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Botulism
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Last updated Apr 9, 2012 Agent and Pathogenesis Epidemiology Botulinum Toxin as a Biological Weapon Emergency Response T herapeutic Botulinum Toxin Clinical Features and Dif f erential Diagnosis Laboratory Diagnosis Prevention and Treatment Issues Inf ection Control (Including Autopsies and Burial) Case Def initions and Public Health Reporting Bibliography Agent and Pathogenesis Agent Pathogenesis

Agent Bot ulinum Toxin

Botulism is an intoxication caused by botulinum toxin, which is produced by Clostridium botulinum and, rarely, by other Clostridium species. Seven antigenically distinct toxin types (A, B, C, D, E, F, G) have been identif ied. T he f ollowing are key characteristics of botulinum toxins (CDC 1998, Hatheway 1998, Lacy 1998, Montal 2010, Schiavo 1994, Sneath 1986). Botulinum toxins are the most lethal toxins known. For type A toxin, the toxic dose is estimated at 0.001 mcg/kg (Franz 1997); the lethal dose f or a 70-kg person by the oral route is estimated at 70 mcg, by the inhalational route 0.80 to 0.90 mcg, and by the intravenous route 0.09 to 0.15 mcg (Sobel 2005). T he toxins are identif ied by neutralization with type-specif ic antitoxin; minor cross-neutralization between

types C and D and between types E and F has been observed (Smith 1988). T he toxins are produced by vegetative cells (ie, germination of spores) and released by cell lysis. Some toxins are f ully activated by the bacteria that produce them (proteolytic strains of type A, B, and F), and some require exogenous proteolytic activation (types E and non-proteolytic types B and F). Types A, B, E, and F cause natural disease in humans. T he vast majority of disease is caused by types A, B, and E; type F rarely occurs (ie, about 1% of US cases [Gupta 2005]). In one study, a novel in vivo mouse assay was used to correlate toxin type and dosage with the duration of muscle paralysis f or types A, B, and E (Keller 2006). Botulinum toxin A produced longer paralysis than botulinum toxin B, consistent with human observations. For type A, duration of paralysis was exponentially related to toxin dose; the paralysis time doubled with every 25% increase of the toxin concentration. For type B, the duration of paralysis was linear relative to the toxin dose. Type E toxin had the shortest duration of action, but unlike the other two toxins, the dose of toxin did not inf luence recovery time. Types C and D cause natural disease in birds, horses, and cattle; strains that produce these types reside in the intestinal tract of certain animals. Contaminated silage has been reported to cause botulism outbreaks among cattle (Myllykoski 2008). Toxin type G has never clearly been shown to cause human disease. Toxin types C, D, and G cause botulism in primates when administered through aerosol challenge. As a result of these experiments, experts generally believe that humans also are susceptible to these types. Botulinum toxins are colorless, odorless, and presumably tasteless. Aerosolized particles of toxin are approximately 0.1 to 0.3 mcm in size (Shapiro 1997). T he toxins are inactivated by heating (>85C f or 5 minutes) (Siegel 1993). In the event of an intentional release of botulinum toxin, the causative organisms may or may not be present.

Clost ridium bot ulinum

T he f ollowing are key microbiologic characteristics of C botulinum (CDC 1998, Hatheway 1998, Smith 1988, Sneath 1986). Gram-positive spore-f orming bacillus (may stain poorly) Somewhat varying strain sizes but generally in the range of 0.5 to 2.0 mcm in width and 1.6 to 22.0 mcm in length (CDC 1998) Straight to slightly curved, with a peritrichous f lagellum Spores are oval, eccentric to subterminal, and usually swell the bacterial cell Strict anaerobe "Sluggishly" motile Produce lipase on egg-yolk agar Ferment glucose and liquef y gelatin (all strains)

Commonly isolated f rom soil and marine and lake sediments T he classif ication of C botulinum strains is based on metabolic activity(groups I to IV) and on toxin types (types A to G) (Hatheway 1998, Smith 1988, Sneath 1986): Group I includes type A strains and proteolytic strains of types B and F. Group II includes type E strains and nonproteolytic strains of types B and F Group III includes nonproteolytic strains of types C and D. Group IV includes only strains that produce type G. Strains that produce more than one toxin type or have genetic sequences encoding more than one toxin have been identif ied (Barash 2004, Fathalla 2008, Kirma 2004). Each group has a dif f erent optimal growth temperature, but there are no colonial morphology f eatures that allow distinction between groups or antigenic types. Genetic homology has been demonstrated within antigenic groups of C botulinum , and there is minimal antigenic cross-reactivity between groups. Antimicrobial susceptibilities of C botulinum strains vary somewhat by group, but most strains are susceptible to penicillin, metronidazole, rif ampin, and erythromycin (Smith 1988). C botulinum spores have the f ollowing f eatures (Smith 1988): Spores may survive boiling f or up to 3 to 4 hours or temperatures of 105o C f or 100 minutes. Spores are readily killed by chlorine (either as chlorinated water or as diluted solutions of hypochlorite). Spores undergo maximum germination when activated by heat. For example, type A strains undergo maximum germination by heat treatment (or "heat shocking") at 80C f or 10 to 20 minutes. Spores are resistant to desiccation and can survive in the dry state f or 30 years or more. Spores are resistant to ultraviolet light, alcohols, and phenolic compounds. T hey are relatively resistant to irradiation. Back to top

Pat hogenesis
Exposure to botulinum toxin occurs through the f ollowing mechanisms (toxin is not absorbed through intact skin): Ingestion of pref ormed toxin Inhalation of pref ormed toxin Local production of toxin by C botulinum organisms in the gastrointestinal tract Local production of toxin by C botulinum organisms in devitalized tissue at the site of a wound Iatrogenic exposure caused by injection of botulinum toxin f or cosmetic purposes or to treat certain musculoskeletal disorders, such as spasticity or blepharospasm (Coban 2010) Following exposure, pathogenesis includes the f ollowing steps (Arnon 2001, CDC 1998, Halpern 1995, Schiavo 1995, Simpson 2004): Botulinum toxin is activated by proteolytic cleavage; the activated structure is a 150-kd polypeptide comprising two chains (a heavy chain [100 kd] and a light chain [50 kd]) that are connected by a single

disulf ide bond. Botulinum toxin enters the circulation and is transported to the neuromuscular junction. At the neuromuscular junction, the heavy chain of the toxin binds to the neuronal membrane on the presynaptic side of the peripheral synapse. T he toxin then enters the neuronal cell via receptor-mediated endocytosis. T he light chain of the toxin crosses the membrane of the endocytic vesicle and enters the cytoplasm. Once inside the cytoplasm, the light chain of the toxin (which is a zinc-containing endopeptidase) cleaves some of the proteins that f orm the synaptic f usion complex. T he synaptic proteins, ref erred to as SNARE proteins, include synaptobrevin (cleaved by toxin types B, D, F, and G), syntaxin (cleaved by toxin type C), and synaptosomal-associated protein (SNAP-25; cleaved by toxin types A, C, E) (Arnon 2001). T he clostridial neurotoxin apparently f irst binds to the SNARE complex bef ore cleavage occurs (Breidenbach 2004). T he synaptic f usion complex allows the synaptic vesicles (which contain acetylcholine) to f use with the terminal membrane of the neuron. Disruption of the synaptic f usion complex prevents the vesicles f rom f using with the membrane, which in turn prevents release of acetylcholine into the synaptic clef t. Without neuronal acetylcholine release, the af f iliated muscle is unable to contract and becomes paralyzed. T he blockade of acetylcholine release lasts up to several months; normal f unctioning slowly resumes either through turnover of SNARE proteins within the cytoplasm or through production of new synapses. Death f rom botulism results acutely f rom airway obstruction or paralysis of respiratory muscles. Death also can result f rom complications related to prolonged ventilatory support and intensive care. Botulinum toxin apparently does not cross the blood-brain barrier; theref ore, central nervous system f unctions remain intact. Back to top Agent and Pathogenesis Epidemiology Botulinum Toxin as a Biological Weapon Emergency Response T herapeutic Botulinum Toxin Clinical Features and Dif f erential Diagnosis Laboratory Diagnosis Prevention and Treatment Issues Inf ection Control (Including Autopsies and Burial) Case Def initions and Public Health Reporting Bibliography Epidemiology

Foodborne Botulism Wound Botulism Inf ant Botulism Adult Intestinal Toxemia Botulism Inhalation Botulism Iatrogenic Botulism Botulism Caused by Other Clostridium Species

