by: sandip harsoda m.sc. microbiology

8/14/2019 By: Sandip Harsoda M.sc. Microbiology

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DIPHTHERIADIPHTHERIA

By: Sandip harsodaBy: Sandip harsodaM.Sc. MicrobiologyM.Sc. Microbiology


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ContentContent

IntroductionIntroduction PathogenesisPathogenesis What are the symptoms of diphtheria?What are the symptoms of diphtheria? Clinical featuresClinical features

ComplicationComplication Diphtheria ToxinDiphtheria ToxinThe nature of immunity to DiphtheriaThe nature of immunity to DiphtheriaTechniques to measure Antibody ResponseTechniques to measure Antibody Response

TreatmentTreatment PreventionPreventionTransmissionTransmission ReferenceReference


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IntroductionIntroduction

Diphtheria is a very contagious and lifeDiphtheria is a very contagious and life

threatening bacterial disease.threatening bacterial disease.

Diphtheria usually attacks the throat andDiphtheria usually attacks the throat and

nose.nose. In more serious cases, it can attack theIn more serious cases, it can attack the

heart and nerves.heart and nerves.

Because of widespread immunization,Because of widespread immunization,diphtheria is very rare in United States.diphtheria is very rare in United States.


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ORGANISM:ORGANISM:Genus :Genus : CorynebacteriumCorynebacteriumSpecies:Species: diphtheriaediphtheriae

GENERAL CONCEPT:GENERAL CONCEPT:

CorynebacteriaCorynebacteria belong to the familybelong to the familyMycobacteriaceaeMycobacteriaceae and are part of the CMNand are part of the CMN

group (group (CorynebacteriaCorynebacteria,, MycobacteriaMycobacteria andandNocardiaNocardia).).


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The family The family MycobacteriaceaeMycobacteriaceae are Gram-positive,are Gram-positive,nonmotile, catalase-positive and have a rodlike tononmotile, catalase-positive and have a rodlike tofilamentous morphology (filamentous morphology (CorynebacteriaCorynebacteria areare

pleomorphic).pleomorphic).

They produce characteristic long chain fatty acids They produce characteristic long chain fatty acidstermed mycolic acids.termed mycolic acids.

C. diphtheriaeC. diphtheriae is a facultatively anaerobic.is a facultatively anaerobic. Characterized by non-encapsulated, non-sporulated,Characterized by non-encapsulated, non-sporulated,

immobile, straight or curved rods with a length of 1 toimmobile, straight or curved rods with a length of 1 to8 m and width of 0.3 to 0.8 m, which form ramified8 m and width of 0.3 to 0.8 m, which form ramifiedaggregations in culture (looking like "Chineseaggregations in culture (looking like "Chinese

characters").characters").


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CONTINUECONTINUE

Scientific classificationScientific classification

Kingdom: BacteriaKingdom: Bacteria

Phylum:Phylum:ActinobacteriaActinobacteria

Order:Order:ActinomycetalesActinomycetales

Family:Family: CorynebacteriaceaeCorynebacteriaceae

Genus:Genus: CorynebacteriumCorynebacterium

Species:Species: diphtheriaediphtheriae


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Corynebacterium diphtheriaeCorynebacterium diphtheriae is ais a

pathogenic bacterium that causespathogenic bacterium that causes

diphtheria.diphtheria.

It is also known as the Klebs-Lffler bacillus,It is also known as the Klebs-Lffler bacillus,because it was discovered in 1884 bybecause it was discovered in 1884 by

German bacteriologists Edwin Klebs andGerman bacteriologists Edwin Klebs and

Friedrich Lffler.Friedrich Lffler.


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Many strains ofMany strains ofC. diphtheriaeC. diphtheriae produce a protein exotoxinproduce a protein exotoxinwith a molecular weight of 62 kilodaltons,which iswith a molecular weight of 62 kilodaltons,which is

responsible for the signs of diphtheria.responsible for the signs of diphtheria.Three subspecies are recognizedThree subspecies are recognized

C. diphtheriae mitis,C. diphtheriae mitis,

C. diphtheriae intermediusC. diphtheriae intermedius

C. diphtheriae gravis.C. diphtheriae gravis.

They differ slightly in their ability to metabolize certain They differ slightly in their ability to metabolize certainnutrients, but all may be toxigenic or non-toxigenic.nutrients, but all may be toxigenic or non-toxigenic.


