TheGIMicrobialAssayPlus(GI-MAP™)

MultiplexDNAStoolTechnologyfortheIntegrativeandFunctionalMedicinePractitioner

Contents

MicrobiologyandDNAAnalysis ................................................................................................................... 3

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

Methodology................................................................................................................................................ 4

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

Pathogens..................................................................................................................................................... 6

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

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

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

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

OpportunisticPathogens............................................................................................................................ 11

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

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

Chemistries................................................................................................................................................. 14

DrugResistanceGenes............................................................................................................................... 16

HerbalAntimicrobialAgents ...................................................................................................................... 16

Conclusions ................................................................................................................................................ 17

CompleteListofTargetAnalytesMeasuredontheGI-MAP ..................................................................... 17

TheGIMicrobialAssayPlus(GI-MAP)

MultiplexDNAStoolTechnologyfortheIntegrativeandFunctionalMedicinePractitioner

MicrobiologyandDNAAnalysis

Inthelastfewdecades,DNAanalysishastransformedthefieldofmicrobiology.TheNationalInstitutesofHealthhavefollowedsuitwithinitiativessuchastheHumanMicrobiomeProject,whichcharacterizedthemicrobiomefromover15habitatsofthebodyinmorethan200healthyhumansubjectsusingDNAanalysis.2Morethaneverbefore,wearekeenlyawareofthehealthbenefitsordiseaserisksbroughtaboutbythemicroorganismsthatinhabitthehumanbody.Culturetechniques,previouslythestandard,leftupto50%ofbacterialspeciesvirtuallyinvisible.3Whennext-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.1Thispanelmeasuresviralcausesofgastroenteritis,unavailablebyothercommonstooltests.ItmeasuresparasitessuchasCryptosporidium,Giardia,andEntamoebahistolytica.TheGI-MAPanalyzesHelicobacterpylorianditsvirulencefactors.ItcandetectopportunisticpathogenssuchasPseudomonasaeruginosa,Klebsiellapneumoniae,Yersiniaenterocolitica,andProteusmirabilus,associatedwithautoimmunemolecularmimicry.Itincludesapanelofsingle-celled,amebicparasitessuchasBlastocystishominis,Dientamoebafragilis,andEntamoebacoli.FungalorganismsaremeasuredbytheGI-MAPsuchasCandida,Geotrichum,andMicrosporidia,withthelatterbeinganewadditiontoDNAstoolanalysis.Finally,theGI-MAPmeasuresstandardmarkersofimmunity,inflammationanddigestionincludinglactoferrin,secretoryimmunoglobulinA(sIgA),anti-gliadinantibody,andpancreaticelastase1.

Disruptionofthegastrointestinalmicrobiomecancause:GastrointestinalsymptomsAbdominalpain4Bloating5Constipation5Crohn’sdisease6,7Diarrhea4,5,8

Foodpoisoning9Gastriccancer10Gastritis10Gastroenteritis11,12Gastroesophagealreflux13,14Irritablebowelsyndrome12,15Smallintestinalbacterialovergrowth(SIBO)16

Gastrointestinalsymptoms,continuedUlcer10Ulcerativecolitis17Vomiting18AutoimmuneConditionsAnkylosingspondylitis19ReactiveandRheumatoidarthritis19-22Thyroiditis(Hashimoto’s,Grave’s)78AllergicDiseaseAsthma23Eczema24-26

Methodology

DiagnosticSolutionsLaboratoryisusinganovelDNAtechniquetodetectacomprehensivelistofstoolbacteria,viruses,fungi,andparasites.ThemethodandinstrumentusedisFDA-clearedforthedetectionof15ofthemostcommoncausesofgastroenteritis-bacteria,parasites,andviruses.1Ithasbeenclinicallyvalidatedforusewithhumanstoolsamplesandsensitivityandspecificitydatacanbefoundbelow.Thisisanautomated,multiplexDNAanalysismethod,allowingforthesimultaneousmeasurementofmultiplebacteria,fungi,parasites,andviruses,allfromasinglesample.1

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.ThisistheonlyFDAclearedDNAtestforgastrointestinalmicrobesavailable.Incontrasttomolecularmethodologiesusedbyotherlaboratories,thismethodwasnotdevelopedin-house.IndependentclinicalvalidationdataonthemethodusedbyDiagnosticSolutionsLaboratoryshowsthatthetestisreliableandreproducible.TheseaddedqualityaspectsarenovelandgivetheclinicianadditionalconfidenceinDNAanalysis,notavailableelsewhereintheintegrativeandfunctionalmedicinemarket.

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

AfteramplifyingtheDNAtargetsfoundinthestoolspecimen,thespecimenundergoeshybridization.Hybridizationisthebinding(likealockandkey)ofonesingle-strandedDNAsegmenttoanothercomplementarypieceofDNA,calledaprobe.ThisstepisimportantforaccurateidentificationofamicrobebasedonitsDNAsignature.AprobeisasegmentofDNAthatseekstojoinwithitscomplementarymatchandischemicallylabelledformeasurement.InthismethodthesampleDNAishybridizedtoprobesattachedtomagneticbeads.Eachbeadalsohasauniquesignature.Soyounotonlyhavethesignalfromthelabeledprobe,butalsodiscriminationbasedontheuniquebead.Thisallowsforaccurateandsensitivedetectionofatargetorganismsinamultiplexassay.MultiplexPCRusesthissamemolecularapproachformanymicrobesatthesametime,allowingforsensitiveandspecificdifferentiationofmanyorganismssimultaneously.

