This document provides information on Corynebacterium diphtheriae, the bacteria that causes diphtheria. It discusses the etiology, pathogenesis, symptoms, epidemiology, and laboratory diagnosis of diphtheria. Key points include: - C. diphtheriae bacteria produce a toxin that is absorbed systemically and can damage organs. Only toxigenic strains can cause severe disease. - Diphtheria usually causes an infection in the throat that produces a gray membrane. Symptoms include sore throat and fever. It is communicable for weeks without antibiotics. - The disease is most common in children under 5. It spreads through respiratory droplets or direct contact. Laboratory diagnosis involves

1. Etiology,
General characters, Pathogenesis,
symptoms, Epidemiology,
Laboratory diagnosis
of
Corynebacterium diphtheriae

2.  The infection usually occurs in the spring or winter
months. It is communicable for 2-6 weeks without
antibiotic treatment.
 Only bacteria infected by a bacteriophage carrying the
TOX gene are capable of producing the toxin, and only
toxigenic strains can cause severe disease. No toxigenic
strains may cause mild clinical disease.
 Infection my be acquired nosocomial or by unhygienic
practices

3.  Corynebacteria / “Coryneform bacteria” –
agroup of
 Non-spore forming,
 Gram- positive bacilli
 Clubbed or irregularly shaped; (coryne =
club)
 Corynebacterium diphtheriae the causative
agent of Diphtheria is the major pathogen
in this group.
General characters

4.  Other pathogenic corynebacteriaare:
 C.Ulcerans: Diphtheria likelessions.
 Corynebacteria Causing Superficial skin
infections:
 C.minutissimumand C.tenuis.
 Diphtheriods: Normal commensals in
throat,skin and conjunctiva.

5. HISTOR
Y
• Hippocrates provided the first clinical
description of diphtheria in the 4th century B.C.
• Bretonneu (1821), a French army surgeon,
described the unique clinical characteristics of
the disease, and used the term ‘dipht`erie’ to
signify the tough leathery pseudomembrane that
occurs in oropharynx and some times in
nasopharynx;
(diphtheros = leather)

6. HISTORY………..contnd
• The bacterium that caused diphtheria was first
described by Klebs in 1883, and was cultivated by
Loeffler in 1884, who applied Koch's postulates and
properly identified Corynebacterium diphtheriae as
the agent of the disease.
• In 1884, Loeffler concluded that C. diphtheriae
produced a soluble toxin, and thereby provided the
first description of a bacterial exotoxin.
• Roux and Yersin (1888) discovered the diphtheria
exotoxin and established its pathogenic effects.
• The antitoxin was described by von Behring(1890).

7.  After infection, the bacilli multiply on
the mucous membrane or skin
abrasion;
 Thetoxigenic strains start producing toxin;
• Diphtheria is a‘toxemia’;
 Thebacteria confine to the site of entry
but the exotoxin is absorbed into the
mucus membrane and causesdestruction
of epithelium and asuperficial
inflammatory response;
Pathogenesis

8.  Thetoxin causeslocal necrotic changes;
 The resulting fibrinous exudate, together
with the epithelial cells, leucocytes,
erythrocytes and bacteria constitute :
“pseudomembrane”
 Any effort to remove it will tear off
capillaries beneath it and causebleeding;
 Mechanical complications are due to
pseudomembrane and systemic effects are
due to thetoxin;

9.  Commonestsite of infection: upper
respiratory tract (fauces,larynx,nose)
 Ocassionally,other cutaneous or
mucocutaneous areas
(otitic/conjunctival/ genito
vulval/vaginal/ prepucial/skin)
 Faucialdiphtheria is the commonesttype;
 Sorethroat is frequently the
presenting symptom;

10. VIRULENCE
FACTORS
• Virulent strains of diphtheria bacilli producea
very powerfulexotoxin.
• The‘virulence’of diphtheria bacilli isdueto
their capacityto-
– Establishinfection and growingrapidly
– Quickly elaborate anexotoxin
• Avirulent strains are common among
convalescents, contacts and carriers, particularly
those with extra-faucialinfection

11. DIPHTHERIA
TOXIN
• Thepathognomonic effects are due to the
toxin;
• Almost all the gravisand intermediusstrains and
80-85%of mitis strains aretoxigenic
• Toxinisaprotein;
• Mol. Wt.:62,000
• Twofragments,Aand B;
• Extremely potent :
– 0.1 μglethal to guineapig
• Inactive when releasedA

12. Toxin– mechanism of action
• Fragment B : binds to a cell surface receptor
and helps in transport of toxin into the cell;
• After entering the cell, A subunit is released ;
• A subunit catalyses the transfer of ‘adenosine
diphosphate ribose (ADPR)’ from NAD+
• ADPRbinds with the elongation factor EF 2
• “ADPR-EF2” complex is inactive
• protein synthesis stops abruptly
• necrotising and neurotoxic effects of the
toxin;

