Diphtheria Paul R. Earl Facultad de Ciencias Biol

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DiphtheriaPaul R. EarlFacultad de Ciencias
BiológicasUniversidad Autónoma de Nuevo LeónSan
Nicolás de los Garza, NL, Mexico
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Indeed diptheria is well understood and
controled, but it will return unless massive
vaccine pressure is continued. Until 1930 it was
a dreaded killer, yet now is nearly eliminated
from the industrial world. Nevertheless, the
economic collapse in the 1990s of the former
USSR, the decline in social conditions and a
faltering vaccine program forecasted a return of
diphtheria. The development of a preventive
vaccine against a high-risk disease is the basic
story of diptherial control.
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Recent large epidemics in Eastern Europe call for
renewed attention to diphtheria. They highlighted
the need for the following 5 major activities in
diphtheria controla) adequate surveillance, b)
high levels of routine immunization in
appropriate age groups, c) prompt recognition
and attention, d) the availability of adequate
supplies of antibiotics and antitoxin, and e)
the rapid case investigation and management of
close contacts as part of outbreak management.
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History. Effective, and in fact
elegant methods of treatment and prevention of
diphtheria have been developed as a consequence
of the research efforts expended on all phases of
diphtheria, the strangler.The grampositive
bacterium that causes diphtheria was first
described by Klebs in 1883 and was cultivated by
Löffler in 1884, who concluded that C.
diphtheriae produced a soluble toxin, and thereby
he first described a bacterial exotoxin. In 1888,
Roux and Yersin demonstrated the presence of the
toxin in the cellfree culture fluid of C.
diphtheriae which caused the systemic
manifestation of diphtheria in laboratory
animals.
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In 1888, Emil Behring joined Koch at the
Institute of Hygiene in Berlin. During the years
1888-1890, Roux and Yersin, working at the
Pasteur Institute in Paris, had shown that
filtrates of diphtheria cultures which contained
no bacilli, contained a toxin, that produced,
when injected into animals, all the symptoms of
diphtheria.
Emil von Behring
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However in 1890 from cultures of diphtheria
bacill, Brieger and Fraenkel prepared a toxin
which they called toxalbumin, which when injected
into guineapigs, immunized them to diphtheria. In
1890, Behring and Kitasato published their
discovery that graduated doses of sterilized
broth cultures of diphtheria or tetanus bacilli
caused the animals to produce substances
(antitoxins) in their blood which could
neutralize the toxins which these bacilli
produced. They also showed that the antitoxins
produced by one animal could immunize another
animal and cure an animal with symptoms of
diphtheria. Behring received the Nobel prize in
1901 for this work.
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In 1913, Schick invented a skin test for
susceptibility (nonimmunity) or immunity to
diphtheria. Diphtheria toxin will cause a local
inflammatory reaction when very small amounts are
injected intracutaneously. The Schick test
involves injecting a very small dose of the toxin
under the skin of the forearm and evaluating the
injection site after 48 hours. A positive test
(inflammatory reaction) indicates susceptibility.
A negative test (no reaction) indicates immunity
(antibody neutralizes toxin).
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This pneumonia is caused by a toxic grampositive
rod. Diphtheria, long known as a child killer,
can cause 5-10 of patients to die, even if
properly treated. Untreated, the disease claims
even more lives. Untreated patients are
infectious for 2-3 weeks. Unless immunized,
children and adults may be infected repeatedly
with the disease. Treatment consists of immediate
administration of diphtheria antitoxoid and
antibiotics. Note that toxin detoxified by
formalin, heat or another agent is the toxoid
used for vaccination for decades. Antibiotic
treatment usually renders patients noninfectious
within 24 hours.
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Corynebacterium diphtheriae causes infection of
the upper respiratory tract in humans and often
has a toxic reaction involving paralysis of the
heart and peripheral nerves. Diphtheria is
wellknown by the formation of a membrane in the
throat. Strains gravis, intermedius and mitis are
recognized with strain belfanti.These strains
produce the identical toxin, but the gravis
strain grows faster, depletes the local iron
supply, and allows earlier and greater toxin
production. Some strains like C. diphtheriae
belfanti may not produce toxin.
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Toxigenicity. The low extracellular
concentrations of iron and infection by a
lysogenic prophage in the bacterial chromosome
are needed for Corynebacterium diphtheriae
production of toxin. The gene for toxin
production is on the prophage chromosome, but a
bacterial repressor protein controls the
expression of this gene. The repressor is
activated by iron. High yields of toxin are
synthesized only by lysogenic bacteria in iron
deficiency.
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The role of ?-phage. Only those strains of
Corynebacterium diphtheriae that that are
lysogenized by a specific ?-phage produce
diphtheria toxin. A phage lytic cycle is not
necessary for toxin production or release. The
phage contains the structural gene for the toxin
molecule. The original proof rested in the
demonstration that lysogeny of C. diphtheriae by
various mutated ?-phages leads to production of
nontoxic but antigenically-related material
(called CRM for "cross-reacting material").
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Even though the tox gene is not part of the
bacterial chromosome, the regulation of toxin
production is under bacterial control since the
DtxR (regulatory) gene is on the bacterial
chromosome and toxin production depends upon
bacterial iron metabolism. Toxin production
depends on the presence of a lysogenic ?- phage
that carries the tox gene. When DNA of the phage
becomes integrated into the bacterial hosts
genome, the bacteria produce the single
polypeptide toxin. The iron-binding repressor is
DtxR. In the presence of ferrous iron, the
DtxR-iron complex attaches to the tox gene
operator, inhibiting transcription.
