GI-MAP™ Multiplex DNA Stool Technology for the

Integrative and Functional Medicine Practitioner

The GI Microbial Assay Plus

Contents

MicrobiologyandDNAAnalysis ..........................................................................................................................................2

Disruptionofthegastrointestinalmicrobiomecancause:......................................................................................3

Methodology ................................................................................................................................................................................3

TargetAnalytes...........................................................................................................................................................................5

Pathogens......................................................................................................................................................................................5

ViralPathogens...........................................................................................................................................................................8

ParasiticPathogens...................................................................................................................................................................9

Parasites(Non-pathogens)................................................................................................................................................. 10

CommensalBacteria.............................................................................................................................................................. 11

OpportunisticPathogens..................................................................................................................................................... 12

GastrointestinalBacteriaasaTriggerforAutoimmunity..................................................................................... 13

FungalOrganisms................................................................................................................................................................... 14

AdditionalTests....................................................................................................................................................................... 15

DrugResistanceGenes ......................................................................................................................................................... 18

HerbalAntimicrobialAgents ............................................................................................................................................. 18

Conclusions ............................................................................................................................................................................... 20

CompleteListofTargetAnalytesMeasuredontheGI-MAP................................................................................ 20

TheGIMicrobialAssayPlus(GI-MAP)MultiplexDNAStoolTechnologyfortheIntegrativeandFunctionalMedicinePractitionerMicrobiologyandDNAAnalysis

Inthelastfewdecades,DNAanalysishastransformedthefieldofmicrobiology.TheNationalInstitutesofHealthhavefollowedsuitwithinitiativessuchastheHumanMicrobiomeProject,whichcharacterizedthemicrobiomefromover15habitatsofthebodyinmorethan200healthyhumansubjectsusingDNAanalysis.3Morethaneverbefore,wearekeenlyawareofthehealthbenefitsordiseaserisksbroughtaboutbythemicroorganismsthatinhabitthehumanbody.Culturetechniques,previouslythestandard,leftupto50%ofbacterialspeciesvirtuallyinvisible.4Whennext-generationmethodsrevolutionizedthisfield,itallowedtheidentificationoftremendousnumbersofpreviouslyunknownorganisms.Anaerobicbacteriamakeupalargepartofthehumanmicrobiomeandcanbeopportunisticandcauseillness.Therefore,inabilitytocultivatetheseorganismsleftalargeblindspotforclinicianswhentryingtodiagnosethesourceofinfection.

TheGastrointestinalMicrobialAssayPlus(GI-MAP)wasdesignedtoassessapatient’smicrobiomefromasinglestoolsample,withparticularattentiontomicrobesthatmaybedisturbingnormalmicrobialbalanceandmaycontributetoperturbationsinthegastrointestinal(GI)floraorillness.Thepanelisacomprehensivecollectionofmicrobialtargetsaswellasimmuneanddigestivemarkers.Itscreensforpathogenicbacteria,commensalbacteria,opportunisticpathogens,fungi,viruses,andparasites.Itprimarilyusesmultiplex,automated,DNAanalysistogiveintegrativeandfunctionalmedicinepractitionersabetterviewintothegastrointestinalmicrobiome.TheGI-MAPmeasurespathogenicorganismsthatcancausehospital-acquiredinfections(HAI)suchasC.difficileornorovirus,foodborneillnesssuchasE.coliorSalmonella,andcommoncausesofdiarrheasuchasCampylobacter,Shigella,androtavirusA.2Thispanelmeasuresviralcausesofgastroenteritis,unavailablebyothercommonstooltests.ItmeasuresparasitessuchasCryptosporidium,Giardia,andEntamoebahistolytica.TheGI-MAPanalyzesHelicobacterpylorianditsvirulencefactors.ItcandetectopportunisticpathogenssuchasPseudomonasaeruginosa,Klebsiellapneumoniae,Yersiniaenterocolitica,andProteusmirabilus,associatedwithautoimmunemolecularmimicry.Itincludesapanelofsingle-celled,amebicparasitessuchasBlastocystishominis,Dientamoebafragilis,andEntamoebacoli.FungalorganismsaremeasuredbytheGI-MAPsuchasCandida,Geotrichum,andMicrosporidia,withthelatterbeinganewadditiontoDNAstoolanalysis.Finally,theGI-MAPmeasuresstandardmarkersofimmunity,inflammationanddigestionincludingcalprotectin,secretoryimmunoglobulinA(sIgA),anti-gliadinantibody,andpancreaticelastase1.

Disruptionofthegastrointestinalmicrobiomecancause:GastrointestinalsymptomsAbdominalpain5Bloating6Constipation6Crohn’sdisease7,8Diarrhea5,6,9Foodpoisoning10Gastriccancer11Gastritis11Gastroenteritis12,13Gastroesophagealreflux14,15Irritablebowelsyndrome13,16Smallintestinalbacterialovergrowth(SIBO)17Gastrointestinalsymptoms,continued

Ulcer11Ulcerativecolitis18Vomiting19AutoimmuneConditionsAnkylosingspondylitis20Reactivearthritis20-22Rheumatoidarthritis23AllergicDiseaseAsthma24Eczema25-27

Methodology

DiagnosticSolutionsLaboratoryisusinganovelDNAtechniquetodetectacomprehensivelistofstoolbacteria,viruses,fungi,andparasites.ThemethodandinstrumentusedisFDA-clearedforthedetectionof15ofthemostcommoncausesofgastroenteritis-bacteria,parasites,andviruses.2Ithasbeenclinicallyvalidatedforusewithhumanstoolsamplesandsensitivityandspecificitydatacanbefoundbelow.Thisisanautomated,multiplexDNAanalysismethod,allowingforthesimultaneousmeasurementofmultiplebacteria,fungi,parasites,andviruses,allfromasinglesample.2

Multiplexpolymerasechainreaction(PCR)meansthatmanygenesareamplifiedatthesametime,asthoughmanyseparatePCRreactionswerehappeningatonce.Thistechniquemakesitpossibletosimultaneouslydetectmanydifferentorganismsinonesample.Multipleprimersandprobesforeachorganismallowforenhancedsensitivityandspecificity.Themethodmeasuresthe16Sor23SribosomalRNA(rRNA)regions,virulencefactors,andviraltargetsformicrobialdetection.Pathogenicbacteriaarereportedasaqualitativeresult(positive/negative)whereasothermicroorganisms(opportunisticpathogens,fungi,andcommensalflora)arereportedwithquantitativeresults.

Turn-around-timewiththistechniquemaybeaslowasonlythreetofourdays.Otherstooltestingoptionsonthemarketcantakeweekstodeliverresults.Theautomatednatureofthismethodminimizesthechanceforhumanerror.DNAanalysisisnotoriousforbeinghighlylaborintensiveandtherearechancesforhumanerrorinextraction,hybridization,andamplification.ThisistheonlyFDA-clearedDNAtestforgastrointestinalmicrobesavailable.Incontrasttomolecularmethodologiesusedbyotherlaboratories,thismethodwasnotdevelopedin-house.IndependentclinicalvalidationdataonthemethodusedbyDiagnosticSolutionsLaboratoryshowsthatthetestisreliableandreproducible.TheseaddedqualityaspectsarenovelandgivetheclinicianadditionalconfidenceinDNAanalysis,notavailableelsewhereintheintegrativeandfunctionalmedicinemarket.

AccuratemeasurementofDNAtargetsreliesontwomolecularmethods:makingcopiesoftargetgenes(amplification)andmatchingsingle-strandedDNAfromthetargetstotheprobesinalock-and-keymanner(hybridization).Afterreceivingstoolspecimens,nucleicacidsareextractedandpurified.ThesamplesundergomultiplexPCR,whichamplifiesmanygenetargets.AmplificationcangeneratethousandstomillionsofcopiesofasingletargetDNAsequence.

