DIPHTHERIA

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Introduction
 Diphtheria - highly infectious childhood disease caused by -
Corynebacterium diphtheria
 Primarily infects - throat and produces toxin (diphtheria toxin) -
causes an exudative pharyngitis and membranous tonsillitis

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Introduction (Cont..)
 Genus Corynebacterium - gram-
positive, noncapsulated, non-
sporing, non-motile bacillus.
A
 Irregularly stained - shows club-
Club-shaped bacilli in methylene
shaped swellings. blue-stained smear

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Introduction (Cont..)
 1. Chinese letter or cuneiform
arrangement: Appear as V- or L-shaped in
smear - bacterial cells divide and daughter
B
cells tend to lie at acute angles to each
Gram-stained smear shows
other - snapping type of division V- or L-shaped bacilli with
cuneiform arrangement
 2. Metachromatic granules: Present at
ends or poles of the bacilli 4
Metachromatic Granules
 Also called polar bodies or Babes–Ernst bodies or volutin granules

 Storage granules of the organism, composed of

polymetaphosphates
 Granules - stained strongly gram-positive compared to remaining
part of the bacilli
 Granules take up bluish purple metachromatic color - stained with
Loeffler’s methylene blue
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Metachromatic Granules (Cont..)
 Better stained with special stains - Albert’s,
Neisser’s and Ponder’s stain
 Granules - well developed on enriched media -
blood agar or Loeffler’s serum slope
C

 Volutin granules - also possessed by -

Corynebacterium xerosis and Gardnerella
vaginalis 6
History
 Ancient disease, known since the time of Hippocrates

 First recognized by Pierre Bretonneau (1826) - (Greek word

diphtheros— meaning leather like) - leathery pseudomembrane
formation over the tonsil
 First observed by Klebs (1883)

 First cultivated by Loeffler (1884) - Klebs-Loeffler bacillus

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Virulence Factors (Diphtheria Toxin)
 Toxin -polypeptide chain, comprises of two fragments—A (active)
and B (binding)
 Fragment B binds to the host cell receptors - helps in entry of
fragment A

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Mechanism of Diphtheria Toxin (DT )
 Fragment A -active fragment - causes ADP ribosylation of
elongation factor 2 (EF-2) → leads to inhibition of EF-2 → leads to
inhibition of translation step of protein synthesis

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Factors Regulating Toxin Production
 Phage coded: β-corynephage - carrying tox gene

 Iron concentration: 0.1 mg per liter

 Other species: Also produced by C. ulcerans and C.

pseudotuberculosis

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Toxoid is used for Vaccination
 Antigenic and antitoxins are protective in nature. However, as

 It is virulent - cannot be given directly for vaccination

 Toxin - converted to toxoid - used for vaccination

 Toxoid - form of toxin – virulence is lost, retaining its antigenicity

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Toxoid is used for Vaccination (Cont..)
 Toxoid formation - promoted by formalin, acidic pH and prolonged
storage
 Park William 8 strain - preparation of vaccine

 LF unit - Limit of flocculation (Lf) unit.

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Pathogenicity and Clinical
Manifestations
 Diphtheria is toxemia but never a bacteremia

 Bacilli are noninvasive - secrete the toxin - spreads via

bloodstream to various organs
 Toxin responsible for all types of manifestations - local
(respiratory) and systemic complications

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Respiratory Diphtheria
 Most common form of diphtheria

 Tonsil and pharynx (faucial diphtheria) - most common sites

followed by nose and larynx
 Incubation period - 3–4 days

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Respiratory Diphtheria (Cont..)
 Faucial diphtheria: Diphtheria toxin
elicits an inflammatory response -
leads to necrosis of the epithelium
and exudate formation and mucosal
ulcers - lined by tough leathery Pseudomembrane covering the tonsils
classically seen in diphtheria
greyish white pseudomembrane
coat 15
Respiratory Diphtheria (Cont..)
 Extension of pseudomembrane -
into the larynx and bronchial
airways - asphyxia
 Bull-neck appearance - Tonsillar
B

swelling and neck edema - foul Bull neck appearance

breath, thick speech, and stridor
(noisy breathing) 16
Cutaneous Diphtheria
 Punched-out ulcerative lesions with necrosis, due to the organism
itself and is not toxin-mediated
 Also - caused by nontoxigenic strains

 Increasing incidence, especially in vaccinated children

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Systemic Complications
 Neurologic manifestations -Toxin mediated non-inflammatory
demyelinating disorder presented with:
 Cranial nerve involvement & Peripheral neuropathy
 Ciliary paralysis

 Myocarditis

 Typically associated with arrhythmias and dilated

cardiomyopathy 18
Laboratory diagnosis of Diphtheria
 Specimen: Throat swab and a portion of pseudomembrane

 Direct smear

 Gram stain: Club shaped gram-positive bacilli with Chinese

letter arrangement

 Albert’s stain: Green bacilli with bluish black metachromatic

granules
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Laboratory diagnosis of Diphtheria
(Cont..)
 Culture media
 Enriched medium: Blood agar,
chocolate agar and Loeffler’s
serum slope
 Selective medium: Potassium A B

tellurite agar and Tinsdale A. Loeffler’s serum slope;

medium, produces black B. Potassium tellurite agar shows black
colonies colonies.

