Clinical manifestations

Presentation and course

Involvement of the nervous system is uncommon with most microorganisms that might reasonably be employed in biological warfare and bioterrorism.

Bacillus anthracis. Severe hemorrhagic meningitis regularly occurs with anthrax infection, often accompanied by severe systemic disease, most likely transmitted by spore inhalation.

All forms of anthrax infection (ie, cutaneous, gastrointestinal, inhalational, and injection) can be complicated by meningitis. With natural anthrax, anthrax bacilli most commonly enter the body via the skin and disseminate to the CNS via the hematogenous or lymphatic routes, leading to fatal bacterial meningitis. The interval from exposure to presentation of cardinal signs (ie, fever, dyspnea, and headache) varies from 2 to 60 days.

Botulinum toxin. Neurologists are most likely to become involved in primarily diagnosing bioterrorist attacks that use botulinum toxin, either through oral ingestion or inhalation (53). The characteristic descending paralysis typically starts in the extraocular and bulbar muscles, with associated autonomic features. Repetitive nerve stimulation usually shows an incremental muscle response. The differential diagnosis is from naturally occurring paralyzing illnesses (eg, Guillain-Barré syndrome, myasthenic crisis, diphtheria, tetrodotoxin, saxitoxin, snake envenomation, and from chemical warfare poisoning by organophosphates). Treatment is supportive.

Bioterrorists are more likely to contaminate the inhaled air of victims rather than contaminate the food supply. No immediate effects are noted after inhalation of botulinum toxin as the latent period varies from 1 to 5 days. The clinical features are predominantly a combination of multiple cranial nerve palsies and skeletal muscular paralysis. The early manifestations of botulin intoxication are usually ophthalmological, with symptoms such as blurred vision, diplopia, and ptosis. Signs include nystagmus, mydriasis, and extraocular muscle dysfunction. Other symptoms, such as dysphagia and dysarthria, are associated with descending flaccid paralysis that typically develops 12 to 72 hours after exposure. The autonomic effects of botulism are manifested by anticholinergic signs and symptoms (eg, dry mouth, ileus, constipation, and urinary retention). Sensory symptoms are absent. Because botulinum toxins do not cross the blood-brain barrier and do not affect the brain, affected individuals are usually alert and oriented. With severe respiratory muscle paralysis, the patient may become cyanotic or exhibit narcosis from CO2 retention. Respiratory paralysis may lead to death.

Prognosis and complications

Anthrax. Untreated, the case fatality from cutaneous anthrax is about 20% to 30%, compared to 25% to 60% with gastrointestinal anthrax, and nearly 100% with inhalational anthrax. With treatment, mortality is less than 1% with cutaneous anthrax (41; 51; 133; 62), whereas mortality remains high, even with aggressive treatment, with gastrointestinal or inhalational anthrax (64; 62). Inhalational anthrax was generally thought to be fatal in 80% to over 90% of cases (100; 14; 01; 133), but the mortality in the 2001 U.S. outbreak has been much better than anticipated (5 of 11 cases or 45%) with the use of aggressive treatment and intensive care unit support, including mechanical ventilation and dialysis as necessary (23; 133). From 2001 to 2014, 8 of 15 (53%) patients with inhalation anthrax survived with early diagnosis, combination antimicrobial drug treatment to eradicate the bacteria and inhibit toxin production, and aggressive pleural effusion management (62). Initiation of antibiotic or anthrax antiserum therapy during the prodromal phase of inhalational anthrax is associated with markedly improved survival compared with initiation of treatment of fulminant cases (64). Nevertheless, anthrax survivors report significant health problems and poor life adjustment 1 year after the onset of bioterrorism-related anthrax infection (108).

Anthrax meningoencephalitis is usually rapidly fatal, with roughly two-thirds of affected patients dying within 24 hours of presentation (80; 95), although there are a small number of reported survivals following anthrax meningoencephalitis (45; 111; 127; 120; 121; 75; 119; 80; 09). In a systematic review, survival of anthrax meningitis was predicted by treatment with a bactericidal agent and the use of multiple antimicrobials (72). There is limited information on long-term outcomes among the few survivors, but several cases were reported to have fully recovered (120; 119; 80; 09).