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Toxin production (toxigenicity) occurs only Toxin production (toxigenicity) occurs only

when the bacillus is itself infectedwhen the bacillus is itself infected

(lysogenized) by a specific virus(lysogenized) by a specific virus

(bacteriophage)carrying the genetic(bacteriophage)carrying the geneticinformation for the toxin (tox gene).information for the toxin (tox gene).

Only toxigenic strains can cause severeOnly toxigenic strains can cause severe

disease.disease.


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DISTINCTIVE PROPERTIES:DISTINCTIVE PROPERTIES:

Corynebacterial cell walls contain thinCorynebacterial cell walls contain thin

spots which leads to some Gram variabilityspots which leads to some Gram variability

and "ballooning" that produces a "club-and "ballooning" that produces a "club-shaped" cell.shaped" cell.

Old cells store inorganic phosphate, whichOld cells store inorganic phosphate, which

can appear as metachromatic granulescan appear as metachromatic granuleswhen stained.when stained.


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PATHOGENESIS:PATHOGENESIS:

C. diphtheriaeC. diphtheriae is the etiologic agent of diphtheria.is the etiologic agent of diphtheria.

These organisms colonize the mucus membranes These organisms colonize the mucus membranesof the respiratory tract and produce the enzymeof the respiratory tract and produce the enzymeneuraminidase which splits N-acetylneuraminicneuraminidase which splits N-acetylneuraminic

acid (NAN) from cell surfaces to produce pyruvateacid (NAN) from cell surfaces to produce pyruvatewhich acts as a growth stimulant.which acts as a growth stimulant.

C.diphtheriaeC.diphtheriae also produces diphthin, which is aalso produces diphthin, which is aprotease that inactivates IgA.protease that inactivates IgA.


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What are the symptoms ofWhat are the symptoms of

diphtheria?diphtheria?

The following are the most common The following are the most common

symptoms of diphtheria.symptoms of diphtheria. breathing difficultybreathing difficulty

husky voicehusky voice

enlarged lymph glandsenlarged lymph glands

increased heart rateincreased heart rate

stridor (a shrill breathing sound heard onstridor (a shrill breathing sound heard on

inspiration)inspiration)


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nasal drainagenasal drainage

swelling of the palate (roof of the mouth)swelling of the palate (roof of the mouth)

sore throatsore throat

low-grade feverlow-grade fever

malaisemalaise

Skin (cutaneous) diphtheria:Skin (cutaneous) diphtheria:

With this type of diphtheria, the symptoms areWith this type of diphtheria, the symptoms are

usually milder and may include yellow spots orusually milder and may include yellow spots or

sores (similar to impetigo) on the skin.sores (similar to impetigo) on the skin.


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Clinical FeaturesClinical Features

The incubation period of diphtheria is 25 The incubation period of diphtheria is 25

days(range, 110 days).days(range, 110 days).

Disease can involve almost any mucousDisease can involve almost any mucous

membrane. For clinical purposes, it ismembrane. For clinical purposes, it isconvenient to classify diphtheria into aconvenient to classify diphtheria into a

number of manifestations, depending on thenumber of manifestations, depending on the

site of disease.site of disease.


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Anterior Nasal DiphtheriaAnterior Nasal Diphtheria

The onset of anterior nasal diphtheria isThe onset of anterior nasal diphtheria isindistinguishable from that of the common coldindistinguishable from that of the common coldand is usually characterized by a mucopurulentand is usually characterized by a mucopurulentnasal discharge (containing both mucus and pus)nasal discharge (containing both mucus and pus)which may become blood-tinged.which may become blood-tinged.

A white membrane usually forms on the nasalA white membrane usually forms on the nasalseptum.septum. The disease is usually fairly mild because of The disease is usually fairly mild because of

apparent poor systemic absorption of toxin in thisapparent poor systemic absorption of toxin in thislocation, and it can be terminated rapidly bylocation, and it can be terminated rapidly by

antitoxin and antibiotic therapy.antitoxin and antibiotic therapy.


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Pharyngeal and Tonsilar DiphtheriaPharyngeal and Tonsilar Diphtheria

The most common sites of diphtheria infection The most common sites of diphtheria infectionare the pharynx and the tonsils.are the pharynx and the tonsils.

Infection at these sites is usually associated withInfection at these sites is usually associated withsubstantial systemic absorption of toxin.substantial systemic absorption of toxin.