AfteramplifyingtheDNAtargetsfoundinthestoolspecimen,thespecimenundergoeshybridization.ItistreatedwithmagneticbeadsimbeddedwithDNAprobes.AprobeisasegmentofDNAthatseekstojoinwithitscomplementarymatchandisradioactivelylabelledformeasurement.Hybridizationisthebinding(likealockandkey)ofonesingle-strandedDNAsegmenttoanothercomplementarypieceofDNA.ThisstepisimportantforaccurateidentificationofamicrobebasedonitsDNAsignature.EachbeadhasauniqueDNAsignaturethatwillbindtotheamplifiedtargetgene,ifitispresentinthestoolspecimen.Thisallowsforaccurateandsensitivedetectionofatargetorganism.MultiplexPCRusesthissamemolecularapproachformanymicrobesatthesametime,allowingforsensitiveandspecificdifferentiationofmanyorganismssimultaneously.

Inthepast,drawbackswithDNAanalysishavebeenitsincrediblesensitivityandpotentialfornon-specificbinding.BecauseofthetremendoussensitivityofDNAanalysis,microbesmightbedetectedinapatientspecimenthatwerenotactuallypresentinhighnumbersatthetimeofstoolcollection.SpecificitycanbeaproblemwithDNAanalysisbecausemicrobesmaybedetectedincorrectlyduetocross-reactivity.WiththeGI-MAPmethod,probesareattachedtodifferentbeadsinsuchawaythatnon-specificbindingisdecreasedandfalsepositivesaredecreased.DiagnosticSolutionsLaboratoryusesseveralothermethodimprovementstofurtherimproveaccuracyandprecisionofthemethod.

OtherstooltestsonthemarketprimarilyuseMatrixAssistedLaserDesorption/IonizationTime-of-Flight(MALDI-TOF)toidentifystoolmicrobes.MALDI-TOFtechnologyusedbyotherlaboratoriesformicrobialdetectionreliesonbacterialcultureofthestoolspecimen.TheorganismsthatareculturedarethenidentifiedusingtheMALDI-TOF.Alimitationofthismethodistherelianceonculturemethods.Microbesinthestoolspecimencangrowordecayaftercollectionandintransit.Thereforetheymaynotrepresentthesampleatthetimeofcollection.Additionally,organismsthatdonotgrowundercultureconditionscannotbeidentified.CollectionofstoolspecimensforDNAanalysisimmediately“freezes”theDNA,sothatitmorecloselyrepresentstheactualmicrobialpopulationsofthepatient’sgastrointestinaltractatthetimeofcollection.

FDA-Cleared GI Pathogens Measured in the GI-MAP Campylobacter

Clostridium difficile, Toxin A/B

Escherichia coli 0157

Enterotoxigenic E.coli (ETEC) LT/ST Shiga-like Toxin producing E.coli (STEC) stx1/stx2

Salmonella

Shigella

Vibrio cholerae

Yersinia enterocolitica

Viruses

Adenovirus 40/41

Norovirus GI/GII

Rotavirus A

Parasites

Cryptosporidium

Entamoeba histolytica

Giardia

TargetAnalytes

Thehumangastrointestinalmicrobiomehousestrillionsofbacteriaandresearchshowsthatthesemicroorganismsareessentialforhumanmetabolism,28nutrition,immunefunction,29andresistancetoinfection.30Over500differentspeciesofmicroorganismsfrom30differentgenerahavebeenidentifiedfromthehumangut.Butinanyoneperson,thereare100million-1trillionmicroorganismspergramoffecalcontent.31Mostmicrobesinthehumangutarebelievedtobebeneficialorcommensal.Therearemicrobesthatcolonizemanypeoplebutonlybecomepathogenicincertainsituations(opportunisticpathogens).Finally,therearepathogensthatarewidelyrecognizedtocausediseaseinthehumanhost.

Althoughtheyareubiquitous,pathogenicbacteriadonotcauseillnessinallpeople.Thisisbecausecommensalgastrointestinalfloracanprotectthehostfrominfection.Whengutmicrofloraprotectstheintestinesfrompathogensandharmfulmicroorganismsitiscalled,“colonizationresistance.”30Animalmodelsshowthatwhennormalgutmicrofloraarelacking,thehostismoresusceptibletoGIinfectionswithSalmonella.Similarly,afterantibiotictreatmentthereisincreasedriskofpathogenicinfections.30Ontheotherhand,commensalbacteriasuchasLactobacillusandBifidobacteriumcanpreventgastrointestinalinfection.Colonizationresistanceexplainswhymostpathogenicbacteriafailtocausediseaseinhealthysubjects.32

Commensalbacterianaturallyinhabitthehumangastrointestinaltractanddonotcausedisease.Manyarebeneficial;theyproduceenzymes,33vitamins,34shortchainfattyacids,35andothermetabolicproductsthatkeepthebowelsandthebodyfunctioningwell.Theincrediblycomplexinteractionbetweenhumanhealthandthegastrointestinalmicrobiomeisthesubjectofmultiplecutting-edgeresearchstudies.36Giventhemetabolic,nutritional,andimmune-enhancingrolesoftheseorganisms,themicrobiomedeservescloseanalysiswhentreatingpatientswithchronicillness.

Pathogens

TheGI-MAPmeasuresbacterialpathogenssuchasCampylobacter,Escherichiacoli(E.coli)O157,EnterotoxigenicE.coli,Shiga-liketoxin-producingE.coli,Clostridiumdifficile,Salmonella,Shigella,andVibriocholerae.TheGI-MAPmethodhasbeenvalidatedandFDA-clearedforthemeasurementoftheseorganisms.Sensitivityandspecificitydatahavebeendeterminedonthesepathogensinhumanstoolsamplesandarelistedbelow.

ThepathogenictargetshavebeenselectedbasedontheirclinicalutilityandanalyticalvalidityasDNAtargets.Forexample,ClostridiumdifficileispositivewhengenesencodingfortoxinsAandBhavebeendetectedwhileotherorganismsaredetectedbasedontheiruniqueDNAsignatures.Inonecomprehensivereviewofrapidmoleculartechnologiescomparedtoconventionalculturetechniques,theauthorsconcludedthattherewassufficientevidencetorecommendtestingwithPCRforCampylobacter,E.coliO157,andSalmonellaandthatitmayyieldbetterresultsthanculturetechniques.37MultiplexPCR,usingthesamemethodasthatusedfortheGI-MAP,waspreferredoverconventionalmicrobiologicaltechniquesin347patientswithgastroenteritis.AuthorsconcludedthatDNAanalysiswasfasterforpathogenidentificationandprovidedclinicianswitha

largerpanelofpathogens,helpingtocontainnosocomialoutbreaksbeforetheyspread.38

Bacterialpathogensareoftenspreadduetocontaminationoffoodandwaterwithfecalmaterialcontainingthesepathogens.ConsultyourPhysician’sDeskReferenceforstandardtreatmentsforthesepathogens.Antibiotictherapyisnotalwaysrecommendedbecauseantibioticresistancecanworsentheinfection.Hydration,probiotics,andsupportivetherapiesforthegut-immunesystemcanhelptoremovethepathogenfromtheGItract.

Thepresenceofapathogendoesnot,byitself,indicatedisease.2Resultsfromlaboratorytestsmustbeinterpretedtogetherwithclinicalsymptomsandhistorybyaqualifiedhealthpractitioner.WithincreasedawarenessofthecomplexityoftheGIenvironment,apathogenislikelytocausediseaseiftherearevulnerabilitiesinthehost’sdefenses.Forexample,imbalancedmicroflora,poorimmunedefenses,poordiet,toxicexposures,antibiotics,orchronicGIsymptomscouldmakeapersonmoresusceptibletoharmfromapathogen.Whereas,anotherpersonmayhaveafecalpathogenbutisingoodhealth.Inhealthypatients,treatingpathogensmaynotbenecessary.However,continuingtosupportabeneficialanddiversemicrobiotaandastronggut-immunesystemwillfurtherprotectthehostfrominfection.29,39

Despitewhattypeofstooltestisused,thetransientnatureofthemicrobiotamustbeacknowledged.Populationsofmicroorganismscanchangedramaticallyinshortperiodsoftime,especiallyunderstress,withtheuseofantimicrobialmedications,orchangesinthediet,etc.Thetransientnatureofgastrointestinalmicroorganismsmakesitevenmoreimportanttousethelabresultstogetherwithsignsandsymptomstodetermineifaparticularlabfindingisindicativeofaclinicalconditionthatrequirestreatment.Clinicalmonitoringandfollow-uptestingandconfirmationbyothertestingmethodshelpstoanalyzethechangestothemicrobiomeovertimeandverifyclinicallyrelevantfindings.2Similarly,apathogenicorganismfindingonatestresultdoesnotnecessarilyindicatetreatment,evenwhentherearesymptomsofdisease.Healthy,immune-competentpeoplecannaturallyeradicateapathogenwithbasichealthcarepracticesandthepassageofafewweeks,makingtreatmentunnecessary.