FDA-Cleared GI Pathogens Measured in the GI-MAP

Inthepast,drawbackswithDNAanalysishavebeenitsincrediblesensitivityandpotentialfornonspecificbinding.BecauseofthetremendoussensitivityofDNAanalysis,othermethodsdetectedmicrobesthatwerenotactuallypresentinhighnumbersatthetimeofstoolcollectionandwhichmaynothavebeenclinicallyrelevant.Cross-reactivitycanalsobeaproblemwithDNAanalysislendingtoissueswithspecificity.WiththeGI-MAPmethod,probesareattachedtodifferentbeadsinsuchawaythatnon-specificbindingisnearlyeliminated,loweringfalsepositiveratesthathavebeenassociatedwithpreviousDNAmethods.DiagnosticSolutionsLaboratoryalsoemploysseveralothermethodimprovementstofurtherimproveaccuracyandprecisionofthemethod.

OtherstooltestsinthemarketprimarilyuseMatrixAssistedLaserDesorption/IonizationTimeof-Flight(MALDI-TOF)toidentifystoolmicrobes.MALDI-TOFtechnologyusedbyotherlaboratoriesformicrobialdetectionreliesonbacterialcultureofthestoolspecimen.Alimitationofthismethodistherelianceonculturemethods.OnlytheorganismsthatgrowcanbeidentifiedusingtheMALDI-TOF.Meaning,anaerobicorganismsandparasitesthatdonotgrowunderroutinecultureconditionscannotbeidentified.CollectionofstoolspecimensforDNAanalysismostcloselyrepresentstheactualmicrobialpopulationsofthepatient’sgastrointestinaltractatthetimeofcollectionversusculturebasedmethods.

TargetAnalytes

Thehumangastrointestinalmicrobiomehousestrillionsofbacteriaandresearchshowsthatthesemicroorganismsareessentialforhumanmetabolism,27nutrition,immunefunction,28andresistancetoinfection.29Over500differentspeciesofmicroorganismsfrom30differentgenerahavebeenidentifiedfromthehumangut.Butinanyoneperson,thereare100million-1trillionmicroorganismspergramoffecalcontent.30Mostmicrobesinthehumangutarebelievedtobebeneficialorcommensal.Therearemicrobesthatcolonizemanypeoplebutonlybecomepathogenicincertainsituations(opportunisticpathogens).Finally,therearepathogensthatarewidelyrecognizedtocausediseaseinthehumanhost.

Althoughtheyareubiquitous,pathogenicbacteriadonotcauseillnessinallpeople.Thisisbecausecommensalgastrointestinalfloracanprotectthehostfrominfection.Whengutmicrofloraprotectstheintestinesfrompathogensandharmfulmicroorganismsitiscalled,“colonizationresistance.”29Animalmodelsshowthatwhennormalgutmicrofloraarelacking,thehostismoresusceptibletoGIinfectionswithSalmonella.Similarly,afterantibiotictreatmentthereisincreasedriskofpathogenicinfections.29Ontheotherhand,commensalbacteriasuchasLactobacillusandBifidobacteriumcanprevent

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

gastrointestinalinfection.Colonizationresistanceexplainswhymostpathogenicbacteriafailtocausediseaseinhealthysubjects.31

Commensalbacterianaturallyinhabitthehumangastrointestinaltractanddonotcausedisease.Manyarebeneficial;theyproduceenzymes,32vitamins,33shortchainfattyacids,34andothermetabolicproductsthatkeepthebowelsandthebodyfunctioningwell.Theincrediblycomplexinteractionbetweenhumanhealthandthegastrointestinalmicrobiomeisthesubjectofmultiplecutting-edgeresearchstudies.35Giventhemetabolic,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.36MultiplexPCR,usingthesamemethodasthatusedfortheGI-MAP,waspreferredoverconventionalmicrobiologicaltechniquesin347patientswithgastroenteritis.AuthorsconcludedthatDNAanalysiswasfasterforpathogenidentificationandprovidedclinicianswithalargerpanelofpathogens,helpingtocontainnosocomialoutbreaksbeforetheyspread.37Bacterialpathogensareoftenspreadduetocontaminationoffoodandwaterwithfecalmaterialcontainingthesepathogens.ConsultyourPhysician’sDeskReferenceforstandardtreatmentsforthesepathogens.Antibiotictherapyisnotalwaysrecommendedbecauseantibioticresistancecanworsentheinfection.Hydration,probiotics,andsupportivetherapiesforthegut-immunesystemcanhelptoremovethepathogenfromtheGItract.

Thepresenceofapathogendoesnot,byitself,indicatedisease.1Resultsfromlaboratorytestsmustbeinterpretedtogetherwithclinicalsymptomsandhistorybyaqualifiedhealthpractitioner.WithincreasedawarenessofthecomplexityoftheGIenvironment,apathogenislikelytocausediseaseiftherearevulnerabilitiesinthehost’sdefenses.Forexample,imbalancedmicroflora,poorimmunedefenses,poordiet,toxicexposures,antibiotics,orchronicGIsymptomscouldmakeapersonmoresusceptibletoharmfromapathogen.Whereas,anotherpersonmayhaveafecalpathogenbutisingoodhealth.Inhealthypatients,treatingpathogensmaynotbenecessary.However,continuingtosupportabeneficialanddiversemicrobiotaandastronggut-immunesystemwillfurtherprotectthehostfrominfection.28,38