The onset of pharyngitis is insidious.The onset of pharyngitis is insidious.

Early symptoms include malaise, sore throat,Early symptoms include malaise, sore throat,anorexia, and low-grade fever.Within 23 days, aanorexia, and low-grade fever.Within 23 days, abluish-white membrane forms and extends,bluish-white membrane forms and extends,varying in size from covering a small patch on thevarying in size from covering a small patch on thetonsils to covering most of the soft palate.tonsils to covering most of the soft palate.


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Laryngeal DiphtheriaLaryngeal Diphtheria Laryngeal diphtheria can be either anLaryngeal diphtheria can be either an

extension of the pharyngeal form or canextension of the pharyngeal form or can

only involve this site.only involve this site.

Symptoms include fever, hoarseness, andSymptoms include fever, hoarseness, anda barking cough.a barking cough.

The membrane can lead to airway The membrane can lead to airway

obstruction, coma, and death.obstruction, coma, and death.


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ComplicationsComplications

Most complications of diphtheria,Most complications of diphtheria,including death, are attributable to effectsincluding death, are attributable to effectsof the toxinof the toxin

The toxin, when absorbed, affects organsThe toxin, when absorbed, affects organsand tissues distant from the site ofand tissues distant from the site ofinvasion.invasion.

The most frequent complications of The most frequent complications of

diphtheriadiphtheria1: Myocarditis1: Myocarditis

2: Neuritis2: Neuritis


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MyocarditisMyocarditis may present as abnormal cardiacmay present as abnormal cardiac

rhythms and can occur early in the course of therhythms and can occur early in the course of theillness or weeks later, and can lead to heartillness or weeks later, and can lead to heartfailure.failure.

NeuritisNeuritis most often affects motor nerves andmost often affects motor nerves andusually resolves completely.usually resolves completely.

Paralysis of the soft palate is most frequentParalysis of the soft palate is most frequentduring the third week of illness.during the third week of illness.

Paralysis of eye muscles, limbs, and diaphragmParalysis of eye muscles, limbs, and diaphragmcan occur after the fifth week.can occur after the fifth week.

Secondary pneumonia and respiratory failure maySecondary pneumonia and respiratory failure mayresult from diaphragmatic paralysis.result from diaphragmatic paralysis.


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Diphtheria ToxinDiphtheria Toxin

Extracellular substance (exotoxin) producedExtracellular substance (exotoxin) producedbyby Coryebacterium diphtheriae.Coryebacterium diphtheriae.

Diphtheria toxin, a protein with a molecularDiphtheria toxin, a protein with a molecular

weight of 62 000.weight of 62 000. Known as an A-B type toxin, and consists ofKnown as an A-B type toxin, and consists of

two fragments designated A and B.two fragments designated A and B.

Fragment B is necessary for binding toFragment B is necessary for binding to

surface receptors and penetration into cells.surface receptors and penetration into cells.


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Fragment A is responsible for its toxicityFragment A is responsible for its toxicityand exerts its action by interferingand exerts its action by interferingenzymatically with protein synthesis,enzymatically with protein synthesis,finally producing the death of the cells.finally producing the death of the cells.

Diphtheria toxin exerts its effects onDiphtheria toxin exerts its effects ondistant tissues and organs, especially thedistant tissues and organs, especially theheart (myocarditis) and the peripheral andheart (myocarditis) and the peripheral andcranial nerves (weakness progressing tocranial nerves (weakness progressing toparalysis).paralysis).


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When treated with formaldehyde and heat,When treated with formaldehyde and heat,

diphtheria toxin loses its ability to bind todiphtheria toxin loses its ability to bind to

cells and its enzymatic activity, but retainscells and its enzymatic activity, but retains

its immunogenicity.its immunogenicity.This treatment converts diphtheria toxin to aThis treatment converts diphtheria toxin to a

toxoid, which is commonly used totoxoid, which is commonly used to

immunize against diphtheria.immunize against diphtheria.


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Mechanism of DiphtheriaMechanism of Diphtheria

ToxinToxin


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The Nature of Immunity toThe Nature of Immunity to

DiphtheriaDiphtheria

Immunity against diphtheria is antibody-Immunity against diphtheria is antibody-

mediated.mediated.

