Laboratory diagnosis, treatment and prevention of Corynebacterium diphtheriae

Specimen

• 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 semisolid transport media such as Amies.

Microscopy

• Smears stained with alkaline methylene blue or Gram stain show beaded rods in typical arrangement.
• Cells often contain metachromatic granules (polymetaphosphate), which stain bluish-purple with methylene blue.
• In Gram staining, purple coloured beaded rods, angular and palisade arrangements that creates a ‘Chinese character’ effect is observed.

Culture

• Specimens should be inoculated to a blood agar plate 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.
• Non β hemolytic region on blood agar.
• Tellurite inhibits the growth of most upper respiratory tract bacteria and gram-negative rods and is reduced by C. diphtheriae, producing characteristic gray to black color on agar.
• Degradation of cysteine by C. diphtheriae cysteinase activity produces a brown halo around the colonies.
• Tinsdale medium is the best medium for recovering C. diphtheriae in clinical specimens, but it has a short shelf life and requires addition of horse serum.

Biochemical testing

Catalase – positive

Oxidase – negative

Cystinase production – positive

Pyrazinamidase activity – positive

Carbohydrate fermentation

Glucose – positive

Maltose – positive

Sucrose – negative

Trehalose – negative

Urease – negative

Nitrate reduction – positive

Gelatin liquefaction – negative

Toxigenicity Testing

• All isolates of C. diphtheriae should be tested for the production of exotoxin.
• The gold standard for detection of diphtheria toxin is an in-vitro immunodiffusion assay by  Elek-Ouchterlony immunodiffusion test (Elek test).
• An alternative method is detection of the exotoxin gene using a polymerase chain reaction (PCR) based nucleic acid amplification method.
• This test can detect the tox gene in clinical isolates and directly in clinical specimens (e.g., swabs from the diphtheritic membrane or biopsy material).
• 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 probably has diphtheria.
• Although this test is rapid and specific, strains in which the tox gene is not expressed can give a positive signal.
• Enzyme-linked immunosorbent assays can be used to detect diphtheria toxin from clinical C diphtheria isolates.
• An immunochromatographic strip assay allows detection of diphtheria toxin in a matter of hours.
• However ELISA and immunochromatographic assay are not widely used.

Antibody detection

• Measurement of antibodies to diphtheria toxin in serum collected before administration of antitoxin may support the diagnosis when cultures are negative.

Treatment of Corynebacterium diphtheriae

• The early administration of specific antitoxin against the toxin formed by the organisms at their site of entry and multiplication is done promptly.
• 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.
• Erythromycin may be preferred to penicillin for elimination of the bacilli from the throat, particularly in treatment of persistent carriers.
• Some strains are tolerant to the bactericidal action of penicillins, and treatment of complicated infections should contain an association with an aminoglycoside.
• Patients should be placed in strict isolation, nursed by staff whose immunization history is documented and have daily platelet counts and electrocardiography.

Prevention and control of Corynebacterium diphtheriae

• Active immunization in childhood with diphtheria toxoid yields antitoxin levels that are generally adequate until adulthood.
• Diphtheria toxoids are commonly combined with tetanus toxoid (Td) and with a cellular 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).
• Young adults should be given boosters of toxoid because toxigenic diphtheria bacilli are not sufficiently prevalent in the population 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 antitoxin 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.
• Bed rest, isolation to prevent secondary spread, and maintenance of an open airway in patients with respiratory diphtheria are all important.