DIPHTHERIA

By: Sandip harsoda

M.Sc. Microbiology
 Content
► Introduction
► Pathogenesis
► What are the symptoms of diphtheria?
► Clinical features
► Complication
► Diphtheria Toxin
► The nature of immunity to Diphtheria
► Techniques to measure Antibody Response
► Treatment
► Prevention
► Transmission
► Reference
 Introduction
► Diphtheria is a very contagious and life
threatening bacterial disease.
► Diphtheria usually attacks the throat and
nose.
► In more serious cases, it can attack the
heart and nerves.
► Because of widespread immunization,
diphtheria is very rare in United States.
► ORGANISM:
   Genus : Corynebacterium
    Species: diphtheriae

► GENERAL CONCEPT:
Corynebacteria belong to the family
Mycobacteriaceae and are part of the CMN
group (Corynebacteria, Mycobacteria and
Nocardia).
► The family Mycobacteriaceae are Gram-positive,
nonmotile, catalase-positive and have a rodlike to
filamentous morphology (Corynebacteria are
pleomorphic).

► They produce characteristic long chain fatty acids

termed mycolic acids.
► C. diphtheriae is a facultatively anaerobic.
► Characterized by non-encapsulated, non-sporulated,
immobile, straight or curved rods with a length of 1 to
8 µm and width of 0.3 to 0.8 µm, which form ramified
aggregations in culture (looking like "Chinese
characters").
 CONTINUE
► Scientific classification
Kingdom: Bacteria
Phylum: Actinobacteria
Order: Actinomycetales
Family: Corynebacteriaceae
Genus: Corynebacterium
Species: diphtheriae
► Corynebacterium diphtheriae is a
pathogenic bacterium that causes
diphtheria.
► It is also known as the Klebs-Löffler bacillus,
because it was discovered in 1884 by
German bacteriologists Edwin Klebs and
Friedrich Löffler.
► Many strains of C. diphtheriae produce a protein exotoxin
with a molecular weight of 62 kilodaltons,which is
responsible for the signs of diphtheria.
► Three subspecies are recognized
C. diphtheriae mitis,
C. diphtheriae intermedius
C. diphtheriae gravis.
► They differ slightly in their ability to metabolize certain
nutrients, but all may be toxigenic or non-toxigenic.
► Toxin production (toxigenicity) occurs only
when the bacillus is itself infected
(lysogenized) by a specific virus
(bacteriophage)carrying the genetic
information for the toxin (tox gene).
► Only toxigenic strains can cause severe
disease.
► DISTINCTIVE PROPERTIES:
   Corynebacterial cell walls contain thin
spots which leads to some Gram variability
and "ballooning" that produces a "club-
shaped" cell.
Old cells store inorganic phosphate, which
can appear as metachromatic granules
when stained.
 PATHOGENESIS:
► C. diphtheriae is the etiologic agent of diphtheria.
   
► These organisms colonize the mucus membranes
of the respiratory tract and produce the enzyme
neuraminidase which splits N-acetylneuraminic
acid (NAN) from cell surfaces to produce pyruvate
which acts as a growth stimulant.
  
► C.diphtheriae also produces diphthin, which is a
protease that inactivates IgA.
 What are the symptoms of
diphtheria?
► Thefollowing are the most common
symptoms of diphtheria.
 breathing difficulty
 husky voice
 enlarged lymph glands
 increased heart rate
 stridor (a shrill breathing sound heard on
inspiration)
  nasal drainage
 swelling of the palate (roof of the mouth)
 sore throat
 low-grade fever
 malaise
• Skin (cutaneous) diphtheria:
With this type of diphtheria, the symptoms are
usually milder and may include yellow spots or
sores (similar to impetigo) on the skin.
 Clinical Features
► The incubation period of diphtheria is 2–5
days(range, 1–10 days).
► Disease can involve almost any mucous
membrane. For clinical purposes, it is
convenient to classify diphtheria into a
number of manifestations, depending on the
site of disease.
 Anterior Nasal Diphtheria
► The onset of anterior nasal diphtheria is
indistinguishable from that of the common cold
and is usually characterized by a mucopurulent
nasal discharge (containing both mucus and pus)
which may become blood-tinged.
► A white membrane usually forms on the nasal
septum.
► The disease is usually fairly mild because of
apparent poor systemic absorption of toxin in this
location, and it can be terminated rapidly by
antitoxin and antibiotic therapy.
 Pharyngeal and Tonsilar Diphtheria

