Abstract

Objective.—This review article is designed to thoroughly familiarize all health care professionals with the history, classification, epidemiology, clinical characteristics, differential diagnosis, diagnostic evaluation (including laboratory-based testing), treatment, and prognosis of botulism. It is especially targeted toward clinical laboratorians and includes a detailed enumeration of the important clinical laboratory contributions to the diagnosis, treatment, and monitoring of patients with botulism. Finally, the bioterrorism potential for botulism is discussed, with an emphasis on the clinical laboratory ramifications of this possibility.

Data Sources.—Included medical periodicals and textbooks accessioned from computerized and manual medical literature searches. More than 1000 medical works published from the 1800s through 2003 were retrieved and reviewed in this process.

Data Synthesis.—Pertinent data are presented in textual and tabular formats, the latter including 6 tables presenting detailed information regarding the clinical parameters, differential diagnosis, diagnostic studies, laboratory testing, and therapeutic approaches to botulism.

Conclusions.—Because botulism is such a rare disease, a keen awareness of its manifestations and prompt diagnosis are absolutely crucial for its successful treatment. The bioterrorism potential of botulism adds further urgency to the need for all health care professionals to be familiar with this disease, its proper evaluation, and timely treatment; the need for such urgency clearly includes the clinical laboratory.

Botulism is a rare disease in the United States and, for that matter, in most of the world. Nonetheless, it warrants a high index of suspicion and ongoing vigilance by the medical community. If not diagnosed and treated in a timely fashion, it results in significant morbidity and mortality. Yet, because of its rarity, most physicians have never seen a case of botulism. Furthermore, in view of the shocking terrorist acts perpetrated in the United States during the fall of 2001 (including the October anthrax attacks), the potential biologic warfare ramifications of botulinum toxin are of great interest to health care workers and the general public; therefore, this topic is also discussed in the following review.

MATERIALS AND METHODS

This review analyzes the world's medical literature relevant to botulism. Computerized (MEDLINE 1966–2001) and multiple manual medical literature searches (Index Medicus encompassing 1986–2003 and the individual bibliographies of more than 500 pertinent articles) were performed, and more than 1000 articles, including some written in languages other than English, were retrieved and reviewed in this process. Some of the non-English articles provided an English abstract, while others did not. Data retrieval from these publications was accomplished by review of available English abstracts and/or actual translation of the reference work. The references cited in the bibliography section represent only a very small fraction of works reviewed for this article and an even much smaller fraction of the total world's literature on botulism; nevertheless, the literature cited herein represents a significant sampling.

HISTORY

Botulism, caused by the strictly anaerobic gram-positive bacillus Clostridium botulinum, was so named in the late 1700s after an outbreak of the disease in southern Germany involving 13 people who had consumed portions of the same large sausage (botulus being the Latin word for sausage).1 By 1817, the German neurologist Justinus Kerner had provided a detailed clinical description of botulism.2 The bacterial agent and toxicologic mechanism of action responsible for botulism were described by Emile van Ermengem in 1895 and published in 18973; van Ermengem thoroughly investigated an 1895 outbreak of the disease in Ellezelles, Belgium, and originally called its etiologic agent Bacillus botulinus.

Ironically, modern medicine is today increasingly utilizing purified botulinum toxin to treat a number of medical conditions characterized by muscle hyperactivity/spasm. These conditions include blepharospasm, strabismus, cervical dystonia, glabellar lines, spastic dysphonia, limb spasticity associated with underlying neurological problems, tremors, chronic anal fissure, hyperhidrosis, and many others. Botulinum toxin therapy is currently officially approved by the Food and Drug Administration for the first 4 of the clinical uses listed (although this list will most likely expand in upcoming years) and is widely used “off label” for numerous other conditions.4,5 

TOXIN TYPES

Clostridium botulinum spores are quite widespread throughout the world, inhabiting soil as well as both freshwater and saltwater mud.1 These very resilient spores are capable of surviving for up to 2 hours at 100°C. Clostridium botulinum organisms elaborate a very potent neurotoxin that is one of the most (if not the most) potent toxins known to man; as little as 10 pg is sufficient to kill a mouse, and the estimated ingested human toxic dose is 1 ng/kg body mass.6–8 This neurotoxin is heat labile and is rapidly inactivated by heating (at 85°C or higher for at least 5 minutes).1 

