What is botulism?

Botulism is a rare, life-threatening, neurotoxin-mediated illness caused by the anaerobic bacteria Clostridium botulinum. The neurotoxin inhibits acetylcholine release at the neuromuscular junction, thereby blocking communication with the muscle cells leading to muscular paralysis. These bacteria themselves do not make people ill.

However, exposure to low-oxygen environments, such as the infant's gastrointestinal tract, canned foods or wounds, make the bacteria produce the neurotoxin. There are seven neurotoxin types (A-G), and human botulism is caused by A, B, E and F. Of these, type A causes the most severe symptoms. The incidence of botulism is low; however, the mortality rate increases in the absence of prompt diagnosis and treatment.¹ 

Why is an early diagnosis important?

An early diagnosis is a prerequisite for successful anti-toxin therapy and patient survival. This underscores the importance of disease awareness and rapid laboratory diagnostic tests in addition to understanding the epidemiology of the disease for planning preventive strategies. Diagnosis relies on clinical suspicion and patient history to initiate treatment, followed by mandatory laboratory testing to confirm the source of the toxin and toxin type and rule out diseases mimicking botulism.²

How can you be exposed to the toxin?

Exposure can be via food containing the preformed toxin due to improper handling and sterilisation of home food while canning or preservation (food botulism); via ingestion of raw honey or dust containing the bacteria, followed by colonisation in the gut (infant botulism); colonisation in the wound or use of contaminated needles among drug abusers (wound botulism); injection of toxin at high concentrations for therapeutic or cosmetic purpose (iatrogenic botulism); and intentional contamination of food with the toxin or via aerosolisation (bioterrorism/inhalation botulism).²

Symptoms

• Muscle weakness and acute afebrile symmetric descending flaccid paralysis
• Slurred speech
• Dilated pupils
• Difficulty swallowing and speaking
• Diplopia
• Shortness of breath
• Dry mouth
• Ophthalmoplegia
• Ptosis³

Diagnosis

Culture

Traditionally, the bacteria are grown in liquid culture under anaerobic conditions, and the neurotoxin is detected in the culture supernatant using the mouse lethality assay. Subsequently, the positive samples are streaked onto solid agar plates, followed by further confirmation of toxin formation with the mouse assay.

Clinical matrices, such as faeces or serum are pre-treated with ethanol to recover bacterial spores and eliminate vegetative bacteria. Non-selective media commonly used to isolate C. botulinum include egg yolk agar (EYA), blood agar, tryptone-peptone-glucose-yeast extract medium and cooked meat medium. A lipase reaction is observed on EYA for C. botulinum. EYA supplemented with inhibitory compounds, including cycloserine, trimethoprim and sulfamethoxazole enable the isolation of lipase-positive C. botulinum and inhibition of non-botulinum faecal flora.

Mouse lethality assay

This has been the gold standard in the detection of the neurotoxin. The sample diluted in phosphate buffer is intra-peritoneally injected into mice. The presence of toxins will show the classic signs of botulism, including fuzzy hair, muscle weakness and respiratory failure, characterised by a wasp-like narrow waist. The symptoms appear within 1-4 days. The toxin type is determined by neutralising the toxin with specific anti-toxins. The mice injected with the anti-toxin will survive, whereas others will develop botulism.

The test is sensitive with the detection limit being 0.01 ng/mL of sample eluate. It has been successful with all sample types, such as faeces, food, gastric, serum, wound and bacterial culture supernatants.

The method is time-consuming, expensive and has ethical concerns owing to animal use. False-positive results have been obtained due to tetanus toxin or endotoxins from gram-negative bacteria.

The traditional mouse assay has been improved via intra-muscular injection of low-volume non-lethal neurotoxin. The site of administration is observed for paralysis, and software is utilised to analyse toe-spread reflex changes. The results can be obtained in a day.^1,2

Cell-based assays

Neuronal cells are cultured and samples containing the neurotoxin are added, followed by a demonstration of neurotoxin activity via receptor binding, internalisation and proteolytic activity (protein breakdown) using various methods, including western blotting and cleavage-specific antibodies.

These assays can have higher sensitivity than mouse assays and take around 3 days to complete. It requires sterile samples, which may hamper the testing of food, clinical or environmental samples. Although some assays have received FDA approval, implementation in diagnostic testing is still pending.¹

Immunoassays

Enzyme-linked immunosorbent assay (ELISA)

ELISA is the most common immunoassay used for the detection of botulinum neurotoxins. The neurotoxin present in the sample binds to the solid matrix pre-coated with mono- or poly-clonal capture antibodies against the different toxin types. Subsequently, a second anti-toxin antibody is used to bind the toxin. An anti-toxin molecule carrying horseradish peroxidase or alkaline phosphatase is used for signal production. The sensitivity of conventional ELISA has been improved using biotinylated antibodies, chromogenic diaphorase system and avidin-enzyme conjugate for signal amplification or using enzyme-linked coagulation assay (ELCA).

Samples, such as purified neurotoxin, toxic C. botulinum cultures and foods associated with botulism outbreaks have been tested with ELISA. Food components, including egg yolk and white, chicken meat and milk can interfere with ELCA; however, ELISA has successfully detected toxins in various samples, such as fish fillets, corned beef, potatoes, pasta products and turkey meat. The performance of ELISA samples using clinical specimens, such as faeces or serum is limited. Faeces are known to interfere with and dramatically reduce the sensitivity of ELISA, which has been improved with prolonged incubation times and using foetal bovine serum to block the interfering substances in the faeces.

