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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