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Nader
Linaabdelhadi
MohammadAlTamary
KhalidSaadeh
Sheet4–C.diphtheria&B.pertussis
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میحرلا نمحرلا هللا مسب Inthislecturewewillbetalkingabouttworodshapedbacteria,oneisGram-positiveandtheotherisGram-negative.Theyaregroupedtogetherbecauseoftheirabilitytoproducetoxins.
Corynebacteriumdiphtheriae:
Generally,diphtheroidsarepartofthenormalfloraoftheupperrespiratorytract,butweareinterestedinC.diphtheriawhichisimplicatedinopportunisticinfectionstoo.
• ThisistheG-positiverod/bacilli.• Itisnotvirulent(thatitdoesnotproducetoxin)exceptwhenitisinfectedwith
bacteriophage,allowingthemtoproducethetoxin(toxigenic).• Theycausecutaneousandrespiratoryinfections*whentheyhavetheabilityto
producethetoxin.
MorphologyCorynebacteria,clubshapedGrampositiverods(wideratoneend)andarearrangedinpalisadesorinV-orL-shapedformations(orChineseletters).
Therodshaveabeadedappearance.Thebeadsconsistofgranulesofhighlypolymerizedpolyphosphate—astoragemechanismforhigh-energyphosphatebonds.
that(i.e.,adyestainmetachromaticallyThegranulesgranulesstainthestainstherestofthecellbluewill
).red
motile-sporeforming,non-Non
Palades:paralleltoeachother
Transmission• HumansaretheonlynaturalhostofC.diphtheriae• Bothtoxigenicandnontoxigenicorganismsresideintheupperrespiratorytract
andaretransmittedbyairborneordroplets(likeotherrespiratorypathogens).• Theorganismcanalsoinfecttheskinatthesiteofapreexistingskinlesion.• Thisoccursprimarilyinthetropicsbutcanoccurworldwideinindigentpersonswithpoorskin
hygiene.
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PathogenesisAswesaidbefore,theyarenotharmful,unlesstheytakethephagefortoxinproduction.Theproductionoftoxinsisthecauseofthedisease.
Noinvasionintotheblood,buthowthesystemicinvolvementispresentindiphtheriadisease?Itisduetotheproducedtoxinwhichwillentertheblood.So,thesystemicsignsandsymptomsarerelatedtothetoxin.
Hereiswhatiswrittenintheslides:
Thepathogenisis:
-Mainlyexotoxinmediated(similartootherG+verods),however,thebug(bacteria)exotoxinproduction.opriort(noinvasiveness)mustestablishitselfinthethroatfirst
.Bfashion(active/binding)-SimilartoothertoxinsitisformedinanA-
2-ribosylationofelongationfactor-bitsproteinsynthesisbyADPDiphtheriatoxininhi-noproteinsynthesisin=hainc2)usedtomaintainelongationofthepeptide-(EF
.eukaryoticcell
-Asmentioned,toxinisencodedonagenetransmittedbytransductiononatemperatephage.
ClinicalFindings/complications
Forrespiratoryinfection,itstartsbyformingathickpseudomembraneinthepharynxwhichcouldextendintothelarynxandmaycauseairwayobstruction.(atumor-likefeatures"#$%)keepinmindtodifferentiatebetweenstrep.throatandthispseudomembranewhichisdirtierlookingandconsistsoffibrin,WBCs,RBCs,bacteriaandexudates,removingoffthismembranecancausebleeding.
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Thisishowthepseudomembranelookslike.Formedbythenon-invasivebacteria.
• Therearethreeprominentcomplications:
)1( Extensionofthemembraneintothelarynxandtrachea,causingairwayobstruction.
)2( Myocarditisaccompaniedbyarrhythmiasandcirculatorycollapse.
(3)Nerveweaknessorparalysis,especiallyofthecranialnerves.
•Theotheraspectsarenonspecific:fever,sorethroat,andcervicaladenopathy.
-Thesystemiceffectsresultedfromthetoxinaremainlyassociatedwithcardiacandneuralsymptoms:
Cardiac:endocarditis,pancarditis(inflammationofendocardium,myocardiumandpericardium)
Diagnosis-diagnosisismainlybyclinicalsuspicionandthetreatmentisbygivinganti-toxinimmediatelywithoutwaitingforlaboratoryresults.