Despitewhattypeofstooltestisused,thetransientnatureofthemicrobiotamustbeacknowledged.Populationsofmicroorganismscanchangedramaticallyinshortperiodsoftime,especiallyunderstress,withtheuseofantimicrobialmedications,orchangesinthediet,etc.Thetransientnatureofgastrointestinalmicroorganismsmakesitevenmoreimportanttousethelabresultstogetherwithsignsandsymptomstodetermineifaparticularlabfindingisindicativeofaclinicalconditionthatrequires

treatment.Clinicalmonitoringandfollow-uptestingandconfirmationbyothertestingmethodshelpstoanalyzethechangestothemicrobiomeovertimeandverifyclinicallyrelevantfindings.1Similarly,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.39

ToxinsAandBarethemajorvirulencefactorsbelievedtoberesponsibleforC.diffinfectionsymptoms.Theyareproinflammatoryandcytotoxic.Theydamagethecytoskeletonofintestinalepithelialcells,permittingfluidinflux,theyopentightjunctionsintheGIlining,andtherebydamagetheGIlining.ToxinsAandBhaveevenshownsystemiceffectsinanimalmodels,suggestingthattheirbioactivitymaynotbelocalizedtotheGItract.ToxinsAandBareencodedbythetcdAandtcdBgenesandarethereforedetectableusingDNAanalysis.40Real-timepolymerasechainreactionisconsideredagoldstandarddiagnosticmethodologyforC.diff.39

Escherichiacoliisalargeandvariedspeciesofbacteriathatincludesmanystrains.Theycolonizehumansandanimalsandarespreadthroughcontaminatedwater,food,orcontactwithinfectedhumansoranimals.41E.colicancauseinfectionsoutsideoftheGItractsuchasurinarytractinfections,meningitis,andintra-abdominalabscess.42

Whiletherearemanyharmless,andevenbeneficial,E.colistrains,therearesixstrainsthatarenotoriousfortheirpathogenicity,especiallyforGIinfections.EnterotoxigenicE.coli(ETEC)cancausetraveler’sdiarrhea.EnteropathogenicE.coliisacauseofchildhooddiarrhea.EnteroinvasiveE.coli(EIEC)canleadtodysenterysimilartothatcausedbyShigella.EnterohemorrhagicE.colicanleadtohemorrhagiccolitisorhemolytic-uremicsyndrome.EIECandEHECcolonizethecolonwhiletheotherscolonizethesmallintestinesandsubsequentlyinitiatediarrhea.42ThestrainsmeasuredontheGI-MAPare:E.coliO157,EnterotoxigenicE.coliLTandST(ETEC),andShiga-liketoxin-producingE.coli(STEC),targetingstx1andstx2genes.

Shiga-liketoxinproducingE.coli(STEC)hasbeeninvolvedinfoodborneillnessoutbreaks.41ItcausesvariousGIillnesses,includingbloodyandnon-bloodydiarrhea.Shigatoxin(stx1)andShigatoxin2(stx2)aregenerallyconsideredtobethevirulentfactorsresponsibleforseriousillnesscausedbySTEC.Stx1

andstx2aregenetictargetsthathelpaccuratelydetectthepresenceofShiga-liketoxinproducingE.coliinstoolsamples.43

ThesamemethodusedintheGI-MAPwasusedtoinvestigatethecauseofanoutbreakinGermanhospitalsinpatientssufferingwithhemolyticuremicsyndrome,presumablyinducedbySTEC.Withrapidscreeningdiagnosticmethodstheywereabletoidentifyanovelserotype--E.coliO104:H4thathadvirulencefactorscharacteristicofbothenterohemorrhagicE.coliandenteroaggregativeE.coli.44

TheserotypeO157:H7hasbeenimplicatedinmanyoutbreaksandcasesofbloodydiarrheaandhemolyticuremicsyndrome42andhasahighprevalenceworldwide.44

EnterotoxigenicE.coliheat-labiletoxin(LT)andheat-stabletoxin(ST)aretheenterotoxinsresponsiblefordiarrhealdiseaseinhumans.ST-producingE.coliiswidelyknowntocausediarrheabutthemechanismisstillunknown.LTactssimilarlytothecholeratoxinbyactivatingadenylatecyclase,leadingtodiarrhea.45

ViralPathogens

Adenovirus,norovirus,androtavirusareviralcausesofgastroenteritisthatarenormallyself-limitinginhealthyindividuals.Whenaclinicianislookingforamicrobialcauseofgastroenteritis,theywouldberemisstooverlookthesevirusesaspossiblecausesofdiarrhea,abdominalpain,andvomiting.Inastudyof4,627patientswithgastroenteritis,PCRstooltechnologydetectednorovirusin36%androtavirusAin31%ofsamples.46Anotherstudyofover300peoplewithacutediarrheaoverthecourseofayearshowed36.0%werepositivefornorovirusand17.3%werepositiveforrotavirus,while5.4%werepositiveforadenovirus.Intotal,virusesaccountedfor58.7%ofcasesofacutegastroenteritis,47pointingtothevalueofviraldetectioninstoolspecimens.

PrevioustestswiththeGI-MAP(unpublished)showedhighincidenceofviralpathogensandevidenceofchroniccarriers.Thismayberelatedtothepersistenceandpervasivenessofviruses.Noroviruswasdetectableforoverthreeyearsingroundwaterandinfectiousforatleast61days.48Therearenostandardtreatmentsforviralgastroenteritisinhealthyhosts.Antiviralsarenotrecommended.49Supportivecareforthegastricmucosa,hydration,andimmune-boostingagentsmaybewarranted.