Botulism. The advent of modern supportive care has improved botulism prognosis so that the case fatality rate for food-borne botulism has decreased from the 50% to 70% range to the 5% to 20% range. Although botulinum neurotoxins bind irreversibly to presynaptic endplates and impair them irreparably, axons can regenerate new endplates, and full recovery can occur, although it may take several months. The case fatality and outcomes from biological warfare or terrorism could easily be much worse than the results obtained with individual sporadic cases or cases from small natural outbreaks because the medical capacity could be overwhelmed and treatment supplies (eg, Botulism Antitoxin Heptavalent [A, B, C, D, E, F, G]) could be exhausted by sudden widescale demand.

Biological basis

Etiology and pathogenesis

Various agents used for biological and chemical warfare are listed in Table 1. Many of them can cause neurologic manifestations. Encephalomyelitic viruses, toxins, and chemicals are more likely to be associated with neurotoxicity. Some of the bacterial infections may be associated with neurologic complications.

Characteristics that make a pathogen a high risk for bioterrorism include a low infective dose, ability to be aerosolized, high contagiousness, and hardiness (ie, survival in a variety of environmental conditions) (03).

Table 1. Potential Biological Warfare or Bioterrorism Agents with Frequent Involvement of the Nervous System

CDC categorization of bioterrorism agents and diseases. The Centers for Disease Control and Prevention (CDC) has a 3-tier categorization system for bioterrorism agents and diseases (CDC: Bioterrorism Agents and Diseases):

Category A. Category A is comprised of high-priority agents that pose a risk to national security because (1) they can be easily disseminated or transmitted from person to person, (2) they have the potential for major public health impact (eg, high case-fatality rates), (3) they will likely cause public panic and social disruption, and (4) they require special measures for public health preparedness. The two Category A diseases with the greatest potential to affect the nervous system as a major feature of the illness are anthrax (Bacillus anthracis) and botulism (Clostridium botulinum toxin). Other Category A diseases or agents include plague (Yersinia pestis), smallpox (variola major), tularemia (Francisella tularensis), and viral hemorrhagic fevers (eg, Ebola, Marburg) (06; 07).

Category B. Category B is comprised of moderate-priority agents that (1) are moderately easy to disseminate, (2) result in moderate morbidity rates and low mortality rates, and (3) require specific enhancements for diagnostic capacity and warrant enhanced disease surveillance. Of these, only viral encephalitis viruses (alphaviruses, such as eastern equine encephalitis, Venezuelan equine encephalitis, and western equine encephalitis) are likely to primarily affect the nervous system.

Category C. Category C is comprised of lower-priority agents, including emerging pathogens that could potentially be engineered for mass dissemination because of availability, ease of production and dissemination, or potential for major health impact (eg, potential for high morbidity or mortality).

Bacillus anthracis (Category A). Insight regarding the mechanism for the spread of B anthracis by inhalation is provided by pathologic studies of victims from the Sverdlovsk epidemic of 1979. The contagion mechanism was traced to the release of aerosols containing this organism at a secret biological-agent production facility in Russia (60). A hematogenous spread involved the central nervous system with resulting vasculitis, hemorrhages, and edema due to the toxins. The risk of hemorrhagic meningitis after exposure to anthrax inhalation is estimated to be as high as 50% (66; 67).

Equine encephalitis (Category B). Venezuelan equine encephalitis virus could be developed for transmission by inhalation with secondary zoonotic transmission cycles sustained by horses and mosquitoes (53), as could eastern and western equine encephalitis viruses.

The Eastern equine encephalitis virus is spread to people by the bite of an infected mosquito (15).

In the United States, most cases occur in eastern or Gulf Coast states.

Although rare, approximately 30% of people with Eastern equine encephalitis die, and many survivors have residual neurologic problems. Eastern equine encephalitis, commonly called Triple E or sleeping sickness (not to be confused with African trypanosomiasis), is caused by a zoonotic mosquito-vectored Togavirus that is present in North, Central, and South America and the Caribbean.

In the United States, Eastern equine encephalitis is often associated with coastal plains, most commonly in East Coast and Gulf Coast states. There are no human vaccines or licensed therapeutic drugs available in the U.S. for Eastern equine encephalitis. Eastern equine encephalitis virus is a potential bioweapon, particularly because it is transmissible by aerosol.