Antibody, called antitoxin, is primarily of theAntibody, called antitoxin, is primarily of the

IgG type.IgG type. Antitoxin is distributed throughout the bodyAntitoxin is distributed throughout the body

and can pass easily through the placenta,and can pass easily through the placenta,

providing passive immunity to the newbornproviding passive immunity to the newborn

during the first few months of life.during the first few months of life.


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Techniques to MeasureTechniques to Measure

Antibody ResponseAntibody Response

Two important properties of diphtheria Two important properties of diphtheria

toxin are utilized to determine the activitytoxin are utilized to determine the activity

of diphtheria antibodies.of diphtheria antibodies.

1.1. The ability to produce an inflammatoryThe ability to produce an inflammatoryreaction.reaction.

2.2. Second property is the capacity ofSecond property is the capacity of

diphtheria toxin to block protein synthesis.diphtheria toxin to block protein synthesis.


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Schick testSchick test

To perform the Schick test, 0.1 ml of To perform the Schick test, 0.1 ml ofdiphtheria toxin (about l/50 of the minimaldiphtheria toxin (about l/50 of the minimallethal dose for a guinea pig) is injectedlethal dose for a guinea pig) is injectedintradermally on the volar surface of theintradermally on the volar surface of theforearm of the person being tested.forearm of the person being tested.

If the person has circulating diphtheriaIf the person has circulating diphtheriaantitoxin at a level of 0.01 to 0.03 IU/ml, theantitoxin at a level of 0.01 to 0.03 IU/ml, theinjected toxin will be neutralized and noinjected toxin will be neutralized and noreaction will occur.reaction will occur.


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A positive reaction signifies lack of antitoxinA positive reaction signifies lack of antitoxin

and is characterized by inflammationand is characterized by inflammation

appearing after 24 to 36 hours andappearing after 24 to 36 hours and

persisting for 4 days or longer.persisting for 4 days or longer. A control test is always performed on theA control test is always performed on the

opposite arm using toxin inactivated byopposite arm using toxin inactivated by

heating to 60C for 15 minutes.heating to 60C for 15 minutes.

A positive reaction to inactivated toxin and aA positive reaction to inactivated toxin and a

positive reaction to toxin indicates anpositive reaction to toxin indicates an

allergic response to toxin.allergic response to toxin.


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Neutralization test onNeutralization test on

animalsanimalsTheThe in vivoin vivo neutralization test is usuallyneutralization test is usually

performed on the depilated skin of rabbits.performed on the depilated skin of rabbits.

Different dilutions of serum mixed with fixedDifferent dilutions of serum mixed with fixed

amounts of diphtheria toxin are injected intoamounts of diphtheria toxin are injected intothe depilated skin of the animal and thethe depilated skin of the animal and the

antitoxin concentration is estimated basedantitoxin concentration is estimated based

on the presence or absence of anon the presence or absence of an

inflammatory reaction.inflammatory reaction.


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AdvantageAdvantage

The test show not only the The test show not only theneutralization of toxin by antibody presentneutralization of toxin by antibody presentin the test serum, but also reaction betweenin the test serum, but also reaction betweenother antigen-antibody systems.other antigen-antibody systems.

DisadvantageDisadvantage

The test is laborious, time-consuming,The test is laborious, time-consuming,expensive, and requires suitable animals.expensive, and requires suitable animals.

Neutralization test onNeutralization test on


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Neutralization test onNeutralization test onmicrocellmicrocell

cultureculture It is based on the observation that theIt is based on the observation that thesurvival of mammalian cells in culture issurvival of mammalian cells in culture is

inhibited by diphtheria toxin.inhibited by diphtheria toxin.

The titration of the antitoxin in the serum The titration of the antitoxin in the serumsamples is done in plastic microtissuesamples is done in plastic microtissue

culture plates, in which dilutions of test seraculture plates, in which dilutions of test sera

are mixed with challenge toxin.are mixed with challenge toxin.


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After a short incubation, Vero (greenAfter a short incubation, Vero (greenmonkey renal epithelium) cell or HeLa cellmonkey renal epithelium) cell or HeLa cellsuspension in a special culture medium issuspension in a special culture medium isadded.added.

After incubation for 3 or 4 days, results areAfter incubation for 3 or 4 days, results areread as a change in the color of theread as a change in the color of thereagents in the microtiter plate wells.reagents in the microtiter plate wells.