14. Symptoms
 Other common symptoms include:
 fever
 chills
 swollen glands in the neck
 a loud, barking cough
 a sore throat
 bluish skin
 drooling
 a general feeling of uneasiness or discomfort
 Additional symptoms may occur as the infection progresses,
including:
 difficulty breathing or swallowing
 changes in vision
 slurred speech
 signs of shock, such as pale and cold skin, sweating, and a rapid
heartbeat

15. Pathophysiology
 Thebacilli continue to produce thetoxin;
 The toxin is absorbed systemically and damages
heart muscle, liver, adrenalsetc.;
 The toxin also cause nerve damage, especially of
soft palate(palatine) and eyemuscles(ciliary);
 Toxin absorption is negligible in case of
skin infection with toxigenicstrains;
 Nontoxigenic strains canalso produce local
diseasebut systemiceffects areabsent;

16. CLINICAL
DISEASES
• Incubation period : usually 3-4days;
• Acute infection : in the form of –
– Membranous tonsillitis
– Nasal infection
– Laryngeal infection
– Skin infection–uncommon;

17. CLINICALDISEASES
• Characteristic feature is:
‘wash –leather’ eleveted
greyish greeen
membrane in the tonsils
with awell defined edge
surrounded by azone of
inflammation;

18. ‘wash –leather’ eleveted greyish greeen membrane in the tonsils
Pseudomembrane

19. CLASSIFICATION BASED ON CLINICAL
SEVERITY
• Malignant or hypertoxic:
– ‘Bull neck’due to marked adenitis inneck;
– Severetoxemia
– Circulatory failure
– Death
– Paralyticsquealaein survivors
• Septic: ulceration, cellulitis andgangrene
around pseudomembrane;
• Hemorrhagic: bleeding from the edgeof
pseudomembrane,epistaxis, purpura etc.

20. Bull neck : due to cervical adenitis and
edema of neck

21. COMPLICATIONS
• Asphyxia : due to mechanicalobstruction
Emergencytracheostomy may be necessary;
• Acute circulatory failure
• Myocarditis
• Postdiphtheritic paralysis-
palatine(soft palate) and ciliary ( eyemuscles)
nerves
Recovery– spontaneous and complete
• Septic : pneumonia and otitis media
• Relapse: in about 1%of cases

22. Epidemiology

23. Contd,,
 Mainly a disease of childhood(pediatrics) in
endemic areas – uncommon below 1st year;
peaks at 5.
 In nature, C.diphtheriae occurs in the
respiratory tract, in the wounds or in the
skin of the infected persons or carriers;
 Transmission is by-
– Droplet dissemination from cases or carriers
– Direct contact
– Occasionally, fomites;
 Nasopharyngeal or cutaneous carriage of
toxigenic or nontoxigenic strains can persist for
life in healthy people;

24. Distribution

25. LABORATORY DIAGNOSIS
• Specimens :
– Swabsfrom – nose, throat or other suspected
lesions;
• Smear examination: Gram stain
– shows beaded rods in typical arrangement;
– Difficult to differentiate from somecommensal
corynebacteria normally found in throat;
– Albert’s stain or Neisser’s stain is usefulfor
demonstrating the granules;

26. Numbers of large-sized Gram-positive rods are
embedded within the pseudomembrane(Gram).
LABORATORYDIAGNOSIS

27. LABORATORYDIAGNOSIS :
CULTURE
• If the swabscannot be inoculated promptly,they
should be kept moistened with serum;
• Inoculate on :
– Loeffler’s serum slope
– Telluriteblood agar or Tinsdalemedium
– Blood agar ( for differentiating Staphylococcalor
Streptococcal pharyngitis that simulate
diphtheria);
• Telluritemedium is particulary useful for isolatingthe
organism from – convalescents, contacts or carriers;

28. MORPHOLOGY
• Special stains for
demonstrating the
granules :
– Albert’s stain
– Neisser’s stain
– Ponder’s stain
• Thebacilli are arranged in
pairs, palisades or small
groups; the bacilli lie at
various angles to eachother,
resembling the letters, VorL;
• Thisis called, “Chineseletter
pattern” or “cuneiform
pattern”;

29. CULTURAL
CHARACTERISTICS
• Aerobe and facultative anaerobe;
• Optimum temperature is370C
• Growth scanty on ordinary media;
• Enrichment with: blood, serum or eggis
necessary for goodgrowth;
• Potassiumtellurite(0.04%) acts asa‘selective
agent’, asit inhibits growth of most oral
commensalsand retards the growth ofCandida
albicans and S.aureus;

30. MEDIA FOR
CULTIVATION
• Blood agar
• Loeffler’s serumslope
• Tellurite blood agar
• Hoyle’s tellurite lysed-blood agar
• Tinsdale’s medium (cystine added to tellurite
containing agar)

31. COLONY
CHARACTERISTICS
• Blood agar : small,
granular and graywith
irregular edges;
Hemolysis mayor may
not present;
• Loeffler’s serum slope:
– Very rapid growth;
– Colonies in 6-8hrs
– Initially circular white
opaque colonies and
acquire yellowish tint on
incubation

