Morphology and Identification

 Corynebacteria are 0.5–1 µm in diameter and several micrometers long. Characteristically, they possess irregular swellings at one end that give them the “club-shaped” appearance (Figure 1). Irregularly distributed within the rod (often near the poles) are granules staining deeply with aniline dyes (metachromatic granules) that give the rod a beaded appearance. Individual corynebacteria in stained smears tend to lie parallel or at acute angles to one another. True branching is rarely observed in cultures.

Fig1. Corynebacterium diphtheriae from Pai medium stained with methylene blue. Typically, they are 0.5–1 × 3–4 µm. Some of the bacteria have clubbed ends (original magnification ×1000).

On blood agar, the C. diphtheriae colonies are small, granular, and gray with irregular edges and may have small zones of hemolysis. On agar containing potassium tellurite, the colonies are brown to black with a brown-black halo because the tellurite is reduced intracellularly (staphylococci and streptococci can also produce black colonies). Four bio types of C. diphtheriae have been widely recognized and each of them produces the potent exotoxin: gravis, mitis, intermedius, and belfanti. These variants have been classified on the basis of growth characteristics such as colony morphology, biochemical reactions, and severity of disease produced by infection. Very few reference laboratories are equipped with methods to provide reliable biotype characterization. The incidence of diphtheria has greatly decreased and the association of severity of disease with biovar is no longer important to clinical or public health management of cases or outbreaks. If necessary, in the setting of an outbreak, immunochemical and preferably molecular methods such as ribotyping can be used to type the C. diphtheriae isolates.

C. diphtheriae and other corynebacteria grow aerobically on most ordinary laboratory media. On Löffler’s serum medium, corynebacteria grow much more readily than other respiratory organisms, and the morphology of organisms is typical in smears made from these colonies.

Corynebacteria tend to pleomorphism in microscopic and colonial morphology. When some nontoxigenic diphtheria organisms are infected with bacteriophage from certain toxigenic diphtheria bacilli, the offspring of the exposed bacteria are lysogenic and toxigenic, and this trait is subsequently hereditary. When toxigenic diphtheria bacilli are serially sub cultured in specific antiserum against the temperate phage that they carry, they tend to become nontoxigenic. Thus, acquisition of phage leads to toxigenicity (lysogenic conversion). The actual production of toxin occurs perhaps only when the pro phage of the lysogenic C. diphtheriae becomes induced and lyses the cell. Whereas toxigenicity is under the control of the phage gene, invasiveness is under the control of bacterial genes.

Pathogenesis

 The principal human pathogen of the genus Corynebacterium is C. diphtheriae, the causative agent of respiratory or cutaneous diphtheria. In nature, C. diphtheriae occurs in the respiratory tract, in wounds, or on the skin of infected persons or normal carriers. It is spread by droplets or by contact to susceptible individuals; the bacilli then grow on mucous membranes or in skin abrasions, and those that are toxigenic start producing toxin.

All toxigenic C. diphtheriae are capable of elaborating the same disease-producing exotoxin. In vitro production of this toxin depends largely on the concentration of iron. Toxin production is optimal at 0.14 µg of iron per milliliter of medium but is virtually suppressed at 0.5 µg/mL. Other factors influencing the yield of toxin in vitro are osmotic pressure, amino acid concentration, pH, and availability of suitable carbon and nitrogen sources. The factors that control toxin production in vivo are not well understood.

Diphtheria toxin is a heat-labile, single-chain, three domain polypeptide (62 kDa) that can be lethal in a dose of 0.1 µg/kg body weight. If disulfide bonds are broken, the molecule can be split into two fragments. Fragment B (38 kDa), which has no independent activity, is functionally divided into a receptor domain and a translocation domain. The binding of the receptor domain to host cell membrane proteins CD-9 and heparin-binding epidermal growth fac tor (HB-EGF) triggers the entry of the toxin into the cell through receptor-mediated endocytosis. Acidification of the translocation domain within a developing endosome leads to creation of a protein channel that facilitates movement of Fragment A into the host cell cytoplasm. Fragment A inhibits polypeptide chain elongation—provided nicotinamide adenine dinucleotide (NAD) is present—by inactivating the elongation factor EF-2. This factor is required for translocation of polypeptidyl-transfer RNA from the acceptor to the donor site on the eukaryotic ribosome. Toxin Fragment A inactivates EF-2 by catalyzing a reaction that yields free nicotinamide plus an inactive adenosine diphosphate-ribose-EF-2 complex (ADP-ribosylation). It is assumed that the abrupt arrest of protein synthesis is responsible for the necrotizing and neurotoxic effects of diphtheria toxin. An exotoxin with a similar mode of action can be produced by strains of Pseudomonas aeruginosa.