Clostridium botulinum is a phylogenetically very heterogenous species, actually originally categorized as consisting of 7 different serotypes, each producing an antigenically distinct neurotoxin (designated A, B, C-α, D, E, F, and G; an eighth serotype, called C-β, produces C2 toxin, which is not a neurotoxin and apparently does not cause human botulism).9 These antigenic types are distinguished by means of serologic toxin neutralization tests and serve as valuable epidemiologic markers. Types A, B, and F are coded for by genes located on clostridial chromosomal material.9 The production of neurotoxins C, D, and E is coded for by genes carried by bacteriophages9; the gene responsible for type G neurotoxin is present on a plasmid.9 

The vast majority (approximately 98.5% of cases for which a specific toxin type is established) of human botulism is caused by botulinum toxin types A, B, and E.10–12 Very rare cases of types C, D, and F have been reported to cause disease in humans, although they are much more important causes of botulism in animals other than man, especially birds.13–18 It is not entirely clear whether actual human botulism has been caused by type G toxin, although Sonnabend et al19 did report its identification in association with sudden death in 5 humans. Interestingly, some toxigenic strains of clostridial species other than C botulinum are capable of producing botulinum neurotoxin. These strains are rarely encountered in clinical medicine and include Clostridium butyricum (produces type E toxin) and Clostridium baratii (produces type F toxin).20–30 Indeed, a recent report described 34 cases of toxin type E–producing C butyricum botulism in India.30 Finally, recent RNA/DNA phylogenetic studies have resulted in significant taxonomic change for the genus Clostridium, including reclassification of the type G neurotoxin serotype of C botulinum to a new species, called Clostridium argentinense.31,32 

Mechanism of Toxin Action

Regardless of toxin type, each botulism neurotoxin is a dichain peptide molecule with a molecular mass of approximately 150 000 d.6,9,22,33 Botulinum neurotoxin is actually initially produced as a single inactive polypeptide, which is then cleaved by a clostridial trypsinlike protease into 2 polypeptide chains linked by a disulfide bond; the heavy chain (100 000 d) is responsible for binding onto target neural tissue, while the 50 000-d light-chain subunit is directly responsible for neurotoxic effects.8,33 Remarkably like the tetanus toxin (tetanospasmin) in structure and function, the botulinum neurotoxin is a metalloproteinase that enters nerve cells and blocks neurotransmitter release by zinc-dependent cleavage of protein components of the neuroexocytosis apparatus.8,33 Specifically, botulinum toxin types A and E target a protein known as SNAP-25, and toxin types B, D, F, and G target the protein VAMP/synaptobrevin. Toxin C targets both SNAP-25 and syntaxin.33 Target protein cleavage significantly disrupts release of the neurotransmitter acetylcholine. In its native state, this neurotoxin is bound to nontoxic proteins, which greatly enhances toxin molecular stability.9 Botulinum toxin introduced into the body (potentially by a number of mechanisms but most commonly by absorption from the gastrointestinal tract) is carried throughout the body by the bloodstream. Toxin binds to nerve-ending receptors, becomes internalized within the neuron, and causes an irreversible blockade of cholinergic transmission at the following sites: all ganglionic synapses, all postganglionic parasympathetic synapses, and all neuromuscular junctions. The far-reaching results at these sites include widespread flaccid paralysis and potentially devastating autonomic nervous system perturbations.

Geographical Distribution of Toxin Types

As stated earlier, clostridial organisms producing neurotoxins A, B, and E account for nearly all known human botulism on a worldwide basis; this is certainly also true in the United States. Type A neurotoxin producers are more commonly found west of a line running north to south through the Dakotas, Nebraska, Kansas, Oklahoma, and Texas. Type B producers are found primarily east of this line, while type E producers are found along the shores of the Great Lakes and in Alaska.12,34 Of course, there are exceptions to these generalizations regarding distribution of organisms; type A cases have been reported in the eastern half of the United States, and type B cases have been recorded in the western half.34 

CLINICAL FORMS

Botulism is today divided into 5 clinical forms: classic or food-borne botulism, infant botulism, wound botulism, hidden botulism, and inadvertent botulism.35,36 