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Laboratory diagnosis of Diphtheria
(Cont..)
 Identification

 Biochemical tests such as sugar fermentation tests using Hiss’s

serum sugar media

 Automated identification systems such as MALDI-TOF or VITEK

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Laboratory diagnosis of Diphtheria
(Cont..)
Diphtheria toxin demonstration
 In vivo tests (Guinea pig inoculation): Subcutaneous and
intracutaneous tests
 In vitro tests:
 Elek’s gel precipitation test
 Detection of tox gene-by PCR
 Detection of toxin-by ELISA or ICT
 Cytotoxicity on cell lines. 22
Elek’s gel precipitation test

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Elek’s gel precipitation test (Cont..)
 Isolates 1 to 4 are toxigenic strains
 Isolates 1 and 2: Precipitation bands crossed over – toxins are not-
identical - strains are unrelated
 Isolate 2 and 3: Partial fusion of precipitation bands - strains are
partially related to each other
 Isolates 3 and 4: Precipitation bands fused with each other -
strains are completely related
 Isolate 5 : non-toxigenic strain (no precipitation band is formed).

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Typing of C. diphtheriae
 Useful for epidemiological studies, to know the relatedness
between the isolates
 Biotyping - use in the past - four biotypes —gravis, intermedius,
mitis and belfanti
 Vary in virulence and toxin production –gravis 100% toxigenic and
more virulent
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Epidemiology
 Incidence decreasing – widespread vaccination coverage

 Source of infection - Carriers (95%) & cases (5%)

 Carriers (0.1% to 5%)

 Temporary (persist for a month) or chronic (persist for a year)

 Nasal carriers (more dangerous - frequent shedding) & throat

carriers
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Epidemiology (Cont..)
 Transmission - respiratory droplets or rarely by contact with
infected skin
 Reservoir - Humans are the only reservoir
 Age - Common age affected - 1–5 years
 Shift from preschool to school age with immunization
 Newborns - maternal antibodies protective
 Global situation - Due to wide spread immunization, cases were
drastically declined by >95% over last 3 decades
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Epidemiology (Cont..)
 Resurgence of diphtheria

 In 2019, outbreaks – Tamil Nadu, Kerala and Karnataka and few

other states
 Majority (>70%) of cases - children 5–10 years or more - low
coverage of diphtheria vaccine especially the booster doses -
primary cause of its resurgence
 Waning immunity in adults - minor cause contributes to adult
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diphtheria
Treatment of Diphtheria
 Treatment - started immediately on clinical suspicion of diphtheria

 Antidiphtheritic serum or ADS (antitoxin): Passive immunization -

antidiphtheritic horse serum - neutralizes the toxin

 A test dose should be given to check for hypersensitivity

 It is given either IM or IV and the dose depends on stage of

illness:
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Treatment of Diphtheria (Cont..)
 Early stage (< 48 hours): 20,000–40,000 units

 If pharyngeal membranes present: 40,000–60,000 units

 Late stage (> 3 days, with bull neck): 80,000–120,000 units

 Human antitoxin therapy is under development

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Treatment of Diphtheria (Cont..)
 Antibiotics: Penicillin or erythromycin is the drug of choice

 If given early (<6 h of infection), before the toxin release

 Prevent further release of toxin by killing the bacilli

 Treatment of cutaneous diphtheria

 Treatment of carriers: Drug of choice is erythromycin

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Prophylaxis - Post-exposure Prophylaxis
 For close contacts (e.g. household), booster dose of diphtheria
vaccine + penicillin G (single dose) or erythromycin (7–10 days) is
recommended.

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Prophylaxis - Vaccination
 Protective titer >0.01 Unit/mL of antitoxin

 Vaccine not effective for - cutaneous diph. & carrier state

 Types of Vaccine:

 Single vaccine: Diphtheria toxoid (alum or formal precipitated)

 Combined vaccine:

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Prophylaxis – Vaccination (Cont..)
 Combined vaccine:
 DPT: Contains DT (diphtheria toxoid), Pertussis (whole cell) &
TT
 DaPT: Contains DT, TT and acellular pertussis (aP)
 Td: Contains tetanus toxoid and adult dose (2 Lf ) of diphtheria
toxoid
 Pentavalent vaccine: DPT + hepatitis B and Haemophilus
influenzae type b
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Prophylaxis – Vaccination (Cont..)
 Administration of Diphtheria Vaccine:

 Schedule: Under National Immunization Schedule (NIS) of India

2020:
 Children: Total seven doses are given.
 Three doses at 6, 10 and 14 weeks of birth
 Booster doses at 16–24 months & 5 years
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Prophylaxis – Vaccination (Cont..)
 Site: deep intramuscularly (IM) at anterolateral aspect of thigh

 Thiomersal (0.01%) - preservative

 Storage: - kept at 2–8oC; if accidentally frozen - discarded

 Dose: The usual dose (given to children) - 25 Lf units, adult dose -

2 Lf units

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Prophylaxis – Vaccination (Cont..)
 Adult immunization:

 Td vaccine recommended for adults >18 years who have completed

their primary vaccination schedule - booster dose once in every 10
years till the age of 65
 Adults who have not completed their primary vaccination
schedule: 3 doses of Td given at 0, 1 month, and 1 year.
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Adverse Reactions following DPT
Administration
 Mild - Fever and local reaction (swelling and indurations)

 Severe: Whole cell killed vaccine of B. pertussis is

encephalitogenic. Hence, DPT is not recommended after 7 years of
age
 Absolute contraindication to DPT
 Hypersensitivity to previous dose
 Progressive neurological disorder 38
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