diphtheria

 Coryneform bacteria :

Gram-positive,  non-acid fast, non-motile

Irregular stained with granules

Club shaped swelling at one or both ends so the name

Important pathogen

Diptheros meaning leather

Corynebacterium diphtheriae :

Caused diphtheria

slender rods

clubbing at both ends

pleomorphic

non-capsulate/ acid-fast, gram-positive

granules are composed of polymetaphosphate

staining with Loeffler’s methylene blue show bluish-purple metachromatic granules with polar bodies

CLASSIFICATION :

Corynebacterium diphtheria and diphtheroid (look like C.diphtheria ) are gram-positive, club-shaped rods.

some are saprophytic

some produce disease in animals

C. diphtheria is the most important pathogen in the group

Morphology and cultural characteristics :

small gram-positive Bacilli; arrangement = palisade or Chinese letters

growth on blood agar – raised, translucent, grey colonies

Classification McLeod :

Classified in 3 types

gravis : produce most serious hemorrhagic; paralytic complications- epidemic

intermedius : hemorrhagic

mitis: obstructive complications, endemic geographic locations differ, testing for toxigenic is more important

 

Corynebacterium :

3 morphological types of C.diphtheria are found on tellurite containing media :

Mitis: black colonies with a grey periphery

Gravis: large, grey colonies

Intermedius: small, dull grey to black

all produce an immunologically identical toxin

CULTURAL CHARCTERISTICS :

Incubation 35-37⁰C for 24 hours

they prefer a pH of 7.8 – 8.0 for good growth

they require access to oxygen

Catalase positive

RESISTANCE :

can be killed at 58⁰C in 10 minutes

100⁰C in 1 minute

survive in blankets, floor dust, toys inanimate objects

What is Diphtheria …?

An infection of local tissue of upper respiratory tract with the production of toxin which causes systemic effects on the heart and peripheral tissues.

Pharyngeal diphtheria :

Insidious onset of exudative pharyngitis

Exudates spread to form adherent “pseudomembrane”

can lead to respiratory obstruction and death by asphyxiation

Fever not high, but the patient appears toxic

Staining methods :

Gram’s method

Albert’s stain

Neisser’s stain

Pander’s stain

TOXIN :

Pathogenicity associated with toxin

Gravis/ intermedius 95-99% are toxigenic

Miltis 80-85%

Some abundant others poorly

Toxin production park William 8

Toxin: M.W. 62,000

Toxin contains 2 components: A & B

A produce toxigenicity by the proteolytic effect

B produces binding 

Toxin + Formalin = Toxoid (antigenic, not toxigenic)

Toxin inhibits protein synthesis

Fragment A catalyzes the transfer of ADP ribose from the Nicotinamide adenine dinucleotide to the eukaryotic elongation factor 2 

Causes involvement with affinity.

Myocarditis

Adrenals

Nerve endings 

CLINICAL FEATURES :

Malaise, sore throat, fever

Adherent grey pseudo membrane

Nasal ulcers

Obstruction of larynx and lower airways

Difficulty in swallowing

Lead to Myocarditis, Peripheral neuritis

Paralysis of limbs

Diphtheria- Pseudo-membrane : this may obstruct the airway and result in suffocation.

Corynebacterium :

To prove that an isolate can cause diphtheria, one must demonstrate toxin production.

This is most often done on an Elek plate:

The organism is streaked on a plate containing low iron.

A filter strip containing anti-toxin antibody is placed perpendicular to the streak of the organism

Diffusion of the antibody into the medium and secretion of the toxin into the medium occur.

At the zone of equivalence, a precipitate will form.

Animal testing (obsolete)

PATHOGENICITY :

Bacteria invade, colonise, proliferate

Bacteria are lysogenised by beta phage

Produce toxin

Kills epithelial and neutrophils

Produce pharyngitis and cutaneous lesions

Incubation 3 – 4 days/ one day

Faucal/ Nasal/ Laryngeal/ Otic/ Conjuctival/ Genital/ Vulvae and cutaneous manifestations

Diphtheria is a toxemic condition

Malignant sever toxaemia, Adenitis Bull. Neck circulatory failure, septic gangrene, pseudo membrane, haemorrhagic Epistaxis, purpura, general bleeding tendency, Asphyxia, acute circulatory failure, paralysis pneumonia, septic shock, otitis media, toxaemia, necrotic changes, death in guinea pigs.

C. diphtheria :

C. diphtheria occurs in the respiratory tract, in wounds, or on the skin of infected preson or normal carriers.

It is spread by droplets or by direct contact

Portal of entry : respiratory tract or skin abrasions.

Diphtheria bacilli colonize and grow on mucous membranes and start to produce toxin, which is then absorbed into the mucous membranes, and even spread by the bloodstream.

TOXICITY AND DIPHTHERIA TOXIN :

Local toxigenic effects : elicit inflammatory responses and necrosis of the faucial mucosa cells formation of pseudo membrane (composed of bacteria, lymphocytes, plasma cells, fibrin, and dead cells), causing respiratory obstruction.

Systemic toxigenic effects: necrosis in heart muscle, liver, kidneys and adrenals. Also produces neural damage.

COMPLICATIONS :

Asphyxia – causing mechanical obstruction

May need tracheotomy

Circulatory failure

Post diphtheria paralysis

NON TOXIGENIC CLINICAL MANIFESTATIONS :

Bacteria can produce :

Endocarditis

Meingitis

Cerebral abscess

Osteoarthritis

DIAGNOSIS OF DIPHTHERIA :

A) LABORATORY DIAGNOSIS :

Specific treatment is more important than Laboratory diagnosis : Isolation of diphtheria bacilli; testing for toxigenicity

B) COLLECTION OF SPECIMENS :

Throat swabs; smear examinations ( Gram’s staining; albert’s & Ponder’s staining); immuno-fluroscent methods; culture on Loeffer’s serum slope & blood agar.

VIRULENCE TESTS :

In Vivo and In Vitro

In vivo in animals

Subcutaneous tests (inject broth from culture into two Guinea pigs, 0.8 ml)

One animal given 500 units of antitoxin before 

Other no antitoxin

Animal not given antitoxin will die

In Vitro : Elek’s gel precipitation testing 

Filter paper impregnated with diphtheria antitoxin 1000 units/ml 

Tested on the horse serum agar

Positive/ negative. Test strains tested for immunodiffusion

Line of precipitation – test positive; other methods testing in tissue cultures.

Schick test : injection of toxin intradermal route

Produces redness/ erythematic in 2-4 days

No reaction – protective immunity present

EPIDEMIOLOGY :

Eradicated in developed nations

Children between 2-5 years

A systematic carriers

Person to person contact

Carriers spread

Prolonged contact

PROPHYLAXIS :

Immunization

Active – Passive

Both passive and Active

Herd Immunity

Schick test

Immunization with Antitoxin