Food-Borne Botulism

The oldest recognized form of botulism is the classic or food-borne type. This form occurs after ingestion of food containing preformed neurotoxin, produced by clostridial organisms that contaminate inadequately processed food. The archetypical example of food-borne botulism is that resulting from ingestion of improperly prepared home-canned foodstuffs. Clostridium botulinum spores are quite hardy and may survive the home-canning process at temperatures cooler than 120°C. (This problem is exacerbated by preparing food at high altitudes, where the boiling temperature is <100°C.)36 Foodstuff factors promoting C botulinum spore germination are low acidity (pH > 4.6), low oxygen tension, and high water content. Historically, foods most often implicated in the transmission of botulism have been low-acid, home-canned vegetables (especially corn, beets, carrots, asparagus, and beans), potatoes, and fish; high-acid foods are uncommonly associated with botulism.36 However, food-borne botulism in the United States today is uncommonly due to the ingestion of traditional home-canned foods, but is instead increasingly associated with fermented fish, seal, and whale meats (in Native American communities), as well as mainstream commercially prepared foods consumed at home and in restaurants. Commercially prepared foods that have been implicated include baked potatoes, cheese sauce, garlic oil, and beef stew.36 

Food-borne botulism is still the most common form observed on a worldwide basis, with high-incidence countries including Poland, China, West Germany, France, and the United States.37 In Europe, food-borne botulism is much more commonly associated with meats, especially ham and sausage, than is the case in the United States.37 Since 1980, the most common type of botulism occurring in the United States is not the food-borne type, but rather the infant form.9,35–38 In the 7-year period from January 1, 1990, to December 31, 1996, 123 American cases of food-borne botulism were reported to the Centers for Disease Control and Prevention (CDC) (mean, 17.6 cases per year).36 However, during the year 2002, 28 cases of food-borne botulism were diagnosed in the United States.39 

Infant Botulism

Infant botulism (also called intestinal botulism) was first described in 1976.40–44 Since 1980, it has become the most common form of botulism reported in the United States.36 Between 1976 and 1996, 1442 cases of infant botulism were reported to the CDC, and in 2002, 69 cases were reported.36,39 Unlike food-borne botulism, the infant form is a combination infection/intoxication, including ingestion of C botulinum spores, germination within the gastrointestinal tract, and in vivo production of toxin; these events occur in part due to the lack of a protective gastrointestinal bacterial flora and in part due to the relatively reduced levels of clostridial-inhibiting bile acid as compared to the adult gastrointestinal tract.36 A significant risk factor for the development of infant botulism is honey consumption; 15% to 25% of honey products harbor botulinum spores (especially type B). It is recommended that children younger than 1 year should not be fed honey.36 For more details on the clinical manifestations of infant botulism, see “Clinical Manifestations.”

Wound Botulism

Wound botulism was first described in 1943 and was considered quite rare until 1991, when its recognized incidence increased dramatically. A significant number of new cases involve intravenous drug use; needle puncture sites may become infected with multiple organisms, including C botulinum.36,45–47 Under conditions of tissue necrosis and anaerobiosis, such as those seen in a subcutaneous abscess, C botulinum spores can germinate and produce neurotoxin in vivo, with the same clinical features as those seen in food-borne botulism, except for a lack of acute gastrointestinal signs and symptoms.

Hidden Botulism

Hidden botulism (also known as intestinal colonization botulism) refers to those cases of botulism in adults for which there is no readily apparent source of botulinum toxin. Many of these patients may actually represent an adult form of infant botulism (also known as “adult form of intestinal botulism”). Clostridium botulinum organisms are present within the gastrointestinal tracts of these patients, where they proliferate and produce neurotoxin in vivo.36,48 These patients usually have some abnormality of their gastrointestinal tracts, such as previous surgery, achlorhydria, inflammatory bowel disease, or recent antibiotic therapy exposure.36,48 

Inadvertent Botulism

Inadvertent botulism, the most recent type to be recognized by the medical community, is either an iatrogenic disease (resulting from the therapeutic use of botulinum toxin for any of the medical conditions mentioned in the “History” section)4,5,36,49 or occurring as an accidental exposure in laboratory workers.50 Indeed, the literature includes a recent report of full-blown botulism resulting from therapeutic botulinum toxin use in at least 2 patients.49 Three cases of botulism have been reported in laboratory workers who apparently contracted the disease by inhalation of the toxin; an inhalational route provides a potential basis for the use of botulinum toxin as an agent of bioterrorism or biocrime warfare (see “Biological Warfare and Botulinum Toxin” for more on bioterrorism).50,51 

