Laboratory diagnosis of botulism: differential and microbiological
Botulism – an acute infectious disease caused by a gram-positive bacterium Clostridium botulinum. The microorganism secretes a toxin that affects the nervous system by blocking the acetylcholine receptors of nerve fibers. As a result, develops paralysis of different muscles. Respiratory disorders, swallowing, myocarditis with botulism can cause death.
Infection most often occurs by eating spoiled canned food containing botulinum toxin or bacteria spores. In the form of spores, a microorganism capable of long time to survive in adverse conditions. Under high temperatures Clostridium die.
The course of the disease and development of complications depend on the severity of symptoms and timing of specific therapy. 20-70% of cases, botulism is fatal.
The method allows to determine the presence of botulinum toxin in biological fluids of the patient. For this control group of laboratory animals (rats, mice or Guinea pigs) are injected intraperitoneally filtrate of the test material.
The second group of animals gets together with the material of protivobotulinicheskoy serum, the third injected the filtrate is pre-heated to 100 0C for 30 min. the Diagnosis is considered established if mice that received untreated leachate die due to paralysis. The creation of the second experimental group is necessary in order to determine the type of toxin.
To determine the pathogen, the biomaterial is placed in a nutrient medium and cultured:
- for the types of bacteria A, B, C, D, F, the optimum temperature is 30-40 0C,
- for serovar E 25-370C,
- for serovar G 30-370C.
It is best to grow clostridia in meat environments with a pH of 7.3 to 7.6. For bacterial cultures peculiar sharp smell of rancid butter. A colony of micro-organism has an irregular shape with multiple processes and branches. In agar, colonies look like lumps of cotton wool with filamentous appendages. On gelatine media, the bacteria initially forming a rounded, transparent colonies, which are then given the spinous processes become brownish, turbid. In broth they look like a compact formation at the bottom, and the liquid becomes colorless.
A single bacterium has the form of sticks with rounded ends with a length of 4,4-8,6 µm and width of 0.3-1.3 µm. Clostridium polymorphic, sometimes there are very short or long and thin bacteria. They are painted by Gram. Bacteria spores are in the shape of a tennis racket.
Microbiological diagnosis of botulism by planting the stool of the patient to the nutrient medium may be in error, as in the human intestine live vegetative forms of clostridia that have similar cultural characteristics. Therefore, it is recommended to combine bacteriological test with other diagnostic methods.
Application ELISA allows rapid diagnosis of botulism. The material for the study is venous blood. Immunological tests are highly accurate.
The method consists in that the blood is added labeled antibodies enzymes. They react with bacterial antigens, forming immune complexes. In this case changes the activity of enzymes and therefore the color of the sample. Technicians then measure the intensity of the color. Comparing the result with the reference data, they conclude that the presence or absence in the blood of specific antibodies to the botulinum toxin.
The method of dot-immunoassay
This analysis on botulism conducted by applying the test material on a nitrocellulose membrane filter containing labeled antibodies protivobotulinicheskoy. The filter is then treated with a developer. If in the biological fluids of the patient was toxin, the areas, which cause the sample will be darkened. The level of intoxication is determined by comparing the result with the control dial.
The advantages of the method lie in the speed – analysis requires no more than 3 hours, and opportunities to explore the botulism contamination not only human biomaterials, but also food.
Peculiarities of diagnostics of botulism
Differential diagnosis of botulism difficult early in the disease when patients show symptoms of food poisoning: vomiting, diarrhea, General intoxication of the organism. But these symptoms quickly give way to neurological disorders. At the initial stage of the disease botulism is suspected can experienced doctor at survey.
It is necessary to conduct dif. the diagnosis of botulism with the following pathologies:
- myasthenia gravis,
- diphtheria polyneuritis,
- viral encephalitis,
- acute violation of cerebral circulation,
- the Guillain-Barre syndrome,
- atropine poisoning,
- syndrome Wernicke – lack of vitamin B1,
- mushroom poisoning.
The main criteria by which to determine botulism:
- the preservation of consciousness;
- the absence of fever;
- symmetrical manifestations of paralysis;
- are only motor function, sensitivityis normal;
- does not change the composition of the CSF;
- not affected psyche.
Great importance is the medical history of the patient. If a few days before the first symptoms people ate mushrooms, homemade preserves, smoked or dried fish, it is impossible to reject the possibility of infection with Clostridium. The incubation period of the disease varies from several hours to 2-3 days, but rarely lasting more than a day.
A sign, allowing to correctly diagnose botulism, is present in blood, urine, feces, vomit of a bacterial toxin.
Additionally, we conducted a General analysis of blood and urine. The findings are nonspecific but help assess the severity of the patient's condition.
Laboratory diagnosis of botulism includes a study of stool, gastric lavage, vomitus, blood, urine and food residue. Modern methods allow for more timely and with high accuracy to determine the disease.