The Western equine encephalomyelitis virus is an alphavirus of the family Togaviridae.

The Western equine encephalomyelitis virus is an arbovirus (arthropod-borne virus) transmitted by mosquitoes of the genera Culex and Culiseta.

The virus is transmitted to people and horses by bites from infected mosquitoes (Culex tarsalis and Aedes taeniorhynchus), particularly during wet summer months; birds are the natural reservoir of the virus.

In North America, Western equine encephalomyelitis occurs primarily in U.S. states and Canadian provinces west of the Mississippi River. The disease also occurs in South America. Western equine encephalomyelitis is commonly a subclinical infection in adult humans, but the disease can cause serious sequelae in infants, children, and the elderly. The overall mortality of Western equine encephalomyelitis is low (approximately 4%) and is associated mostly with infection in the elderly. Approximately 15% to 20% of horses that acquire the virus die or need to be euthanized.

There are no human vaccines or licensed therapeutic drugs available in the U.S. for Western equine encephalomyelitis. Western equine encephalitis virus was one of more than a dozen agents the United States researched as potential biological weapons before the nation suspended its biological weapons program.

Venezuelan equine encephalitis is a mosquito-borne disease endemic in regions of Central and South America that causes sporadic outbreaks of equine and human encephalitis. The virus induces neural necrosis and edema in affected mammals.

Toxins. The term “toxin” refers to a toxic substance of biological origin, although simple toxins can be synthesized in the laboratory or produced by genetic modification in other species.

Bacterial toxins are of two general types: proteinaceous exotoxins (eg, tetanus, diphtheria, or botulinum toxins) are part of a defensive system of bacteria (to avoid capture, ingestion, etc.) and so are typically secreted into the surrounding medium, in contrast to endotoxins (eg, lipopolysaccharides in the outer membrane of gram-negative bacteria) that are only released when the bacterium disintegrates.

Mycotoxins are diverse and could be used by terrorist organizations to poison food and water sources, although dispersal in indoor air appears to be the most effective method for a bioterrorist attack (84). Crude concentrated or dried extracts of readily grown fungal cultures can simultaneously produce several toxins with synergistic actions, but mycotoxin bioterror weapons are more likely to involve the liver (eg, aflatoxins, fumonisin B1), kidneys (ochratoxin A, fumonisin B1), lungs (fumonisin B1), gastrointestinal tract (trichothecenes type A, eg, T-2 toxin; type B, eg, deoxynivalenol [DON]), and bone marrow (trichothecenes type A, eg, T-2 toxin) than the nervous system (83; 63; 114; 96; 84; 70).

As chemical products of living organisms, toxins when employed by people for a nefarious purpose occupy an ill-defined “no man’s land” between chemical warfare and biological warfare agents. From a pharmacological and toxicological point of view, toxins could be considered chemical weapons, but most experts and the U.S. Army classify toxins as biological weapons.

Toxins most relevant to bioterrorism include ricin, botulinum, Clostridium perfringens epsilon toxin, conotoxins, shiga toxins, saxitoxins, tetrodotoxins, mycotoxins, nicotine (03), and brevetoxin. Of these, botulinum, brevetoxin, conotoxins, nicotine, saxitoxins, and tetrodotoxin are most likely to involve the nervous system.

(1) Botulinum toxin (Category A). It is feasible to deliver botulinum toxin as an aerosolized biological weapon. Botulinum toxins are a series of seven related protein neurotoxins, serotypes A through G.

Each serotype is a protein of approximately 150 kD produced by a separate strain of the gram-positive anaerobic bacterium Clostridium botulinum. Botulinum toxins block acetylcholine release from the presynaptic nerve terminal in the peripheral nervous system, leading to muscle paralysis. Botulinum toxin A is the most toxic serotype for humans. It can be readily aerosolized and persists in this state for weeks in still water and food but degrades on exposure to heat, acidity, or air for more than 12 hours.

Botulinum toxin binds to high-affinity recognition sites on the cholinergic nerve terminals (there may be a separate one for each serotype). The toxin enters the nerve terminal by receptor-mediated endocytosis but then is extruded into the cytoplasm. In the cytoplasm, the toxin cleaves SNARE proteins (ie, proteins that mediate vesicle fusion with the presynaptic membrane), preventing the cell from releasing vesicles of neurotransmitter, interrupting neurotransmission, and causing paralysis (55; 38).

