Corynebacterium diphtheriae - Laboratory Diagnosis

Lab Diagnosis of Corynebacterium diphtheriae

Initial treatment of Corynebacterium diphtheriae is based on clinical symptoms and treatment is started before the lab report. Hence, lab diagnosis is carried out for epidemiological purposes than for treatment.

Specimen

Specimen collected for diagnosis of Corynebacterium diphtheriae

• Swab(s) from the nose, throat, biopsy tissue, etc.

• pieces of pseudomembrane

* At least two swabs are collected- one for direct smear and the other for culture

Microscopy

• Microscopy after gram staining reveals gram-positive bacilli in short chains

• Albert, Neisser, or Ponder stain of direct smear shows metachromatic granules

  Microscopy is not specific because other species of Corynebacterium are present as commensal in the throat

Culture

The culture of Corynebacterium diphtheria can be done in

• Non-selective media: BA

• Selective media: Tellurite blood agar

• Enriched media: Loeffler medium, Tinsdale medium

  * In Tellurite blood agar, reduces tellurite and produces grey or grey-black colonies measuring 0.5-2mm in diameter after 24-48 hours of incubation

  * In tinsdale medium colonies are grey-black, raised, and surrounded by a dark brown area after 24-48 hours of incubation. The brown color is due to H2S production from the cystine interacting with tellurite. Commercial diphtheroids and other RT commensals colonies lack surrounding brown halo.

Biochemical test

Some biochemical tests performed for Corynebacterium diphtheriae are

• Catalase => positive

• Oxidase => negative

• Nitrate test => positive

• ferments glucose and maltose with acid production

• few strains gravis and mitis biovars ferment sucrose

• Corynebacterium diphtheriae gravis ferments starch with acid production

Identification of bacteria

• 3 Corynebacterium diphtheriae biotypes: mitis, intermedius, gravis

• potentially toxigenic spp, have systinase but no pyrazinamidase activity

Toxigenicity testing

The following toxigenicity tests can demonstrate the production of toxins by Corynebacterium diphtheriae.

In vivo test:

1. subcutaneous test

2. Intradermal test

Subcutaneous test:

• growth from an overnight culture of Corynebacterium diphtheriae on Loeffler’s slope is emulsified in 2-4 ml broth

  Two guinea pigs are injected subcutaneously with 0.8ml of the emulsion. One of the guinea pigs is protected with 500 µl of diphtheria antitoxin injected intraperitoneally 18-24 hours before the test. Another guinea pig is not protected

• If the test stain is virulent, the unprotected animal will die within 4 days. The protected guinea pig shall remain normal

Intradermal test:

• 0.1 ml of the emulsion(obtained from 18 hours of growth of test bacteria cultured on Loeffler's medium slope) is injected intradermally into shaven sites on each 2 guinea pigs

  The control animal is given 500 µl of antitoxin the previous day. Another animal is given 50 µl of antitoxin intraperitoneally 4 hours after the skin test to prevent death

• Toxigenically indicated by an inflammation reaction at the injection site progressing to necrosis in 48-72 hours in test animals. No change in the control of animal

In vitro test:

1. Elek’s gel ppt test

2. Tissue culture test

Elek’s gel ppt test:

• - It is an immuno-precipitation test for demonstration of the biological activity of the toxin.

Procedure:

• Using sterile forceps, soak a strip of filter paper in diphtheria antitoxin diluted to 1000 units per ml

• Allow the strip to drain

• Lay the strip in a petri dish. Leave the petri dish in an incubator for drying or for about 20 minutes

• Prepare serum culture medium by adding 3 ml of clear sterile serum to 15 ml sterile cooled (50-55°C) protease peptone agar or Columbia agar.

• Pour the serum agar medium into a petri dish containing an antitoxin strip and allow the medium to set firmly

• Dry the medium at 35-37°C for 20-30 minutes and should not exceed 60 minutes.

• Heavily inoculate test and control microorganism right angle to antitoxin strip and incubate at 35-37°C overnight

• Precipitation lines are seen when viewed through a low-powered hand-magnifying lens

Tissue culture test:

• Eukaryotic cell lines eg: African green monkey kidney, and Chinese hamster ovary are sensitive to diphtheria toxin

• Inocular of test microorganisms in cell culture monolayer

• The toxin produced by microorganisms diffuses and kills cells in the monolayer

Molecular test

• PCR detects toxigenic strains of Corynebacterium diphtheriae from the clinical specimen

• detects diphtherial toxin gene (TOX).

Sckick’s test:

The procedure for Sckick's test for Corynebacterium diphtheriae is as follows:

• Inject 0.1 ml of highly purified toxin into 1 forearm

• Inject 0.1ml of heat-inactivated toxin into another forearm as a control

• Four types of reaction are observed:

  * Positive reaction

  * Negative reaction

  * Pseudoimmune reaction

  * Combined reaction

Positive reaction

• the local inflammatory reaction that reaches maximum intensity in 4-7 days in the arm and reduces gradually

• indicates the absence of an immunity to Corynebacterium diphtheriae

Negative reaction

• Absence of any inflammatory reaction

• indicates the presence of diphtheria antitoxin

• individual immune to Corynebacterium diphtheriae infection

Pseudoimmune reaction

• although the immune, allergic reaction is observed in both the test and control arm

• inflammation reaches a peak in 36 hours and subsides in 72 hours in both arms

• both immune and hypersensitive

Combine reaction

• develops inflammation in the rest arm, which increases the intensity in 4-7 days

• In the control arm, inflammation is seen maximum at 48-72 hours and subside

• the person is not immune and hypersensitive