*note:peoplewithpreviouscutaneousformofdiphtheria(skindiphtheria)haveanti-bodiesagainstthetoxin,sotheycanbeprotectedagainstthepseudomembraneformationinthethroat.
LaboratoryDiagnosisstrongclinicalsuspicion(throatpseudomembrane)withsystemiceffects>>immediatetreatmentwithantitoxin.
• Fordiphtheriathepresenceoftheorganismisnotenough,weneedtofindthetoxin,becausethereisatoxigenicstrains.
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• Duetothequicknatureoftoxinmediateddisease,thedecisiontotreatwithanantitoxinshouldbeclinicalandnotwaitforlabconfirmation.
2typesofmediaforculturewhichareselectiveforC.diphtheria:
•AthroatswabshouldbeculturedonLoeffler’smedium(creamcoloredcoloniesareshownintheslant),atelluriteplate(blackcoloniesseenatelluriumsaltthatisreducedtoelementaltelluriumwithintheorganismthusblackcoloredcolonies),andabloodagarplate.
• Thetypicalgray-blackcoloroftelluriuminthecolonyisatelltalediagnosticcriterion.
• IfC.diphtheriaeisrecoveredfromtheculturesthenwecanconfirmtoxin(eitheranimalinoculation,antibody-basedgeldiffusionprecipitintestorPCRtestforthepresenceofthegene).
•SmearsofthethroatswabshouldbestainedwithbothGramstainandmethyleneblue.
•Althoughthediagnosisofdiphtheriacannotbemadebyexaminationofthesmear,thefindingofmanytapered,pleomorphicGram-positiverodscanbesuggestive.
• Themethylenebluestainisexcellentforrevealingthetypicalmetachromaticgranules(theclubshapeisduetothesegranules).
-Again,culturingofC.diphtheriaisnotenough,weneedtoknowifitistoxigenicornot.
Thegoldstandardforthedetectionofdiphtheriatoxinistheimmuno-precipitationtest(Elektest).Analternativemethodisthedetectionoftheexotoxingeneusingapolymerasechainreaction(PCR)whichisafasterapproach.Basically,Elektestshowsapositiveresultwhenimmune-precipitationlinesaredetected.ThisisaYouTubevideoaboutElektest:https://youtu.be/-HHSC9Q9314.
Treatment-rememberthenon-invasivecolonizationandthecirculatingtoxin.
1(ANTITOXIN)Thetreatmentofchoiceisantitoxin,whichshouldbegivenimmediatelyonthebasisofclinicalimpression(notonlabconfirmation,thistakeswhiletogetbothisolationoforganismanddetectionoftoxin).
Cultureresultstakeupto48hours
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• Theneedforimmediatetreatmentwithantitoxinisduetothetoxin’sRAPIDandIRREVERSIBLEactiononcells,thusantitoxinwillworkonunboundtoxininthebloodonly
2(ANTIBIOTICS)TreatmentwithpenicillinGorerythromycinisalsorecommendedwithantitoxinbutnotasasubstitute.
• Antibioticswillreducebacterialcountandtoxinproduction,theywillalsoreducethechanceofacarrierstate.
PreventionThevaccine(DTaP)ispartofthenationalvaccinationprogramworldwideandinJordan.Whichconsistsofinactivateddiphtheriatoxin,tetanustoxoid(inactivatedtoxin)andinactivatedpertussistoxin.Thevaccineisgiven3timesatthefirstageafterbirth,thenatthesecondyear,andthenbetween4to6years.Butimmunityfordiphtheriaandpertussisarenotlifelong,theystayfor10years,soadultsreceiveaboosterdoseevery10years.Theadult’sboosterdoseisabbreviatedbysmallletters(dtap).
•Diphtheriaisnowrareintheworldduetoitsinclusioninthescheduledvaccineregiment(DTaP)withdiphtheriatoxoid.
•Inwarzonesorareaswithlapseinimmunization,reemergence(andatypicalsymptoms)areontherise.
•formaldehydetreatmentofthetoxin,destroysthetoxinbutleavestheantigenicityintact.
•Immunizationconsistsofthreedosesgivenat2,4,and6monthsofage,withboostersat1and6yearsofage.
•Becauseimmunitywanes,aboosterevery10yearsisrecommended.
•Immunizationdoesnotpreventnasopharyngealcarriageoftheorganism.
Bordetellapertussis:bacilli= rod-shaped
•B.pertussisisthecauseofwhoopingcough(pertussis).