Clinical Manifestations

The principal features of food-borne botulism are summarized in Table 1.9,36–38,52–54 Wound botulism exhibits almost identical clinical parameters, except that it usually lacks the initial gastrointestinal manifestations of food-borne botulism (such as nausea, vomiting, abdominal discomfort, and diarrhea) and often includes fever and a slightly longer incubation time.45–47,52,53 The clinical features of infant botulism are summarized in Table 2.9,36,40–44 Botulism asserts its dominant clinical manifestations by the emergence of a symmetrical, flaccid paralysis that descends over time from the head to the upper extremities and trunk and, finally, to the lower extremities; its most fearful complication of ventilatory (respiratory) collapse is seen in severe cases of the disease and is caused by paralysis of the diaphragm and intercostal muscles.51 Interestingly, the very first clinical manifestations of botulism almost always include ophthalmologic signs and symptoms, commencing with blurred vision and ptosis in virtually all patients.54 Autonomic nervous system dysfunction, especially cardiovascular lability, is frequently present in botulism and warrants special vigilance when caring for these patients. Cognitive and sensory functions in botulism are nearly always totally spared.55,56 

Table 1. 

Symptoms and Signs of Food-borne and Wound Botulism*†

Symptoms and Signs of Food-borne and Wound Botulism*†
Symptoms and Signs of Food-borne and Wound Botulism*†
Table 2. 

Clinical Features of Infant Botulism*

Clinical Features of Infant Botulism*
Clinical Features of Infant Botulism*

In summary, one can categorize botulism as a rapidly developing, symmetrical, descending paralysis (prominently featuring bulbar palsies) in a patient with intact sensory and cognitive functions who is typically afebrile. Cardinal early manifestations of botulism include blurred vision, ptosis, diplopia, dysarthria, dysphonia, and dysphagia. The differential diagnosis for botulism in adults/ noninfant children and infants is provided in Table 3.1,36,39,54 ,Table 4 summarizes strategies useful in the diagnosis of botulism.9,36,54,57–62 Electrophysiologic studies show that in cases of botulism, nerve conduction velocity is normal and sensory nerve function is normal; however, one sees a pattern of brief, small amplitude motor potentials and facilitation to repetitive stimuli.51,63 These studies are helpful in differentiating botulism from the Eaton-Lambert myasthenic syndrome. Single-fiber electromyography may be especially helpful in some cases.

Table 3. 

Differential Diagnosis for Botulism in Adults/Noninfant Children and Infants*

Differential Diagnosis for Botulism in Adults/Noninfant Children and Infants*
Differential Diagnosis for Botulism in Adults/Noninfant Children and Infants*
Table 4. 

Diagnosis of Botulism*

Diagnosis of Botulism*
Diagnosis of Botulism*

DIAGNOSIS, INCLUDING THE LABORATORY'S ROLE

The diagnosis of botulism is made by a health care professional who is quite familiar with its clinical manifestations and maintains a very high index of clinical suspicion. History and physical examination findings are therefore of paramount importance in the early diagnosis and management of this potentially lethal disease.

General Principles

Nearly all community hospital clinical laboratories (and probably many academic hospital clinical laboratories) lack the capabilities needed to analyze specimens from patients suspected of having botulism. Appropriate samples must therefore usually be sent to a reference laboratory, typically a state health department laboratory facility or the CDC. Laboratory evaluation includes anaerobic culture and toxin assays of appropriate samples. Specimens should be obtained and sent to a reference laboratory after consultation with the state epidemiologist, the state health department, and the CDC (emergency telephone number for the CDC: [770] 488-7100).64 

Initial specimen handling and processing, as would occur at the referring hospital level, should be approached as a biosafety level (BSL) 2 endeavor using a class II biological safety cabinet with the protection of gloves, laboratory coats, and face shield. Specimen types likely to be received include blood (for serum), gastric contents/vomitus, stool or enema material, wound tissue or culture swab, and food material. Wound specimens, tissue, and/ or culture swabs should be maintained and shipped at room temperature, but all other specimen types should ideally be maintained and shipped at 4°C.65 