Bacteria & Bacterial Toxins in the GI-MAP Sensitivity Specificity Shigella 97.70% 97.80% Campylobacter 97.50% 97.80% Yersinia enterocolitica N/A 100.00% Enterotoxigenic E. Coli (ETEC) LT/ST N/A 97.30% E. coli 0157 88.20% 98.80% Shiga-like toxin producing E. Coli (STEC) stx 1/stx 2 100.00% 99% Salmonella 82.10% 99.10% Clostridium difficile Toxin A/B 97.70% 94.90% Vibrio cholerae N/A 100.00%

Clostridiumdifficile(C.difficileorC.diff)isawell-knownpathogenthatcancausecolitisandClostridiumdifficile-associateddiarrheaorCDAD.Itcommonlypresentswithmildtomoderatediarrheaandoccasionallyabdominalcramping.C.diffisabletocolonizetheGItractafteradisturbanceofthemicrobiota,generallyafterantibiotictherapy.C.diffreleasestoxinsthatcauseinflammationanddamagetotheGIlining.Itinfectsnearly20%ofhospitalizedpatients,makingitthemostcommonnosocomialinfection.40

ToxinsAandBarethemajorvirulencefactorsbelievedtoberesponsibleforC.diffinfectionsymptoms.Theyareproinflammatoryandcytotoxic.Theydamagethecytoskeletonofintestinalepithelialcells,permittingfluidinflux,theyopentightjunctionsintheGIlining,andtherebydamagetheGIlining.ToxinsAandBhaveevenshownsystemiceffectsinanimalmodels,suggestingthattheirbioactivitymaynotbelocalizedtotheGItract.ToxinsAandBareencodedbythetcdAandtcdBgenesandarethereforedetectableusingDNAanalysis.41Real-timepolymerasechainreactionisconsideredagoldstandarddiagnosticmethodologyforC.diff.40

Escherichiacoliisalargeandvariedspeciesofbacteriathatincludesmanystrains.Theycolonizehumansandanimalsandarespreadthroughcontaminatedwater,food,orcontactwithinfectedhumansoranimals.42E.colicancauseinfectionsoutsideoftheGItractsuchasurinarytractinfections,meningitis,andintra-abdominalabscess.43

Whiletherearemanyharmless,andevenbeneficial,E.colistrains,therearesixstrainsthatarenotoriousfortheirpathogenicity,especiallyforGIinfections.EnterotoxigenicE.coli(ETEC)cancausetraveler’sdiarrhea.EnteropathogenicE.coliisacauseofchildhooddiarrhea.EnteroinvasiveE.coli(EIEC)canleadtodysenterysimilartothatcausedbyShigella.EnterohemorrhagicE.colicanleadtohemorrhagiccolitisorhemolytic-uremicsyndrome.EIECandEHECcolonizethecolonwhiletheotherscolonizethesmallintestinesandsubsequentlyinitiatediarrhea.43ThestrainsmeasuredontheGI-MAPare:E.coliO157,EnterotoxigenicE.coliLTandST(ETEC),andShiga-liketoxin-producingE.coli(STEC),targetingstx1andstx2genes.

Shiga-liketoxinproducingE.coli(STEC)hasbeeninvolvedinfoodborneillnessoutbreaks.42ItcausesvariousGIillnesses,includingbloodyandnon-bloodydiarrhea.Shigatoxin(stx1)andShigatoxin2(stx2)aregenerallyconsideredtobethevirulentfactorsresponsibleforseriousillnesscausedbySTEC.Stx1andstx2aregenetictargetsthathelpaccuratelydetectthepresenceofShiga-liketoxinproducingE.coliinstoolsamples.44

ThesamemethodusedintheGI-MAPwasusedtoinvestigatethecauseofanoutbreakinGermanhospitalsinpatientssufferingwithhemolyticuremicsyndrome,presumablyinducedbySTEC.Withrapidscreeningdiagnosticmethodstheywereabletoidentifyanovelserotype--E.coliO104:H4thathadvirulencefactorscharacteristicofbothenterohemorrhagicE.coliandenteroaggregativeE.coli.45

TheserotypeO157:H7hasbeenimplicatedinmanyoutbreaksandcasesofbloodydiarrheaandhemolyticuremicsyndrome43andhasahighprevalenceworldwide.45

EnterotoxigenicE.coliheat-labiletoxin(LT)andheat-stabletoxin(ST)aretheenterotoxinsresponsiblefordiarrhealdiseaseinhumans.ST-producingE.coliiswidelyknowntocausediarrheabutthemechanismisstillunknown.LTactssimilarlytothecholeratoxinbyactivatingadenylatecyclase,leadingtodiarrhea.46

ViralPathogens

Adenovirus,norovirus,androtavirusareviralcausesofgastroenteritisthatarenormallyself-limitinginhealthyindividuals.Whenaclinicianislookingforamicrobialcauseofgastroenteritis,theywouldberemisstooverlookthesevirusesaspossiblecausesofdiarrhea,abdominalpain,andvomiting.Inastudyof4,627patientswithgastroenteritis,PCRstooltechnologydetectednorovirusin36%androtavirusAin31%ofsamples.47Anotherstudyofover300peoplewithacutediarrheaoverthecourseofayearshowed36.0%werepositivefornorovirusand17.3%werepositiveforrotavirus,while5.4%werepositiveforadenovirus.Intotal,virusesaccountedfor58.7%ofcasesofacutegastroenteritis,48pointingtothevalueofviraldetectioninstoolspecimens.

PrevioustestswiththeGI-MAP(unpublished)showedhighincidenceofviralpathogensandevidenceofchroniccarriers.Thismayberelatedtothepersistenceandpervasivenessofviruses.Noroviruswasdetectableforoverthreeyearsingroundwaterandinfectiousforatleast61days.49Therearenostandardtreatmentsforviralgastroenteritisinhealthyhosts.Antiviralsarenotrecommended.50Supportivecareforthegastricmucosa,hydration,andimmune-boostingagentsmaybewarranted.

Adenoviruses40and41causegastroenteritis.Theyareacommoncauseofdiarrheaininfantsandchildrenbutcanalsoaffectadults.Thesepathogenscanreplicatereadilyintheintestine.Theyaretheonlyadenovirustypesthatareshowntobecausativeagentsofgastrointestinaldisease.However,otheradenovirusesmaycausegastroenteritis.Feverandwaterydiarrheaareusuallylimitedto1-2weeks.Adenoviruses40and41mayalsobepresentinthestoolofasymptomaticcarriersandmaynotrequiretreatment.50Adenoviruses40and41belongtothelargergroupofadenoviruses,including52differentserotypes,knowntocauseavarietyofillnessesfromrespiratorytractinfections(commoncold,sorethroat,bronchitis,pneumonia)tobladderinfectionandcystitis.Theyarehardyvirusesthataretransmittedthroughclosecontactsuchastouchinganinfectedpersonorsurface,thenshakinghandsortouchingyoureyes,noseormouth.Otherroutesoftransmissionincludeblood,airparticles(coughingorsneezing)andtheoral-fecalroute.Adenovirusesrarelycausesevereillness,butinfantsandthosewithweakenedimmunesystemshaveahigherriskofdevelopingamoreseriousillnessfromtheinfection.

