotherdiagnosispurposessuchasCRCandthosewhoaresuspectedofinfectionbutfoundtobenegativebyotheravailablemethods.
PredominanceofBlastocystisST3wassimilartoapreviousstudyconductedamongpatientsinahospitalinSingapore[24].
BlastocystisST3wasreportedtobetheonlysubtypeofhumanorigin,whiletherestbeingzoonotic[25].
AsanearlierreportsuggestedapossiblecorrelationbetweenST3andpathogenicpotential[26],itiscrucialforsubtypeidentification.
Thiswillenableustounderstandbetter,thepossiblepathogenicroleofthesesubtypesplaytotheworseningofcancer.
Further-more,theoccurrenceofothercasessuchasacuteurti-cariaandgastrointestinalsymptomswerereportedinpatientswithST3infection[27].
WeobservedaverylowprevalenceofST1andST2inbothCRCandhealthyindividuals.
Thesesubtypesweremainlyassoci-atedwithzoonotictransmission.
Similarly,ST4,ST6andST7werenotfoundinthesepatientgroupsastheyaremostlyzoonoticmicroorganisms[28].
n=36,16.
18%n=17,7.
69%n=17,8.
33%n=10,4.
52%n=43,21.
08%**n=22,9.
95%0510152025ColorectalcancerNormalBlastocystisinfection(%)Colonicwashouts+FaecalsamplesFaecalsamplesColonicwashoutsFigure1PercentageofBlastocystisinfectionincolonicwashoutsandfaecalsamplescollectedfromCRCandnormalindividuals.
**p<0.
01isthecomparisondonebetweenCRCpatientsandnormalindividuals.
Table2BlastocystisgenotypesfoundinCRCpatientsandnormalindividualsBlastocystissubtypeColorectalcancerpatients(n=204)%(No.
positive)Healthyindividuals(n=221)%(No.
positive)Subtype14.
41(9)*2.
71(6)Subtype20.
49(1)**0.
90(2)*Subtype312.
75(26)#3.
17(7)Subtype1+20.
98(2)**0(0)Subtype2+31.
96(4)*1.
81(4)Subtype50.
49(1)**1.
36(3)*p<0.
05;**p<0.
01arelevelsofsignificancebetweensubtype3Blastocystisandothersubtypes.
#p<0.
05isthesignificantdifferenceinBlastocystis(subtype)infectionbetweenCRCpatientsandhealthyindividuals.
Kumarasamyetal.
Parasites&Vectors2014,7:162Page4of5http://www.
parasitesandvectors.
com/content/7/1/162ConclusionInconclusion,thisstudydemonstratedthatcolonicwash-outscanbeabetteralternativetofecalsamplestoexam-ineforBlastocystisinfectionespeciallyinCRCcases.
OurstudyshowsthatBlastocystisinfectioniscommoninCRCpatientsanditindicatessubtype3aspredominantamongtheseindividuals.
However,thepathogenicroleofthisparasiteinCRCpatientsisstillunclear.
Therefore,furtherstudyhastobeconductedtodeterminethecorrelationbetweenthegenotypeandsymptomatology.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterest.
Authors'contributionsVK,SKG,ACRandURKparticipatedinthedesignofthestudy.
VKdidthedataandsamplecollectionsfromthepatients.
VK,SKG,ACRandURKdidthedataanalysis.
VKwrotethemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgmentsWearegratefultopersonnelatUniversityofMalayaMedicalCentreforcontinuessupportandinassistingussamplecollection,andtotheDepartmentofParasitologyofUniversityofMalayaforprovidingfacilitiesforustoconducttheresearch.
FundingforthisstudywasprovidedbyHighImpactResearchGrantUM.
C/625/1/HIR031andUniversityofMalayaPostgraduateResearchGrant(PPP)–ResearchPG108-2012B.
Authordetails1DepartmentofParasitology,FacultyofMedicine,UniversityofMalaya,KualaLumpur50603,Malaysia.
2DepartmentofSurgery,FacultyofMedicine,UniversityofMalaya,KualaLumpur50603,Malaysia.
3DepartmentofBiomedicalScience,FacultyofMedicine,UniversityofMalaya,KualaLumpur50603,Malaysia.
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1186/1756-3305-7-162Citethisarticleas:Kumarasamyetal.
:AdvantageofusingcolonicwashoutsforBlastocystisdetectionincolorectalcancerpatients.
Parasites&Vectors20147:162.
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