1. Diphtheria Paul R. Earl Facultad de Ciencias Biológicas Universidad Autónoma de Nuevo León San Nicolás de los Garza, NL, Mexico

2. 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.

3. Recent large epidemics in Eastern Europe call for renewed attention to diphtheria. They highlighted the need for the following 5 major activities in diphtheria control: a) 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.

4. 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.

5. In 1888, Emil Behring joined Koch at the Institute of Hygiene in Berlin. During the years , 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.
In 1890, von Behring and Kitasato succeeded in immunizing guinea pigs with a heat-attenuated toxoid that raised protective antitoxins. Regardless, this modified toxin was found to cause severe local reactions in humans and could not be used as a vaccine. In 1909, Theobald Smith in the US showed that diphtheria toxin neutralized by antitoxin formed a Toxin-Anti-Toxin complex, TAT, that remained immunogenic and eliminated local reactions. TAT sometimes was highly toxic, and secondly toxoid was produced in horse serum which can sensitize some individuals to the serum.
Emil von Behring

6. 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.
Earlier in 1898, Behring and Wernicke had found that immunity to diphtheria could be produced by the injection into animals of diphtheria toxin neutralized by diphtheria antitoxin, as Smith in had suggested that such toxin-antitoxin mixtures might be used to immunize man against this disease. It was Behring, however, who announced, in 1913, his production of a mixture of this kind, and subsequent work which modified and refined the mixture originally produced by Behring resulted in the modern methods of immunization which have largely banished diphtheria

7. 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).
In 1929 by adding formaldehyde, Ramon demonstrated the conversion of diphtheria toxin to its nontoxic, but antigenic equivalent: the toxoid. In 1951, Freeman found that the pathogenic strains of C. diphtheriae are lysogenic. Toxin producers are infected by a temperate  phage. The gene for toxin production is located on the chromosome of the  phage.

8. This pneumonia is caused by a toxic grampositive rod
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.

9. 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.

10. 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.
Toxin is synthesized in high yield after the supply of Fe++ and Fe+++ has become exhausted. Remarkably, C. diphtheriae will synthesize diphtheria toxin as 5% of its total protein, when starved for iron.
The tox gene is regulated by a mechanism of negative control wherein a repressor molecule, product of the DtxR gene, is activated by iron. The active repressor binds to the tox gene operator and prevents transcription. When iron is removed from the repressor (under growth conditions of iron limitation), derepression occurs, the repressor is inactivated and transcription of the tox genes can occur. Iron is referred to as a corepressor since it is required for repression of the toxin gene.

11. 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").
CRMs have shorter chain length than the diphtheria toxin molecule but cross react with diphtheria antitoxins due to their antigenic similarities to the toxin. The properties of CRMs established beyond a doubt that the tox genes resided on the phage chromosome rather than the bacterial chromosome.

12. 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 host’s 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.
The toxin has an active A domain, a binding B domain, and a hydrophobic segment, the T domain. The A domain catalyzes the transfer of an adenosine diphosphate-ribose molecule to one of the elongation factors responsible for protein synthesis. This transfer can inactivate the factor and inhibit protein synthesis, which can cause cell death. Cell death by toxin is the keynote of the pathogenesis, and the prevention of diphtheria by vaccination with toxoid is the keynote of successful epidemiologic control.

13. 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.
Polyneuropathy occurs in 10% of cases of average severity and up to 75% of severe cases. Bulbar symptoms often occur in the first 2 weeks of illness like difficulty in swallowing and paresis of the palatal and ocular muscles. Respiratory dysfunction is the most severe symptom of toxicosis. Bulbar symptoms can progress to paralysis of the proximal and then distal skeletal muscles. However, most patients regain complete neurologic function.

14. The heart, kidneys and peripheral nerves can be involved
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.
Affected nerves shows myelin sheath and axon degeneration. The large myelinated fibers are affected, demonstrating segmental demyelination and macrovacuolization. Both the motor and sensory fibers of the peripheral nerves demonstrate fatty degenerative changes and disintegration of the medullary sheaths due to demyel-ination by toxin. The anterior horn cells and posterior columns of the spinal canal can be involved, and the central nervous system may develop signs of hemorrhage, meningitis and encephalitis. Nonetheless, the polyneuro-pathy is reversable.
At autopsy, the heart is pale brown, soft and enlarged with a characteristic streaky appearance. Neutral fat accumulations are observed in approximately 50% of patients, with extensive hyaline degeneration and necrosis with inflammatory changes. EM demonstrates swollen disorganized mitochondria containing dense osmophilic granules.

15. 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.

16. Elek-Ouchterlony toxigenicity test
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 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.
One well in the center of the Petri dish with antitoxin and 6 surrounding wells if positive for specific toxin will produce precipitin lines in possibly 24 hourse. Sometimes paper disks are used rather than wells cut out with a testtube. Small dishes of 4.5 cm take 3 ml of agar, and disks or wells can be 9 mm apart.

17. 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.
Usually infants are immunized with a trivalent vaccine containing diphtheria toxoid, pertussis vaccine, and tetanus toxoid (DPT). Toxoid is given in 2 or 3 doses 1 month apart at 3-4 months of age. A booster injection is given a year later. Several booster shots can be given during childhood.
The morbidity and mortality of many diseases has been remarkably reduced by appropriate vaccinactions. Diphtheria is one of the best known and is now combined with several other diseases including inactivated polio. On December 13, 2002, the US Food and Drug Administration licensed a combined diphtheria and tetanus toxoids and acellular pertussis adsorbed hepatitis B (recombinant) and inactivated poliovirus vaccine for use in infants ages 2, 4 and 6 months. Combination of hepatitis A vaccine and HBV was safe and effective. Those vaccines to be combined in the future are MMR-varicella, pneumococcal-meningococcal.

18. 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.
In countries where pertussis is no longer a public health problem, bivalent vaccine in its pediatric form (DT) may be used for booster doses in preschool children. The adult form (Td) should be used for booster doses in children aged seven years and over and in adolescents and adults. The following 3 DTP immunization schedules are in widespread use: a) 3 doses: 3 primary doses of DTP vaccine given during the 1st year of life, b) 4 doses: primary series of 3 doses reinforced with a booster dose usually administered around the 2nd or 3rd year of life and c) 5 doses: primary series of 3 doses reinforced with a 1st booster dose in the 2nd year of life and a 2nd booster given before entering school at the age of 4-6 years. Diphtheria toxoid is also included in the pentavalent DTP–HBV–Hib vaccine. Booster doses: A regime of routine booster doses approximately every 10 years is indicated for the maintenance of immunity.

19. 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.

20. 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.
Random amplified polymorphic DNA (RAPD) is also used convenially since often groups by ribotypes can be correlated with RAPDs as in the illustration from Kombarova et al. Emerg Infect Dis, Vol. 7, 2001.

21. Section of the figure a has RAPD bands for Corynebacterium diphtheriae gravis and mitis groups, whereas the same strains are ribotyped in Section b.

22. 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.
For the finished genome, see

23. 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.