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•Itisstillseenespeciallyininfantsunder2months(receivednoorlittleprotectionfrommother,usuallytypicalwhoopingcoughisseen)
•ImportantProperties:
•B.pertussisisaGram-negativerod,alsosmallcoccobacillusshape,encapsulated.
Pathogenesis&EpidemiologyAlthoughitisagramnegativeandhasLPS,themainvirulencefactoristheproductionofexotoxins.5wellstudiedvirulencefactorsareinvolvedinthepathogenesis.
Firstofall,thebacteriaisnon-invasive,soitneedsafactorthatwillhelptoattachandcolonizethepharynxtoestablishthedisease.
{1}Filamentoushemagglutinin,istheproteinthatthebacteriumusestoattachitselftotheciliaoftheepithelialcells,damagesthesecellsaswell.(nocilia=nomoreclearingofmucus)(antibodiesagainstthisproteinareprotective).**nomucusclearance
{2}Pertussistoxinstimulates(byenzymaticADPribosylationofG-proteins)theintracellularcAMP,oncecAMPrises(similartothediarrheamechanismbycholera)itincreasesextracellularsecretions(nowalotmorerespiratorysecretionsarebeingproduced).**overproductionofmucus
-Nomoreclearingofmucus+alotmoremucusisbeingproduced=>BothcontributetothePROLONGEDseverecoughofpertussis.
(theonlymechanismlefttoclearairwaysistoforcefullycoughitout)
-pertussistoxinisthemainfactorinthevaccineconcerningB.pertussis.ThepertussistoxinispartoftheDTaPvaccine(allthreecomponentsofthisvaccinesareA-Bconfigurationtoxins).Thevaccinemainlycontains3ofthe5factors,2ofwhicharefilamentoushemagglutininandpertussistoxin,thethirdoneisanyoftheremaining.
{3}Theorganismsalsosynthesizeandexportadenylatecyclase.Thisenzyme,whentakenupbyphagocyticcellscaninhibittheirbactericidalactivity.Bacterialmutantsthatlackcyclaseactivityareavirulent.**evadedimmunecelldestruction.
{4}Trachealcytotoxinisafragmentofthebacterialpeptidoglycan,thistoxin,actsalongsidewithendotoxintoinducenitricoxide,whichkillstheciliatedepithelialcells.
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Note: infection with this organism will cause leukocytosis with lymphocytosis (which is more commonly present within viral infections). Patientswithpertussisexhibitahighnumberoflymphocytesintheirblood*lymphocytosis),thisisduetoPertussistoxininhibitionofsignaltransduction(byribosylationwithADPonGproteins)ofchemokines,whichinturncausesaninhibitionoflymphocytesenteringthelymphtissueandremainingintheblood.
ClinicalFindings
-Whoopingcoughmainlyaffectschildrenaftertheageof6monthsbecausetheacquiredmaternalantibodies(IgG)willbedepletedandnotsignificant.-Itisof4stages:
•Whoopingcoughbeginswithmildsymptoms(sorethroat,rhinorrhea,sneezing,coughing,(lowgradefever)thendevelopsintoanacutetracheobronchitisfollowedbyasevereparoxysmal(suddenoutbursts)cough,whichlastsfor1to4weeks.
•Theparoxysmalpatternischaracterizedby:aseriesofhackingcoughs,productionoflargeamountsofmucus(productive/wet),endedbyinspiratory(tryingtocatchtheirbreath)whoops,thecharacteristicnoiseisduetonarrowingoftheglottis.
•Theorganismisrestrictedtotherespiratorytractandbloodculturesarenegative,butwithpronouncedleukocytosiswithupto70%lymphocytes.
•Althoughcentralnervoussystemanoxiaandexhaustioncanoccurasaresultoftheseverecoughing,deathismainlyduetopneumonia.
•Theclassicpictureofwhoopingcoughdescribedaboveoccursprimarilyinyoungchildren.
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-uptothreeweeksofincubationperiod>>Catarrhalstage(flulikesymptoms)>>paroxysmalstage(patientsmayexperienceasmanyas20-30paroxysmwith20-25coughscontinuouslydailywhichmaycausevomiting,convulsionsandcyanosis.
*thisisthestageinwhichpatientsseekhospitalizationandimprovement.
Clinicalfindingsinadults
Adultshavelargerairwayssotheymaynotreallydevelopthewhoopingcoughcharacteristicasinchildren.Adultsinfecteddevelopwhatiscalledachronic*100-daycough*whichisnon-productive(notthatmuchmucus).