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Symptoms.
The characteristic diphtheric membrane in
the throat is thick, leathery, grayish-blue or
white, and composed of bacteria, necrotic
epithelium, macrophages and fibrin. The membrane
firmly adheres to the underlying mucosa and
forceful removal causes bleeding. Spreading of
the membrane down the bronchial tree can occur,
causing respiratory tract obstruction and
dyspnea. Guard against respiratory obstruction
and respiratory muscle dysfunction. Upper
respiratory tract symptoms occur including nasal
discharge and sore throat, involving fever and
membrane development on the tonsils. A moderate
elevation in leukocytes and a mild proteinuria is
common.
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The heart, kidneys and peripheral nerves can be
involved. Cardiac enlargement is common and
caused by inflamation by toxin, occurring often
within 1-2 weeks of the onset of illness when
respiratory symptoms are improving. These
symptoms include arrhythmias and congestive heart
failure caused by a dilated cardiomyopathy. The
kidneys can have interstitial edema and necrosis
by toxin. Multiple diphtheritic organ failure and
heart failure cause some deaths.
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Treatment. Many antibiotics bring
about cures efficiently including often
penicillin, although erythromycin might be
preferred. Erythromycin--Adult Dose 500 mg PO/IV
q6h for 14 d if tolerated. Vancomycin (Vancocin)
Adult Dose 1 g IV infused over 1 h q12h. Rifampin
(Rifadin) Adult Dose 600 mg orally.Antitoxin can
be lifesaving but must be given early in the
infection and with antibiotics like penicillin G.
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Elek-Ouchterlony toxigenicity test. The
detection of toxin from corynebacteria is the
most important step for identification. This is
an immunodiffusion test in agar known since 1948.
Traditionally, toxin production was demonstrated
by injecting toxin material into guineapigs and
watching to see if they died. The
Elek-Ouchterlony plate test for biologic activity
of the toxin, an immunoprecipitation test, was
developed to replaced the in vivo guineapig test.

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Vaccination. Acquired immunity to
diphtheria is due primarily to toxin-neutralizing
antibody (antitoxin). Passive immunity in utero
is acquired transplacentally and can last a year.
Individuals that have fully recovered from
diphtheria may continue to harbor the organisms
in the throat or nose for weeks. Healthy carriers
spread the disease, and toxigenic bacteria were
maintained in the population. Before mass
immunization of children, carrier rates of C.
diphtheriae of 5 or higher were observed.
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WHO perspective. Suggestions by the World
Health Organization (WHO) follow. The priority
for every country is to reach at least 90
coverage with the 3 primary doses of
diphtheria-tetanus-pertussis vaccine (DTP) as
early as possible in the schedule. DTP is the
core vaccine in childhood immunization services.
Since 1990, the global coverage for this triple
vaccine has only been around 80. Additional
doses of DTP should be given after completion of
the primary doses. However, the need and timing
for such additional booster doses should be
addressed by individual national programs.
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The public health burden of diphtheria has been
low in most developing countries because most
children have acquired immunity through
subclinical or cutaneous infection. Still, recent
outbreaks of diphtheria have been observed in the
newly independent States of the former Soviet
Union, Algeria, China, Iraq, Jordan, Lao People's
Democratic Republic, Lesotho, Mongolia, Sudan,
Thailand and the Yemen Arab Republic, showing the
importance of immunizing children in all
countries. These recent outbreaks among adults
show the need, still incompletely met in many
countries, to maintain immunity against the
disease throughout life.
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PCR and other tests. Other recent
tests for toxigenicity include PCR detection of
the A fragment of the toxin and rapid enzyme
immunoassay using a monoclonal antibody to the A
fragment. PCR detection and the enzyme
immunoassay (ELISA) test reportedly give
identification results within a few hours. The
usual PCR methods like amplified fragment length
polymorphisms (AFLP), pulsed field gel
electrophoresis (PFGE) and ribotying (ribosome
DNA) are routinely used by reference
laboratories.
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Section of the figure a has RAPD bands for
Corynebacterium diphtheriae gravis and mitis
groups, whereas the same strains are ribotyped in
Section b.
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The restriction enzyme BstEii has been used in
ribotyping to produce 400-1,500 bp fragments (bp
base pairs). Biotypes of C. diphtheriae like
gravis and mitis develop patterns then called G1
and M1, etc. Cluster analysis is then applied,
and a similarity matrix among strains is set up.
It has been astutely remarked that differences
among groups G1-G5 could be due to a/
prophage, b/ inversions, c/ deletions, e/
insertion sequences, f/ transposons or g/
plasmid.
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For the detection of C. diphtheriae, IgG
antitoxin antibodies (IgG-DTAb) in human serum
can be used. Four different methods a/passive
hemagglutination (PHA), b/ latex agglutination
test (LA), c/ toxoid enzyme-linked immunosorbent
assay (Toxoid-ELISA), and d/ toxin-binding
inhibition enzyme-linked immunosorbent assay
(ToBI-ELISA). As the external
standardisation the neutralisation test for C.
diphtheriae toxin in Vero cells (TN Vero) was
used. For internal standardisation of IgG-DTAb
titres, use the WHO standard serum of human
diphtheria.