Foodborne Bot ulism

Foodborne botulism is caused by ingestion of f ood contaminated with pref ormed botulinum toxin and subsequent absorption of toxin through the gastrointestinal tract. T he f ollowing steps are necessary f or a f ood item to cause botulism (CDC 1998): T he f ood item must be contaminated with C botulinum spores, which are normally f ound in soil (and may be f ound in water). T he spores must survive f ood preservation methods. Adequate conditions f or spore germination and neurotoxin production must be present. T he f ood must not be reheated adequately (>85C f or 5 minutes) to inactivate the heat-labile toxin bef ore the f ood is consumed (Siegel 1993). Generally, adequate conditions f or germination and neurotoxin production include the f ollowing, although various caveats exist (CDC 1998, Smith 1988,Solomon 2001): An anaerobic environment Nonacidic pH (generally 4.6 to 4.8; pockets of dif f erent pH may be present within a single f ood source and allow toxin to be produced in a f ood that overall has an acidic pH) Minimum temperature of 10C (the optimum temperature f or growth of proteolytic strains is close to 35C; some nonproteolytic strains of types B, E, and F can produce toxin at ref rigeration temperatures [3C to 4C]) Availability of water with limited solute concentration Toxin types A, B, and E account f or most cases of f oodborne botulism, and toxin types tend to be geographically distributed within the United States. T he outbreaks reported to the Centers f or Disease Control and Prevention (CDC) between 1950 and 1996 (CDC 1998) were distributed as f ollows: 144 (86%) of 167 type A outbreaks occurred west of the Mississippi River 37 (61%) of 61 type B outbreaks occurred east of the Mississippi River 56 (84%) of 67 type E outbreaks occurred in Alaska Type F f oodborne botulism has rarely been reported in humans (CDC 1998, Midura 1972). Botulism can be recurrent, although only a f ew such cases have been reported. One case arose f rom repeated ingestion of home-prepared hot chili pepper sauce (Bilusic 2008). Another report describes recurrent wound botulism among injecting drug users in Calif ornia (Yuan 2011). T he median number of cases of f oodborne botulism reported to the CDC annually between 1973 and 1996 was 24 (range, 8 to 86 cases) (Shapiro 1998). T he mean number of f oodborne botulism outbreaks per year between 1950 and 1996 was 9.4, with a mean number of 2.5 cases per outbreak (CDC 1998).

Between 1990 and 2000, the median number of botulism events per year was 14 (range, 9 to 24) and the median number of cases per event was 1 (range, 1-17) (Sobel 2004). During this time period, the highest incidence rates were in Alaska (19 per million population), Idaho (0.6 per million population), and Washington (0.3 per million population). Improperly home-canned or home-prepared f oods (particularly vegetables) continue to account f or most of the f ood vehicles associated with f oodborne botulism in the United States (Sobel 2004). Over the past 20 years, a wide variety of commercially produced (preserved and nonpreserved) f oods have caused botulism outbreaks. Examples include f oil-wrapped baked potatoes, sauteed onions held under a layer of butter, garlic in oil, commercially produced cheese sauce, commercially prepared chili, hazelnut yogurt, jarred peanuts, matambre (Argentine meat roll) sealed in heat-shrinked plastic wrap, commercially prepared carrot juice, green-olive paste, and canned chili sauce (Angulo 1998, CDC 2007, Chou 1988, Kalluri 2003, MacDonald 1985: Type A botulism f rom sauteed onions, O'Mahony 1990, Pingeon 2011, St Louis 1988, Sheth 2008, Townes 1996, Villar 1999). A variety of salted, f ermented, smoked, and canned f ish sources have been implicated in type E botulism outbreaks in the United States and elsewhere (King 2009, Lindstrom 2006, Sobel 2007, Telzak 1990). Foodborne botulism is a signif icant public health problem among Alaskan natives and is usually associated with consumption of f ermented meat f rom aquatic mammals (eg, whales, seals, walruses, and beavers) and f ish (Fagan 2011, McLaughlin 2004, Shaf f er 1990, Wainwright 1988). T he incidence of disease among Alaskan natives appears to be decreasing but continues to be more than 800 times higher in this population compared with the general US population (Fagan 2011). Occasionally, unusual f ood preparation methods (particularly f or home-prepared products) can lead to botulism. For example, an outbreak in Turkey (eastern Anatolia) in 2005 was associated with eating suzme (yogurt buried under soil) (Akdeniz 2007). Outbreaks of botulism in prisons have been attributed to drinking pruno (an alcoholic beverage concocted by prisoners f rom f ood scraps such as potato peelings and apples that are allowed to f erment unref rigerated) (Vugia 2009). Sales of minimally heated, chilled f oods have grown recently in Western countries, such as the United States and the United Kingdom, and have raised concerns about the potential f or f oodborne botulism (Peck 2006). Waterborne botulism has not been reported, most likely because botulinum toxin is rapidly inactivated by standard treatment of potable water and a very large amount of toxin would be needed to contaminate a water supply because of the dilution f actor (Arnon 2001, Siegel 1993). However, water may serve as a source of contamination f or other f ood items. For example, an investigation by the US Food and Drug Administration (FDA) of a canning f acility in Michigan f ound that some cans of green beans were contaminated with viable neurotoxin-producing C botulinum . Further investigation demonstrated that C botulinum spores were present in the cooling water system (Sachdeva 2010). Back to top

Wound Bot ulism

Wound botulism is caused by inf ection of a contaminated wound with C botulinum and subsequent absorption into the circulation of locally produced toxin. C botulinum is a natural contaminant of soil throughout the United States (Smith 1978). Wound botulism has been recognized with increasing f requency among injecting drug users, particularly in Calif ornia, where the disease has been associated with use of black tar heroin (Davis 2008, MacDonald 1985: Botulism and botulism-like illness in chronic drug users, Passaro 1998, Werner 2000, Yuan 2011). Similarly, in the United Kingdom, bacterial inf ections (particularly wound botulism) have

increased markedly since 2000 among injecting heroin users (Brett 2005: Sof t tissue inf ections caused by spore-f orming bacteria in injecting drug users in the United Kingdom). T he authors of this study observed that the major risk f actor was skin- or muscle-popping. Cases also have been reported in Germany (Preuss 2006, Schroeter 2009) and in Sweden, where real-time polymerase chain reaction (PCR) was used to diagnose a case of type E wound botulism (Artin 2007). Wound botulism in injecting drug users can be misdiagnosed as drug intoxication (Royl 2007); however, presenting f eatures can alert physicians to the correct diagnosis (Sam 2010, Wenham 2008). Botulism should be considered in injecting drug users who present with dysarthria and dysphagia (Preuss 2006). Wound botulism may occur f ollowing traumatic injury to an extremity, such as a compound f racture, laceration, puncture wound, gunshot wound, severe abrasion ("road rash"), or crush injury (Merson 1973, Werner 2000). Sinusitis associated with intranasal cocaine use has been the source of wound botulism in a f ew cases (Kudrow 1988, MacDonald 1985: Botulism and botulism-like illness in chronic drug users, Roblot 2011, Werner 2000). A f ew cases have occurred postoperatively (usually f ollowing intra-abdominal procedures) and an abscessed tooth was the source of C botulinum inf ection in one case (Nystrom 2011, Weber 1993). Between 1943 (when the condition was f irst recognized) and 1985, 33 cases of wound botulism were reported to the CDC. Between 1986 and 1996, 78 cases were reported and most were associated with injecting drug use (CDC 1998). Back to top

Inf ant Bot ulism

Most pediatric cases of botulism occur in inf ants (ie, inf ant botulism), although f oodborne and wound botulism also can af f ect the pediatric population. Inf ant botulism is caused by ingestion of C botulinum spores. T he spores subsequently colonize the gastrointestinal tract, germinate, and produce toxin, which is absorbed into the circulation. Most inf ants are well bef ore illness onset (Wigginton 1993). T he disease characteristically begins with lethargy and poor f eeding (with or without constipation), f ollowed by neuromuscular paralysis, hypotonia, or weakness (Clemmens 2007). Constipation may be subtle or overt. T he source of spores f or most cases remains unknown, although the most common sources of inf ection f or inf ants appear to be honey and environmental exposure (Arnon 1979, Brook 2007, Nevas 2005). Inf ant f ormula was postulated to be the source f or one case (Brett 2005: A case of inf ant botulism with a possible link to inf ant f ormula milk powder). Other risk f actors identif ied in one study f or inf ants 2 months of age and older included breast-f eeding, less than one bowel movement per day in the 2 months bef ore illness onset, and ingestion of corn syrup (Spika 1989). In that study, the only identif ied risk f actor among inf ants less than 2 months old was living in a rural area or on a f arm. Between 1976 (when inf ant botulism was f irst recognized) and 1996, 1,442 cases were reported to the CDC (CDC 1998). Cases were reported f rom 46 states, with Delaware, Hawaii, Utah, and Calif ornia having the highest incidence rates (9.0, 8.8, 6.3, and 5.7 per 100,000 live births, respectively). Almost half of all cases were reported f rom Calif ornia (680 cases; 47.2%). T he mean age at onset was 13 weeks (range, 1 to 63 weeks). Analysis of inf ant botulism cases occurring globally f rom 1996 through 2008 revealed 524 cases in 26

countries representing f ive continents. T he f act that most countries have not reported cases of inf ant botulism suggests that the disorder is underreported, under-recognized, or both, because the organism is present worldwide and cases of f oodborne botulism have been reported in many of these countries (Koepke 2008). Five cases of inf ant botulism caused by C baratii type F have been identif ied; the youngest patient was just 38 hours old at presentation (Barash 2005). A review of charts of inf ant patients in Calif ornia who were treated with the orphan drug Human Botulism Immune Globulin on the basis of clinical presentation but did not ultimately have laboratory-conf irmed botulism (32 of the 681 who were treated) demonstrated that these patients f ell into f ive categories: spinal muscular atrophy type I (f ive patients), metabolic disorders (eight patients), inf ectious diseases (three patients), miscellaneous (seven patients; includes Miller Fisher variant of Guillain-Barre syndrome, neuroblastoma stage III, and cerebral inf arctions, among others), and probable inf ant botulism lacking laboratory conf irmation (nine patients) (Francisco 2007). Back to top