LaboratoryDiagnosis
Diagnosisshouldbedoneasearlyaspossibletostarttreatmentwithantibiotics.
•Theorganismcanbeisolatedfromnasopharyngealswabstakenduringtheparoxysmal(cough)stage.
• Bordet-Gengoumediumusedforthispurposecontainsahighpercentageofblood(20%–30%)toinactivateinhibitorsintheagar.
• Theorganismisthenidentified(fromtheabovegrowthmedium)bydetectingitsantigens(eitherbyagglutinationorbyfluorescentantibodystains).
• Thereasonfordependingonantigendetectionisduetotheslownatureofgrowthforthisorganism,rapiddiagnosisismandatedandthusdirectfluorescent-antibodystainingofthenasopharyngealspecimenscanbeusedfordiagnosis.
• Polymerasechainreaction–basedtestsarehighlyspecificandsensitiveandshouldbeusedifavailable.
Testresultstakeupto1weeksofasterapproachesareneededsuchasdifluorescenceantigentesting,PCR
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Treatment
Azithromycin(macrolide)isthedrugofchoice.Basically,erythromycinisthedrugofchoiceforbothC.diphtheriaandB.pertussis.
• Itisessentialtotreatearly,Azithromycinwillreducethebacterialloadandreducethechangeofcomplications,otherwiseitwillhavelittleeffectonprogressionofthediseaseonceithasreachedfurtherstages(thetoxinalreadycauseddamagetothemucosa (.
• Supportivecare(e.g.,oxygentherapyandsuctionofmucus)duringtheparoxysmalstageisimportant,especiallyininfants.
PreventionVaccinebased:eitheranacellularone(contains5purifiedantigenproteins,nocells,thisisthemostusedvaccine)orkilledvaccinecontaininginactivatedB.pertussisorganisms.
•Themainimmunogeninacellularvaccineistheinactivatedpertussistoxin.
(pertussistoxoid)thetoxoidinthevaccineispertussistoxinthathasbeeninactivatedgeneticallybyintroducingtwoaminoacidchanges,whicheliminatesitsADP-ribosylatingactivitybutretainsitsantigenicity.
•Itisthefirstvaccinetocontainageneticallyinactivatedtoxoid.
•Theotherantigensintheacellularvaccinearefilamentoushemagglutinin,pertactin,andfimbriaetypes2and3.
•Theacellularvaccinehasfewersideeffectsthanthekilledvaccinebuthasashorterdurationofimmunity.
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CorynebacteriumdiphtheriaeBordetellapertussis
*causesDiphtheria*causespertussis(whoopingcough)
Corynebacteriumdiphtheriae Bordetellapertussis-G-positiverod,notcapsulated-notvirulent,unless(bacteriophage>phage>bacteria>toxigenic)-clubshaped-granulesstainmetachromatically-Non-sporeforming,non-motile-humansaretheonlynaturalhostandreservoir.-transmittedbyairbornedroplets.-notinvasive.Systemiceffectsareproducedbytoxins.-pseudomembraneformation>whichleadstoairwayobstruction.-cardiacandneuralcomplications-diagnosis is based on clinical suspicion,laboratoryfindingsforconformation-cultureintelluriteplate>blackdots-Elektest:fortoxindetection-toxin>antitoxinBacteria>erythromycinandpenicillinG-DTaPcontainingdiphtheriatoxoid
-G-negativerod,encapsulated-5virulencefactors(filamentoushemagglutinin,pertussistoxin…)-notinvasive-thecauseofwhoopingcough-increasesmucusproduction-lymphocytosis-mainlychildren-4stages-adultsformiscalled:chronic100-daycough-deathismainlyduetopneumonia-patients diagnosed with whooping cough>antibiotics-culturelastsfor1week-Bordet-Gengoumedium-azithromycin(macrolide)anderythromycin-acellular vaccine (proteins only). The mainimmunogeninacellularvaccineistheinactivatedpertussistoxin
ThingsincommonBotharerod-shaped,themajorvirulencefactoristhetoxinproducedwhichisthemaincauseof
systemicsignsandsymptoms,non-invasivebacteria,samemodeoftransmissionwhichisair-bornedropletsandbotharepresentintheDTaPvaccine.Thisvaccineisgivenindosesandthereisa
boosterdoseevery10yearsbecausethevaccinedoesnotgivealifelongimmunity