Pathology

 Diphtheria toxin is absorbed into the mucous membranes and causes destruction of epithelium and a superficial inflammatory response. The necrotic epithelium becomes embedded in exuding fibrin and red and white cells so that a grayish “pseudomembrane” is formed—commonly over the tonsils, pharynx, or larynx. Any attempt to remove the pseudomembrane exposes and tears the capillaries and thus results in bleeding. The regional lymph nodes in the neck enlarge, and there may be marked edema of the entire neck, with distortion of the airway, often referred to as “bull neck” clinically. The diphtheria bacilli within the membrane continue to produce toxin actively. This is absorbed and results in distant toxic damage, particularly parenchymatous degeneration, fatty infiltration, and necrosis in heart muscle (myocarditis), liver, kidneys (tubular necrosis), and adrenal glands, sometimes accompanied by gross hemorrhage. The toxin also produces nerve damage (demyelination), often resulting in paralysis of the soft palate, eye muscles, or extremities.

Wound or skin diphtheria occurs chiefly in the tropics, although cases have also been described in temperate climates among alcoholic, homeless individuals, and other impoverished groups. A membrane may form on an infected wound that fails to heal. However, absorption of toxin is usually slight and the systemic effects negligible. The small amount of toxin that is absorbed during skin infection promotes development of antitoxin antibodies. The “virulence” of diphtheria bacilli is attributable to their capacity for establishing infection, growing rapidly, and then quickly elaborating toxin that is effectively absorbed. C. diphtheriae does not need to be toxigenic to establish localized infection—in the naso pharynx or skin, for example—but nontoxigenic strains do not yield the localized or systemic toxic effects. C. diphtheriae does not typically invade deep tissues and practically never enters the bloodstream. However, notably over the last two decades, reports of invasive infections such as endocarditis and septicemia due to nontoxigenic C. diphtheriae have increased.

Clinical Findings

When diphtheritic inflammation begins in the respiratory tract, sore throat and low-grade fever usually develop. Prostration and dyspnea soon follow because of the obstruction caused by the membrane. This obstruction may even cause suffocation if not promptly relieved by intubation or tracheostomy. Irregularities of cardiac rhythm indicate damage to the heart. Later, there may be difficulties with vision, speech, swallowing, or movement of the arms or legs. All of these manifestations tend to subside spontaneously.

In general, var gravis tends to produce more severe disease than var mitis, but similar illness can be produced by all types.

Diagnostic Laboratory Tests

These serve to confirm the clinical impression and are of epidemiologic significance. Note: Specific treatment must never be delayed for laboratory reports if the clinical picture is strongly suggestive of diphtheria. Physicians should notify the clinical laboratory before collecting or submitting samples for culture.

Dacron swabs from the nose, throat, or other suspected lesions must be obtained before antimicrobial drugs are administered. Swabs should be collected from beneath any visible membrane. The swab should then be placed in semi solid transport media such as Amies. Smears stained with alkaline methylene blue or Gram-stain show beaded rods in typical arrangement.

Specimens should be inoculated to a blood agar plate (to rule out hemolytic streptococci) and a selective medium such as a tellurite plate (eg, cystine-tellurite blood agar [CTBA] or modified Tinsdale’s medium) and incubated at 37°C in 5% CO2 . Plates should be examined in 18–24 hours. In 36–48 hours, the colonies on tellurite medium are sufficiently definite for recognition of C. diphtheriae. On cystine tellurite agar, the colonies are black with a brown halo.

A presumptive C. diphtheriae isolate should be subjected to testing for toxigenicity. Such tests are performed only in reference public health laboratories. There are several methods, as follows:

1. Modified Elek immunoprecipitation method described by the World Health Organization Diphtheria Reference Unit.

A filter paper disk containing antitoxin (10 IU/disk) is placed on an agar plate. The cultures to be tested (at least 10 colonies should be chosen) for toxigenicity are spot inoculated 7–9 mm away from the disk. After 48 hours of incubation, the antitoxin diffusing from the paper disk has precipitated the toxin diffusing from toxigenic cultures and has resulted in precipitin bands between the disk and the bacterial growth.

 2. Polymerase chain reaction (PCR)-based methods have been described for detection of the diphtheria toxin gene (tox). PCR assays for tox can also be used directly on patient specimens before culture results are available. A positive culture result confirms a positive PCR assay. A negative culture result after antibiotic therapy along with a positive PCR assay result suggests that the patient prob ably has diphtheria.

 3. Enzyme-linked immunosorbent assays can be used to detect diphtheria toxin from clinical C. diphtheriae isolates.

4. An immunochromatographic strip assay allows detection of diphtheria toxin in a matter of hours. This assay is highly sensitive.

