25 Articles

Botulinum From to Medicine

LARRY E. DAVIS, MD, Albuquerque, New Mexico Although thousands of people in the world each year continue to be poisoned with -food-borne, infantile, or wound botulism-the is now sufficiently understood to allow it to be used as a medicinal agent to paralyze specific muscles, giving temporary symptomatic relieffrom a variety of dystonic neurologic disorders. I review some ofthe epidemiologic, clinical, and pathophysiologic aspects of botulinum toxin and how the neurotoxin may act as a poison or a medicine. (Davis LE: Botulinum toxin-From poison to medicine. West J Med 1993 Jan; 158:25-29)

In the past several years, we have seen the following pa- spores can germinate, with progeny bacteria producing the tients: A 43-year-old man who became flaccidly para- botulinum toxin.2 lyzed two days after eating his wife's home-canned chilies; a The DNA necessary for Cbotulinum to produce the toxin previously healthy 4-month-old baby girl in whom diffuse may be in the host genome or come from either a bacterio- weakness and constipation developed; a 50-year-old drug phage or a plasmid. Removal ofthe phage or plasmid from C addict who subcutaneously inoculated heroin and in whom botulinum can result in a nontoxigenic bacterium.4 Why C moderately severe weakness ofall limbs and dysphasia devel- botulinum normally produces the botulinal toxin is unknown oped; and a 45-year-old woman with severe blepharospasm as infection ofvertebrates is not part ofthe normal clostridial who had pronounced relief of involuntary eyelid closures life cycle, and it has no known role in the growth or physiol- after her physician injected botulinum toxin (Botox) into the ogy of the organism.5 orbicularis oculi muscles. All four persons had exposure to the botulinum toxin. The first patient had food-borne botu- Botulinum Toxin lism, the baby had infantile botulism, the heroin addict had Botulinum toxin is a family of serologically closely re- "wound" botulism, and the last patient was therapeutically lated -A, B, C1, D, E, F, and G. The primary administered botulinum A toxin. In this article I review how structure of most serotypes has been determined. The the botulinum toxin can cause these clinical events. are mainly produced by different strains ofclostridia,2 but six other groups of physiologically distinct , such as Botulinum Clostridium barati and Clostridium butyricum, have pro- are gram-positive, rod-shaped, duced botulinum type E and F toxins.6 On a milligram-per- spore-forming, strictly anaerobic bacteria that are widely kilogram basis, they are probably the most potent biologic distributed in soil and water. I Strains of Cbotulinum produce toxins that affect . Ithas been estimated that 0.025 ng seven different neurotoxins and one binary toxin. Toxin types or 2 x 10' molecules of the botulinal toxin are sufficient to A, B, E, and F are the main toxins that affect humans. I Toxin kill a mouse.7 For comparison, it is estimated that 5 x 1013 types C and D cause botulism in and .2 Clos- molecules of a- (cobra ) and 2 x 1018 tridium botulinum strains that produce toxin types A, B, and molecules of cyanide are required to kill a mouse.8 For hu- F are found generally in soil of geographic areas having low mans, about 0.3 ng or 1011 molecules of botulinum toxin rainfall and moderate temperatures. Type A-producing bac- reaching neuromuscular junctions are enough to cause clini- teria are found primarily west of the Mississippi River and cal botulism.9 type B east ofthe Mississippi River.2 Type E-producing bac- Botulinum toxin is odorless and tasteless. Unlike Cbotu- teria are found in the marine life and sediment around fresh linum spores, the toxin is heat-labile and is denatured by water, especially in Alaska and the Great Lakes region.2 heating above 80°C.3 It can resist destruction by stomach Clostridium botulinum is a spore-forming bacterium. The acid and digestion by in the gastrointestinal tract. spores are capable of surviving 100°C for at least six hours, Botulinum toxin shares many properties with the other but they are killed at temperatures of 120°C lasting five bacterial toxins such as and . l, minutes.3 The killing temperature of spores is important to Type A toxin is produced as a single-chain polypeptide with a the canning industry and was responsible for the practice of molecular weight of 150,000.11 Later it is transformed into canning home foods in a pressure cooker. Home canning its active structure by nicking with a to produce a di- done after simply boiling the food is not sufficient to kill the chain polypeptide of about 100,000 and 50,000 molecular spores. Under anaerobic or semianaerobic conditions, the weight.12 There are three major domains in the molecule

