Clostridium botulinum - Tests for diagnosis of botulism: Overview.
Diagnostic tests for botulism
Detection of botulinum toxin in samples or food without culture (see recommended sample in the test section "Test offered by IVAMI and samples required").
This is recommended in a patient with clinical evidence of botulism.
Detecting botulinum toxin can be performed in a liquid such as serum obtained from blood. It can also be detected from the remains of food eaten that has caused a case or an outbreak of botulism. To perform the test using remains of food it is necessary to obtain an extract filtrate from it. In cases of infant botulism or intestinal botulism can be detected the presence of toxin in the feces of children or patients, but consider more advisable to perform a preliminary culture of faeces (see below ). Botulism toxin detection in any of the cases is performed by inoculation in micethat develop paralytic symptoms, followed by death, if inoculated with botulinum toxin. To confirm that the mice have died from the inoculated botulinum toxin, it is necessary a neutralization test, which confirms that there botulinum toxin was present and identifies the type of botulinum toxin present. This neutralization test is performed facing the inoculated product (person or animal serum, extract from ingested food, ...) to specific antisera of each type of toxin.
Prior to the neutralization test step it is necessary to calculate the minimum lethal dose (MLD). Thus we calculate the maximum (highest) dilution which causes the death of the infected animals, and the inoculated volume has caused the death of the animals contain a lethal minimum dose (LMD).
To identify the type of toxin, once known the "Minimum Letal dose", this is faced with different anti-toxin botulinum type antisera. These mixtures will be inoculated in equal volume to experimental mice and those which survive, have been inoculated with the mixture of "minimum lethal dose" plus antiserum to a type that has been able to neutralize.
Detection of toxin in samples / products after preculture (see Recommended sample after in the the test offered by IVAMI and samples required).
When the bacteria Clostridium botulinum, which produces the toxin, can be found in the sample it is recommended prior cultivation, to investigate the toxin after having cultivated the sample. This is the case of feces of a patient with infant botulism or intestinal botulism, the remains of food eaten in which had proliferated bacteria (e.gr., can prerserves), a suspected food that might contain the bacteria (e.gr., a bulged preserves), a food under control to exclude the presence of this bacterium (e.gr., sausages, hams, cans), or other samples such as fresh water sludges or aquatic marine sediments, etc., from areas where mortality has been observed in waterfowl.
During culture in the laboratory, in suitable culture media, the toxin is produced if there is a bacteria and obtaining a culture filtrate can be useful to investigate its presence, inoculating the filtrate to mice. If inoculated mice are affected indicate the likely presence of Clostridium botulinum in the sample o growth in culture. However, mice may have died from another cause, so necessary, before issuing the report, must be check that they have really died for having been inoculated with botulinum toxin.
To confirm the presence of botulinum toxin in the culture neutralization tests using specific antisera for each type of botulinum toxin (neutralization test) can be performed, or detection for the presence of Clostridium botulinum genes and its type in the culture medium (molecular detection by PCR).
Neutralization test is performed facing the inoculated sample (culture filtrate) to specific antisera for each type of toxin.
Prior to the neutralization test step it is necessary to calculate the minimum lethal dose (MLD). Thus the (highest) maximum dilution which causes the death of the inoculated animals is calculated, and the inoculated volume has caused the death of the animals contain a minimum lethal dose.
To identify the type of toxin, once known the "minimum lethal dose", confrontation the minimum lethal dose with different types of anti-botulinum sera is performed. These mixtures will be inoculated in equal volume to experimental mice and those who survive, have been inoculated with the mixture of "minimum lethal dose" plus antiserum to a type that has been able to neutralize.
The molecular detection by PCR avoids the time required for calculating the minimum lethal dose (MLD) and neutralization test.
Detection of anti-botulinum toxin antibodies (see recommended sample after in the section Test offered by IVAMI and samples required)
Detection of anti-botulinum toxin antibodies have interest in the following cases:
- Patients treated with diluted botulinum toxin, such as those receiving botox for aesthetic or medical treatments (e. gr. pain due facial trigeminal neuralgia), to detect the presence of antibodies that prevent its action.
- Patients with suspected infant botulism or adult botulism who has not been able to find the bacterium Clostridium botulinum toxin in feces or serum.
- Vaccinated individuals who are interested check the status of protection.
Clostridium botulinum toxin, at very high dilutions, has been used by local administration to treat spastic processes. These processes have been shown to be a useful remedy. These processes are usually chronic spastic thus requiring that the toxin is administered permanently. Therefore, resistance can arise during treatment due to progressive immunization of the patient throughout the treatment, in which case the effect would be limited. To detect this immunization is required measure accurately and sensitively the existence of antibodies against botulinum toxin A and/or B.
The accepted reference method for detecting and quantifying antibodies to botulinum toxin is the neutralization test in mice (Mouse Neutralization Assay), in which a dilution of botulinum toxin, quantified by the Lethal Dose 50% for mouse (DL50), mixed with various base 2 or base 4 dilutions of the serum/plasma, and after incubation each are inoculated intraperitoneally to groups of mice. The highest dilution of test serum that reduces toxicity correspond to the antibody titer against the corresponding botulinum toxin. This dilution, compared to an international standard allows obtaining results in international units (IU/mL) (1 IU is defined as the amount of antibody that neutralizes 10,000 LD50 of toxins A or B, or 1000 LD50 type E). The amount of toxin used in the tests is one that is neutralized by 0.02; 0.005 and 0.0125 IU/mL of antitoxin for types A, B and E, respectively (Hatheway et al. 1984). Sera that protect the mice titer of 1: 4 are reported as <0.08 IU / mL for type A, or <0.02 IU / mL for type B. The test is laborious, expensive and long duration performing, so alternatives have been sought enmzimoinmunoanálisis based methods (ELISA) using microplates coated with botulinum toxin. However, the values obtained by ELISA sometimes not fully correlate with neutralization test in mice.
Prior to the neutralization test step it is necessary to calculate the minimum lethal dose (DLM) of toxin. The calculation of the minimum lethal dose is performed by base 10 dilutions of the culture filtrate, diluting half with physiological saline to obtain the same dilution as the toxin mixed with serum or plasma from patient, and inoculating each dilution mixture to laboratory mice. Thus the (highest) maximum dilution which causes the death of the infected animals is calculated. The inoculated volume has caused the death of the animals contain a minimum lethal dose (MLD).
Once known the minimum lethal dose, calculate the minimum non-lethal dose (dmnm) corresponding to the minimum amount of toxin in the presence of a constant amount of antitoxin, that donot kill inoculated mice. This amount of toxin is being neutralized by the corresponding units of anti-A antiotoxina or anti-B antitoxin. Minimum non-lethal dose is called because it is the minimum amount of toxin that causes no death of the mice in the presence of antitoxin .