Viral Toxins in the GI-MAP Sensitivity Specificity Adenovirus 40/41 100.00% 100.00% Rotavirus A 94.70% 99.80% Norovirus GI/GII 93.50% 98%

NorovirusGI&GII,orNorwalkvirus,isthemostcommoncauseofnon-bacterialgastroenteritisintheworld.Itiswidelyknownforcausingthestomachfluoncruiseships.51Threegenotypesofthisdiversevirus,GI,GII,andGIV,caninfecthumans.GenotypegroupII,genotype4(GII.4)isthemostcommonandaccountsforthemajorityofoutbreaksaroundtheworld.52Norovirus,whichcanhaveasuddenorgradualonset,typicallydevelops24-48hoursaftercontactwithaninfectedpersonoringestionofcontaminatedfoodorwater.Symptomsincludenauseaandvomiting,diarrhea,abdominalcramps,low-gradefever,muscleaches,fatigue,andheadache.Norovirusisgenerallyshort-lived,lastingabout24-72hoursbutitishighlycontagiousduetoitsstabilityintheenvironmentandresistancetoheat,cold,anddisinfectantsolutions.Itcansurviveonhardsurfacesforweeksandupto12daysoncontaminatedfabrics.53Infectionaffectsthemicrovilliofthesmallintestine,notthecolon.Thoseinfectedcanshedthevirusforuptotwoweeksafterrecovery,continuingtospreadthevirus.

Norovirusesarethemostcommoncauseofsporadicdiarrheaincommunitysettingsandcauseuptohalfofalloutbreaksofgastroenteritis.54Treatmentsfornorovirusincludehydrationandelectrolytesprimarily,andinsomecasesantiemeticsfornauseaandvomiting,andanalgesicsforpainandheadache.Intravenousfluidandelectrolytesmaybeneededinextremecases.PCRisahighlysensitiveandspecificmethodfordetectionofnorovirus.55

Rotavirusisthemostcommoncauseofsevere,waterydiarrheaininfantsandchildren,especiallyinthedaycaresetting.ThemostcommontypeisrotavirusA,whichaccountsformorethan90%ofinfectionsinhumans.Symptomsusuallybeginwithin2daysofexposuretoaninfectedperson.Low-gradefever,vomiting,abdominalcramps,anorexia,andwaterydiarrheaarecharacteristicsigns.Dehydrationcanbeamajorrisk.56Theinfectioncanpersistfor3to8daysandusuallyresolvesonitsown.However,forthosewithweakenedimmunesystems,theinfectioncancausehospitalizationanddeath.Byagefive,almosteverychildhasbeeninfected.57Adultscanalsobecomeinfectedbutthesymptomsaregenerallylesssevere.Treatmentconsistsmainlyofrehydrationtherapy.

ParasiticPathogens

Aparasiteisanorganismthatlivesandfeedsonahostorganismattheexpenseofthehost.Someparasitescancauseinfectiousdiseaseinhumansbutothersdonot.Parasitescanliveinsidethegut,removingvitalnutrients,anddamagingthegutlining.Someparasiticinfectionsareeasilytreatedandothersarenot,withsymptomsrangingfrommilddiscomforttosevereproblems,includingdeath.Itiscommonlythoughtthatparasiticinfectionsoccurmostlyinunderdevelopedcountries,buttheseinfectionsalsoaffectpeopleindevelopedcountriesincludingtheUnitedStates.Infact,suchpathogenscansurviveintheirhostsandcausehealthproblemsthatmaybehardtoidentify.Parasiticpathogensthatinfectthegastrointestinaltracttypicallycauseawidevarietyofsymptomssuchasdiarrhea,constipation,abdominalcramping,bloating,gas,nausea,andvomiting.Inimmunosuppressedpatients,symptomsmayinvolvethecentralnervoussystem.

Contaminatedfoodanddrinkingwaterpresentthehighestriskforparasitetransmission,butlakes,swimmingpools,andsexualcontactarealsowaysapersoncancontractthesepathogens.Thefecal-

oralrouteisacommonwaythatparasiticpathogensarespread.Therefore,poorhygieneoranyconceivablecontactwithfecalmaterialcouldresultinparasiticinfection.Treatmentsshouldbespecificandbasedonthetypeofparasiteidentified.Effortsshouldbemadetointerrupttheparasite’slifecycletopreventreinfection.Oncesymptomsaregone,itisimportanttoretesttomakesuretheparasitehasbeeneradicated.

Cryptosporidiumisnotoriousforbeingspreadbyswimmingpools.AnumberofCryptosporidiumoutbreakshaveoccurredaftercontaminationofpublicswimmingfacilities.Cryptosporidiumcancausegas,bloating,diarrhea,andabdominalpain.Inahealthy,immune-competentperson,thisisaself-limitinginfectionandcanbeclearedwithin2-3weeks.

Entamoebahistolytica(E.histolytica)isadisease-causingparasitethatcanaffectanyone,althoughitismorecommoninthosewholivedortravelledintropicalareaswithpoorsanitaryconditions.Diagnosiscanbedifficultsince,underamicroscope,itlookssimilartootherparasitessuchasEntamoebadisparandEntamoebahartmanii.Thelattertwoparasitesgenerallydonotcauseillness.E.histolyticaistransmittedviatheoral-fecalrouteorfromcontaminatedfoodorsurfaces.Infectedpeopledonotalwaysbecomesickandsymptomsareoftenmildincludingstomachcrampsandloosestools.Thisparasitecaninfecttheliverorspreadtootherpartsofthebodyincludingthelungsandbrain,althoughthisisnotascommon.Researchhasshownthatinasmallpercentageofpatientswithamebicliverabscess,theinfectioncancausebrainabscesswiththepatientpresentingwithcentralnervoussystemsymptoms.58TreatmentforinfectionwithE.histolyticaincludesantiparasiticdrugtherapyandmayincludeacombinationbasedontheseverityofinfection.

Parasites in the GI-MAP Sensitivity Specificity Giardia lamblia 100.00% 98.20% Cryptosporidium 87.50% 100.00% Entamoeba histolytica 100.00% 99.40%

Parasites(Non-pathogens)Non-pathogenicparasitesarepresentinthegastrointestinaltractandgenerallyareself-limitinganddonotcauseillness.However,someresearchshowsanassociationbetweennon-pathogenicparasitesandgastrointestinalsymptoms.59Therefore,testingofthesemicroorganismsmaybeusefulinsomecases.Recentresearchshowscertainparasites,suchasBlastocystishominis,asanemergingpotentialpathogen.60

Blastocystishominisisfoundthroughouttheworldinbothpeoplewithandwithoutsymptoms.CommonsignsofinfectionwithBlastocystisincludediarrheaorwaterystools,abdominalpain,analitching,constipation,excessgas,anddermatologicissues.Someresearchrecommendstreatmentforpeoplewithgastrointestinalanddermatologicsymptomsbutnotreatmentforthosewhoareasymptomatic.61TheremayalsobeanassociationbetweenBlastocystisandchronicdigestivedisorders,suchasirritablebowelsyndrome.62

EntamoebacoliandE.hartmanniareintestinalamebaethatarefoundinthelargeintestine.Theygenerallyarenotconsideredpathogenic.However,whentheseamebaearefoundinstoolsamplesitcanindicatethepresenceofotherpotentiallypathogenicorganisms.

CommensalBacteria

Trillionsofmicroorganismsinhabitthehumanintestinetomakeupacomplexecosystemthatplaysanimportantroleinhumanhealth.Thegutmicrobiotaisdiverse,variesamongindividuals,andcanchangeovertime,especiallyduringdevelopmentalstagesandwithdisease.ThepredominantclassesofbacteriainthegutareFirmicutes,Bacteroidetes,Actinobacteria,andProteobacteria.ThefungithatarepartofthegutfloraincludeCandida,Saccharomyces,Aspergillus,andPenicillum.

Thesecommensal(friendly)bacteriacoexistwiththeirhumanhostandperformmanyimportantfunctions.Theyextractnutrientsandenergyfromourdiets,maintaingutbarrierfunction,producevitamins(biotinandvitaminK),andprotectagainstcolonizationbypotentialpathogens.30Researchhasdemonstratedthemicrobiota’scapacitytointeractwiththeimmunesystemasanimportanthealthbenefit.63Themicrobiotaalsohasanti-inflammatoryandantioxidantactivity.64Itisessentialthatcommensalbacteriaarediverseandbalancedsincedisruptiontothenormalbalance(ordysbiosis)hasbeenassociatedwithobesity,malnutrition,inflammatorybowelandotherautoimmunediseases,neurologicaldisorders,andcancer.65AlimitedlistofcommensalfloraisincludedintheGI-MAPtestasageneralscreenforlevelsofnormal,protectivefloraortomonitorprobioticsupplementation.TheseincludeBacteroidesfragilis,LactobacillusandBifidobacteriaaswellasE.coli.

