diphtheria and it's causative agent The pathogenesis and Pathogenicity Elek test and immunotoxin examination . its culture and growth morphology . and treatment and control . CORYNEBACTERIA diphtheriae .

1. Al-Balqa Applied University
Higher Studies College
Department of Medical Laboratory Sciences
Course of Advanced Diagnostic Microbiology
Instructor by : Dr. Nasser El-Banna
Student name : Majd Ghawanmeh
Diphtheria

2. Outline :
• Diphtheria
• The pathogen and its general characteristics.
• The source and transmission.
• Pathogenesis and Clinical findings.
• Diagnostic and laboratory tests.
• Treatment.
• Prevention and control.
• References.

3. Diphtheria :
it is a potentially endemic, communicable,
fatal contagious bacterial infection that
mainly affects the pharyngeal or cutaneous
Regions, this disease has a presentation of
local inflammation of the throat with a
pseudomembrane caused by dead cells
and exudate, and general symptoms that
occur as a result of absorption of the toxins

4.  The pathogen and its general characteristics
• Corynebacterium diphtheriae also known as Klebs-Löffler bacillus is a
Gram-positive bacillus responsible for causing diphtheria The genus and
species names are derived from the Greek word: korynee (“club”) after the
microscopic appearance of the organisms and diphtheria (“leather hide”)
for the pseudomembrane that is the hallmark of respiratory tract
infection. (as in slid 6) .
• Gram-positive, non-spore-forming, non-motile, non-capsulated .
• Aerobes or facultative anaerobes, Ferment glucose, starch glycogen.
• Club-shaped ,Chinese letters.
• Urease negative, oxidase negative, Catalase positive.

5. Source and transmission
The source of infection is carriers who harbor the organisms in the
oropharynx or skin. Human to human transmission is spread by respiratory
droplets (coughs and sneezes), secretions, or direct contact with infected
cutaneous lesions and to a lesser extant by fomites .

6.  Pathogenesis and Clinical findings.
 C.diphtheriae toxins has a local and systemic effects in the
case of diphtheria disease.
• Local infection :
Locally secreted toxin can damage or destroy mucosa cells at
the site of infection in the throat and upper respiratory tract
resulting in production of thick gray adherent pseudomembrane
over the tonsils and throat is the most prominent specific sign
(consist of bacteria and dead epithelial and white blood cells)
 Laryngeal involvement lead to obstruction of the airways
If not treated early, the extension of pseudomembrane may lead
to laryngeal obstruction, asphyxia, and death.

7. • Systematic intoxication :
The toxin also released into and disseminated via the
blood stream.
Certain body tissue cells are more vulnerable than other,
like cardiac muscle motor nerves and adrenal glands
That's lead to this complication:
myocarditis, peripheral neuritis and death usually
From heart related complications.

8. C. diphtheriae is not an invasive organism. The pathogenesis of
diphtheria is based on its potent exotoxin carried by lysogenized
strains of C. diphtheriae.
• Mode of action of Diphtheria toxin
Like other exotoxins (botulinum, tetanus, cholera toxin), diphtheria toxin also has
A and B subunits. The A or active subunit possesses the toxic activity, and the B
or binding subunit is responsible or binding the exotoxin to specific receptors.
The toxin binds to the cell surface via its binding subunit (B), and the active
subunit (A) enters the cell. The active subunit is an enzyme that catalyzes the
addition of ADP-ribose (ADP-R) to elongation factor-2 (EF-2). This inactivates
EF-2, and protein synthesis is inhibited, leading to the death of host cells

9. Mechanism of Diphtheria toxin (Image source: Ref-3)

10.  Laboratory diagnosis of C.diphtheriae
 Laboratory diagnosis of Corynebacterium diphtheriae involves isolation of the organism and
subsequent demonstration of toxin production.
 Sample: Swabs (preferably two) from the lesion of throat, larynx or nasal cavity; one for direct
examination and another for culture or a portion of the pseudomembrane.
 Direct examination
Smears of the throat swab should be
stained with both Gram stain and
methylene blue or Albert stain.
Appearance of many tapered, irregularly
stained, pleomorphic (typically arranged in
Chinese letter) gram-positive rods suggest.

11. Club shaped Corynebacterium diphtheriae in
Methylene Blue Staining
Source: PHIL Photo ID# 7323
Typical metachromatic granules are seen in
Albert stain. C.diphtheriae appears as green
bacilli with bluish-black metachromatic
granules in Albert stain.(source fig 4).
• Club-shaped due the presence of metachromatic granules at its ends.
• Cells are arranged singly, in “palisades” of parallel cells, or in pairs that remain
connected after cell division to form V, or L shapes (Chinese letters).

12.  Culture
C. diphtheriae is a fastidious organism, so it does not grow on the ordinary medium.
• Blood tellurite agar: a selective and differential medium for C. diphtheriae.
After 48-72 hours, colonies of C. diphtheriae appear as small, grey, or black with a
raised center.(inhibit flora)
• Tinsdale’s Agar: After incubation for at least 48 hours, colonies of
C. diphtheriae appear black with dark brown halos.
• Loeffler’s medium: containing serum and eggs, stimulates the growth
of C. diphtheriae and stimulates the production of metachromatic
granules within the cells. Primary plating in Loeffler’s medium is not
recommended due to the growth of commensals.

13. Tinsdale’s Agar
Blood
tellurite
agar
Image source: (in References 5 )

14. Demonstrating its toxicity
• Elek immunodiffusion test: It is the most common in vitro assay
for determining toxigenicity of C. diphtheriae. This test is based
on the double diffusion of diphtheria toxin and antitoxin in an
agar medium. A sterile, antitoxin-saturated filter paper strip is
embedded in the culture medium, and C diphtheriae isolates are
streak-inoculated at a 90° angle to the filter paper. The production
of diphtheria toxin can be detected within 18 to 48 hours by the
formation of a toxin-antitoxin precipitin band in the agar.
 Detection of toxin gene by polymerase chain
reaction (PCR).

16. Non toxic strain

17.  Treatment and immunization
Treatment is achieved by suppression of bacterial growth by administration
of penicillin and erythromycin antibiotics that eliminates C. diphtheriae and
terminate the production of toxin.
Prevention and control
• Active immunization is achieved by the administration of diphtheria
toxoid(formaldehyde inactivated diphtheria toxin).
In endemic areas vaccine is started at 3 months and 3 doses are given
at 6-8 weeks interval and a booster dose(DT) at 6 years. It is often
administered in combination with tetanus,and pertussis vaccine(DPT)
• Passive immunization is considered as an emergency measure when
susceptible person is exposed to infections and in such case 500-100units
• of antitoxin is administered subcutaneously after skin test (Schick test)

18. References
• Microbiology/practical by Leena Adnan .
• A concise review of clinical laboratory science / [edited by]
Joel Hubbard.—2nd ed.
• Jamal, S. B., Tiwari, S., Silva, A., Azevedo, V., Jamal, S. B., Tiwari, S.,
Silva, A., & Azevedo, V. (2017). Pathogenesis of Corynebacterium
diphtheriae and available vaccines: An Overview. Global Journal of
Infectious Diseases and Clinical Research, 3(1), 020–024.
• Todar's Online Textbook of Bacteriology / Diphtheria/
page 1/Figure 1.
• https://microbeonline.com/corynebacterium-diphtheriae-
properties-pathogenesis-diagnosis/#General_properties