DIPHTHERIA
A.GENERAL COMMENTS
B.HISTORY
C.PROPERTIES OF THE ORGANISM
D.HABITAT
E.TRANSMISSION
F.VIRULENCE FACTORS
1.DIPHTHERIA TOXIN
a.RELATION TO LYSOGENY
b.REGULATION OF TOXIN FORMATION
c.MODE OF ACTION OF TOXIN
d.TOXIN STRUCTURE AND INTERACTION WITH MAMMALIAN CELLS
G.PATHOGENESIS AND CLINICAL MANIFESTATIONS
H.DIAGNOSIS
I.TREATMENT
J.EPIDEMIOLOGY
K.PREVENTION
DIPHTHERIA
A. GENERAL
1. Contagious disease of humans, C. d. colonizes mucous membranes of pharynx (sometimes extends to larynx and trachea.
Colonization of subcutaneous tissue of skin can result in cutaneous diphtheria
2. Important today as model for toxigenic disease
B. HISTORY
1. 1 century ago in most countries one of the most dreaded diseases
2. Coming to understand diphtheria and devising means of combating diphtheria death stands as most important model investigation of etiology of infectious disease
[1876-Robert Koch demonstrated that bacteria can cause disease]
1821 Pierre Bretonneau: Diphtheria characterized by formation of tightly adhering membranous growth (pseudomembrane) on mucous membranes of throat, sometimes extending into the trachea
Called the sickness diphtheritis (Gk. diphthera, skin or membrane)
BEGINNING OF SEPARATION OF 1 THROAT INFECTION FROM ANOTHER
Bretonneau asked: ?Can diphtheria be transferred from one human to another?
1879-Trendelenburg- showed injection of pseudomembrane material -->rabbits-->pseudomembrane formation
Loeffler: Looked for bacterial etiology (in Koch's lab); cultured diphtheria bacillus from clinical cases; animal work led to discovery that diphtheria bacilli tended to remain at site of injection which autopsy revealed damage of organs far from infection site.
1888 Roux and Yersin: connected diphtheria bacillus at superficial location and damage to organs and subsequent death to the filterable heat-labile toxin in culture liquid - injection of this toxin into appropriate animals caused all of the systemic manifestations of diphtheria
1890: Behring & Kitasato: Discovered that antibodies prepared against diphtheria toxin could neutralize its toxicity- thus providing means to save patients by passively immunizing against diphtheria toxin
Serum of immunized animals protected susceptible animals against the disease.
Antitoxin first given to diphtheritic child on Christmas night 1891
Ramon and Glenny: found prolonged incubation of DT with formalin under alkaline conditions converted DT into nontoxic antigen [Basis of today's vaccine against diphtheria]
1951-Freeman discovered that all toxigenic strains of C. diphtheriae are lysogenic
C. PROPERTIES OF THE ORGANISM
1. MORPHOLOGY (Figure 1)
Gm+, non-motile rods, non-spore-forming, club shaped; Chinese letters - pleomorphic
2. OTHER PROPERTIES
Grows aerobically at 37C
3. ANTIGENIC STRUCTURE (as relates to virulence)
K antigen - surface protein; antiphagocytic
D. HABITAT
1. Nose, throat, nasopharynx, skin
E. TRANSMISSION
1. Exogenous route
Upper respiratory tract --> droplet nuclei
Cutaneous disease- organism from case or carrier colonizes skin and enters wound
F. VIRULENCE FACTORS
Diphtheria Toxin (Figure 2)
1. Relation to lysogeny (Figure 3)
Only strains lysogenic for -prophage (or related temperate phages) produce DT
Structural gene for toxin (tox) on phage genome and expressed under certain nutritional conditions.
2. Regulation of Toxin Formation
tox gene controlled by metabolism and physiological state of bacterium
Fe3+ concentration impt factor
Toxin synthesized synthesized only after exogenous Fe supply exhausted
Fe regulates transcription of tox by interacting with regulatory protein
3. Mode of action of toxin
Pure DT lethal at 0.1 ug/kg
Toxin kills cultured cells from susceptible animals (cells from insensitive animals affected only at very high toxin concentrations.
DT kills sensitive cells by blocking protein synthesis
DT converted to enzyme that inactivates eukaryotic EF-2, the elongation factor required for translocation of polypeptidyl-tRNA from acceptor to the donor site on the eukaryotic ribosome
Inactivation involves transfer of ADP-ribosyl group of NAD to specific site on EF-2
(Prokaryotic elongation factor not affected)
NAD + EF-2 ADP-ribosyl-EF-2 + nicotinamide + H+
(equilibrium far to right so rxn irreversible under physiological conditions.
Reaction called ADP-ribosylation: This activity found in other bacterial toxins (eg Ps. aeruginosa exotoxin A, cholera toxin, Pertussis toxin)
4. Toxin structure and interaction with mammalian cells
Toxin Structure
a. AB toxin: A chain has enzymatic activity, B chain binding and entry
b. Toxin must be “nicked” for activity (trypsin-like enzyme)
c. A chain must gain entry into cytosol in order to inhibit protein synthesis
Interaction with mammalian cells
a. Attachment to receptors:
b. Endocytosis: entry involves exposure to low pH with endosomal vesicles
c. Membrane insertion: at low pH hydrophobic sequences become exposed and toxin inserts into membrane
d. Release of A into the cytosol: disulfide bridge reduced in cytosol releasing A chain
F. PATHOGENESIS & CLINICAL MANIFESTATIONS (Figure 4)
Human Disease
1. Usually begins in respiratory tract
2. Virulent diphtheria bacilli lodge in throat of susceptible individual
3. Multiply in superficial layers of mucous membrane
4. Elaborate toxin which causes necrosis of neighboring tissue cells
5. Inflammatory response eventually results in pseudomembrane (Figure 5)
Usually appears first on tonsils or posterior pharynx and spreads upward or down
In laryngeal diphtheria, mechanical obstruction may cause suffocation
Regional lymphnodes in neck often enlarged (bull neck)
6. In tropics cutaneous lesions infected with toxigenic C. diphtheriae
G. DIAGNOSIS
Culture id, test for toxigenicity (Figure 6)
H. TREATMENT
1. Once toxin inside cell can no longer be neutralized by antitoxin
2. Antitoxin therapy must be started without delay
Time factor critical - clinician may start antiserum without waiting for results of culture tests.
Antiserum derived from horses so may test for sensitivity to horse proteins
3. Antibiotics (penicillin, erythromycin, tetracycline) administered in conjunction with antitoxin in order to reduce organism load
I. EPIDEMIOLOGY
1. Newborn born to resistant mothers acquire temporary immunity lasting `1-2 years (transplacental antitoxin)
2. Persons who recover may continue to harbor organism in nose or throat for weeks or months.
J. PREVENTION
1. Man only reservoir
2. Disease still prevalent where immunization not practiced
3. All toxigenic strains elaborate same antigenic type of toxin - so should be possible to eradication disease
4. Vaccine is toxoid
DT treated with dilute formaldehyde under suitable conditions. Converted to toxoid which is devoid of toxicity but is indistinguishable antigenically from toxin.