Bacteroidesfragilisisahumancommensalbacteriumthatcolonizesthelowergastrointestinaltractinmammals.Bacteroidesspeciesaresomeofthefirstmicroorganismstocolonizethehumangutandarepresentinhighnumbers.B.fragilisisaverycommon,important,Gram-negativeanaerobeyetitaccountsforonlyapproximately0.5%oftheBacteroidesspeciesfoundinthegut.66Initsusualroleasacommensalgutbacterium,B.fragilishasbeneficial,immunomodulatoryactivity.However,ifB.fragilisentersthebloodstream,asaresultofintestinalpermeability,traumaorsurgery,itcancauseseriousinfections.67

B.fragilishasbeenthesubjectofrigorousinvestigationinrecentyearsbecauseitappearstohaveaprotectiveeffectagainstinflammationandpossiblyagainstautoimmunedisorders.B.fragilisrepairsdefectsinthegutbarrierbyinfluencingtightjunctionproteinsandcytokineexpression.35Whenautistic-likemiceweregivenBacteroidesfragilis,itnormalizedintestinalpermeability,restoredmicrobialbalance,andremovedbehavioralandcognitivesymptoms.35B.fragilishasalsobeenshowntocorrectgastrointestinalpathologyinanimalmodelsofcolitis59andinhibitneuroinflammationinmousemodelsofmultiplesclerosis.60Itsanti-inflammatoryactivityisattributedtoasurfacemoleculecalledpolysaccharideAwhichpromotesregulatoryTcellsandanti-inflammatorycytokinesthroughtoll-likereceptor2(TLR2)signaling.67

BifidobacteriaandLactobacillusareanaturalpartoftheflorainthehumanbody.Theyareoftendescribedasbeneficialorcommensalbacteria.Theyaregiventherapeuticallyasprobiotics.Thesebeneficialbacteriapromotegooddigestion,regularity,boosttheimmunesystem,68andhelpcontrolintestinalpH.69BifidobacteriaandLactobacillushelppreventtheovergrowthofCandidaalbicans,E.coli,andotherpathogenicbacteria.32,70

OpportunisticPathogens

TheGI-MAPwasdesignedtodetectpathogenicandopportunisticorganismsthatmaybecausingsymptomsorillness.ManybacteriameasuredontheGI-MAPareopportunisticpathogens,meaningthattheyonlycausediseaseandillnessinsomeindividuals,particularlytheimmune-compromised.Manypeoplecomeintocontactwithopportunisticpathogensandexperiencenosymptoms,probablybecauseopportunistsaresuppressedbythebalanceofcommensalbacteria.32Overgrowthandexcessivecolonizationbyopportunisticbacteriamayoccurwhenthecommensalbacteriaareimpairedbypoordiet,antibioticuse,parasiticinfection,oraweakenedimmunesystem.Opportunisticpathogensarenotrecognizedbystandardmedicalauthoritiestocauseillness,andfindingmeasurablequantitiesinthestoolmaybeconsideredclinicallyinsignificant.ExamplesareCitrobacterspeciesorMorganellaspecies.

However,certainopportunisticpathogensmayberecognizedintheintegrativeandfunctionalmedicalfieldascreatingimbalanceinthegutmicrobiotaorotherwisepreventingproperhealingoftheGImucosalbarrier.Someoftheseorganismshavebeenimplicatedincontributingtoextra-intestinaldisease.Klebsiella,CitrobacterandYersiniaspeciesarebelievedtosetoffsystemicautoimmunediseaseincertainpatients.H.pyloriisoneexception.Widelylaudedasapathogen,itisplacedhereinthecategoryofOpportunisticPathogensbecauseofrecentsuggestionsaboutitsprotectiveeffectsanddwindlingevidencethatitispathogenicinallwhoarecolonized.

Helicobacterpylori(H.pylori)anditsvirulencegenes,cagA(cytotoxin-associatedproteinA)andvacA(vacuolatingtoxin)areincludedontheGI-MAP.Helicobacterpylorihasbeenevolvingwithhumanbeingsforwellover50,000years,sincetheymigratedoutofAfrica.11H.pyloricolonizationhasbeenimplicatedinavarietyofgastroduodenaldiseasesincludinggastritis,gastriccancer,andduodenalandpepticulcer.71H.pylorihasalsobeendetectedbystoolPCRincasesofdyspepsia,abdominalpain,andchronicgastrointestinalsymptoms.72-74Itisinfamousforitscausallinktoulcersandgastriccancer,whichresultedinaNobelprizeawardedtoRobinWarrenandBarryMarshallin2005.However,somesourcesaresuggestingitsrole,atleastinpart,asacommensalorganism.H.pylorimayprotectitshostfromcertainatopicdisorders,15aswellasotherdiseasessuchasesophagealcancer75reflux,andobesity.15

PopulationdatashowsthatH.pylorivirulencevariesgeographically.Itisassociatedwithhighratesofcancerincertainregions,butnotinothers.ThedifferencemaylieinH.pylori’sgenetics.11HostimmunestatusandacidsecretionseemtobeotherimportantfactorscontributingtoH.pylori’scolonizationandpathogenesis.71TheH.pylorivirulencefactorsthataremostwellrecognizedarevacAandcagA.

ThepresenceofcagA-positiveH.pyloristrainshasbeensignificantlyassociatedwithgastriccancerandpepticulcer.76ThegenecodesforatypeIVsecretionsystemwhichallowsthebacteriumtoinjectthecagAproteinintothehostcell.Onceinsidethehost’sgastricepithelialcells,cagAcandisruptcellsignaling,leadingtoabnormalproliferation,motility,andchangesinthecytoskeleton.76Thesechangestonormalcellsignalingcaninitiatecancer.

ThepresenceofvacAhasbeenassociatedwithgastriccancer,pepticulcer,andduodenalulcer.76ThevacAgeneispresentinallstrainsofH.pyloributispolymorphic,whichleadstodifferentlevelsofvacuolatingtoxin.VacAtoxinsinteractwithcertainreceptorsonhostcells,settingoffachainofeventsincludingmitochondrialdamage,inhibitionofT-lymphocytes,andinterferenceofantigenpresentation.76

NumerouspaperssuggesttheclinicalutilityofPCRtestingforH.pylori.DetectionofH.pyloriinbiopsyspecimensbyPCRhasprovensuperiortoothermethods.72,74,77Ithasshownsensitivityandspecificityreachingthatofthediagnostic“goldstandard,”whichisendoscopywithbiopsyandureasetest.72-74H.pylorigenotypingmaybeusefulforresistantH.pyloriinfectionsthathavefailedtorespondtotripleantibiotictherapy.72InonestudyofRT-PCR,authorsstateditwasa“highlyaccuratenoninvasivemethodtodetectH.pyloriinfectioninstoolandatthesametimeallowsforculture-independentclarithromycinsusceptibilitytesting.”72

H.pylorimaybeasymptomaticandrequirenotreatmentoronlysupportivecaretoimprovetheintestinalmucosaandgastrointestinallining.InformationonthecagAandvacAgenesmayhelpdeterminewhethertreatingapositiveH.pyloriresultisnecessary.

Pseudomonasspeciesaregram-negativebacteriafoundwidelyintheenvironment.Pseudomonasaeruginosaisthemostcommonspeciescausinginfectionandcanaffecteveryportionoftheintestine.Inthegastrointestinaltractitcancauseinflammation,epithelialbarrierdysfunction,tightcelljunctioninterruption,andintestinalpermeability.78Thisbacteriumexhibitsenhancedvirulencewithstress,trauma,surgery,andcancer.78Symptomsofentericinfectionincludefever,dehydration,abdominaldistention,diarrhea,andphysicalfindingsofShanghaifever.79Theinfectionusuallyaffectsyoungchildrenandadultswithhematologicmalignanciesandneutropenia.OutsidetheGItract,itcancauseurinarytractinfections,dermatitis,bacteremia,boneandjoint,respiratory,andsystemicinfectionsespeciallyinimmune-compromisedindividuals.