Adenoviruses40and41causegastroenteritis.Theyareacommoncauseofdiarrheaininfantsandchildrenbutcanalsoaffectadults.Thesepathogenscanreplicatereadilyintheintestine.Theyaretheonlyadenovirustypesthatareshowntobecausativeagentsofgastrointestinaldisease.However,otheradenovirusesmaycausegastroenteritis.Feverandwaterydiarrheaareusuallylimitedto1-2weeks.Adenoviruses40and41mayalsobepresentinthestoolofasymptomaticcarriersandmaynotrequiretreatment.49

Adenoviruses40and41belongtothelargergroupofadenoviruses,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.50Threegenotypesofthisdiversevirus,GI,GII,andGIV,caninfecthumans.GenotypegroupII,genotype4(GII.4)isthemostcommonandaccountsforthemajorityofoutbreaksaroundtheworld.51Norovirus,whichcanhaveasuddenorgradualonset,typicallydevelops24-48hoursaftercontactwithaninfectedpersonoringestionofcontaminatedfoodorwater.Symptomsincludenauseaandvomiting,diarrhea,abdominalcramps,lowgradefever,muscleaches,fatigue,andheadache.Norovirusisgenerallyshort-lived,lastingabout24-72hoursbutitishighlycontagiousduetoitsstabilityintheenvironmentandresistancetoheat,cold,anddisinfectantsolutions.Itcansurviveonhardsurfacesforweeksandupto12daysoncontaminatedfabrics.52Infectionaffectsthemicrovilliofthesmallintestine,notthecolon.Thoseinfectedcanshedthevirusforuptotwoweeksafterrecovery,continuingtospreadthevirus.

Norovirusesarethemostcommoncauseofsporadicdiarrheaincommunitysettingsandcauseuptohalfofalloutbreaksofgastroenteritis.53Treatmentsfornorovirusincludehydrationandelectrolytesprimarily,andinsomecasesantiemeticsfornauseaandvomiting,andanalgesicsforpainandheadache.Intravenousfluidandelectrolytesmaybeneededinextremecases.PCRisahighlysensitiveandspecificmethodfordetectionofnorovirus.54

Rotavirusisthemostcommoncauseofsevere,waterydiarrheaininfantsandchildren,especiallyinthedaycaresetting.ThemostcommontypeisrotavirusA,whichaccountsformorethan90%ofinfectionsinhumans.Symptomsusuallybeginwithin2daysofexposuretoaninfectedperson.Lowgradefever,vomiting,abdominalcramps,anorexia,andwaterydiarrheaarecharacteristicsigns.Dehydrationcanbeamajorrisk.55Theinfectioncanpersistfor3to8daysandusuallyresolvesonitsown.However,forthosewithweakenedimmunesystems,theinfectioncancausehospitalizationanddeath.Byagefive,almosteverychildhasbeeninfected.56Adultscanalsobecomeinfectedbutthesymptomsaregenerallylesssevere.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,thisisaselflimitinginfectionandcanbeclearedwithin2-3weeks.

Entamoebahistolytica(E.histolytica)isadisease-causingparasitethatcanaffectanyone,althoughitismorecommoninthosewholivedortravelledintropicalareaswithpoorsanitaryconditions.Diagnosiscanbedifficultsince,underamicroscope,itlookssimilartootherparasitessuchasEntamoebadisparandEntamoebahartmanii.Thelattertwoparasitesgenerallydonotcauseillness.E.histolyticaistransmittedviatheoral-fecalrouteorfromcontaminatedfoodorsurfaces.Infectedpeopledonotalwaysbecomesickandsymptomsareoftenmildincludingstomachcrampsandloosestools.Thisparasitecaninfecttheliverorspreadtootherpartsofthebodyincludingthelungsandbrain,althoughthisisnotascommon.Researchhasshownthatinasmallpercentageofpatientswithamebicliverabscess,theinfectioncancausebrainabscesswiththepatientpresentingwithcentralnervoussystemsymptoms.57TreatmentforinfectionwithE.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.58Therefore,testingofthesemicroorganismsmaybeusefulinsomecases.Recentresearchshowscertainparasites,suchasBlastocystishominis,asanemergingpotentialpathogen.59

Blastocystishominisisfoundthroughouttheworldinbothpeoplewithandwithoutsymptoms.CommonsignsofinfectionwithBlastocystisincludediarrheaorwaterystools,abdominalpain,analitching,constipation,excessgas,anddermatologicissues.Someresearchrecommendstreatmentforpeoplewithgastrointestinalanddermatologicsymptomsbutnotreatmentforthosewhoareasymptomatic.60TheremayalsobeanassociationbetweenBlastocystisandchronicdigestivedisorders,suchasirritablebowelsyndrome.61

EntamoebacoliandE.hartmanniareintestinalamebaethatarefoundinthelargeintestine.Theygenerallyarenotconsideredpathogenic.However,whentheseamebaearefoundinstoolsamplesitcanindicatethepresenceofotherpotentiallypathogenicorganisms.