As ELISA is simple, rapid and easy to perform, it can be used as an initial screening tool in suspected cases of botulism. A drawback is the unavailability of good-quality antibodies.²

Lateral flow assays

Dipstick-type lateral flow assays are useful for toxin testing as they are easy, rapid (<30 min) and require no additional equipment. However, their use is limited because of low sensitivity. These assays are useful as initial screening tools; however, negative results must be confirmed using the mouse assay.²

Immuno-PCR

Immuno-PCR has demonstrated high specificity and sensitivity to detect the different neurotoxins. In this method, the coupling of nucleic acid reporters and detector antibodies is amplified to generate a signal on binding the neurotoxin target. Compared with conventional ELISA, a 10⁵-fold increase in sensitivity is observed upon signal amplification. Complex matrices have not been analysed using this method.¹

Centrifugal microfluidic immunoassays

Neurotoxins are captured on microspheres coated with antibodies, tagged with antibodies labelled with quantum dots and filtered through a density medium to eliminate matrix and inhibiting particles. Fluorescence quantification is performed following excitation with a laser. This method is rapid and can be used with various sample matrices as no sample preparation is required.¹

Nucleic acid testing

PCR is sensitive, rapid, and specific and does not involve animals. It relies on the detection of bot (botulinum neurotoxin gene), but it does not detect gene activity or the toxin. Colonies or liquid cultures are screened for the presence of a bot, thereby indicating the presence of the bacteria. Multiplex PCR assays have simultaneously detected botA, botB, botE, and botF in a single reaction, thereby saving reagent cost, labour and time. The multiplex PCR products are identified using gel electrophoresis or hybridisation onto membranes coated with cDNA probes specific to the PCR products.¹

Endopeptidase assay

This assay is based on the specific zinc endopeptidase activity of the neurotoxin with selected targets in the synaptic cleft. This assay involves the selective breakdown of synaptic proteins, which is detected either by targeting the cleaved peptide or via observing the fluorescence emitted after cleavage of the quenched chromophore-tagged peptide.

This assay can replace the mouse assay as it is more sensitive and detects only the biologically active toxin. It is specific and no cross-reactivity with other botulinum toxins or the tetanus toxin has been reported.²

Genetic characterisation of C. botulinum

Pulse-field gel electrophoresis (PFGE)

Specific restriction enzyme-digested genomic DNA is electrophoresed yielding a unique fingerprint pattern comprising 5-15 fragments with a size range of 10-1000 kbp for each bacterium. This method is useful to diagnose the source of the outbreak by comparing the C. botulinum isolates obtained from patients and suspected food items. Although this method is reproducible and discriminates between different strains, it is time-consuming and is subjected to DNA degradation by DNases.²

Ribotyping

This method involves the analysis of bacterial rRNA gene restriction patterns that are hybridised with labelled cDNA probes designed for Escherichia coli rRNA genes. Compared with PFGE, it is an ideal tool for phylogenetic analysis as it demonstrates significant power to differentiate between bacterial species, although low power to distinguish between strains. This method is reproducible, but DNA degradation may hamper the typing of some strains. RiboPrinter is an automated ribotyping system that is used for C. botulinum.²

Amplified fragment length polymorphism

The genomic DNA is digested using two restriction enzymes, followed by ligation of restriction site-specific adapters and amplification of a subset of fragments using PCR. This method is rapid, reproducible, discriminates between groups I and II C. botulinum strains and is unaffected by DNA degradation.²

Randomly amplified polymorphic DNA (RAPD) analysis

RAPD involves PCR with universal primers that randomly anneal under less stringent conditions. Although this method is easy to perform and rapid, it lacks reproducibility due to the use of non-specific primers that randomly anneal. The discriminatory power is high for group II strains, whereas it is low for group I strains.²

Repetitive element sequence-based PCR

PCR is performed using repetitive extra-genic elements as targets with single or multiple consensus primers, and the PCR amplification products’ number and size produce a species-specific fingerprint. This method is rapid, and unlike RAPD, it is highly reproducible as the primers are specific. This method could discriminate group II type B and E toxin-producing strains to the strain level; in contrast, all group I strains were discriminated only up to the toxin type level in clinical isolates.²

Electrochemical assays and biosensors

Biosensors are coated with SNARE complex proteins, and once it is cleaved by the neurotoxin, the changes in the coating are determined by measuring the electrochemical properties to determine the presence of enzymatically active neurotoxin. These assays are sensitive and results are once the prepared sample is placed in the instrument, the results are obtained within minutes.¹

Differential diagnosis

Botulism can be confused with other similar diseases, including stroke, Guillain–Barré syndrome and myasthenia gravis, which can be differentiated with a brain scan, cerebrospinal fluid examination, and the tensilon test, respectively.⁴

Summary

Awareness of the clinical signs and symptoms consistent with the illness is crucial for prompt anti-toxin treatment or a public health response. Rapid detection of the neurotoxin and the organism producing it is important; thus, the turnaround testing time is crucial while developing laboratory tests.

Efforts have been made to reduce the use of the mouse assay and several molecular biology tools have been developed. The rapidity and sensitivity of these technologies need improvement wherein all seven toxin types are detected in one test. The tests must be validated using various complex matrices, including faeces and food. An attempt should be made to isolate toxin-producing organisms from the patient and the source, enabling our understanding of the epidemiology of the disease and planning preventive strategies.