Klebsiellaspeciesaregram-negativebacterianormallyfoundintheintestinaltractthatareassociatedwithawiderangeofsmallintestinaldisordersincludingalterationsofmotility,diarrhea,gas,abdominalpain,andbloating.Itsovergrowthinthesmallintestinecanalsocausehistaminosisandgutinflammationthroughthereleaseofhistaminebythebacteria.80Thosewithahistoryoflong-termantibioticuseareatrisk.

GastrointestinalBacteriaasaTriggerforAutoimmunityOpportunisticgastrointestinalpathogensaregainingattentionfortheirabilitytoinitiateautoimmunethyroiditisandinflammatoryarthritissuchasrheumatoidarthritisandankylosingspondylitis.Klebsiellaspecies,Proteusmirabilis,Citrobacterspecies,andYersiniaarebacteriathatcouldcontributetoinflammatoryarthritisinsusceptibleindividuals.YersiniaenterocoliticainfectionhasbeenassociatedwithHashimoto’sthyroiditisandGrave’sdisease81andhigherantibodiestoYersiniaenterocoliticahavebeenfoundinthesepatients.82Enterovirusisalsoassociatedwithimmunogenicthyroiditis.83Analysisofgastrointestinalmicrobesisrecommendedinchronicautoimmunedisordersthatdon’trespondtotheusualtherapies.Inhealthyindividuals,opportunisticpathogensshouldnotpresentaproblem.Ahealthygastrointestinalbarrier,84goodlevelsofcommensalflora,andstrongimmunedefensesinthegutshouldeliminatethepotentialpathogenwithinafewweeks,causinglittletonosymptoms.However,whentheintestinalbarrierisbreached,normallyharmlessopportunisticmicrobescanpassthroughthebarrier,creatingextraintestinalinfectionandillness.Intestinalpermeability,orleakygut,hasbeendocumentedinanumberofautoimmunediseases:ankylosingspondylitis,rheumatoidarthritis,celiacdisease,inflammatoryboweldisease,IgAnephropathy,nonalcoholicsteatohepatitis,andmultiplesclerosis.85,86Patientswiththeseconditionsordocumentedintestinalpermeabilitymaybeatriskifgutmicrobiotaareimbalanced.

Sometheoriesofmicrobial-initiatedautoimmunediseasearemolecularmimicry,thebystandereffect,andthehygienehypothesis.Molecularmimicryisacommonexplanationforhowamicrobialinfectioncaninitiateautoimmunedisease,presumablyduetoantibacterialandcross-reactiveautoantibodies.23Itisbelievedthatmicrobialantigensresembleself-antigens.Thesecross-reactionsessentially“confuse”theimmunesystemwhichmistakenlymountsanattackagainstself-tissues.Thebystandereffecttheoryproposesthatmicroorganismsdamageself-tissues,exposingself-antigenstoimmuneattack.Finally,thehygienehypothesispresumesthatdecreasedexposuretomicrobesincreasestheTh1responsewhichcanleadtoautoimmunity.85Spondyloarthropathiesareafamilyofchronic,multi-system,inflammatorydiseasesinvolvingthesacroiliacjointsandaxialskeletonandtheymayhaveaninfectioustrigger.20Theyinclude:ankylosingspondylitis,arthritisassociatedwithulcerativecolitisorCrohn’sdisease,psoriaticarthritis,andreactivearthritis.Alloftheseshareageneticpredispositionandallarecharacterizedbyenthesitis,orinflammationofthesiteswhereligamentsandtendonsinsertintothebone.20TheyareusuallyrheumatoidfactornegativeandtheyshowanassociationwithhumanleukocyteantigenB27(HLA-B27).AprominenthypothesisisthatHLA-B27mayresembleoractasareceptorforbacterialantigens,triggeringtheautoimmuneattackonself.20Reactivearthritiscanbebroughtonbygenito-urinaryinfectionswithProteusmiribalis87,88orgastrointestinalinfectionswithbacterialagentssuchasChlamydia,Salmonella,Shigella,Campylobacter,Yersinia21,22andClostridiumdifficile.ParasitessuchasStrongyloidesstercoralis,Giardialamblia,Ascarislumbricoides,andCryptosporidiumspeciescanalsoresultinreactivearthritis.89,90Aggressivecasescouldevolveintoankylosingspondylitis.21SubstantialdatasupportsacausativeroleforProteusmirabilisinrheumatoidarthritiswhileankylosingspondylitisandCrohn’sdiseasehavebeenrelatedtoKlebsiellamicrobialinfections.23EvidenceofSalmonellahasbeenfoundincasesofankylosingspondylitis.91,92OtherdatashowsabnormalserumantibodyresponsestoKlebsiellaandProteusmirabilisinthespondyloarthropathies,90highlevelsofIgGantibodiestoKlebsiellainpatientswithankylosingspondylitis,Crohn’sdisease,andulcerativecolitis,andantibodiestoProteusinrheumatoidarthritis.86Whileculturesofsynovialfluiddonotyieldgastrointestinalmicrobes,thereisevidenceofbacterialantigenandimmuneresponsesinthesynoviumofthejoint,suggestingthatmicrobesdoplayaroleinthepathology.93Fecalstudieshavenotbeenusedtoprovidefirmevidenceofthecausativerelationshipofstoolmicrobeswithautoimmunesyndromes.However,stooltestingforopportunisticpathogensseemsareasonableavenueinchronic,intractable,andpainfulautoimmuneconditions,especiallyifonsetcloselyfollowedagastrointestinalinfection.

FungalOrganismsFungalorganismsareapartofthenormalhumandigestivetract,butfungalovergrowthcancauseillnessinsusceptiblepeople.Commonsymptomsassociatedwithfungalovergrowtharegas,bloating,constipation,diarrhea,eczema,andothersignsoffungalinfectionsuchasathlete’sfoot,vaginalyeastinfections,thrush,andjockitch.Stooltesting,usingGI-MAP,forfungisuchasCandida,Microsporidia,andGeotrichumcanoftenrevealahiddensourceofcontinualfungalgrowth–thegut.Fungalovergrowthisusuallycontrolledwithadietlowinsugarsandstarches.Insomecasesantifungalmedicationsarenecessary.

Microsporidiaspecieswerefirstidentifiedasparasitesofthesilkworm,butarenowrecognizedasfungi.Theyareoftendifficulttodiagnosebutsignificantprogresshasbeenmadewithmoleculardiagnosticsfordetectionoftheseorganisms.94TheseopportunisticpathogensofteninfectimmunosuppressedindividualssuchasthosewithHIVinfection,organtransplantation,orchemotherapy,butcanalsoinfecthealthypeople.Commonsymptomsincludediarrheaandwastingduetoentericinfection,butthespectrumofrelateddiseasesduetothesepathogensalsoincludessinusitis,bronchitis,pneumonia,nephritis,myositis,hepatitis,encephalitis,andotherbraininfections.94Treatmentoftenincludesantifungalmedicationsalongwithdietandnutritionalinterventionstohelpwithchronicdiarrhea.

AdditionalTestsTheGI-MAPincludesmarkersofimmunefunction,inflammation,digestion,andgliadinsensitivity,andmetabolicactivityofthegastrointestinalbiome.Thesemarkerswereselectedfortheirclinicalutility.Calprotectinandelastasehaveastrongfoundationofclinicalevidencetosupporttheiruseinclinicalcare.Calprotectinhelpstheintegrativeandfunctionalmedicinepractitionermeasurethelevelofimmuneactivationinthegut,oftenassociatedwithinfectionand/orinflammatoryboweldisease.Pancreaticelastase1isanexcellentglobalmarkerofpancreaticexocrinefunctionandcanbeanindicatorofpoordigestivecapacityorpancreatitiswhenextremelylow.SecretoryIgAisthebody’sfirstlineofdefenseinthegut.Aportionofthisimmunoglobulinmightbedirectedtowardgliadin,indicatinganimmunereactiontothecommonproteininwheatandotherfieldgrassgrains.Beta-glucuronidaseisanenzymeproducednaturallyincellsoftheliver,kidney,andintestinalepithelium.However,thisenzymeisalsoproducedexcessivelybybacteriaknowntobepathogenic,andhighlevelsmaybeanindicationofadversemetabolicactivityoftheintestinalmicrobiome.