Clinical Specimens in Suspected Cases of Botulism

Food-Borne Specimens

Demonstration of C botulinum neurotoxin in patient serum and/or gastrointestinal tract or recovery of C botulinum in stool by anaerobic culture constitutes the laboratory's gold standard for diagnosing food-borne botulism. Demonstration of toxin in the suspected foodstuff helps implicate the food as a source in an outbreak, but by itself is only considered indirect evidence to support a suspected case of botulism. Toxin excretion may continue up to 1 month after onset of illness, and stool cultures may remain positive for a similar period.

Infant Botulism

Demonstration of botulinum neurotoxin in patient gastrointestinal tract contents and/or serum, as well as by fecal cultures for C botulinum organisms, constitutes the diagnostic gold standard; toxin is less likely to be detected in serum of infants with this diagnosis.

Infant botulism can generally be excluded by the demonstrated absence of neurotoxin in serum and/or gastrointestinal tract contents and by 2 or more fecal cultures negative for C botulinum.

Wound Botulism

Wound botulism is ideally diagnosed by demonstrating the presence of wound and/or serum neurotoxin, as well as a wound culture positive for C botulinum organisms.

Specimen Types

Ideally, 15 to 20 mL of serum (or at least 2 mL of serum from infants) should be obtained from blood collected in a “tiger”-top or red-top tube; 25 to 50 g of stool (walnut-sized sample or as much stool as possible if from infants) or any return from a sterile water/saline enema; any vomitus material; any wound tissue and/or culture swabs; and suspected foodstuff should be collected. Food should be submitted in its original container. If the original container is not available, the food should be put into a sterile, unbreakable, leak-proof container. In the rare event of a suspect commercial food product, the unopened food container should be sent immediately to the Food and Drug Administration.

Specimen Transport

Clinical specimens should be packaged in an insulated container with refrigerant and transported to the laboratory as quickly as possible. All specimens for isolation of C botulinum should be collected in an anaerobic transport medium. If there is an unavoidable delay in transport, serum or feces may be frozen and shipped on dry ice; freezing clinical specimens will not interfere with toxin detection but may compromise microbiologic culture detection of clostridia.

Toxin Demonstration in Reference Laboratory

Mouse Toxigenicity Assay

The most sensitive and specific test for toxin is still the mouse bioassay.22,61 (Serum from patients with acute inflammatory polyneuropathy can produce paralysis in mice, so the test is not absolutely specific for botulism.66)

In this assay, a culture supernatant or a serum specimen containing neurotoxin is injected intraperitoneally into one mouse, while a second mouse is challenged with the culture supernatant/serum after first being treated with specific antitoxin against botulinum toxin. If the first mouse develops flaccid paralysis and dies, while the second is protected, presence of botulinum toxin is confirmed. It should be noted that the mouse toxigenicity bioassay requires up to 4 days to complete.51,59 

Other Methods of Toxin Determination

In addition to the time-tested mouse neutralization test, newer techniques have been developed during the last 2 decades58,67–73; these tests include in vitro assays, such as gel hydrolysis and the enzyme-linked immunosorbent assay. They are certainly more rapid than the mouse bioassay, and claims have been made that they are equally sensitive and specific. Their use at present is limited and still considered experimental. (Assays are also available to measure antibodies directed against botulism toxins, but these are also restricted to reference laboratories.74,75)

Microbiological Identification of C botulinum

Culture

Clostridium botulinum can be cultured on commercially available anaerobic media, such as anaerobic blood agar, Brucella agar with 5% sheep blood, and phenyl ethyl alcohol blood agar, at 35–37°C in an anaerobic atmosphere.65 

Colonial Morphology

Colonies of C botulinum are gray-white with a circular/irregular edge and are usually β-hemolytic.65 

Gram Stain

Clostridium botulinum is a gram-positive, straight rod (0.3–2.0 μm × 1.5–20 μm) with oval subterminal spores that resemble a tennis racquet. Spores may be demonstrated by phase-contrast microscopy of wet mounts, where they appear mature and refractile. To demonstrate spores, it is helpful to inoculate a chopped meat medium and incubate it anaerobically for 5 to 7 days at 30°C. Organisms from the growth are examined by Gram stain or phase-contrast microscopy.