From the Neurology Service, Veterans Affairs Medical Center, and Departments of Neurology and Microbiology, University ofNew Mexico School ofMedicine, Albuquerque. This work was supported in part by Research Service, United States Department of Veterans Affairs. Reprint requests to Larry E. Davis, MD, Veterans Affairs Medical Center, 2100 Ridgecrest Dr SE, Albuquerque, NM 87108. 26 BOTULINUM TOXIN

tal acetylcholine release by the presynaptic cholinergic ABBREVIATIONS USED IN TEXT synapse.1",6 There is evidence that the inhibitory effect of ADP = adenosine diphosphate the toxin occurs selectively at vesical release sites on the CDC = Centers for Disease Control membrane of the presynaptic terminal. The blockage of the acetylcholine release results in muscle weakness. The biochemical basis for the blockade of acetylcholine Channel-forming release is still unknown, but related bacterial toxins provide d some clues. For many bacterial toxins, the toxic molecule Internal _ appears to be an or regulatory . For example, toxin appears to be an adenylate cyclase. ' The chol- domain era toxin is an adenosine diphosphate (ADP)-ribosylating regulatory protein that stimulates adenylate cyclase.'8 The is an ADP-ribosylating regulatory protein that inhibits adenylate cyclase.'8 The botulinum C2 toxin (not a neurotoxin) appears to be an ADP-ribosyltransferase en- zyme."9 Thus the botulinal neurotoxins are likely to be en- zymes that amplify inhibitory molecules or regulatory that act to reduce quantal acetylcholine release.5 The blockade of quantal acetylcholine release by botuli- nal toxin may last several months. Synaptic acetylcholine Binding-site transmission may recover through a turnover of critical pre- * domain synaptic molecules by the cell or by the production of a new on synapse through the sprouting of motoneuron axon terminals with the development ofan adjacent new synapse on the same Figure 1.-The botulinum toxin molecule is composed of a heavy and a light muscle fiber.20'2' chain with a total molecular of weight 150,000. Food-borne Botulism (Figure 1): The receptor binding site is at the carboxyter- Food-borne botulism accounts for most of the cases of minus of the heavy chain (B3). The channel-forming domain botulinal intoxication. Food-borne botulism occurs when a is the aminoterminus of the heavy chain. The internal toxin person eats preformed toxin. Home foods, especially fruits domain resides in the lighter chain, and the two chains are and vegetables that are improperly processed, account for held together with a bond. 90% of the cases of botulism. The pathophysiology of this toxin involves several steps: Most outbreaks of type E botulism involve or fish systemic absorption, binding to the nerve terminal, internal- ization, and synaptic . The toxin reaches the lym- phatic channels first and then the bloodstream either by absorption through the upper gastrointestinal tract (food- borne and infantile botulism) or through tissue absorption <= Botulinum toxin (wound botulism).9 The toxin then circulates in the blood until it reaches cholinergic synapses in the peripheral ner- vous system. The toxin appears not to cross the blood-brain barrier so central cholinergic synapses are Endocytotic vesicle Quantal not involved.'3 The toxin binds to the presynaptic side of the acetylcholine cholinergic synapse.'1" This binding step involves attach- blocking ment of the carboxyterminus of the heavy chain of the toxin molecule molecule to receptors on the presynaptic side of the syn- apse.'4 It has been estimated that there are about 150 to 600 Precursor binding sites per micrometer of presynaptic membrane.8 Enzyme 3 molecule Binding ofthe botulinal toxin to its receptor is ofhigh affinity. amplification'~ Type-specific botulinum is capable of neutralizing the toxin during the period (about '/2 hour) between binding Figure 2.-Botulism poisoning involves 3 stages: binding of toxin to tissues, of internalization of toxin, and internal synaptic poisoning. The internal poisoning and internalization."5 Internalization the toxin occurs step appears to involve the light chain acting as an enzyme to convert host through receptor-mediated endocytosis.' In a final step, the proteins into inhibitory molecules or regulatory proteins that act to inhibit botulinal toxin light chain crosses the membrane ofthe endo- quantal acetylcholine release (from Simpson5). cytic vesicle and enters the cytoplasm of the presynaptic terminal. As the pH in the vesicle falls below 4.0, a con- products.22 In Alaska, botulism among Native Americans formational change in the toxin molecule occurs. The has occurred from eating uncooked, dried, or fermented heavy chain enters the vesical membrane to form a channel fish.23 through which the light chain then passes into the cytoplasm After toxin ingestion, the mean