Adult Int est inal Toxemia Bot ulism

T he pathogenesis of intestinal botulism in adults is similar to that of inf ant botulism. Disease is caused by ingestion of C botulinum spores, with subsequent colonization of the gastrointestinal tract. Spores germinate and produce toxin, which is then absorbed into the circulation. Only a f ew cases have been recognized, and most have occurred postoperatively or in adults with underlying pathology of the gastrointestinal tract such as Crohn's disease (Bartlett 1986, Chia 1986, Grif f in 1997, Shapiro 1998, Sheppard 2012). As of early 2012, cases had been reported f rom Canada, Iceland, Italy, Japan, and the United States (Sheppard 2012). Several cases caused by type F toxin produced by C baratii have been reported to the CDC (McCroskey 1991), and cases caused by C butyricum producing type E toxin also have been recognized (Fenicia 1999). A review of type F adult botulism in the United States between 1981 and 2002 demonstrated the f ollowing f indings (Gupta 2005): T hirteen cases of adult type F botulism were reported to the CDC during the study period, representing 1% of US cases. A toxigenic C baratii organism producing type F toxin was isolated in 8 (80%) of 10 positive stool cultures. Type F toxin was identif ied in serum f or nine of the cases. In 5 (42%) of 12 cases, a history of gastrointestinal disease or an invasive gastrointestinal procedure was present bef ore illness onset. Also in 5 (42%) of 12 cases, antimicrobials were reportedly taken bef ore illness onset. A possible f ood source was only identif ied in one instance. Back to top

Inhalat ional Bot ulism

Disease is caused by inhalation of aerosolized pref ormed botulinum toxin with subsequent absorption through the lungs into the circulation. T hree cases of inhalational botulism were reported in 1962 in veterinary technicians in Germany who were working with aerosolized botulinum toxin in animals (Arnon 2001). Symptoms occurred about 72

hours af ter exposure. Inhalational disease also has been produced experimentally in animals. One study, involving primates, demonstrated that illness occurred 12 to 80 hours af ter exposure (Franz 1993). Another study, involving mice, demonstrated that f ollowing inhalational challenge, the maximum concentration of botulinum toxin in blood occurred at 2 hours postexposure (Park 2003). A mouse study characterized the pathological consequences of inhalational botulinum toxin exposure in mice given prophylactic pentavalent (ABCDE) toxoid. T he authors f ound that the mice sustained severe histopathological lung damage despite protection f rom the lethal neurotoxic ef f ects. Signs included "thickening of the alveolar septa and perivascular areas with a generalized spreading interstitial edema and a moderate intra-alveola/intrabronchiola hemorrhage" (Taysse 2005). T hese f indings suggest a direct toxic af f ect of botulinum toxin on lung tissues; however, more research is needed to better def ine this potential ef f ect. Back to top

Iat rogenic Bot ulism

Iatrogenic botulism is caused inadvertently f ollowing injection of botulinum toxin f or therapeutic or cosmetic reasons (Sobel 2005). See the section: T herapeutic Botulinum Toxin f or more inf ormation. Four cases of iatrogenic botulism occurred in December 2004 in Florida f ollowing cosmetic injection with a botulinum toxin that was not approved f or use in humans (see Dec 15, 2004, CIDRAP News story). T he injections contained much higher concentrations of botulinum toxin than the FDA-approved product Botox. A research f irm in Arizona sold the raw botulinum toxin to healthcare practitioners as a Botox substitute. Another report identif ied f our patients who developed iatrogenic botulism af ter receiving therapeutic doses of botulinum toxin f or spasticity and blepharospasm; all recovered (Coban 2010). Back to top

Bot ulism Caused by Ot her Clostridium Species

C butyricum producing type E toxin has been reported to cause intestinal botulism in inf ants and young adults in Italy and f oodborne botulism in Asia (Aureli 1986, Fenicia 1999, Schechter 1999). C baratiiproducing type F toxin has caused intestinal botulism in inf ants and adults; in the latter it is usually associated with gastrointestinal pathology, recent gastrointestinal surgery, or recent use of antimicrobial agents (Barash 2005, Gupta 2005, McCroskey 1991, Schechter 1999). Back to top Agent and Pathogenesis Epidemiology Botulinum Toxin as a Biological Weapon Emergency Response T herapeutic Botulinum Toxin Clinical Features and Dif f erential Diagnosis

Laboratory Diagnosis Prevention and Treatment Issues Inf ection Control (Including Autopsies and Burial) Case Def initions and Public Health Reporting Bibliography Botulinum Toxin as a Biological Weapon Historical Perspective Mechanisms and Outbreak Features Pediatric Considerations

Hist orical Perspect ive

Botulinum toxin poses a signif icant bioweapon threat "because of its extreme potency and lethality; its ease of production, transport, and misuse; and the need f or prolonged intensive care among af f ected persons" (Arnon 2001). However, some experts believe that the potential of botulinum toxin as a bioweapon is limited because of challenges with stabilizing the toxin f or aerosol dissemination (Arnon 2001). Past ef f orts to weaponize botulinum toxin include the f ollowing: T he United States produced botulinum toxin as a potential biological weapon beginning in World War II; however, the US of f ensive biological weapons program ended af ter the 1972 Biological and Toxin Weapons Convention (BT WC). T he f ormer Soviet Union conducted research on use of botulinum toxin as a biological weapon as late as the early 1990s, despite having signed the BT WC. At the time of the Gulf War, Iraq had produced 19,000 L of concentrated botulinum toxin, some of which was loaded into military weapons (Z ilinskas 1997). T he Japanese cult Aum Shinrikyo attempted to use aerosolized botulinum toxin in Japanese cities on at least three occasions between 1990 and 1995. T he C botulinum used in these attempts was collected f rom soil in northern Japan. T hese attacks f ailed because of f aulty microbiological technique, def icient aerosol-generating equipment, or internal sabotage (Arnon 2001). Back to top

Mechanisms and Out break Feat ures

T he two most likely mechanisms f or use of botulinum toxin as a terrorist weapon include deliberate contamination of f ood or beverages or via an aerosol release (Villar 2006). Because f ood products are of ten widely distributed, contamination of a commercially produced f ood or beverage product could result in a high number of casualties and f atalities across the country. In addition, such a bioterrorist act would produce severe civic disruption, economic loss, and social anxiety. According to the CDC, potentially contaminated f ood or beverage items need to be heated at 85C (185F) f or 5 minutes prior to consumption to ensure that toxin is destroyed (CDC: Botulism: control measures overview f or clinicians). Concern has been raised that typical temperatures employed f or pasteurization of commercially available beverage products (such as milk) may not suf f iciently denature all botulinum toxin in the product.

Mathematical modeling suggests that 1 g of botulinum toxin added to commercially distributed milk consumed by 568,000 people could result in 100,000 cases of botulism (Wein 2005). Ten grams of toxin added to the same quantity of milk could result in over 500,000 cases in the exposed population. One study reported that conventional milk pasteurization (63C, 30 min) inactivated botulinum toxin serotype A but did not inactivate botulinum toxin serotype B, indicating that serotype B toxin is potentially heat stable in milk (Rasooly 2010). However, another study f ound that standard high-temperature short-time (HT ST ) pasteurization (heating milk to 72C and holding it steady at this temperature f or at least 15 seconds) inactivates at least 99.99% of botulinum toxin types A and B, suggesting that standard pasteurization conditions would reduce activity of these toxins much more dramatically than originally thought (Weingart 2010). An aerosol release could also lead to high numbers of casualties, although the event would be more localized. Experts have estimated that 1 g of aerosolized botulinum toxin could kill up to 1.5 million people (Shapiro 1997). Aerosolized particles of botulinum toxin are approximately 0.1 to 0.3 mcm in size (Shapiro 1997). Despite these estimates, some experts discount the potential of botulinum toxin as a bioweapon because the toxin may not be very stable in an aerosolized f orm (Arnon 2001). Although contamination of a water supply is f easible, this approach is unlikely since a large amount of toxin would be needed to initially contaminate water. In general, deliberated contamination of water with potential bioterrorism agents may not be very ef f ective f or the f ollowing reasons: dilution of the agent in a large body of water; direct inactivation f rom chlorine or other disinf ectants; nonspecif ic inactivation by other mechanisms (such as hydrolysis, sunlight, or microbes); f iltration; and the relatively small amount of water that is actually ingested f rom the source (Khan 2001). Botulinum toxin is naturally inactivated in f resh water within 3 to 6 days, and toxin is rapidly (within 20 minutes) inactivated by standard potable water treatment (Siegel 1993). A 2005 study f ound that two of seven small-scale water purif ication devices tested were able to ef f ectively eliminate botulinum toxin f rom water. T hose based on f iltration (pore size 0.2 to 0.4 mcm) or irradiation f rom a UV-lamp (254 nm) f ailed to remove the toxin f rom inoculated water. Reverse osmosis and experimental sand f iltration ef f ectively eliminated the toxin (Horman 2005). It is unlikely that therapeutic botulinum toxin could be used in a terrorist attack, because a vial of the currently licensed preparation contains only about 0.3% of the estimated human lethal inhalational dose and 0.005% of the estimated lethal oral dose (Arnon 2001). T he f ollowing f eatures of a botulism outbreak would suggest deliberate toxin release (Arnon 2001). An outbreak involving a larger number of cases than previous outbreaks An outbreak caused by an unusual toxin type (ie, C, D, F, or G) or an outbreak involving type E toxin without an apparent aquatic source Multiple simultaneous outbreaks with or without an apparent source. For aerosol release, cases would not have a common f ood exposure but would have been in a common geographic location during the week bef ore symptom onset Back to top