32. COLONY
CHARACTERISTICS
• Tellurite bloodagar:
– Growth slow; colonies seenafter 48hrs;
– Thecolonies are brown to blackwith abrown-
blackhalo becausethe tellurite isreduced to
metallic tellurium;
– Staphylococcusalsoproduce such colonies
A diagrammatic representation

33. COLONY
CHARACTERISTICS
• Tinsdale’s medium (also
contain cystine in
addition to tellurite):
– Grey black colonies with
dark brown haloes
indicate C.diphtheriae
and C.ulcerans (these
contain cystinase)

34. Feature gravis intermedius mitis
Morphology
shot rods,
few granules
some degree of
pleomorphism
long barred forms
poor granulation
Pleomorphism
long curved
prominent granules
Pleomorphism
Colony on
tellurite blood
agar (48 hrs)
Daisy head colony
(flat colony with raised
dark centre and crenated
edge; radial striations)
Frog's egg colony
(dull granular centre
with glistening
periphery and
lighter ring near edge)
Poached egg colony
(shiny , flat with central
elevation)
Consistency of
the colonies
Brittle
not easily emulsifiable intermediate
soft, buttery
easily emulsifiable
Hemolysis Variable nonhemolytic hemolytic
Glycogen/
starch
fermentation
Positive Negative Negative

35. LABORATORYDIAGNOSIS :
CULTURE
• Processing:
– Serumslope mayshowgrowth in 4-8 hrs but if
negativemayneed to be incubated for 24hrs;
– Smearmayshow ‘diphtheria-like’ organisms;
– Byabout 48hrs,Tellurite plates will yieldgrowth;
– Theisolate must be submitted for –‘Virulencetests’ or
‘Toxigenicity tests’before the bacteriological diagnosisis
complete;

36. VIRULENCE
TESTS
• In vivo methods:
– Subcutaneous test
– Intracutaneous test
• In vitro methods:
– Elek’s gel precipitation test
– Tissueculture test

37. SUBCUTANEOUSTEST
Emulsify the growth form an overnight culture
of Loeffler’s serum slope in 2-4 ml broth
0.8 ml injected subcutaneously
Into two guinea pigs
Protected with
500 IU of antitoxin
18-24 hrs previously
Unprotected
Die in 4 daysif the strainis
Virulent; autopsy shows
Characteristic features
Remain healthy
Disadvantage : Death of theanimal
Control animal
Testanimal

38. INTRACUTANEOUS
TEST
0.1 ml of emulsion broth inoculated
intracutaneously in to two guinea pigs
Control animal Testanimal
Should receive 500 IU
Of antitoxin previous day
Give50 IU of antitoxin
Intraperitoneally
4 hrs after skintest
(To prevent death)
Inflammatory reaction
Progressto necrosis in 48-72hrs
NOCHANGE

39. INTRACUTANEOUS
TEST
• Animal does not die;
• Rabbits may also be used;
• Asmany as10 strains canbe tested
simultaneously;

40. Elek’sgel precipitationtest
• In vitro test;
• Arectangular strip of filter paper issaturated
with the diphtheria antitoxin(1000units/ml);
• Thisstrip isplacedon : agar platewith 20%
horse serum, while the medium issetting;
• Thecultures to be tested are streaked atright
anglesto the filter paperstrip;
• Apositive and negative control should beput;

41. Incubate the plate for 24-48 hrs at 370C
TP N

42. After incubation – line of precipitation can be observed
Where the toxin and antitoxin meet at optimum conc.
The lines of precipitation will indicate that
the test strain is toxigenic
P T N

43. Tissueculture test
• Bacteria incorporated into an agar overlayof
cell culture monolayers;
• Thetoxin, if produced, diffuses into thecells
below and kills them;

44. Medications
 Specific measure : prompt administration of
antitoxin to neutralize the circulatingtoxin;
 – Dose:20,000-1,00,000 units
– Half the dosegivenIV
– Antitoxin treatment is generally not indicated
for cutaneous diphtheria
 Antibiotics : Penicillin or Erythromycin for 14days;
 Complete bed rest;
 Supportive therapy and treatment of complications
 Erythromycin: for treatment ofcarriers.

45. Active immunisation -toxoids
• Adsorbed toxoid –givenasIM injections
• Recommendedvaccines:
– Asatrivalent preperation : DPT(adsorbed
Diphtheria/Pertusis/Tetanus)
– Adsorbed Diphtheria/Tetanus (DT)
– Adsorbed low dosediphtheria vaccinefor adults(d)
– AdsorbedTetanus/low dosediphtheria vaccine(Td)
for adults;
– Aquadraple vaccinecontaining DPT+inactivated
polio vaccineisalsoavailable;

46. Active immunisation- schedules
• Primary immunisation:
– 3 dosesof DPTbegening at 4th week of age,8th
and 12th week under Routine Immunization
schedule(Govt. of Tanzania)
• Booster (DPT)at 15-18 months of age;
– Further booster, as‘DT’ at – 5 yearsof age;
• Dosage: 0.5 ml
– 10-25 Lf units of toxoid -
recommendedfor children