Laboratory Diagnosis of Clostridium botulinum infections

Laboratory Diagnosis

The clinical diagnosis of botulism requires a high degree of clinical suspicion. The diagnosis is suspected in an afebrile patient with progressive descending paralysis, especially in the presence of gastrointestinal manifestations. Laboratory diag-nosis of the condition rests on demonstration of C. botulinum bacilli or toxins:

a)        Diagnosis of food-borne botulism is made by demonstration of C. botulinum in food by culture and by demonstration of toxin in food or feces.

b)       Diagnosis of infant botulism is confirmed by isolation of bacilli and detection of botulinum toxin in feces of the patient.

c)        Diagnosis of wound botulism is made by isolation of or by detection of toxin of botulinum in wound pus and exudates.

◗ Specimens

Feces, vomitus, or gastric aspirate are collected for diagnosis of all the forms of botulism. Tissue from wound is collected for wound botulism.

◗ Microscopy

Gram staining of the smears made from food and other specimens may show Gram-positive spore-bearing C. botulinum.

◗ Demonstration of toxin

C. botulinum toxin can be demonstrated in foods, feces, andother specimens by serum toxin bioassay, enzyme-linked immunosorbent assay, and polymerase chain reaction.

Serum toxin bioassay: The toxin is demonstrated in food orfeces by neutralization test in mice. In this method, food filtrate in sterile saline is inoculated intraperitoneally into two mice. One mouse is protected with polyvalent botulinum antitoxin (control animal) and another is not protected by any antitoxin (test animal). If test animal dies but control animal remains healthy, the test is considered positive and is suggestive of the presence of toxin in the specimen. This is a useful test to dem-onstrate toxin during early stages of food botulism. However, enzyme-linked immunoassays and polymerase chain reaction are still at the experimental stage.

◗ Culture

In food-borne botulism, C. botulinum may be isolated from suspected contaminated food and from the feces by culture. Food culture is performed first by heating the specimens at 80°C for 10 minutes. This is done to destroy all the vegeta-tive forms of the bacteria. The heated specimens are then cul-tured on RCM media and incubated in anaerobic conditions followed by subculture on blood agar. Anaerobic incubation facilitates germination of the spores to vegetative forms of the bacteria.

◗ Identification of bacteria

The identifying features of C. botulinum colonies are summarized.