Pediat ric Considerat ions

In the event of an aerosol release of botulinum toxin, children may be at an even greater level of risk than adults, since children have a higher number of respirations per minute and consequently could have an increased level of exposure to toxin (AAP 2000). Signs and symptoms of botulism in children f ollowing a bioterrorist attack (ie, aerosol or f oodborne exposure) would be similar to those seen in adults. Ensuring adequate intensive care resources f or the pediatric population in the event of a bioterrorism attack involving an agent such as botulinum toxin should be an important priority in bioterrorism preparedness planning. However, these analyses pertain to military uses of botulinum toxin to immobilize an opponent (William C. Patrick, unpublished data, 1998). In contrast, deliberate release of botulinum toxin in a civilian population would be able to cause substantial disruption and distress. For example, it is estimated that a point-source aerosol release of botulinum toxin could incapacitate or kill 10% of persons within 0.5 km downwind (William C. Patrick, unpublished data, 1998). In addition, terrorist use of botulinum toxin might be manif ested as deliberate contamination of f ood. Misuse of toxin in this manner could produce either a large botulism outbreak f rom a single meal or episodic, widely separated outbreaks (Arnon 2001). In the United States, the CDC maintains a well-established surveillance system f or human botulism based on clinician reporting that would promptly detect such events (Arnon 2001). Back to top Agent and Pathogenesis Epidemiology Botulinum Toxin as a Biological Weapon Emergency Response T herapeutic Botulinum Toxin Clinical Features and Dif f erential Diagnosis Laboratory Diagnosis Prevention and Treatment Issues Inf ection Control (Including Autopsies and Burial) Case Def initions and Public Health Reporting Bibliography Emergency Response Botulism Surveillance Botulism Outbreak or Intentional Dissemination Emergency Response to a Mass Exposure International Public Health Concerns

Bot ulism Surveillance

T he CDC maintains an intensive surveillance system f or botulism in the United States. Cases are identif ied through f ollow-up of requests f or botulinum antitoxin.

Cases also may come to detection through requests f or laboratory testing of f ood or clinical specimens. Arrangements f or laboratory testing are made through state public health laboratories. T hese laboratories either have the capability to test specimens directly or they collect and submit specimens to another laboratory f or testing (usually at the CDC). All positive specimens identif ied through state public health laboratories are reported to the CDC on at least an annual basis. All state health departments have 24-hour emergency phone lines f or reporting cases of botulism (CDC: Emergency response). Requests to the CDC f or antitoxin are usually made through the state epidemiology of f ices, although some requests are made directly to the CDC by clinicians caring f or suspect botulism patients. T he authors of a report published in 2012 observed: "T he identif ication of epidemiologic linkages between f oodborne botulism cases is a critical part of diagnostic evaluation and outbreak detection. Investigation of an intentionally contaminated f ood item with a long shelf lif e and widespread distribution may be delayed until an astute physician suspects f oodborne botulism; suspicion of f oodborne botulism occurs more f requently when more than one case is hospitalized concurrently. In an ef f ort to augment national botulism surveillance and antitoxin release systems and to improve f ood def ense and public health preparedness ef f orts, medical organizations and Homeland Security of f icials should emphasize the education and training of medical personnel to improve f oodborne botulism diagnostic capabilities to recognize single f oodborne botulism cases and to look f or epidemiologic linkages between suspected cases" (Newkirk 2012). Back to top

Bot ulism Out break or Int ent ional Disseminat ion

A single case of f oodborne botulism (or botulism f rom an unknown source) is considered an outbreak (MacDonald 1986) and is a public health emergency. Suspected cases should be reported immediately to state or local public health of f icials. Public health of f icials will: (1) assist with appropriate laboratory testing to conf irm the diagnosis, (2) authorize use of antitoxin, (3) conduct aggressive surveillance f or other cases, and (4) immediately begin an epidemiologic investigation to identif y the source or vehicle (such as a contaminated commercial product) or to determine if there is evidence to suggest a bioterrorism-related event. Original specimens should be preserved and their custody documented, pursuant to public health and regulatory investigation procedures as well as potential criminal investigation procedures (ASM 2013). Public health of f icials will coordinate notif ication of local FBI agents as appropriate. If available evidence suggests the potential f or a continued increase in cases while the investigation proceeds, involved hospitals should establish communication networks between the emergency department, the intensive care unit, and those services likely to be involved in managing cases (eg, inf ectious disease, pulmonary, respiratory therapy, critical care, neurology). T hese networks should f ocus on establishing policies and procedures f or handling large numbers of patients (see below). Back to top

Emergency Response t o a Mass Exposure

In the event of a mass exposure, such as a widespread aerosol release of botulinum toxin, the f ollowing steps would be necessary. Rapid administration of antitoxin to ill persons: Although antitoxin does not reverse existing paralysis, once administered it binds to any toxin remaining in the circulation and, theref ore, can mitigate

progression of disease, increase the likelihood of survival, and decrease the duration of mechanical ventilatory support (if respiratory f ailure occurs). Release of antitoxin and coordination of administration would be perf ormed by local/state public health of f icials in conjunction with the CDC. Rapid mobilization of mechanical ventilators: Adequate supportive care resources, including those f or inf ants and children, would be critical to successf ul management of any mass-exposure botulism outbreak. Two articles published in 2009 provide tools f or management of botulism mass casualty incidents. One involves an algorithm f or the evaluation and management of botulism patients in a triage setting (Rega 2009), and the other of f ers a short questionnaire that can assist with screening of potential casualties (BurkholderAllen 2009). Back to top

Int ernat ional Public Healt h Concerns

A large outbreak in T hailand (209 cases) in 2006 emphasized the need f or addressing global policy issues concerning outbreaks in developing countries, including health inf rastructure, communication and response systems, stockpiles of medication and supplies, decision algorithms f or notif ication, and international response to public health emergencies (Ungchusak 2007). Back to top Agent and Pathogenesis Epidemiology Botulinum Toxin as a Biological Weapon Emergency Response T herapeutic Botulinum Toxin Clinical Features and Dif f erential Diagnosis Laboratory Diagnosis Prevention and Treatment Issues Inf ection Control (Including Autopsies and Burial) Case Def initions and Public Health Reporting Bibliography T herapeutic Botulinum Toxin T herapeutic Uses Special Considerations

Therapeut ic Uses
Patients with a range of spastic or autonomic neuromuscular disorders may benef it f rom small amounts of purif ied botulinum toxin injected into af f ected muscles (Schantz 1992). T here are two types of therapeutic botulinum toxin: purif ied botulinum toxin type A (Botox, produced by Allergan, Inc) (Allergan,

Inc) and purif ied botulinum toxin type B (Myobloc, produced by Elan Pharmaceuticals, Inc) (FDA: Myobloc labeling inf ormation). Examples of conditions that can be treated with botulinum toxin include: Spasmodic torticollis Strabismus Blepharospasm Laryngeal dystonia Focal dystonias of the hand Limb spasticity Hemif acial spasm Cerebral palsy Migraine headache Hyperhydrosis (severe underarm sweating) Post-stroke spasticity Urinary incontinence in adults with overactive bladder caused by neurologic disease In April 2002, the FDA approved use of botulinum toxin type A f or cosmetic purposes ( Allergan, Inc, FDA: Botox Cosmetic labeling inf ormation). Back to top

Special Considerat ions

T herapeutic botulinum toxin contains about 0.3% of the estimated lethal human inhalational dose and only 0.005% of the estimated lethal human oral dose; theref ore, this f orm of toxin is not likely to be used as a bioterrorist weapon (Arnon 2001). However, iatrogenic cases of botulism have been reported. A report published in 2010 identif ied f our patients who developed iatrogenic botulism f ollowing treatment with botulinum toxin f or musculoskeletal disorders (Coban 2010). One patient required intensive care, but all f our survived. An unlicensed, highly concentrated preparation of botulinum toxin caused botulism in f our adult patients undergoing cosmetic procedures. Af f ected patients may have received doses 2,857 times the estimated human lethal dose by injection. Pretreatment serum levels in three of the f our patients were f rom 21 to 43 times the estimated human lethal dose (Chertow 2006). Following protracted hospital courses, prolonged mechanical ventilation, and physical rehabilitation, all f our of these patients survived. Back to top Agent and Pathogenesis Epidemiology Botulinum Toxin as a Biological Weapon Emergency Response T herapeutic Botulinum Toxin