SecretoryImmunoglobulinA(sIgA)isanantibodyproteinsecretedintothegastrointestinaltractasafirstlineofimmunedefenseagainstpathogenicmicroorganisms.95Thisimmunoglobulininfluencesthegutmicrobiome95andhelpstomaintainbarrierfunction96byformingcomplexeswithgutpathogensandallergens,preventingthemfrompenetratingtheintestinalbarrier.ImpairmentofsecretoryIgAmayincreasetheriskofinfectious,allergic,andinflammatorydiseasesoftheintestine.97ChronicstressmayalsodisruptlevelsofsIgA.ElevatedlevelsofsIgAmayindicateanactivatedimmuneresponsetochronicinfectionsorinflammatoryreactions.

Thepresenceoffecalanti-gliadinantibodiescanindicateanimmuneresponsetogluteninthediet.Gliadinisacomponentofgluten,theproteinfoundinwheatandotherfieldgrassgrainssuchasbarley,maltandrye.Becausegliadincouldstimulateintestinalimmunityandincreaselevelsoffecalanti-gliadinantibodyevenwhenserumconcentrationsareundetectable,98,99itisoftenusedasmarkerfornon-celiacglutensensitivity.Highlevelsoffecalanti-gliadinantibodiescanprovideclinicianswithaneffectivetreatmentstrategy:agluten-freediet.

Fecalpancreaticelastase-1isanaccuratefunctionalscreeningmarkerforpancreaticexocrineinsufficiency.Pancreaticelastaseisanenzymeproducedbythepancreastohelpbreakdownproteins.Pancreaticinsufficiencyoccurswhenthepancreasisnotworkingwellandbecomesinflamed(pancreatitis).Thiscanimpairthebody’sabilitytoabsorbnutrientsfromfood,includingfat-solublevitamins.100Thistestalsoaccuratelypredictsapatient’sresponsetopancreaticenzymesupplementation,especiallyinpatientswithunexplaineddiarrheaandsuspectedpancreaticinsufficiency.101Thefecalpancreaticelastase-1testmayalsobeusefulformonitoringdiabeticsbecausebothinsulinandnon-insulin-dependentdiabetescanimpairpancreaticfunction.102

Fecalcalprotectinisthemoststudiedmarkerofgastrointestinalinflammation103andthegoldstandardmarkerforthediagnosisandmonitoringofinflammatoryboweldisease(IBD).104ItisusedtodiscriminateIBDfromirritablebowelsyndrome(IBS).103,104Calprotectinisacalcium-bindingproteinthatisfoundathighconcentrationinneutrophils.Calprotectinisalsofoundinmonocytes,macrophages,andgutepithelialcells.105InIBD,thereisamigrationofinflammatorycellssuchasneutrophilstotheinflamedintestinalmucosa.Becauseleukocytesareshedintotheintestinallumen,pro-inflammatoryproteinssuchascalprotectincanbeidentifiedandmeasuredinstoolspecimens.106Fecalcalprotectinlevelsareproportionaltothelevelofneutrophilinfiltrationandinflammationinthegut.105Calprotectinhasbeenshowntocorrelatewithhistologicandendoscopicmeasuresofinflammatoryboweldiseaseseverity.106Itisnon-invasive,stable,104andshowsaconsiderablesensitivityandspecificityof93%and96%,respectively,whenusedtoscreenforIBDactivity.107Highcalprotectincanalsobedetectedincolorectalcancers,diverticulardisease,andinfectiousgastroenteritis.103WhenIBDissuspectedbasedonclinicalpresentation,afecalcalprotectinlevel<50ug/gstoolsuggestsIBS,notIBD.Calprotectinlevelsbetween50and150ug/gindicateGIinflammationanddeservetreatmentandfollow-uptesting.Calprotectinlevelsgreaterthan150ug/gsuggestorganicdiseasesuchasIBDorcolorectalcancerandfollow-upcolonoscopyisrecommended(SeeFigure1).103Fecalcalprotectincanelevatewithenteropathycausedbyexcessivenon-steroidalanti-inflammatorymedicationuse.105Forthisreason,itmaybebeneficialtotemporarilydiscontinueNSAIDs,whenpossibleinselectpatients,priortomeasuringfecalcalprotectin.105Figure1.ThealgorithmusedtodifferentiateIBDfromIBSusingfecalcalprotectin.AdaptedfromWalshametal.103

Beta-glucuronidaseisanenzymeproducedbycellsintheliver,kidney,intestinalepithelium,endocrine,andreproductiveorgans.108However,themajorproducersofbeta-glucuronidasearethesebacteria:Bacteroidesfragilis,Bacteroidesvulgatus,Bacteroidesuniformis,Clostridiumparaputrificum,Clostridiumclostridioforme,Clostridiumperfringens,Escherichiacoli,Eubacterium,Peptostreptococcus,Ruminococcus,andStaphylococcus.Itisfoundin97%ofE.colistrains.109TheenzymehydrolyzesB-glucuronidetomakeglucuronicacidandanaglycone,suchasimine,thiol,oralcohol.Glucuronidationbywayofbeta-glucuronidaseisamajorrouteofdetoxificationinthehumanbody.109However,thisenzymecanalsoconvertpro-carcinogenstocarcinogeniccompounds.108Highlevelsoffecalbeta-glucuronidasecanindicateunfavorablechangesinthecolon.Whentheenzymeiselevatedinplasma,thereisanincreasedriskofhormone-sensitivecancers,suchasthoseofthebreastorprostate.108Evidenceofincreasedenzymaticactivityofintestinalmicroorganismsmaysuggestincreasedriskofdigestivetractcancer.110ToxinsstimulateB-glucuronidaseactivityanddietaryredmeatandproteinincreasestheenzyme.AntibioticsincreaseB-glucuronidaselevels.Alow-calorie,vegetariandietcanreducefecalB-glucuronidaselevels.108Steatocrithasbeenusedwidelysince1981todetectsteatorrheainpatientswithpancreaticinsufficiencyandsmallintestinalmalabsorption.111Itisasimpletestthatusescentrifugationtoseparatethesolid,aqueous,andlipidlayersofthestool.Thelipidlayerismeasuredinthesteatocritandthismakesupthetotalfecalfat.111Acidificationofthestooldramaticallyimprovedtheperformanceofthismethod.Theacidsteatocritmethodhasbeenshowntocorrelatewellwith24-hourand72-hourfecalfats.112,113Zonulinisaproteinsecretedbyintestinalcellsthatregulatesintercellulartightjunctions.1,114Tightjunctionsaretheconnectionsbetweenepithelialcellsthatmakeupthegastrointestinallining.Zonulinincreasesintestinalpermeabilityinthejejunumandileum115andisconsideredabiomarkerforbarrierpermeability.1,114Tightjunctionscanbeopenedorclosed,dependingonthephysiologicalneed.Zonulin’sroleistoopentightjunctionsinthegut.Inthecaseofentericinfections,highzonulincan“openthefloodgates”andflushoutbacteriaandtoxins.1Certaingutbacteriaandgliadin(themainstapleproteinfromwheat)canactivatethezonulinsystem.114,116Theintestinalbarrierisacriticalinterfacebetweenthelumenofthegutandtheinternalmilieu.Dysfunctionofthisbarrierisbelievedtoinitiateimmunedysfunctionbecauseitallowsmacromoleculesfromthegutlumentopassintothebloodstream.117Intestinalpermeability,alsoknownas“leakygut,”hasbeenassociatedwithinflammatoryboweldisease,celiacdisease,foodallergy,irritablebowelsyndrome,criticalillness,autoimmunediseases,118andobesityandmetabolicdisease.119Inmanycases,permeabilityprecedesdisease.1Zonulinregulatesbarrierpermeability.Serumzonulincorrelateswithintestinalpermeabilityandlactulose/mannitoltestsforintestinalpermeability.115,120Highserumzonulinhasbeenassociatedwithceliacdisease,type1diabetes,120insulinresistanceandtype2diabetes,115cancers,neurologicalconditions,andautoimmunediseases.1