incubation period is 2 (Figure 2). days with a range of 1/2 to 6 days.24 In general, the longer the On entrance of the light chain into the cytoplasm, the incubation period, the milder the signs and symptoms. The toxin induces dysfunction of the presynaptic terminal. Botu- classic clinical features of botulism include a descending linum toxin blocks stimulus-induced and spontaneous quan- symmetric paralysis of all skeletal muscles and many smooth THE WESTERN JOURNAL OF MEDICINE * JANUARY 1993 * 158 * 1 27 muscles.2 24 Limb muscles become weak or completely para- testinal tract. Because most cases of botulism belong lyzed. Cranial nerve musculature is usually involved, result- to types A, B, and E, it is possible to give equine anti-A, B, E ing in an external ophthalmoplegia, dysarthria, dysphasia, serum to patients.2 One vial (10 ml) should be given intra- ptosis, and facial weakness. In addition, patients have a dry muscularly and the second vial intravenously. The antitoxin mouth and often fixed dilated pupils. Weakness ofrespiratory is not available in hospitals or at most state laboratories. In muscles may be severe enough to require intubation and me- general, the antitoxin must be obtained from regional chanical ventilation. Smooth muscle paralysis typically in- branches of the Centers for Disease Control (CDC). If botu- volves the gastrointestinal tract and bladder, resulting in lism is suspected, the physician should contact the botulism constipation and at times paralytic ileus and urinary reten- officer at the CDC ([404] 639-3753, days; or [404] 639- tion. Occasionally paresthesias or asymmetric limb weak- 2888, evenings and weekends). At that time, the public ness may develop.25 The signs and symptoms of botulism health officer can release the antitoxin from the nearest CDC usually progress over one to three days. There are several branch to be placed on the next available commercial airline. important negative clinical features ofbotulism. Because the Equine botulinal antitoxin is extremely potent and is ca- toxin inactivates cholinergic synapses of the peripheral ner- pable of neutralizing 10 to 1,000 times the amount of toxin vous system, patients usually have normal sensation, normal that would be expected in the blood of most patients.29 Be- mentation, normal memory, and normal temperature, blood cause the antitoxin is of equine origin, the patient's pressure, and heart rate.2 should be tested with antitoxin first. There is about a 4% The botulinum toxin has been shown in experiments to incidence of subsequent serum sickness.30 bind to cholinergic synapses in brain homogenates and to Botulinal antitoxin will not reverse existing clinical weak- block cholinergic transmission.5 Patients do not show signs ness as the toxin has already been internalized and is not of cholinergic blockade, however.26 accessible to antibody neutralization. Nevertheless, there The difference may reflect the inability of the toxin to cross does appear to be value in neutralizing any toxin that is still in the blood-brain barrier or to block completely central ner- the circulation. In one study, patients receiving the antitoxin vous system cholinergic transmission. on day 1 oftheir symptoms had fewer days on a respirator and Electrophysiologic studies are often helpful in the diagno- shorter hospital stays than those receiving antitoxin after the sis of botulism. A modest increment in M-wave amplitude first day.3" To date, patients with botulism have not had a with rapid repetitive stimulation often occurs and may pro- notable improvement from the administration of drugs that vide an early clue to the cause of the weakness.27'28 Less increase the release ofacetylcholine from normal cholinergic specific changes include low-amplitude and short-duration synapses (guanidine, or 3,4-diaminopyridine) or that inhibit motor unit action potentials with small M-wave amplitudes the breakdown of acetylcholine in the synaptic cleft (pyrido- on an electromyogram. Single-fiber electromyography may stigmine) 32,33 show increased jitter and blocking.2" Motor conduction Because weakness may progress for several days, it is velocities and distal latencies are normal. Patients with botu- important to place patients in intensive care and monitor lism usually have a normal hemogram, urinalysis, and cere- them carefully. Should respirations begin to fail, intubation brospinal fluid. and assisted ventilation are required. The respiratory weak- The differential diagnosis of food-borne botulism in- ness is often prolonged, necessitating a tracheostomy. Pa- cludes the Guillain-Barre syndrome, diphtheric polyneurop- tients may have an ileus for several days, so feeding through a athy, paralysis, curare poisoning, poliomyelitis, nasogastric tube should not begin until