The color change is due to the metabolic The color change is due to the metabolicformation of acid, which changes the pH.formation of acid, which changes the pH.


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Vero cells are more sensitive to diphtheriaVero cells are more sensitive to diphtheria

toxin.toxin.

They have large numbers of binding sites They have large numbers of binding sites

(receptors) and they take up the toxin in a(receptors) and they take up the toxin in ahighly specific, time- and temperature-highly specific, time- and temperature-

dependent manner.dependent manner.

When a serum dilution contains antitoxin inWhen a serum dilution contains antitoxin in

excess, the cells continue to grow, and theexcess, the cells continue to grow, and thecolor of the medium changes from red tocolor of the medium changes from red to

yellow.yellow.


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Passive hemagglutinationPassive hemagglutination

The passive hemagglutination (HA) test is The passive hemagglutination (HA) test is

frequently used to test for diphtheriafrequently used to test for diphtheria

antibody.antibody.

In the HA test, sheep, turkey, horse, orIn the HA test, sheep, turkey, horse, orhuman red cells (previously treated withhuman red cells (previously treated with

tannic acid or diazotized benzidine andtannic acid or diazotized benzidine and

sensitized with diphtheria toxoid) aresensitized with diphtheria toxoid) are

agglutinated by diphtheria antibody.agglutinated by diphtheria antibody.


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TreatmentTreatment

Treatment with erythromycin orally Treatment with erythromycin orallyor by injection (40 mg/kg/day;or by injection (40 mg/kg/day;

maximum, 2 gm/day) for 14 days.maximum, 2 gm/day) for 14 days.

Penicillin G daily, intramuscularly.Penicillin G daily, intramuscularly.The disease is usually not contagiousThe disease is usually not contagious

48 hours after antibiotics are48 hours after antibiotics are

instituted.instituted.


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How can diphtheria beHow can diphtheria be

prevented?prevented?There is a vaccine for diphtheria.There is a vaccine for diphtheria.

The diphtheria vaccine is usually given in aThe diphtheria vaccine is usually given in acombination shot with tetanus and pertussiscombination shot with tetanus and pertussis

vaccines, known as DTP vaccine.vaccines, known as DTP vaccine.

A child should have received four DTP shotsA child should have received four DTP shotsby 18 months of age, with a booster shot atby 18 months of age, with a booster shot atage 4 years to 6 years.age 4 years to 6 years.

After that, diphtheria and tetanus boostersAfter that, diphtheria and tetanus boostersshould be given every 10 years to provideshould be given every 10 years to providecontinued protection.continued protection.


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How is diphtheriaHow is diphtheria

transmitted?transmitted? The diphtheria bacterium can enter the The diphtheria bacterium can enter the

body through the nose and mouth.body through the nose and mouth.

It can also enter through a break in theIt can also enter through a break in the

skin.skin. It is transmitted from person to person byIt is transmitted from person to person by

respiratory secretions or droplets in therespiratory secretions or droplets in theair.air.

After being exposed to the bacterium, itAfter being exposed to the bacterium, itusually takes 2 to 4 days for symptoms tousually takes 2 to 4 days for symptoms todevelop.develop.


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ConclusionConclusion

Thus diphtheria, which is caused by Thus diphtheria, which is caused by

Corynebaterium diphtheriaeCorynebaterium diphtheriae can create severecan create severe

fatal problem if proper vaccination and care isfatal problem if proper vaccination and care is

not taken.not taken.

After 1980, less than 10 cases per year haveAfter 1980, less than 10 cases per year have

been reported in US. After 1995, no case there.been reported in US. After 1995, no case there.

We have more than 100 cases per year evenWe have more than 100 cases per year even

today in city of Rajkot.Poor immunizationtoday in city of Rajkot.Poor immunization

coverage, under nutrition, damp climate andcoverage, under nutrition, damp climate and

congested living are probable responsiblecongested living are probable responsible

factors.factors.


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ReferenceReference

www.dhpe.org/infect/dip.www.dhpe.org/infect/dip.

www.cdc.gov/nip/publications/pink/dip.www.cdc.gov/nip/publications/pink/dip.

www.mayoclinic.comwww.mayoclinic.com.. www.www.diphtheriadiphtheria.net ..net .

www.google.comwww.google.com
http://www.mayoclinic.com/http://www.mayoclinic.com/http://www.mayoclinic.com/


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by: sandip harsoda m.sc. microbiology

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