► The most common sites of diphtheria infection

are the pharynx and the tonsils.
► Infection at these sites is usually associated with
substantial systemic absorption of toxin.
► The onset of pharyngitis is insidious.
► Early symptoms include malaise, sore throat,
anorexia, and low-grade fever.Within 2–3 days, a
bluish-white membrane forms and extends,
varying in size from covering a small patch on the
tonsils to covering most of the soft palate.
► The membrane is greyish-green, or black.
► There is a minimal amount of mucosal
erythema surrounding the membrane.
► Extensive membrane formation may result
in respiratory obstruction.
► If enough toxin is absorbed, develop
severe prostration, striking pallor, rapid
pulse, stupor, and coma, and may even
die within 6 to 10 days.
 Laryngeal Diphtheria
► Laryngeal diphtheria can be either an
extension of the pharyngeal form or can
only involve this site.
► Symptoms include fever, hoarseness, and
a barking cough.
► The membrane can lead to airway
obstruction, coma, and death.
 Cutaneous (Skin) Diphtheria
► In the United States, cutaneous diphtheria
has been most often associated with
homeless persons.
► Skin infections may be manifested by a
scaling rash or by ulcers with clearly
demarcated edges and membrane, but
any chronic skin lesion may harbor C.
diphtheriae along with other organisms.
 Complications
► Most complications of diphtheria,
including death, are attributable to effects
of the toxin
► The toxin, when absorbed, affects organs
and tissues distant from the site of
invasion.
► The most frequent complications of
diphtheria
1: Myocarditis
2: Neuritis
Myocarditis may present as abnormal cardiac
rhythms and can occur early in the course of the
illness or weeks later, and can lead to heart
failure.
Neuritis most often affects motor nerves and
usually resolves completely.
► Paralysis of the soft palate is most frequent
during the third week of illness.
► Paralysis of eye muscles, limbs, and diaphragm
can occur after the fifth week.
► Secondary pneumonia and respiratory failure may
result from diaphragmatic paralysis.
 Diphtheria Toxin
► Extracellularsubstance (exotoxin) produced
by Coryebacterium diphtheriae.
► Diphtheria toxin, a protein with a molecular
weight of 62 000.
► Known as an A-B type toxin, and consists of
two fragments designated A and B.
► Fragment B is necessary for binding to
surface receptors and penetration into cells.
► Fragment A is responsible for its toxicity
and exerts its action by interfering
enzymatically with protein synthesis,
finally producing the death of the cells.
► Diphtheria toxin exerts its effects on
distant tissues and organs, especially the
heart (myocarditis) and the peripheral and
cranial nerves (weakness progressing to
paralysis).
► When treated with formaldehyde and heat,
diphtheria toxin loses its ability to bind to
cells and its enzymatic activity, but retains
its immunogenicity.
► This treatment converts diphtheria toxin to a
toxoid, which is commonly used to
immunize against diphtheria.
Mechanism of Diphtheria
Toxin
 The Nature of Immunity to
Diphtheria
► Immunity against diphtheria is antibody-
mediated.
► Antibody, called antitoxin, is primarily of the
IgG type.
► Antitoxin is distributed throughout the body
and can pass easily through the placenta,
providing passive immunity to the newborn
during the first few months of life.
 Techniques to Measure
Antibody Response
► Two important properties of diphtheria
toxin are utilized to determine the activity
of diphtheria antibodies.
1. The ability to produce an inflammatory
reaction.
2. Second property is the capacity of
diphtheria toxin to block protein synthesis.
 Schick test
► To perform the Schick test, 0.1 ml of
diphtheria toxin (about l/50 of the minimal
lethal dose for a guinea pig) is injected
intradermally on the volar surface of the
forearm of the person being tested.
► If the person has circulating diphtheria
antitoxin at a level of 0.01 to 0.03 IU/ml, the
injected toxin will be neutralized and no
reaction will occur.
►A positive reaction signifies lack of antitoxin
and is characterized by inflammation
appearing after 24 to 36 hours and
persisting for 4 days or longer.
► A control test is always performed on the
opposite arm using toxin inactivated by
heating to 60°C for 15 minutes.
► A positive reaction to inactivated toxin and a
positive reaction to toxin indicates an
allergic response to toxin.
 Neutralization test on
animals
► The in vivo neutralization test is usually
performed on the depilated skin of rabbits.
► Different dilutions of serum mixed with fixed
amounts of diphtheria toxin are injected into
the depilated skin of the animal and the
antitoxin concentration is estimated based
on the presence or absence of an
inflammatory reaction.
Advantage
The test show not only the
neutralization of toxin by antibody present
in the test serum, but also reaction between
other antigen-antibody systems.