Clinical Features and Dif f erential Diagnosis Laboratory Diagnosis Prevention and Treatment Issues Inf ection Control (Including Autopsies and Burial) Case Def initions and Public Health Reporting Bibliography Clinical Features and Dif f erential Diagnosis Clinical Features Dif f erential Diagnosis

Clinical Feat ures

Botulism is characterized by acute af ebrile descending symmetric paralysis. Recovery occurs over weeks to months and of ten requires extensive supportive care. Disease generally begins with evidence of cranial nerve dysf unction and then progresses to muscle weakness (proximal muscle groups are af f ected f irst and may be more severely involved). Severity of disease ranges f rom mild cranial nerve dysf unction to complete f laccid paralysis. Paralysis of pharyngeal or respiratory muscles may result in the need f or prolonged mechanical ventilation. Severity of disease correlates with the amount of toxin absorbed into the circulation. Several studies have shown that a shorter incubation period correlates with more severe disease (MacDonald 1985: Type A botulism f rom sauteed onions, Tacket 1984). Similarly, a study of botulism cases in Japan revealed that patients who had shorter incubation periods had a signif icantly higher risk of death (Nishiura 2007). Disease caused by toxin type A tends to be more severe than disease caused by toxin type B or E (Shapiro 1998). Among more than 200 patients in an outbreak in T hailand, respiratory f ailure was less likely to develop in those who did not manif est nausea or vomiting and did not have urinary retention requiring catheterization. Nausea or vomiting and any cranial neuropathy with urinary retention or dif f iculty swallowing were symptoms most predictive of respiratory f ailure (Wongtanate 2007). One study of injecting drug users who had wound botulism f ound that longer time f rom presentation in the emergency department to administration of antitoxin and longer time f rom presentation to wound drainage were independently associated with increased length of stay in intensive care (Of f erman 2009). Death can result f rom airway obstruction or paralysis of respiratory muscles. Death also can result f rom complications related to prolonged ventilatory support and intensive care, such as aspiration pneumonia and other inf ectious conditions. Bef ore mechanical ventilation was widely available, the case-f atality rate was about 60% (Shapiro 1998). T he case-f atality rate currently is low owing to adequate supportive care; overall the rate is 5% to 10% f or f oodborne disease and somewhat higher f or wound botulism (Shapiro 1998, Werner

2000). In the event of a mass exposure (such as a bioterrorism attack), clinical resources could be overwhelmed rapidly and the case-f atality rate could be much higher. A retrospective study of hospitalized f oodborne botulism cases in the Republic of Georgia, 19802002, f ound that patients with shortness of breath and impaired gag ref lex and without diarrhea were 23 times more likely to die than were patients without this syndrome (Varma 2004). In this case series, the incubation period was similar among those who died and those who survived, as was the likelihood of receiving antitoxin.
Clinica l Fe a t ure s o f Fo o dbo rne a nd Wo und Bo t ulis m

Cha ra ct e ris t ic

Fe a t ure s

Incubation perioda

Dependent on level of toxin exposure For f oodborne botulism, 2 hr8 days For wound botulism, 4-14 days Unknown f or inhalational botulism; estimated to be 24-36 hr; the only three reported cases in humans had an incubation period of 72 hr Nausea (88%)c Dry mouth (82%) Blurred vision (78%) Dysphonia (76%) Dysphagia (75%) Weakness (72%) Fatigue (69%) Dyspnea (65%) Dysarthria (63%) Double vision (60%) Dizziness (56%) Vomiting (52%)c Constipation (related to autonomic dysf unction) (45%) Sore throat (40%) Abdominal cramps or abdominal pain (40%)d Diarrhea (35%)c Paresthesias (29%)

Symptoms (compiled f rom reports of f oodborne botulism outbreaks caused by toxin types A, B, and E)b

Signs (compiled f rom cases of types A and B botulism reported to CDC in 1973 and 1974)d

Alert mental status (90%) Weakness of upper extremities (75%) Ptosis (73%) Weakness of lower extremities (69%) Extraocular muscle weakness (65%) Diminished gag ref lex (65%) Facial nerve dysf unction (63%) Dilated or f ixed pupils (44%) Diminished or absent deep tendon ref lexes in af f ected groups (40%) Nystagmus (22%) Ataxia (17%) Other considerations: ~Patients generally af ebrile ~Mental status generally intact, although patients may appear lethargic or have dif f iculty communicating because of bulbar dysf unction ~Sensory exam generally normal Normal CSF glucose, protein, cell count Normal CBC Normal imaging of brain and spine (ie, CT scan or MRI) Characteristic EMG f indings e: ~Incremental response (f acilitation) to repetitive stimulation (not always present and of ten seen only at 50 Hz) ~Short duration of motor unit potentials (MUPs); polyphasic MUPs ~Decreased amplitude of compound muscle action potentials (CMAPs) af ter a single nerve stimulus (most prominent in proximal muscle groups) ~Normal sensory nerve f unction ~Normal nerve conduction velocity (motor and sensory) Respiratory f ailure (which may require prolonged ventilatory support); in some outbreak settings, up to 30%-40% of patients required mechanical ventilation Aspiration pneumonia (among patients with respiratory f ailure)f Residual f atigue, dry mouth or eyes, dyspnea on exertion up to several years af ter initial presentationg 5%-10% f or f oodborne botulismi 15%-44% f or wound botulismj

Laboratory f eatures

Complications

Case-f atality rateh

Back to top

Dif f erent ial Diagnosis

Dif f e re nt ia l Dia gno s is o f Bo t ulis m

Co ndit io n

Fe a t ure s t ha t dis t inguis h e a ch co ndit io n f ro m bo t ulis m a

Guillain-Barre syndrome (GBS) (particularly Miller Fisher variant)

Classic GBS results in ascending paralysis Miller Fisher variant may be descending and may have pronounced cranial nerve involvement; it usually includes a triad of ophthalmoplegia, ataxia, and aref lexia (5% of GBS cases are of the Miller Fisher variant)b Abnormal CSF protein 1-6 wk af ter illness onset (although may be normal early in clinical course) Paresthesias commonly occur (of ten stocking/glove pattern) EMG shows abnormal nerve conduction velocity; f acilitation with repetitive nerve stimulation does not occur (as with botulism) History of antecedent diarrheal illness (suggestive of Campylobacter inf ection, which accounts f or about one third of GBS cases) Outbreaks of GBS do not occur (unlike botulism) Dramatic improvement with edrophonium chloride (ie, a positive Tensilon test), although some botulism patients may exhibit partial improvement f ollowing administration of edrophonium chloride (ie, a borderline Tensilon test) EMG shows decrease in muscle action potentials with repetitive nerve stimulation Ascending paralysis Paresthesias are common Caref ul examination reveals presence of tick attached to skin Recovery occurs within 24 hr af ter tick removal EMG shows abnormal nerve conduction velocity and unresponsiveness to repetitive stimulation Usually does not involve cranial nerves Commonly associated with carcinoma (of ten oat cell carcinoma of lung) Although EMG f indings are similar to those in botulism, repetitive nerve stimulation shows much greater augmentation of muscle action potentials, particularly at 20-50 Hz Increased strength with sustained contraction Deep tendon ref lexes of ten absent; ataxia may be present Usually does not involve cranial nerves Paralysis usually asymmetric Brain imaging (CT or MRI) usually abnormal Sensory def icits common Altered mental status may be present Febrile illness CSF shows pleocytosis and increased protein Altered mental status may be present Paralysis of ten asymmetric History of shellf ish (ie, clams, mussels) or puf f er f ish ingestion within several hours bef ore symptom onset Paresthesias of mouth, f ace, lips, extremities commonly occur History of recent exposure to belladonna-like alkaloids Fever Tachycardia Altered mental status History of recent exposure to aminoglycoside antibiotics More likely to occur in the setting of renal insuf f iciency Most commonly seen with neomycin Most commonly associated with other neuromuscular blocking agents such as succinylcholine and paralytics

Myasthenia gravis

Tick paralysis c

Lambert-Eaton syndrome

Stroke or CNS mass lesion

Poliomyelitis

Paralytic shellf ish poisoning or ingestion of puf f er f ish Belladonna toxicity

Aminoglycoside toxicity

Other toxicities (hypermagnesemia, organophosphates, nerve gas, carbon monoxide)

History of exposure to toxic agents Carbon monoxide toxicity: altered mental status may occur, cherry-colored skin Hypermagnesemia: history of use of cathartics or antacids may be present, elevated serum magnesium level Organophosphate toxicity: f ever, excessive salivation, altered mental status, paresthesias, miosis CNS inf ections (particularly brainstem inf ections) Inf lammatory myopathy Hypothyroidism Diabetic neuropathy Viral inf ections Streptococcal pharyngitis (pharyngeal erythema and sore throat can occur in botulism owing to dryness caused by parasympathetic cholinergic blockade)

Other conditions

Back to top Agent and Pathogenesis Epidemiology Botulinum Toxin as a Biological Weapon Emergency Response T herapeutic Botulinum Toxin Clinical Features and Dif f erential Diagnosis Laboratory Diagnosis Prevention and Treatment Issues Inf ection Control (Including Autopsies and Burial) Case Def initions and Public Health Reporting Bibliography Laboratory Diagnosis Specimen Collection and Transport Laboratory Biosaf ety Laboratory Response Network Diagnostic Tests f or Detection of Botulinum Toxin and C botulinum

Specimen Collect ion and Transport

Specimen collection and transport procedures f or testing related to diagnosing botulism are outlined in the f ollowing table.