The latter two assays are not widely available.

Resistance and Immunity

Because diphtheria is principally the result of the action of the toxin formed by the organism rather than invasion by the organism, resistance to the disease depends largely on the availability of specific neutralizing antitoxin in the blood stream and tissues. It is generally true that diphtheria occurs only in persons who possess no antitoxin antibodies (IgG) (or less than 0.1 IU/mL). Assessment of immunity to diphtheria toxin for individual patients can best be made by review of documented diphtheria toxoid immunizations and primary or booster immunization if needed.

Treatment

 The treatment of diphtheria rests largely on rapid suppression of toxin-producing bacteria by antimicrobial drugs and the early administration of specific antitoxin against the toxin formed by the organisms at their site of entry and multiplication. Diphtheria antitoxin is produced in various animals (horses, sheep, goats, and rabbits) by the repeated injection of purified and concentrated toxoid. Treatment with anti toxin is mandatory when there is strong clinical suspicion of diphtheria. From 20,000 to 120,000 units are injected intra muscularly or intravenously depending on the duration of symptoms and severity of illness after suitable precautions have been taken (skin test) to rule out hypersensitivity to the animal serum. The antitoxin should be given intravenously on the day the clinical diagnosis of diphtheria is made and need not be repeated. Intramuscular injection may be used in mild cases. Diphtheria antitoxin will only neutralize circulating toxin that is not bound to tissue.

Antimicrobial drugs (penicillin, macrolides) inhibit the growth of diphtheria bacilli. Although these drugs have virtually no effect on the disease process, they arrest toxin production and assist public health efforts. They also help to eliminate coexistent streptococci and C. diphtheriae from the respiratory tracts of patients or carriers. Antimicrobial resistance to these agents is rare.

Epidemiology, Prevention, and Control

Before artificial immunization, diphtheria was mainly a dis ease of small children. The infection occurred either clinically or subclinically at an early age and resulted in the widespread production of antitoxin in the population. An asymptomatic infection during adolescence and adult life served as a stimulus for maintenance of high antitoxin levels. Thus, most members of the population, except children, were immune.

By age 6–8 years, approximately 75% of children in devel oping countries where skin infections with C. diphtheriae are common have protective serum antitoxin levels. Absorption of small amounts of diphtheria toxin from the skin infection presumably provides the antigenic stimulus for the immune response; the amount of absorbed toxin does not produce disease.

By the late 20th century, most developed countries predicted the elimination of diphtheria as a result of successful childhood vaccination strategies. However, from 1990 to 1998, a resurgence of epidemic diphtheria, primarily among adults, occurred in the Russian Federation and the Newly Independent States (NIS) of the former Soviet Union. This likely resulted from reduced vaccination coverage, among other social factors. Currently, India has the largest total number of reported cases of diphtheria, followed by Indonesia and Madagascar. These outbreaks clearly emphasize the importance of maintaining global immunization.

Active immunization in childhood with diphtheria toxoid yields antitoxin levels that are generally adequate until adulthood. Young adults should be given boosters of toxoid because toxigenic diphtheria bacilli are not sufficiently prevalent in the population of many developed countries to provide the stimulus of subclinical infection with stimulation of resistance. Levels of antitoxin decline with time, and many older persons have insufficient amounts of circulating anti toxin to protect them against diphtheria.

The principal aims of prevention are to limit the distribution of toxigenic diphtheria bacilli in the population and to maintain as high a level of active immunization as possible.

To limit contact with diphtheria bacilli to a minimum, patients with diphtheria should be isolated. Without treatment, a large percentage of infected persons continue to shed diphtheria bacilli for weeks or months after recovery (convalescent carriers). This danger may be greatly reduced by active early treatment with antibiotics.

Diphtheria toxoids are commonly combined with tetanus toxoid (Td) and with acellular pertussis vaccine (DaPT) as a single injection to be used in initial immunization of children (three doses in the first year of life, 15–18 months of age, and 4–6 years of age). For booster injection of adolescents and adults, only Td toxoids or Td toxoids combined with acellular pertussis vaccine (Tdap) (for a one-time injection for those individuals who received whole-cell pertussis vaccine as children) are used; these combine a full dose of tetanus toxoid with a 10-fold smaller dose of diphtheria toxoid to diminish the likelihood of adverse reactions.

All children must receive an initial course of immunizations and boosters. Regular boosters with Td are particularly important for adults who travel to developing countries, where the incidence of clinical diphtheria may be 1000-fold higher than in developed countries, where immunization is universal.

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دراسة حديثة تكشف ما تفعله الشاشات بـ"قلوب الأطفال" !

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