Spore Selection Techniques

Isolation of C botulinum from highly contaminated specimens such as feces or foods is facilitated by treatment of specimens by heat (80°C) or ethanol.65 Ethanol may be more effective than heat if the specimen contains relatively heat-sensitive serotypes, such as C botulinum type E. For alcohol to be effective, the specimen should be well homogenized.

Presumptive Identification

Clostridium botulinum can be presumptively identified following aerotolerance testing, Gram staining, spore staining, growth on egg yolk agar (lipase-positive, variable for lecithinase activity), carbohydrate fermentation tests, and demonstration of specific volatile metabolic products from chopped meat medium as determined by gas-liquid chromatography.

Definitive Identification

Definitive identification involves demonstration of toxigenicity. The isolate must be transferred to a reference laboratory under anaerobic conditions and examined using the neurotoxin neutralization test.

Table 5 summarizes the clinical laboratory role in diagnosing and managing botulism.

Table 5. 

The Clinical Laboratory's Role in Diagnosing and Managing Botulism

The Clinical Laboratory's Role in Diagnosing and Managing Botulism
The Clinical Laboratory's Role in Diagnosing and Managing Botulism

TREATMENT AND PROGNOSIS

Rapid diagnosis and the availability of well-coordinated, multidisciplinary supportive-care capabilities constitute the most effective measures in the treatment of botulism (whatever its type).22,51 Indeed, the keystone for such supportive care is ventilatory support; protection and control of the airway by intubation and mechanical ventilation may not be necessary in mild cases of botulism, but they should be available and used whenever appropriate because these measures become an absolute necessity in cases of severe botulism. Although it is true that botulinum neurotoxins bind irreversibly to presynaptic endplates and irreparably impair these structures, axons possess a remarkable regenerative capability by which they can sprout new endplates if the patient can be successfully shepherded through the acute botulism crisis.22 Thus, supportive care is generally necessary for several days to several weeks until the patient is capable of survival without these measures. Antibiotics and debridement have very limited but nonetheless bona fide roles, principally in botulism of the wound type, to treat infection and clean up an injury site; however, aminoglycosides, clindamycin, and polymyxin B should especially be eschewed because they have intrinsic neuromuscular blocking properties. Equine-derived antitoxin preparations have a number of possible side effects, including serum sickness and anaphylaxis,76–79 and are not recommended for use in infant botulism but do have a role in the treatment of food-borne and wound botulism in adults and noninfant children, especially if administered within 24 hours of symptom onset.22,76 Recommended adult dose is one 10-mL vial22; this antitoxin is available in the United States from the CDC (emergency telephone: [770] 488-7100).64 A human botulism immune globulin preparation is currently available from the California Department of Health Services (telephone: [510] 540-2646) as an investigational product primarily for use in infants, but also for noninfant patients who cannot tolerate equine-derived products.79  Table 6 summarizes therapeutic strategies used in the treatment of botulism.

Table 6. 

Summary of Multidisciplinary Treatment Parameters for Botulism*

Summary of Multidisciplinary Treatment Parameters for Botulism*
Summary of Multidisciplinary Treatment Parameters for Botulism*

Prognosis

The prognosis for botulism has improved greatly during the past 50 years, coincident with the advent of modern supportive care measures provided in the intensive care setting, especially featuring pulmonary function protection with the mechanical ventilator. Thus, the case fatality rate for food-borne botulism has decreased from the 50% to 70% range (worse for C botulinum neurotoxin type A) to the 5% to 20% range seen today.22,80 Infant botulism and wound botulism currently have case fatality rates of approximately 15% and 1%, respectively.79,81 It should be stressed that survival of an episode of botulism provides essentially no immunity and therefore offers no protection from future bouts of this disease.22 

IMMUNOLOGY OF BOTULINUM TOXIN (IMMUNOGENICITY, ACTIVE AND PASSIVE IMMUNIZATION)