Collect ion and Transport of Laborat ory Specimens f or t he Diagnosis of Bot ulism

Spe cim e n

Clinica l Indica t io n

Co lle ct io n a nd T ra ns po rt

Serum

Intentional release, f oodborne botulism, autopsy specimens

Collect >20 mL whole blood bef ore administration of antitoxin using red-top or separator tube (no anticoagulant) Ship >10 mL serum at 4 o C Do not ship whole blood, which tends to become hemolyzed during transit Notif y testing lab if patient has received "stigmine drugs" or a Tensilon test Keep specimen ref rigerated at all times Collect 30 cc whole blood (bef ore antitoxin administration) Ship at 4o C Sera submitted f or toxin detection should not be hemolyzed Notif y testing lab if patient has received "stigmine drugs" or a Tensilon test Keep specimen ref rigerated at all times Collect exudate, tissue, or swabs Ship at room temperature in anaerobic transport system Obtain 10-50 g of stool (as little as "pea-size" f or inf ant botulism); transport at 4o C Enema f luid (20 cc) can be collected as an alternative to stool, using minimal amount of sterile nonbacteriostatic water; ship at 4o C Intestinal f luid collected at autopsy (20 cc); ship at 4o C Collect within 72 hr of symptom onset Obtain 20 cc of vomitus; ship at 4o C Obtain 20 cc of gastric f luid (living cases or at autopsy); ship at 4o C Serum, according to methods outlined above Contents f rom dif f erent sections of small and large intestines (10 g per sample in separate containers) Gastric contents as indicated, according to methods outlined above Tissue samples as indicated, according to methods outlined above Obtain 10-50 g of implicated or suspect f ood; ship at 4o C in original container Place individually in leak-proof sealed transport devices Obtain anaerobic swab; ship at room temperature

Wound botulism (critical specimen f or conf irmation)

Wound/tissue

Wound botulism

Stool, enema f luid, intestinal f luid

Intentional release, f oodborne botulism, inf ant botulism, wound botulismb

Gastric f luid, vomitus

Foodborne botulism, intentional release

Specimens to collect at autopsy

Intentional release, f oodborne botulism, inf ant botulism

Food samples (epidemiologically implicated) Nasal swab

Intentional release, f oodborne botulism, inf ant botulism

Intentional releasec

Environmental sample

Intentional release, inf ant botulism

Collect as appropriate: ~Environmental swab; ship at room temperature ~Soil (50-100 g) ~Water (>100 mL)

2001
b A wound may not be the actual source of inf ection/intoxication. cToxin may be present on nasal mucosa f or up to 24 hr af ter inhalational exposure (Franz 1997).

Guidelines have been published f or packing and shipping of inf ectious substances, diagnostic specimens, and biological agents f rom suspected bioterrorism (ASM 2012). C botulinum is classif ied under World Health Organization (WHO) risk group 4. Cultures that are reasonably suspected to contain C botulinum must be transported as "inf ectious substances." In addition, the US Department of Transportation (DOT ) regulations and International Air Transport Association (IATA) rules require training of all individuals involved in the transport of dangerous goods, including inf ectious substances (DOT 2008, IATA 2012). Once botulinum toxin is identif ied, samples may be regulated as select agents and subject to additional transport requirements (see below). Chain of custody should be documented f or material that may constitute evidence of criminal activity. Back to top

Laborat ory Biosaf et y

Botulinum toxin and Clostridium species that produce botulinum toxin are classif ied as select agents and theref ore are regulated under 42 CFR part 73 (Possession, Use, and Transf er of Select Agents and Toxins), which was published in f inal f orm in the Federal Register in March 2005 (HHS 2005). As specif ied in the Public Health Security and Bioterrorism Preparedness and Response Act of 2002, 42 CFR part 73 provides requirements f or laboratories that handle select agents (including registration, security risk assessments, saf ety plans, security plans, emergency response plans, training, transf ers, record keeping, inspections, and notif ications). C botulinum toxin detection should be perf ormed only by trained individuals at laboratory response network (LRN) ref erence or higher laboratories. Sodium hypochlorite (0.1%) or sodium hydroxide (0.1 N) inactivate the toxin and are recommended by the CDC f or decontaminating work surf aces and spills of cultures or toxin (CDC 2009). Biosaf ety recommendations f rom the FDA f or laboratories that test f or C botulinum include the f ollowing (Solomon 2001): Place biohazard signs on doors to restrict entrance and keep the number of people in the laboratory to a minimum. All workers should wear laboratory coats and saf ety glasses. Never pipette anything by mouth; use mechanical pipettes. Use a biohazard hood f or transf er of toxic material if possible. Centrif uge toxic materials in a hermetically closed centrif uge with saf ety cups. Personally take all toxic material to the autoclave and see that it is sterilized immediately. Do not work alone in the laboratory or animal rooms af ter hours or on weekends. Have an eye wash f ountain and f oot-pedaled f aucet available f or hand washing. Allow no eating or drinking in the laboratory. In a very visible location, list phone numbers where therapeutic antitoxin can be obtained.

In a very visible location, list phone numbers where therapeutic antitoxin can be obtained. Reduce clutter in the laboratory to a minimum and keep all equipment and other materials in their proper place. Back to top

Laborat ory Response Net work

T he LRN is a network of more than 150 national and international laboratories. T he network includes f ederal, state and local public health, military, f ood testing, environmental, veterinary, and international laboratories (CDC: Facts about the Laboratory Response Network, CDC: T he Laboratory Response Network). T he LRN structure f or bioterrorism designates laboratories as sentinel, ref erence, or national. Designation depends on the types of tests a laboratory can perf orm and how it handles inf ectious agents to protect workers and the public. Sentinel laboratories, formally called level A laboratories represent an estimated 25,000 hospitalbased laboratories that have direct contact with patients. In an unannounced or covert terrorist attack, sentinel laboratories could be the f irst f acilities to encounter suspicious specimens. T hese laboratories generally have at least BSL-2 containment capabilities. A sentinel laboratory's responsibility is to rule out B anthracis or ref er a suspicious sample to the nearest LRN ref erence laboratory. Sentinel laboratories use the ASM [American Society f or Microbiology] Sentinel Level Clinical Microbiology Laboratory Guidelines to rule out microorganisms that might be suspected as agents of bioterrorism (ASM). Reference laboratories, sometimes ref erred to as "conf irmatory ref erence," can perf orm tests to detect and conf irm the presence of a threat agent. T hese laboratories ensure a timely local response in the event of a terrorist incident. Rather than having to rely on conf irmation f rom laboratories at the CDC, ref erence laboratories are capable of producing conclusive results; this allows local authorities to respond quickly to emergencies. T hese are mostly state or local public health laboratories but also include military, international, veterinary, agriculture, and f ood- and water-testing laboratories. Ref erence laboratories operate with BSL-3 containment f acilities that have been given access to nonpublic testing protocols and reagents. One of the roles of the LRN ref erence laboratories is to provide guidance, training, outreach, and communications to the sentinel laboratories in their jurisdictions. National laboratories have unique resources to handle highly inf ectious agents and the ability to identif y specif ic agent strains through molecular characterization methods. T hese laboratories also are responsible f or methods development, biof orensics, and select-agent activity. Back to top