CommensalBacteria

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

Thesecommensal(friendly)bacteriacoexistwiththeirhumanhostandperformmanyimportantfunctions.Theyextractnutrientsandenergyfromourdiets,maintaingutbarrierfunction,producevitamins(biotinandvitaminK),andprotectagainstcolonizationbypotentialpathogens.29Researchhasdemonstratedthemicrobiota’scapacitytointeractwiththeimmunesystemasanimportanthealthbenefit.62Themicrobiotaalsohasanti-inflammatoryandantioxidantactivity.63Itisessentialthatcommensalbacteriaarediverseandbalancedsincedisruptiontothenormalbalance(ordysbiosis)hasbeenassociatedwithobesity,malnutrition,inflammatorybowelandotherautoimmunediseases,neurologicaldisorders,andcancer.64AlimitedlistofcommensalfloraisincludedintheGI-MAPtestasageneralscreenforlevelsofnormal,protectivefloraortomonitorprobioticsupplementation.TheseincludeLactobacillusandBifidobacteriaaswellasE.coli.

BifidobacteriaandLactobacillusareanaturalpartoftheflorainthehumanbody.Theyareoftendescribedasbeneficialorcommensalbacteria.Theyaregiventherapeuticallyasprobiotics.Thesebeneficialbacteriapromotegooddigestion,regularity,boosttheimmunesystem,65andhelpcontrolintestinalpH.66BifidobacteriaandLactobacillushelppreventtheovergrowthofCandidaalbicans,E.coli,andotherpathogenicbacteria.31,67

OpportunisticPathogens

TheGI-MAPwasdesignedtodetectpathogenicandopportunisticorganismsthatmaybecausingsymptomsorillness.ManybacteriameasuredontheGI-MAPareopportunisticpathogens,meaningthattheyonlycausediseaseandillnessinsomeindividuals,particularlytheimmune-compromised.Manypeoplecomeintocontactwithopportunisticpathogensandexperiencenosymptoms,probablybecauseopportunistsaresuppressedbythebalanceofcommensalbacteria.31Overgrowthandexcessivecolonizationbyopportunisticbacteriamayoccurwhenthecommensalbacteriaareimpairedbypoordiet,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.10H.pyloricolonizationhasbeenimplicatedinavarietyofgastroduodenaldiseasesincludinggastritis,gastriccancer,andduodenalandpepticulcer.68H.pylorihasalsobeendetectedbystoolPCRincasesofdyspepsia,abdominalpain,and

chronicgastrointestinalsymptoms.69-71Itisinfamousforitscausallinktoulcersandgastriccancer,whichresultedinaNobelprizeawardedtoRobinWarrenandBarryMarshallin2005.However,somesourcesaresuggestingitsrole,atleastinpart,asacommensalorganism.H.pylorimayprotectitshostfromcertainatopicdisorders,14aswellasotherdiseasessuchasesophagealcancer72reflux,andobesity.14

PopulationdatashowsthatH.pylorivirulencevariesgeographically.Itisassociatedwithhighratesofcancerincertainregions,butnotinothers.ThedifferencemaylieinH.pylori’sgenetics.10HostimmunestatusandacidsecretionseemtobeotherimportantfactorscontributingtoH.pylori’scolonizationandpathogenesis.68TheH.pylorivirulencefactorsthataremostwellrecognizedarevacAandcagA.

ThepresenceofcagA-positiveH.pyloristrainshasbeensignificantlyassociatedwithgastriccancerandpepticulcer.73ThegenecodesforatypeIVsecretionsystemwhichallowsthebacteriumtoinjectthecagAproteinintothehostcell.Onceinsidethehost’sgastricepithelialcells,cagAcandisruptcellsignaling,leadingtoabnormalproliferation,motility,andchangesinthecytoskeleton.73Thesechangestonormalcellsignalingcaninitiatecancer.

ThepresenceofvacAhasbeenassociatedwithgastriccancer,pepticulcer,andduodenalulcer.73ThevacAgeneispresentinallstrainsofH.pyloributispolymorphic,whichleadstodifferentlevelsofvacuolatingtoxin.VacAtoxinsinteractwithcertainreceptorsonhostcells,settingoffachainofeventsincludingmitochondrialdamage,inhibitionofT-lymphocytes,andinterferenceofantigenpresentation.73

NumerouspaperssuggesttheclinicalutilityofPCRtestingforH.pylori.DetectionofH.pyloriinbiopsyspecimensbyPCRhasprovensuperiortoothermethods.69,71,74Ithasshownsensitivityandspecificityreachingthatofthediagnostic“goldstandard,”whichisendoscopywithbiopsyandureasetest.69-71H.pylorigenotypingmaybeusefulforresistantH.pyloriinfectionsthathavefailedtorespondtotripleantibiotictherapy.69InonestudyofRT-PCR,authorsstateditwasa“highlyaccuratenoninvasivemethodtodetectH.pyloriinfectioninstoolandatthesametimeallowsforculture-independentclarithromycinsusceptibilitytesting.”69

H.pylorimaybeasymptomaticandrequirenotreatmentoronlysupportivecaretoimprovetheintestinalmucosaandgastrointestinallining.InformationonthecagAandvacAgenesmayhelpdeterminewhethertreatingapositiveH.pyloriresultisnecessary.