Fecalzonulinisavailableforinvestigationalusebuthasnotbeencorrelatedwithcirculating(serum)levelsasofthiswriting.Serumzonulinmayconstitutezonulinsecretionnotonlyfromintestinalcells,butalsofromextraintestinaltissuessuchastheliver,heartandbrain.121Stoolmaythereforepresentanappropriatespecimenforanalyzingonlyintestinalproductionofzonulin.Fecalzonulinhasbeenusedinhumanstudiesasamarkerofintestinalpermeability.Inathletes,fecalzonulinlevelsimproved(decreased)after14weeksofprobioticsupplementation.114Treatmentwithzeoliteloweredstoollevelsofzonulininathletesandpresumablyimprovedintestinalbarrierfunction.122

DrugResistanceGenesDrugresistancegenesaregenescarriedbybacteriathatconferaspecialresistanceorprotectionfromcertainantibiotics.Formostantibioticsthereareseveraldifferentgenes.Thegenetypeisdependentonthemodeofresistanceandtheorganism(s)itmaybefoundin.IntheGI-MAP,theantibioticanddrugresistancegenesforanantibioticaremeasuredbasedonthepathogenicorganismfoundtobepositiveinthefecalsample.Resultswillbereportedforthegenotypicresistance,meaningtheARgenespecifictothepositiveorganism,andglobally,meaningallotherARGenesforthatdrugfoundthroughoutthemicrobiota.

HerbalAntimicrobialAgentsBotanicalandvolatileoilextractshavealonghistoryoftraditionaluseasnaturalantimicrobials.Naturalagentssuchasberberine,garlic,oliveleaf,caprylicacid,wormwood,blackwalnut,uvaursi,citrusseedextract,andTribulusterrestrisprovideabroadspectrumofactivityagainstthemostcommonpathogensthatcausegastrointestinalillnessanddysbiosis.Antimicrobialherbsdonotposethesameriskformicrobialresistance,123-125_ENREF_100_ENREF_104ascomparedtoantibiotics,becausemultipleactiveingredientsfromthewholeplantworktogetherinsynchrony.Theirlonghistoricalusesuggestslowriskofadverseeffects.

Serumzonulinishighinanumberofimmune-mediatedconditions:1Autoimmunediseases

CeliacdiseaseAnkylosingspondylitisInflammatoryboweldiseaseType1diabetesRheumatoidarthritisSystemiclupuserythematous

CancersBrain(gliomas)BreastLungadenocarcinomaOvarianPancreatic

NeurologicaldiseasesMultiplesclerosisChronicinflammatorydemyelinatingpolyneuropathySchizophrenia

AntimicrobialAgent

DescriptionandClinicalUse

Berberine BerberinehasshowneffectivenessagainstETEC-associateddiarrheaandhasbeenstudiedextensivelyforitsantibacterialeffect.126Itshowsantimicrobialactivityagainstfungi,protozoans,helminths,viruses,andchlamydia.127

Garlic Garlichasshownactivityagainstbacteria,protozoa,helminths,viruses,andfungi.128,129Itstronglysuppressedgram-negativediarrheagenicpathogens(Shigella,Salmonella,Proteusmirabilis,andE.coli)isolatedfromstoolsamples.130AqueousgarlicextractinhibitedE.coliO157:H7andE.coliLF82andenhancedthegrowthofLactobacillusreuteriinvitro.131Thissuggeststhatantimicrobialherbsmaysparebeneficialflora.

Oliveleaf Oliveleafhasantibacterial,antifungal,132,133andantiviralproperties.134-136

Caprylicacid CaprylicacidreducesCampylobacterandSalmonellainthegastrointestinaltractandstoolofpoultrywhenaddedtothefeedorwater.137-139_ENREF_65_ENREF_66Caprylicacidhasantiviralandantifungalproperties.140,141_ENREF_12

Wormwood Artemesiaannua(wormwood)demonstratessignificantantimicrobialeffectsandhasbeenusedinthetreatmentofmalariaandparasiticgastrointestinalinfections.

Blackwalnut Juglansnigra(BlackWalnut)hasalonghistoryofuseasanintestinalantiparasitic(i.e.vermifuge,anthelminthic),antibacterial,andantifungal.

Uvaursi Arctostaphylosuva-ursileaveshavebeenusedworldwideasadiuretic,astringent,antiseptic,andtreatmentforurinarytractandgastrointestinalinfections.

Tribulus TribulusterrestriscontainsXsteroidalsaponinsthatshowantibacterialandantiviraleffects.

Citrusseedextract GrapefruitandothercitrusseedextractshavelongbeenusedasantisepticsandareusedclinicallytoreducefungalovergrowthbysuchcommonorganismsasCandidaandGeotrichum.Citrusseedextractalsohasdemonstratedantibacterialaction,mostnotoriouslywithhemolyticcoliformbacteria.

ConclusionsTheGI-MAPcanbeusedinthedetectionandidentificationofgastrointestinalmicrobialnucleicacidsandhasbeenclinicallyvalidatedforthedetectionofgastrointestinalpathogensthatcauseinfectiouscolitisorgastroenteritis.2Thistechnologyhasbeenusedtoidentifyandcontrolpathogenoutbreaksbecauseofitsrapidturn-around-time.2ItmeasuresasubstantiallistofopportunisticpathogensaswellasalistofFDA-clearedpathogens,includingnoveltargetssuchasviruses,Microsporidia,andpathogenicvirulencefactors.Chronicgastrointestinalsymptoms,intestinalpermeability,hormonalimbalance,andfoodsensitivitiesmaytracetheiroriginstoimbalancedgutmicrobesasarootcause.Further,chronicinflammatoryarthritiscouldhaveamicrobialcomponentthatmaywarrantinvestigationbystoolstudies.Thisstooltestoffersintegrativeandfunctionalmedicinepractitionerssuperiorsensitivityandspecificitytohelpresolvepersistentandcomplexillnesses.Sincetheimmunesystem,theintestinalbarrier,andmicrobialdiversityareintimatelyinterwoven,thoroughunderstandingofourgutmicrobiomeholdspromisefornewapproachestotreatandpreventdisease.142

CompleteListofTargetAnalytesMeasuredontheGI-MAPBacterialpathogens:CampylobacterC.diffToxinA&B**E.colio157**EnterotoxigenicE.coliLT&ST(ETEC)**Shiga-likeToxinproducingE.colistx1&stx2(STEC)**SalmonellaShigellaVibriocholeraYersiniaenterocoliticaViralpathogens:Adenovirus40&41**NorovirusGI&GII**RotavirusA**ParasiticpathogensCryptosporidium**Entamoebahistolytica**

GiardiaAdditionaltargetsBacteria:Helicobacterpyloriandvirulencefactors,cagAandvacA**EnterococcusLactobacillus**Bifidobacter**Bacteroidesspp.BacteroidesfragilisgrpE.coli(total)Citrobacterspp.CitrobacterfreundiiProteusspp.ProteusmirabilusProteusvulgarisPseudomonasspp.**PseudomonasaeruginosaMorganellaspp.Staphylococcusspp.(aureus)Streptococcusspp.Klebsiellaspp.**KlebsiellapneumoneiaeParasites:Blastocystishominis**DientamoebafragilisEndolimaxnanaEntamoebacoli**Entamoebahartmanni**ChilomastixmesnelliCyclosporacayetanenensisPentatrichomonashominisLDTfungi/yeast:Microsporidiaspp.includingEnterocytozoonbieneusiandEncephalitozoonintestinalis**CandidaalbicansCandidaspp.Geotrichumspp.Trichosporonspp.Othertests:SecretoryIgA(sIgA)Anti-gliadinsIgAPancreaticelastase1BetaglucuronidaseCalprotectinZonulin(availableasanadd-ontest)SteatocritOccultblood

Organismswith**arelistedwithcitationsinthispaper.

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