Diagnost ic Test s f or Det ect ion of Bot ulinum Toxin and C bot ulinum
According to the ASM guidelines, LRN sentinel laboratories "should not attempt to culture, identif y the organism, or attempt to perf orm toxin analysis." Furthermore, LRN sentinel laboratories should not accept environmental or animal specimens; such specimens should be f orwarded directly to the state health department laboratory (ASM 2012). Only certain LRN ref erence laboratories have the capability to perf orm mouse bioassay testing. T he mouse bioassay is currently the primary diagnostic method used f or detection and identif ication of botulinum toxin. Other methods (see below) are still considered investigational. Mice are injected intraperitoneally with the patient sample, stool or f ood extract, culture f iltrate, or other sample and observed f or up to 4 days. Control mice are injected with a mixture of the sample combined with neutralizing antibody to

dif f erent toxin types. Signs of botulism intoxication usually are evident in 6 to 24 hours. As little as 0.03 ng of toxin can be detected by this method (CDC 1998, Shantz 1992). One report of a cohort of clinically def ined wound botulism cases f ound that the serum mouse bioassay was only 68% sensitive in conf irming inf ection (Wheeler 2009). T he authors pointed out that physicians should be aware of the test's limitations and base their f inal diagnosis on clinical criteria when the mouse bioassay produces negative results. Culture f or C botulinum f or stool or gastric specimens has been used f or diagnosis, in addition to toxin testing (CDC 1998). Isolates are tested f or neurotoxin by the mouse bioassay. An activation step with trypsin is required to detect toxin f rom some group II strains. Isolation of C botulinum f rom stool or a wound is considered diagnostic in patients with signs and symptoms of botulism. Nasal swabs could potentially be collected in the event of an aerosol exposure (CDC: Specimen selection table, Franz 1997). As with other types of potential bioterrorism exposures, the sensitivity and diagnostic value of nasal culture is unknown. Nasal swabs should only be used as part of an epidemiologic investigation or on the basis of recommendations made by the CDC in the event of a bioterrorist attack. Serological assays f or botulinum toxin antibody are not usef ul as a measure of exposure, which does not typically induce an antibody response. Detailed methods f or testing f ood samples have been published by the FDA's Center f or Food Saf ety and Applied Nutrition (CFSAN) (Solomon 2001). Detection of botulinum toxin in an epidemiologically implicated f ood item conf irms the diagnosis of botulism. Since C botulinum is widely distributed in nature, the organism may be present in f ood without producing toxin or causing disease. T heref ore, positive culture results f rom f ood, in the absence of detectable toxin, must be interpreted within the context of other epidemiological f indings. Pulsed-f ield gel electrophoresis (PFGE), randomly amplif ied polymorphic DNA analysis, and automated ribotyping methods have been compared f or epidemiological typing of C botulinum type E using clinical and f ood isolates associated with f our botulism outbreaks that occurred in the Canadian Artic. A modif ied PFGE protocol was judged to be the most usef ul method f or typing epidemiologically related type E strains, based on its ability to type all strains reproducibly and with an adequate level of discrimination (Leclair 2006) Investigators have identif ied high-af f inity monoclonal antibodies (mAbs) that specif ically bind botulism toxins type A and B. T hese have been used to develop highly sensitive sandwich immunoassays, which appear to be promising alternatives to the mouse bioassay (Scotcher 2010, Stanker 2008, USDA 2009). A "ruggedized" real-time PCR assay called R.A.P.I.D. f or use by f irst-responders and in military f ield hospitals and other rough environments is commercially available but not FDA approved (Idaho Technology). Other tests f or botulinum toxin (considered investigational): Other enzyme-linked immunoassays (ELISA) (Dezf ulian 1991, Ferreira 2001, Ferreira 2003, Wictome 1999) An immuno-PCR assay that measures antigen-antibody reactions using a conjugated reporter DNA molecule f ollowed by PCR amplif ication (Chao 2004) Time-resolved f luorescence assays f or C botulinum A/B neurotoxin (Peruski 2002) Matrix-assisted laser desorption/ionisation-time of f light mass spectrometry (MALDI-T OF MS) (Barr 2005, Cruzan 2006, Darby 2001, Wilkes 2006)

An optical immunoassay f or rapid detection of neurotoxins A, B, E, and F (Ganapathy 2008) A micromechanosensor f or detection of botulinum toxin type B (Liu 2003) Lateral f low devices f or environmental testing (Alexeter Technologies,New Horizon Diagnostics, Osborn Scientif ic Group) A botulinum neurotoxin serotype A assay with a large immuno-sorbent surf ace area (BoNT /A ALISSA) that captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a f luorogenic substrate (Bagramyan 2008) Other tests f or C botulinum (organism) PCR assays have been used f or the detection of C botulinum toxin genes in animal, f ood, and f ecal samples (Craven 2002, Dahlenborg 2001, Fenicia 2007, Franciosa 1994, Lindstrom 2001). One report published in 2009 described a set of real-time PCR tests f or detecting botulinum neurotoxin genes f or A, B, E, and F toxins produced by C botulinum , C baratii, and C butyricum (Fach 2009). PCR-based assays detect genetic sequences of the organism, not the toxin molecule itself . T his is important to consider, since the organism may not be present in clinical specimens or may not be involved in an intentional release of botulinum toxin. Subtyping methods f or C botulinum , such as ribotyping, have been described (Skinner 2000). An amplif ication method that analyzes variable number tandem repeat regions in C botulinum has been shown to be capable of discriminating among type A strains and may provide laboratories with a rapid, highly discriminatory diagnostic tool f or use in botulism outbreaks (Macdonald 2008). Back to top Agent and Pathogenesis Epidemiology Botulinum Toxin as a Biological Weapon Emergency Response T herapeutic Botulinum Toxin Clinical Features and Dif f erential Diagnosis Laboratory Diagnosis Prevention and Treatment Issues Inf ection Control (Including Autopsies and Burial) Case Def initions and Public Health Reporting Bibliography Prevention and Treatment Issues T herapy f or Botulism Botulinum Toxoid Research on New T herapies and Vaccines

Therapy f or Bot ulism

Supportive care is the mainstay f or treatment of botulism; prolonged intensive care, mechanical ventilation, and parenteral nutrition may be required. Botulinum antitoxin can be administered to treat f orms of botulism other than inf ant botulism and is most ef f ective if given early in the clinical course (Sobel 2009: Diagnosis and treatment of botulism). Although antitoxin will not reverse existing paralysis, it will prevent additional nerve damage if given bef ore all circulating toxin is bound at the neuromuscular junction. For botulism cases other than inf ant botulism, the CDC provides heptavalent botulinum antitoxin (HBAT, Cangene Corporation) through a CDC-sponsored FDA Investigational New Drug (IND) protocol. HBAT replaced bivalent botulinum antitoxin AB in March 2010 (CDC 2010). T he HBAT FDA IND treatment protocol includes specif ic, detailed instructions f or intravenous administration of antitoxin and return of required paperwork to the CDC. HBAT contains equine-derived antibody to the seven known botulinum toxin types (A through G) with the f ollowing nominal potency values: 7,500 U anti-A; 5,500 U anti-B; 5,000 U anti-C; 1,000 U anti-D; 8,500 U anti-E; 5,000 U anti-F; and 1,000 U anti-G. In the setting of a bioterrorist attack, where cases may have been exposed to unusually large amounts of toxin, additional doses of antitoxin may be necessary. Alternatively, the patient's serum could be retested f or the ongoing presence of circulating toxin (Arnon 2001); however, this process would take time. T he scarcity of antitoxin would limit the capacity to provide additional doses. In cases of wound botulism, the wound should be surgically debrided and antibiotics should be administered (usually penicillin). Botulism immune globulinintravenous (human) (BIG-IV) f or treatment of inf ant botulism was licensed by the FDA in October 2003 as BabyBIG. A 5-year randomized, double-blind, placebo-controlled trial of BIG-IV treatment f or inf ant botulism in Calif ornia demonstrated that it signif icantly: (1) shortened duration of hospitalization (f rom a mean of 5.7 weeks to 2.6 weeks), (2) shortened time spent in intensive care (f rom 5.0 weeks to 1.8 weeks), and (3) decreased mean hospital costs per patient by $88,000 (Arnon 2006). BIG-IV is available as a public-service orphan drug and may be obtained by contacting the Calif ornia Department of Human Services, Inf ant Botulism Treatment and Prevention Program (Arnon 2006, Calif ornia Department of Health Services). T he circumstances that enabled the creation of BIG-IV have been presented as a possible paradigm f or development of other "orphan" drugs (drugs used to treat relatively f ew patients) (Arnon 2007).

Availabilit y of Bot ulinum Ant it oxin

Antitoxin should be requested as soon as the diagnosis of botulism is suspected, since conf irmation of botulism may take several days and antitoxin is most ef f ective if given within 24 hours af ter symptom onset (Tacket 1984). Antitoxin f or use in the United States is of equine origin and only available through the CDC via state and local health departments (except in Calif ornia and Alaska, where antitoxin release is controlled by the state health departments). Requests f or antitoxin usually are made through contact with state epidemiology of f ices. In addition to resources at the state level, epidemiologists at the CDC are available 24 hours a day to provide advice

regarding use of antitoxin. Antitoxin (supplied by the CDC) is maintained at quarantine stations located in airports in various metropolitan areas, Once antitoxin is requested f or a patient with suspected botulism, it generally can be delivered within a f ew hours (Shapiro 1997, Sobel 2009: Diagnosis and treatment of botulism).