Pseudomonasspeciesaregram-negativebacteriafoundwidelyintheenvironment.Pseudomonasaeruginosaisthemostcommonspeciescausinginfectionandcanaffecteveryportionoftheintestine.Inthegastrointestinaltractitcancauseinflammation,epithelialbarrierdysfunction,tightcelljunctioninterruption,andintestinalpermeability.75Thisbacteriumexhibitsenhancedvirulencewithstress,trauma,surgery,andcancer.75Symptomsofentericinfectionincludefever,dehydration,abdominaldistention,diarrhea,andphysicalfindingsofShanghaifever.76Theinfectionusuallyaffectsyoungchildrenandadultswithhematologicmalignanciesandneutropenia.OutsidetheGItract,itcancauseurinarytractinfections,dermatitis,bacteremia,boneandjoint,respiratory,andsystemicinfectionsespeciallyinimmune-compromisedindividuals.

Klebsiellaspeciesaregram-negativebacterianormallyfoundintheintestinaltractthatareassociatedwithawiderangeofsmallintestinaldisordersincludingalterationsofmotility,diarrhea,gas,abdominalpain,andbloating.Itsovergrowthinthesmallintestinecanalsocausehistaminosisandgutinflammationthroughthereleaseofhistaminebythebacteria.77Thosewithahistoryoflong-termantibioticuseareatrisk.

GastrointestinalBacteriaasaTriggerforAutoimmunityOpportunisticgastrointestinalpathogensaregainingattentionfortheirabilitytoinitiateautoimmunethyroiditisandinflammatoryarthritissuchasrheumatoidarthritisandankylosingspondylitis.Klebsiellaspecies,Proteusmirabilis,Citrobacterspecies,andYersiniaarebacteriathatcouldcontributetoinflammatoryarthritisinsusceptibleindividuals.YersiniaenterocoliticainfectionhasbeenassociatedwithHashimoto’sthyroiditisandGrave’sdisease78andhigherantibodiestoYersiniaenterocoliticahavebeenfoundinthesepatients.79Enterovirusisalsoassociatedwithimmunogenicthyroiditis.80Analysisofgastrointestinalmicrobesisrecommendedinchronicautoimmunedisordersthatdon’trespondtotheusualtherapies.Inhealthyindividuals,opportunisticpathogensshouldnotpresentaproblem.Ahealthygastrointestinalbarrier,81goodlevelsofcommensalflora,andstrongimmunedefensesinthegutshouldeliminatethepotentialpathogenwithinafewweeks,causinglittletonosymptoms.However,whentheintestinalbarrierisbreached,normallyharmlessopportunisticmicrobescanpassthroughthebarrier,creatingextraintestinalinfectionandillness.Intestinalpermeability,orleakygut,hasbeendocumentedinanumberofautoimmunediseases:ankylosingspondylitis,rheumatoidarthritis,celiacdisease,inflammatoryboweldisease,IgAnephropathy,nonalcoholicsteatohepatitis,andmultiplesclerosis.82,83Patientswiththeseconditionsordocumentedintestinalpermeabilitymaybeatriskifgutmicrobiotaareimbalanced.Sometheoriesofmicrobial-initiatedautoimmunediseasearemolecularmimicry,thebystandereffect,andthehygienehypothesis.Molecularmimicryisacommonexplanationforhowamicrobialinfectioncaninitiateautoimmunedisease,presumablyduetoantibacterialandcross-reactiveautoantibodies.22Itisbelievedthatmicrobialantigensresembleself-antigens.Thesecross-reactionsessentially“confuse”theimmunesystemwhichmistakenlymountsanattackagainstself-tissues.Thebystandereffecttheoryproposesthatmicroorganismsdamageself-tissues,exposingself-antigenstoimmuneattack.Finally,thehygienehypothesispresumesthatdecreasedexposuretomicrobesincreasestheTh1responsewhichcanleadtoautoimmunity.82Spondyloarthropathiesareafamilyofchronic,multi-system,inflammatorydiseasesinvolvingthesacroiliacjointsandaxialskeletonandtheymayhaveaninfectioustrigger.19Theyinclude:ankylosingspondylitis,arthritisassociatedwithulcerativecolitisorCrohn’sdisease,psoriaticarthritis,andreactivearthritis.Alloftheseshareageneticpredispositionandallarecharacterizedbyenthesitis,orinflammationofthesiteswhereligamentsandtendonsinsertintothebone.19TheyareusuallyrheumatoidfactornegativeandtheyshowanassociationwithhumanleukocyteantigenB27(HLA-B27).AprominenthypothesisisthatHLA-B27mayresembleoractasareceptorforbacterialantigens,triggeringtheautoimmuneattackonself.19Reactivearthritiscanbebroughtonbygenito-urinaryinfectionswithProteusmiribalis84,85orgastrointestinalinfectionswithbacterialagentssuchasChlamydia,Salmonella,Shigella,Campylobacter,Yersinia20,21andClostridiumdifficile.ParasitessuchasStrongyloidesstercoralis,Giardialamblia,Ascarislumbricoides,andCryptosporidiumspeciescanalsoresultinreactivearthritis.86,87Aggressivecasescouldevolveintoankylosingspondylitis.20SubstantialdatasupportsacausativeroleforProteusmirabilisinrheumatoidarthritiswhileankylosingspondylitisandCrohn’sdiseasehavebeenrelatedtoKlebsiellamicrobialinfections.22EvidenceofSalmonellahasbeenfoundincasesofankylosingspondylitis.88,89OtherdatashowsabnormalserumantibodyresponsestoKlebsiellaandProteusmirabilisinthespondyloarthropathies,87highlevelsofIgGantibodiestoKlebsiellainpatientswithankylosingspondylitis,Crohn’sdisease,andulcerativecolitis,andantibodiestoProteusinrheumatoidarthritis.83Whileculturesofsynovialfluiddonotyieldgastrointestinalmicrobes,thereisevidenceofbacterial