Disadvantage
The test is laborious, time-consuming,
expensive, and requires suitable animals.
 Neutralization test on
microcell
culture
► It is based on the observation that the
survival of mammalian cells in culture is
inhibited by diphtheria toxin.
► The titration of the antitoxin in the serum
samples is done in plastic microtissue
culture plates, in which dilutions of test sera
are mixed with challenge toxin.
► After a short incubation, Vero (green
monkey renal epithelium) cell or HeLa cell
suspension in a special culture medium is
added.
► After incubation for 3 or 4 days, results are
read as a change in the color of the
reagents in the microtiter plate wells.
► The color change is due to the metabolic
formation of acid, which changes the pH.
► Vero cells are more sensitive to diphtheria
toxin.
► They have large numbers of binding sites
(receptors) and they take up the toxin in a
highly specific, time- and temperature-
dependent manner.
► When a serum dilution contains antitoxin in
excess, the cells continue to grow, and the
color of the medium changes from red to
yellow.
Passive hemagglutination
► The passive hemagglutination (HA) test is
frequently used to test for diphtheria
antibody.
► In the HA test, sheep, turkey, horse, or
human red cells (previously treated with
tannic acid or diazotized benzidine and
sensitized with diphtheria toxoid) are
agglutinated by diphtheria antibody.
 Treatment
► Treatment with erythromycin orally
or by injection (40 mg/kg/day;
maximum, 2 gm/day) for 14 days.
► Penicillin G daily, intramuscularly.
► The disease is usually not contagious
48 hours after antibiotics are
instituted.
 How can diphtheria be
prevented?
► There is a vaccine for diphtheria.
► The diphtheria vaccine is usually given in a
combination shot with tetanus and pertussis
vaccines, known as DTP vaccine.
► A child should have received four DTP shots
by 18 months of age, with a booster shot at
age 4 years to 6 years.
► After that, diphtheria and tetanus boosters
should be given every 10 years to provide
continued protection.
 How is diphtheria
transmitted?
► The diphtheria bacterium can enter the
body through the nose and mouth.
► It can also enter through a break in the
skin.
► It is transmitted from person to person by
respiratory secretions or droplets in the
air.
► After being exposed to the bacterium, it
usually takes 2 to 4 days for symptoms to
develop.
 Conclusion
► Thus diphtheria, which is caused by
Corynebaterium diphtheriae can create severe
fatal problem if proper vaccination and care is
not taken.
► After 1980, less than 10 cases per year have
been reported in US. After 1995, no case there.
We have more than 100 cases per year even
today in city of Rajkot.Poor immunization
coverage, under nutrition, damp climate and
congested living are probable responsible
factors.
 Reference
► www.dhpe.org/infect/dip.
► www.cdc.gov/nip/publications/pink/dip.
► www.mayoclinic.com.
► www.diphtheria.net .
► www.google.com