bowel sounds have myasthenia gravis, and the Eaton-Lambert syndrome. If returned. Frequently the bladder paralysis necessitates the there is a cluster ofpatients with similar symptoms, botulism use of an indwelling urinary catheter. It would be ideal to should be high in the differential diagnosis. remove unabsorbed toxin from the gastrointestinal tract by The laboratory diagnosis of botulism is made by analyz- the use of laxatives or enemas, but if gastrointestinal tract ing specimens of serum (20 to 30 ml), stool (25 grams), and paralysis has occurred, this should not be undertaken. suspected food for the botulinum toxin.2 Despite modern Recovery from botulism is slow. Patients may be in the serologic tests such as the radioimmunoassay, the standard hospital for as long as six months and may be on a respirator and most sensitive diagnostic test for the botulinum toxin is a for weeks to a few months. Even after hospital discharge, biologic test involving mice. The number oftoxin molecules patients often complain of fatigue or dyspnea on exertion for needed to paralyze mouse neuromuscular junctions and to at least a year. Muscle strength tests and pulmonary function kill mice from respiratory failure is lower than the minimum tests are usually normal, however.34 Patients who recover amount of toxin that currently is needed for detection by from botulism do not have to botulinum toxin, standard serologic tests using antibodies against the botuli- since the intoxication results from insufficient antigen to num toxin. The mouse test involves intraperitoneal inocula- stimulate the immune system. The mortality from food- tion ofa mouse with the patient's serum or extracts ofstool or borne botulism has decreased in the United States primarily food specimens to determine whether the mouse becomes because of better intensive care. From 1900 to 1950 about paralyzed and dies.2 Heating the specimen to 100°C or com- 60% ofpatients died, whereas from 1970 to 1975 only 21% of bining it with specific botulinum antitoxin should prevent the persons died of food-borne botulism.35 's death. Treatment should begin as soon as the diagnosis of botu- Infantile Botulism lism is made. In many situations, a firm clinical diagnosis of Infantile botulism results from the ingestion of clostridial botulism can be made even before the laboratory confirms spores that then colonize the gut to produce their neurotoxin the diagnosis. The treatment offood-borne botulism involves directly in the gut.36 The normal gastrointestinal tract of three steps: neutralize the circulating toxin; support the pa- children and adults is remarkably resistant to colonization by tient; and remove excess toxin, ifpossible, from the gastroin- clostridial bacteria. The gastrointestinal tract of infants 1 28 BOTULINUM TOXIN

week to 11 months of age may, however, occasionally allow Wound Botulism colonization of this organism. Factors influencing the vul- Wound botulism is the least common type of botulinal nerability of the infant gut to colonization are poorly under- intoxication. Wound botulism occurs when Cbotulinum rep- stood, but some infections occur when breast-feeding is licates in an abscess that has an anaerobic environment pro- changed to bottle-feeding. Under rare circumstances, adults ducing botulinal toxin that becomes systemically absorbed. have suffered a similar colonization of the gastrointestinal Wound botulism is a rare disease in the United States and tract by clostridial organisms. In general, these adults have occurs most often from subcutaneous abscesses or deeper had their gut flora and pH altered by a surgical procedure or wound infections in muscle, nerves, and soft tissues.49 At drugs.37 present, most cases occur in drug abusers who subcutane- The average age of patients with infantile botulism is 3 ously inject street drugs ("skin popping")50 or following months, but cases occur in infants I to 11 months.3839 Infants penetrating trauma where the wound becomes infected.51 may have mild or severe disease.40 Constipation from a par- The incubation period from infected wounds varies from 4 to tially paralyzed ileus usually develops first and can persist as 17 days.51 long as three weeks before other symptoms appear.38'41 A Signs and symptoms consistent with botulism generally flaccid paralysis then develops, and the infants have a weak develop over several days. Electrodiagnostic study findings cry, poor suck, and a decreased interest in their environment. are usually similar to those seen in food-borne botulism.50 Loss of head control is a common early sign. Cranial nerve The diagnosis of wound botulism is best made by the palsies are common, often producing ptosis, dilated pupils, isolation of C botulinum organisms from the infected ab- facial diplegia, and