antigenandimmuneresponsesinthesynoviumofthejoint,suggestingthatmicrobesdoplayaroleinthepathology.90Fecalstudieshavenotbeenusedtoprovidefirmevidenceofthecausativerelationshipofstoolmicrobeswithautoimmunesyndromes.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.91TheseopportunisticpathogensofteninfectimmunosuppressedindividualssuchasthosewithHIVinfection,organtransplantation,orchemotherapy,butcanalsoinfecthealthypeople.Commonsymptomsincludediarrheaandwastingduetoentericinfection,butthespectrumofrelateddiseasesduetothesepathogensalsoincludessinusitis,bronchitis,pneumonia,nephritis,myositis,hepatitis,encephalitis,andotherbraininfections.91Treatmentoftenincludesantifungalmedicationsalongwithdietandnutritionalinterventionstohelpwithchronicdiarrhea.

Chemistries

TheGI-MAPincludesmarkersofimmunefunction,inflammation,digestion,gliadinsensitivity,andmetabolicactivityofgastrointestinalmicrobiome.Thesemarkerswereselectedfortheirclinicalutility.Lactoferrinandelastasehaveastrongfoundationofclinicalevidencetosupporttheiruseinclinicalcare.Lactoferrinhelpstheintegrativeandfunctionalmedicinepractitionermeasurethelevelofimmuneactivationinthegut,oftenassociatedwithinfectionand/orinflammatoryboweldisease.Pancreaticelastase1isanexcellentglobalmarkerofpancreaticexocrinefunctionandcanbeanindicatorofpoordigestivecapacityorpancreatitiswhenextremelylow.SecretoryIgAisthebody’sfirstlineofdefenseinthegut.Aportionofthisimmunoglobulinmightbedirectedtowardgliadin,indicatinganimmunereactiontothecommonproteininwheatandotherfieldgrassgrains.Beta-glucuronidaseisanenzymeproducednaturallyincellsoftheliver,kidney,andintestinalepithelium.However,thisenzymeisalsoproducedexcessivelybybacteriaknowntobepathogenic,andhighlevelsmaybeanindicationofadversemetabolicactivityoftheintestinalmicrobiome.

Humanlactoferrinisaglycoproteinsecretedbythemucosalcellsofthegastrointestinaltractasaresponsetoinflammatorysignals.Thisisthebody’sdefensemechanismagainstintestinalinflammation.Themeasurementoffecallactoferrinisanon-invasivebiomarkerthatisusedtohelpadoctordistinguishbetweenaninflammatoryboweldiseaseandanon-inflammatoryconditionsuchasirritablebowelsyndrome.92Anelevatedfecallactoferrinlevelinthestoolindicatesinflammation,oftenduetoinfection,ispresentandactiveinthegastrointestinaltract.Inpatientswhohaveundergonea

colectomy,fecallactoferrinisalsoasensitiveandspecifictestthatcandetectinflammationintheilealpouch(pouchitis)93andprovideclinicianswithgreaterconfidencewhenprescribingantibiotics.Fecallactoferrinmaybehelpfulinmonitoringmucosalhealinginresponsetospecifictherapies.94

Fecalpancreaticelastase-1isanaccuratefunctionalscreeningmarkerforpancreaticexocrineinsufficiency.Pancreaticelastaseisanenzymeproducedbythepancreastohelpbreakdownproteins.Pancreaticinsufficiencyoccurswhenthepancreasisnotworkingwellandbecomesinflamed(pancreatitis).Thiscanimpairthebody’sabilitytoabsorbnutrientsfromfood,includingfat-solublevitamins.95Thistestalsoaccuratelypredictsapatient’sresponsetopancreaticenzymesupplementation,especiallyinpatientswithunexplaineddiarrheaandsuspectedpancreaticinsufficiency.96Thefecalpancreaticelastase-1testmayalsobeusefulformonitoringdiabeticsbecausebothinsulinandnon-insulin-dependentdiabetescanimpairpancreaticfunction.97

Steatocritisasimpletestthatusescentrifugationtoseparatethesolid,aqueous,andlipidlayersofthestool.Thelipidlayerismeasuredinthesteatocritandthismakesupthetotalfecalfat.Acidificationofthestoolhasdramaticallyimprovedtheperformanceofthismethod.Theacidsteatocritmethodhasbeenshowntocorrelatewellwith24-hourand72-hourfecalfats.125,126Fecalsteatocrithasbeenusedwidelysince1981todetectsteatorrheainpatientswithpancreaticinsufficiencyandsmallintestinalmalabsorption. 127

SecretoryImmunoglobulinA(sIgA)isanantibodyproteinsecretedintothegastrointestinaltractasafirstlineofimmunedefenseagainstpathogenicmicroorganisms.98Thisimmunoglobulininfluencesthegutmicrobiome98andhelpstomaintainbarrierfunction99byformingcomplexeswithgutpathogensandallergens,preventingthemfrompenetratingtheintestinalbarrier.ImpairmentofsecretoryIgAmayincreasetheriskofinfectious,allergic,andinflammatorydiseasesoftheintestine.100ChronicstressmayalsodisruptlevelsofsIgA.ElevatedlevelsofsIgAmayindicateanactivatedimmuneresponsetochronicinfectionsorinflammatoryreactions.