Long-t erm Out come Following Treat ment

A case-control study of 217 botulism patients provided details about long-term outcome of treated patients (Gottlieb 2007). Of the 211 patients who survived, 68% reported having worse health at the time of interview than 6 years earlier, compared with 17% of 656 controls (matched odds ratio, 17.6; 95% conf idence interval, 10.9-28.4). Nearly twice as many patients as controls (49% vs 25%) reported their current health as f air or poor. Signif icantly more botulism patients than controls reported f atigue, dizziness, dry mouth, and dif f iculty lif ting objects. Botulism patients were signif icantly more likely than controls to report dif f iculty breathing with moderate exertion and were also more likely to report being limited in vigorous activities, walking 3 blocks, and climbing 3 f lights of stairs. Back to top

Bot ulinum Toxoid

For years, the CDC recommended immunization with pentavalent (ABCDE) botulinum toxoid (PBT ) f or vaccination of workers at risk f or occupational exposure to botulinum serotypes A, B, C, D, and E. PBT was available f rom the CDC under an investigational new drug protocol. However, as of November 30, 2011, the CDC no longer of f ers PBT (CDC 2011). According to the CDC, "T his decision was based on an assessment of the available data, which indicated a decline in immunogenicity of some of the toxin serotypes." Back to top

Research on New Therapies and Vaccines Ant ibody Therapeut ics

Humanized monoclonal antibodies, small peptides, peptide mimetics, receptor mimics, and small molecules targeting active sites are candidates f or inhibiting botulinum toxin and may eventually be used in treatment strategies (Adekar 2008, Cai 2007, Nowakowski 2002).

Prot ein and Pept ide Vaccines

Various recombinant vaccines are currently under investigation in animal models (Baldwin 2008, Boles 2006, Lee 2007, Pier 2008, Webb 2007, Yu 2008. Vaccines based on the recombinant carboxy-terminal heavy-chain (Hc) f ragment of the neurotoxin appear to be the most promising (Rusnak 2009, Smith 2009, Yu 2009, Z ichel 2010).

A recombinant botulinum vaccine (rBV A/B) is being developed to protect adults 18 to 55 years of age against types A and B botulism. Toxicity has been evaluated in mice; the rBV A/B vaccine produced no apparent systemic or neurobehavioral toxicity and only transient mild inf lammation at the injection site in the mice studies. T hese results indicate a f avorable saf ety prof ile and support its use in a phase I clinical trial (Shearer 2012).

Viral Vect or Vaccines

Vectored vaccines also have been studied; one report involved Venezuelan equine encephalitis virus as the vector (Lee 2006) and others have involved adenovirus (Xu 2009, Z eng 2007). Back to top Agent and Pathogenesis Epidemiology Botulinum Toxin as a Biological Weapon Emergency Response T herapeutic Botulinum Toxin Clinical Features and Dif f erential Diagnosis Laboratory Diagnosis Prevention and Treatment Issues Inf ection Control (Including Autopsies and Burial) Case Def initions and Public Health Reporting Bibliography Inf ection Control (Including Autopsies and Burial) Isolation Precautions Issues Related to Autopsies and Burial

Isolat ion Precaut ions

In the hospital setting, Standard Precautions are adequate f or patients with botulism, since person-toperson transmission does not occur. In the laboratory setting, sodium hypochlorite (0.1%) or sodium hydroxide (0.1 N) inactivate the toxin and are recommended by CDC f or decontaminating work surf aces and spills of cultures or toxin (CDC 1999: Biosaf ety in microbiological and biomedical laboratories). Back to top

Issues Relat ed t o Aut opsies and Burial

Recent guidelines f rom CDC indicate that Standard Precautions should be used f or postmortem care. T hese include using a surgical scrub suit, surgical cap, impervious gown or apron with f ull sleeve

coverage, a f orm of eye protection (eg, goggles or f ace shied), shoe covers, and double surgical gloves with an interposed layer of cut-proof synthetic mesh (CDC 2004: Medical examiners, coroners, and biologic terrorism). In addition, autopsy personnel should wear N-95 respirators during all autopsies, regardless of suspected or known pathogens. Powered air-purif ying respirators (PAPRs) equipped with N-95 or highef f iciency particulate air (HEPA) f ilters should be considered. Bodies inf ected with biological terrorism agents should not be embalmed (CDC 2004: Medical examiners, coroners, and biologic terrorism). Back to top Agent and Pathogenesis Epidemiology Botulinum Toxin as a Biological Weapon Emergency Response T herapeutic Botulinum Toxin Clinical Features and Dif f erential Diagnosis Laboratory Diagnosis Prevention and Treatment Issues Inf ection Control (Including Autopsies and Burial) Case Def initions and Public Health Reporting Bibliography Case Def initions and Public Health Reporting Botulism Case Def initions Public Health Reporting

Bot ulism Case Def init ions

T he f ollowing case def initions were published in the CDC's Morbidity and Mortality Weekly Report in 1997 (CDC 1997).

Foodborne Bot ulism

Clinical description: Ingestion of botulinum toxin results in an illness of variable severity. Common symptoms are diplopia, blurred vision, and bulbar weakness. Symmetric paralysis may progress rapidly. Laboratory criteria for diagnosis: Detection of botulinum toxin in serum, stool, or patient's f ood or Isolation of C botulinum f rom stool Probable case: A clinically compatible case with an epidemiologic link to a f ood source (eg, ingestion of a

home-canned f ood within the previous 48 hours) Confirmed case: A clinically compatible case that is laboratory-conf irmed or that occurs among persons who ate the same f ood as persons who have laboratory-conf irmed botulism

Inf ant Bot ulism

Clinical description: An illness of inf ants, characterized by constipation, poor f eeding, and "f ailure to thrive" that may be f ollowed by progressive weakness, impaired respiration, and death. Laboratory criteria for diagnosis: Detection of botulinum toxin in stool or serum or Isolation of C botulinum f rom stool Confirmed case: A clinically compatible case that is laboratory-conf irmed, occurring in a child less than 1 year of age

Wound Bot ulism

Clinical description: An illness resulting f rom toxin produced by C botulinum that has inf ected a wound. Common symptoms are diplopia, blurred vision, and bulbar weakness. Symmetric paralysis may progress rapidly. Laboratory criteria for diagnosis: Detection of botulinum toxin in serum or Isolation of C botulinum f rom a wound Confirmed case: A clinically compatible case that is laboratory conf irmed in a patient who has no suspected exposure to contaminated f ood and who has a history of a f resh, contaminated wound during the 2 weeks bef ore onset of symptoms. Back to top

Public Healt h Report ing

According to state disease-reporting requirements, all conf irmed and suspected cases of botulism must be reported immediately to state or local public health of f icials, even af ter normal working hours. Public health of f icials will then contact the CDC through the CDC Emergency Operations Center (telephone number: 770488-7100) to arrange f or clinical consultation if necessary and f or the release of botulinum antitoxin as appropriate (CDC 2003). Back to top Agent and Pathogenesis Epidemiology Botulinum Toxin as a Biological Weapon Emergency Response T herapeutic Botulinum Toxin

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Back to top Oct 10, 2013

Scient ist s f ind new bot ulinum t oxin, wit hhold genet ic det ails
Robert Roos | News Editor | CIDRAP News | Oct 10, 2013 Discovery of the f irst new botulinum toxin in 40 years is coupled with withholding key data f or security reasons. Mar 25, 2013

FOOD SAFETY SCAN: Changes t o f ood saf et y rules, no meat -inspect or f urloughs, bot ulism ant it oxin approved
Mar 25, 2013 Feb 14, 2013

FOOD SAFETY SCAN: Pot ent ial f ood inspect ion cut s, bot ulism ant it oxin recommendat ion
Feb 14, 2013 Dec 13, 2012

Wit h new rules, f ew public healt h labs t o handle riskiest agent s

Robert Roos | Dec 13, 2012 (CIDRAP News) With tougher security requirements set to take ef f ect next April, f ew state public health laboratories plan to maintain stocks of certain pathogens considered most tempting to bioterrorists, according to the Association of Public Health Laboratories (APHL) and of f icials with state labs. Dec 11, 2012

HHS of f ers some f orecast s on medical count ermeasures

Robert Roos | Dec 11, 2012

(CIDRAP News) A new report f rom the US Department of Health and Human Services (HHS) of f ers a f ew predictions on when certain new countermeasures against biological threats will become available, including a f orecast f or two novel inf luenza drugs and possibly a next-generation anthrax vaccine within the next 5 years. Nov 28, 2012

NEWS SCAN: More West Nile cases, psit t acosis in Hong Kong, prison-brew bot ulism
Nov 28, 2012 Oct 25, 2012

NEWS SCAN: Bot ulism out break lessons, more meningit is cases, UK pert ussis, Tdap in pregnant women, Mexico oust s H7N3
Oct 25, 2012 Oct 18, 2012

HHS seeks comment s on risks of H5N1 research

Robert Roos | Oct 18, 2012 (CIDRAP News) Federal health of f icials are inviting the public to weigh in on whether research on H5N1 avian inf luenza viruses, including strains modif ied in the lab to make them more transmissible, is risky enough to require new saf ety regulations and precautions. Oct 10, 2012

Changes in select agent rules concern public healt h labs

Robert Roos | Oct 10, 2012 (CIDRAP News) T he Centers f or Disease Control and Prevention (CDC) has revised its list of potentially dangerous biological agents and toxins and the regulations covering them, and some of the changes have public health laboratories concerned.

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