It is well known that the survivors of botulism, even in its severest forms, do not possess effective immunity to further episodes of this disease. Nonetheless, C botulinum neurotoxins are indeed immunogenic,74,75 and the absence of acquired immunity in survivors of botulism can be explained by the fact that disease is caused by an infinitesimally minute amount of toxin (the human lethal dose is estimated to be 1 ng/kg body mass for type A botulinum toxin)7 that is not sufficient to stimulate an effective immune response.82–84 It is possible to produce a botulinum toxoid vaccine by chemically altering comparatively large amounts of toxin; a pentavalent (types A through E) toxoid vaccine is currently available under Investigational New Drug status from the CDC; in the United States, this vaccine is available solely from the CDC and is intended for use in laboratory workers who work with C botulinum and/or its neurotoxins, but is not currently warranted as an immunization measure for the general population due to the rarity of botulism, scarcity of vaccine, and the consideration that widespread vaccination against botulism would most likely vitiate any later patient benefit from therapeutic botulinum toxin administration.9,22,64 As mentioned, equine-derived, polyvalent antitoxin is also available from the CDC to treat botulism.9,64 However, antitoxin effectively neutralizes only those toxin molecules not already bound by neural tissue, and its effectiveness is greatest when given early during the course of the disease (especially the first 24 hours), when much of the toxin remains in the circulation. Furthermore, equine-derived antitoxin carries with it the risk of side effects, the most worrisome of which include serum sickness and anaphylactic reaction. It is therefore recommended that antitoxin be given only after demonstration of a negative skin test for horse-serum sensitivity.9 Currently available are a human-derived antitoxin preparation, as well as a “despeciated” [F(ab′)a] heptavalent (against types A through G) equine-derived antitoxin77,82; the former appears to have special utility for infants (in whom the safety of equine-derived antitoxin has been questioned), as well as for adults who have been sensitized to equine proteins, while the latter is an investigational product held by the US Army.9,22,79,82 

BIOLOGICAL WARFARE AND BOTULINUM TOXIN

Prior to the tragic events of September 11, 2001, the risks and threats of biological warfare and/or bioterrorism seemed quite remote to most Americans. However, it is sobering to consider that botulinum toxin is a potentially effective agent for the mass destruction of human life in biological warfare/bioterrorism contexts and is indeed categorized as a biothreat level A biological warfare agent.51,82,85–100 

A “successful” agent of bioterrorism should be (a) pathogenic to living beings, (b) environmentally stable, (c) effective at low doses, (d) transmissible via aerosol, (e) capable of causing high rates of morbidity and mortality, and (f) difficult to diagnose and/or treat.51,86,87 Botulinum toxin is not only highly lethal after ingestion of minute amounts (in water and/or food), but can also cause disease via the inhalational route and therefore potentially lends itself to biowarfare/bioterrorism activity.51 Indeed, unsuccessful attempts to utilize botulinum toxin in the bioterrorism context occurred in Japan on at least 3 occasions between 1990 and 1995.51 In 1995, Iraq revealed that it had deployed more than 11 000 L of botulinum toxin into specially designed SCUD missiles.90 In the same year, the Aum Shinrikyo cult in Japan prepared botulinum toxin before its attack on the Tokyo subway system, even though it instead chose to deploy the nerve agent sarin; interestingly, botulinum toxin is 100 000 times more toxic than sarin.82 

Although bioterrorism-related botulinum toxin could be introduced into its target population by contaminating food and/or water supplies, these vectors are associated with significant limitations, including logistical difficulties.51,82,90 Therefore, most health care experts and government officials have focused their strongest bioterrorism-related concerns on the inhalational delivery of botulinum toxin into a target population.51 

Inhalational botulism has a dose-dependent onset that varies from 24 to 36 hours to several days after the exposure event. Disease onset typically occurs between 1 and 5 days after exposure; the latent period is longer for lower-dose exposure and, in general, is longer than is the case for ingested toxin.51 It has been suggested that inhaled, aerosolized toxin is typically not identifiable in either stool or serum.91 Finally, it should be emphasized that botulism (in any form, including inhalational) is a noncontagious disease that cannot be transmitted from affected to nonaffected persons, and that the botulinum toxin cannot pass through intact skin.51 

In the event of a biological attack with botulinum toxin, substantial responsibility for the rapid identification of this agent will rest with the staff of the “front-line” clinical laboratory. The clinical laboratory would be expected to provide critical information and expertise in the initial selection, collection, safe handling, and transport of diagnostic specimens and to ensure forwarding of clinical materials to an appropriate reference laboratory with a biosafety capability appropriate for the safe handling of C botulinum and its neurotoxin.