impaired gag reflex.38 Decreased respira- scess. The infected wound should be searched for with care tions may develop, and respiratory assistance may be re- because it may not be obvious. Irrigating the abscess with a quired. Deep tendon reflexes are decreased or absent. sterile saline solution and subsequent culture ofthe recovered Autonomic involvement usually includes dry mucous mem- fluid in anaerobic culture media may yield the organism. branes, decreased bowel motility, and urinary retention. The Unfortunately, the toxin often is not detected in serum, and in children are usually afebrile. wound botulism, the toxin is never found in stool. The differential diagnosis includes dehydration or elec- The treatment of wound botulism includes debridement trolyte imbalance, diphtheric polyneuropathy, neonatal my- ofthe abscess or infected wound. Penicillin should be admin- asthenia gravis, poliomyelitis, hypothyroidism, tick istered along with the botulinum antitoxin. Patients should paralysis, Werdning-Hoffmann spinal muscular atrophy, be placed in intensive care and given supportive care, includ- Leigh disease (subacute necrotizing encephalomyelopathy), ing endotracheal intubation and mechanical ventilation if congenital myopathy, the Guillain-Barre syndrome, and ex- necessary. posure to toxins such as heavy and organophos- phates.42 Botulinum A Toxin as a Similar to those with food-borne botulism, patients with As the mechanism of action of the botulinum toxin be- infantile botulism usually have reduced amplitude of their came better understood, it was recognized that the toxin evoked compound muscle action potentials and incremental could be used selectively to paralyze muscles.52 Purified type responses following repetitive nerve stimulation.43 A botulinum toxin is now used to treat various neurologic The diagnosis of infantile botulism depends on the recov- disorders including blepharospasm, spasmodic torticollis, ery of clostridial organisms and toxin from the stool.244 hemifacial spasm, strabismus, and laryngeal dystonia or Toxin and organisms may persist in the stool for a long time, spastic dysphonia.5355 The dystonic or spasmodic neuro- even after clinical recovery.45 Toxin is seldom detected in logic illnesses are characterized by involuntary intermittent serum.45 or sustained contractures ofspecific groups ofmuscles. They The treatment of infantile botulism differs from food- have been difficult to treat with other because borne botulism. Botulinal antitoxin has not affected the out- the dosage required for any symptomatic reliefusually weak- come of infantile botulism and is generally not given.46 ens all muscles. Similarly, penicillin or other antibiotic therapy has not af- The administration of minute amounts of botulinal toxin fected the outcome of the disease.3847 Aminoglycoside anti- into specific skeletal muscles causes localized muscle weak- biotics are contraindicated because they increase presynaptic ness or paralysis by blocking acetylcholine release at the muscular blockade and may worsen the paralysis.48 There- neuromuscularjunctions ofonly the treated muscles. In addi- fore, treatment is excellent supportive care in an intensive tion, there is a diminution of the dystonia or tonic muscle care unit. Because the infant stool contains viable clostridia, spasms. Botulinal toxin treatment of these facial or neck care should be taken not to cross-infect other infants in the muscle disorders has given dramatic, albeit temporary, intensive care unit. Infants usually recover spontaneously symptomatic relief to thousands of patients. Botulinal toxin over several weeks. Currently less than 3% of patients in treatment usually gives relief for 4 to 12 months before the hospital die.46 condition returns. At that time, the botulinal toxin is often The age distribution ofthe sudden infant death syndrome readministered to the same muscles. Repeated muscle injec- parallels that of infantile botulism.40 Studies of infants dying tions may provide relief for shorter periods than the initial of the sudden infant death syndrome have shown that about administration. It is not yet clear if botulinal toxin can be 5% have evidence of intestinal infection with C botulinum, readministered indefinitely or whether the effectiveness suggesting that infantile botulism may be one cause of the eventually wears off. syndrome.40'41 It should be noted that Cbotulinum organisms Care must be taken when the toxin is administered be- have also been identified in healthy infants without signs of cause it can diffuse from the inoculation site through tissue to botulism.38 paralyze neuromuscularjunctions ofadjacent muscles, caus- o o 9 THE WESTERN JOURNAL OF MEDICINE * JANUARY 1993 * 158 * 1 29

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