Thepresenceoffecalanti-gliadinantibodiescanindicateanimmuneresponsetogluteninthediet.Gliadinisacomponentofgluten,theproteinfoundinwheatandotherfieldgrassgrainssuchasbarley,maltandrye.Becausegliadincouldstimulateintestinalimmunityandincreaselevelsoffecalanti-gliadinantibodyevenwhenserumconcentrationsareundetectable,101,102itisoftenusedasmarkerfornonceliacglutensensitivity.Highlevelsoffecalanti-gliadinantibodiescanprovideclinicianswithaneffectivetreatmentstrategy:agluten-freediet.

Beta-glucuronidaseisanenzymeproducedbycellsintheliver,kidney,intestinalepithelium,endocrine,andreproductiveorgans. 123Inhealthyindividuals,itplaysaroleincarbohydratedigestion,andglucuronidationbywayofbeta-glucuronidaseisamajorrouteofdetoxificationinthehumanbody. 124However,highlevelsoffecalbeta-glucuronidasecanindicateunfavorablechangesinthecolon.Themajorproducersofbeta-glucuronidasearethesebacteria:Bacteroidesfragilis,Bacteroidesvulgatus,Bacteroidesuniformis,Clostridiumparaputrificum,Clostridiumclostridioforme,Clostridiumperfringens,Escherichiacoli,Eubacterium,Peptostreptococcus,Ruminococcus,andStaphylococcus.Itisfoundin97%ofE.colistrains. 124TheenzymehydrolyzesB-glucuronidetomakeglucuronicacidandanaglycone,suchasimine,thiol,oralcohol.Thisenzymecanalsoconvertpro-carcinogenstocarcinogeniccompounds. 123 Whenbeta-glucuronidaseiselevatedinplasma,thereisanincreasedriskofhormone-sensitivecancers,suchasthoseofthebreastorprostate. 123ToxinsstimulateB-glucuronidaseactivity,anddietaryredmeatandproteinincreasestheenzyme.AntibioticshavealsobeenshowntoincreaseB-glucuronidaselevels.Alow-calorie,vegetariandietmayreducefecalB-glucuronidaselevels. 123

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

HerbalAntimicrobialAgentsBotanicalandvolatileoilextractshavealonghistoryoftraditionaluseasnaturalantimicrobials.Naturalagentssuchasberberine,garlic,oliveleaf,caprylicacid,wormwood,blackwalnut,uvaursi,citrusseedextract,andTribulusterrestrisprovideabroadspectrumofactivityagainstthemostcommonpathogensthatcausegastrointestinalillnessanddysbiosis.Antimicrobialherbsdonotposethesameriskformicrobialresistance,103-105ascomparedtoantibiotics,becausemultipleactiveingredientsfromthewholeplantworktogetherinsynchrony.Theirlonghistoricalusesuggestslowriskofadverseeffects.

AntimicrobialAgent

DescriptionandClinicalUse

Berberine BerberinehasshowneffectivenessagainstETEC-associateddiarrheaandhasbeenstudiedextensivelyforitsantibacterialeffect.106Itshowsantimicrobialactivityagainstfungi,protozoans,helminths,viruses,andchlamydia.107

Garlic Garlichasshownactivityagainstbacteria,protozoa,helminths,viruses,andfungi.108,109Itstronglysuppressedgram-negativediarrheagenicpathogens(Shigella,Salmonella,Proteusmirabilis,andE.coli)isolatedfromstoolsamples.110AqueousgarlicextractinhibitedE.coliO157:H7andE.coliLF82andenhancedthegrowthofLactobacillusreuteriinvitro.111Thissuggeststhatantimicrobialherbsmaysparebeneficialflora.

Oliveleaf Oliveleafhasantibacterial,antifungal,112,113andantiviralproperties.114-116

Caprylicacid CaprylicacidreducesCampylobacterandSalmonellainthegastrointestinaltractandstoolofpoultrywhenaddedtothefeedorwater.117-119Caprylicacidhasantiviralandantifungalproperties.120,121

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.1Thistechnologyhasbeenusedtoidentifyandcontrolpathogenoutbreaksbecauseofitsrapidturn-around-time.1ItmeasuresasubstantiallistofopportunisticpathogensaswellasalistofFDA-clearedpathogens,includingnoveltargetssuchasviruses,Microsporidia,andpathogenicvirulencefactors.Chronicgastrointestinalsymptoms,intestinalpermeability,hormonalimbalance,andfoodsensitivitiesmaytracetheiroriginstoimbalancedgutmicrobesasarootcause.Further,chronicinflammatoryarthritiscouldhaveamicrobialcomponentthatmaywarrantinvestigationbystoolstudies.Thisstooltestoffersintegrativeandfunctionalmedicinepractitionerssuperiorsensitivityandspecificitytohelpresolvepersistentandcomplexillnesses.Sincetheimmunesystem,theintestinalbarrier,andmicrobialdiversityareintimatelyinterwoven,thoroughunderstandingofourgutmicrobiomeholdspromisefornewapproachestotreatandpreventdisease.122

CompleteListofTargetAnalytesMeasuredontheGI-MAPBacterialpathogens:CampylobacterC.diffToxinA&B**E.colio157**EnterotoxigenicE.coliLT&ST(ETEC)**Shiga-likeToxinproducingE.colistx1&stx2(STEC)**SalmonellaShigellaVibrio choleraYersiniaenterocoliticaViralpathogens: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-gliadinsIgAPancreaticelastase1LactoferrinOccultblood

Organismswith**arelistedwithcitationsinthispaper.

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