Specimen Types

In the case of bioterrorist attack via the inhalational route, serum, feces, and environmental swabs should be collected for toxin analysis51,86; it has been reported that detectable botulinum toxin may be present on mucous membranes for 24 hours after inhalation.82 Specimens should be collected before administration of antitoxin. In the case of food-borne illness, specimens should be collected as described under “Diagnosis, Including the Laboratory's Role: Specimen Types.”65,99 Environmental swabs should be collected in plastic containers without transport medium. These samples do not require insulation or refrigerant. Further analysis and identification of the organism and its toxin are as described in the “Microbiological Identification of C botulinum” section.

Biosafety Levels for Laboratories Handling C botulinum and Its Toxin

Recommendations and requirements of the various BSLs in clinical microbiology laboratories are described in the latest edition of Biosafety in Microbiological and Biomedical Laboratories.86 These requirements vary for different organisms, and their levels of sophistication increase progressively, depending on the perceived risk of biologic exposure. For C botulinum and its toxin, the recommended BSL for clinical specimen handling is BSL-2. Any procedures resulting in aerosolization of toxin and/or production of toxin necessitate BSL-3 precautions.

BSL-2

BSL-2 requirements state that all microbiologic laboratory procedures that may generate aerosols (such as blending, shaking, or vortexing) should be conducted in a class II biological safety cabinet, with a laboratory coat, disposable surgical gloves, and a face shield. For other requirements, refer to the publication Biosafety in Microbiological and Biomedical Laboratories.86 

BSL-3

The prescribed practice for BSL-3 is that all manipulations for the open culture be carried out in a biological safety cabinet. For other requirements, see Gilchrist et al.94 The botulinum neurotoxin may be inactivated by 0.2 M sodium hydroxide. Clostridium botulinum is inactivated by a 1:10 dilution of household bleach with a 20-minute contact time. For successful inactivation of both organism and toxin, both bleach and sodium hydroxide must be applied for a total of 40 minutes.

Most clinical laboratories function at BSL-2 and are therefore equipped to handle clinical specimens suspected to contain C botulinum organisms and/or toxins. Once the organism is cultured and preliminarily identified, the culture plates and tube must be transferred immediately to a class II biological safety cabinet and the supervisor notified of the potential biosafety hazard. For many small to mid-sized laboratories, shipment of the specimen and culture to a reference laboratory, such as a public health laboratory, would then be appropriate.

The level of risk of exposure to personnel in state and public health reference laboratories is significantly greater since they work with larger numbers of organisms. If aerosolizing and/or production of toxin are carried out, BSL-3 capability is mandatory for these facilities.

In the event of a true bioterrorist release of an agent, the Federal Bureau of Investigation requires a chain-of-custody protocol. Federal Bureau of Investigation personnel may elect to personally escort the clinical specimen or isolate to the receiving reference laboratory, or they may authorize signature-traced shipment by a commercial carrier. All specimens must be packaged, labeled, and shipped in accordance with requirements specified in Biosafety in Microbiological and Biomedical Laboratories with a proper “infectious substance” label.86 

For further information on bioterrorism, the reader is directed to several excellent recent references.51,85–100 The CDC offers invaluable guidance in the evaluation of bioterrorism-related events and can be reached by telephone at (770) 488-7100 and by e-mail at healthalert@cdc.gov on a 24 hour per day, 7 day per week basis.64 

SUMMARY

This review was intended to familiarize the laboratorian with C botulinum and its toxins. The potential biological warfare/bioterrorism implications of botulinum toxin are also discussed, and the interested reader is directed to several pertinent recent medical references.

Although the medical literature on C botulinum and its toxins is truly enormous, the references cited herein provide a meaningful framework for understanding this most fascinating pathogen.

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The authors have no relevant financial interest in the products or companies described in this article.

Author notes

Reprints: James G. Caya, MD, Medical Assessment and Consultation, SC, 8156 Stagecoach Rd, Cross Plains, WI 53528 (jgcayamac@aol.com)