Botulinum toxin consists of two polypeptide subunits: A and B chains. The B subunit binds to receptors on the axons of motor neurons. The toxin is taken into the axon by receptor-mediated endocytosis, and then the A chain acts to prevent fusion of the synaptic vesicle with the cell membrane, thus, preventing the release of acetylcholine. This mechanism of blocking neuromuscular transmission is called presynaptic inhibition. The presynaptic inhibition interrupts both cholinergic autonomic (muscarinic) and motor (nicotinic) neurotransmission, producing the cranial neuropathies and skeletal muscle paralysis seen in clinical botulism. Recovery only occurs after the neuron generates a new axon terminal, which can take months.

The estimated lethal dose of type A toxin is 1.3 to 2.1 ng/kg intravenously or intramuscularly, 10 to 13 ng/kg when inhaled, or 1000 ng/kg orally.

(2) Brevetoxins (Category C). Brevetoxins are a group of tasteless and odorless neurotoxic cyclic polyether compounds produced naturally by species of dinoflagellates (Karenia sp.). Brevetoxins bind to voltage-gated sodium channels in nerve cells, leading to disruption of normal neurologic processes and causing neurotoxic shellfish poisoning. The typical natural route of human exposure is by ingestion of contaminated shellfish. Ingestion of these toxins produces a combination of gastrointestinal and neurologic signs and symptoms; gastrointestinal symptoms include abdominal pain, vomiting, and diarrhea, and the most disabling neurologic signs and symptoms include vertigo and ataxia. Inhalational exposure may cause respiratory symptoms, such as cough, dyspnea, and bronchospasm. Toxicity can also result from dermal exposure.

(3) Conotoxins (Category C). Conotoxins, the venom of various species of cone snails, are pharmacologically and chemically diverse.

Conotoxins affect neurotransmitter receptors (eg, nicotinic, adrenergic, NMDA, and serotonergic) and ion channels (eg, sodium, potassium, and calcium).

The most lethal effect of conotoxins to humans is diaphragmatic paralysis and respiratory arrest. Most conotoxins are not a bioterrorism threat, but some could be weaponized and used as an aerosol (eg, a-conotoxins, k-conotoxins, and o-conotoxins).

(4) Saxitoxins (Category C). There are more than 50 structurally related neurotoxins (known collectively as "saxitoxins") produced by protists, algae, and cyanobacteria. Saxitoxin, the best-known paralytic shellfish toxin, is one of the most potent known natural toxins. Saxitoxin acts as a selective, reversible, voltage-gated sodium channel blocker. Saxitoxin binds directly in the pore of the channel protein, occluding the opening, and preventing the flow of sodium ions through the membrane.

(5) Tetrodotoxin (Category C). Tetrodotoxin is an extremely potent toxin naturally found mainly in the liver and sex organs (gonads) of some fish (eg, puffer fish, globefish, and toadfish) and in some amphibian, octopus, and shellfish species.

Tetrodotoxin binds to the voltage-gated sodium channels in nerve cell membranes and blocks the passage of sodium ions (responsible for the rising phase of an action potential) into the neuron (81). This mechanism of action was established in 1964 by Toshio Narahashi (1927-2013), John W Moore (1920-2019), and William R Scott at Duke University (92; 91).

Brevetoxins, saxitoxins, tetrodotoxin, and some conotoxins all act to block voltage-gated sodium channels.

Epidemiology

The most likely first indicator of a biological warfare event would be a marked increase in the number of patients presenting with clinical features caused by the disease agent, beyond what would be expected for natural outbreaks. Patients presenting en masse with a similar pulmonary syndrome should alert the clinician to the possibility of aerosolized toxin exposure (134).

If an attack with a toxin were to occur, epidemiological clues would include the clustering of cases (ie, more than expected with the natural occurrence of the disease), more severe disease than typical, and unusual routes of exposure (eg, inhalation rather than ingestion for some toxins).

Anthrax bioweapon or bioterrorism. Anthrax is rarely found in most developed countries because of vaccines for animals and at-risk people. There has been a dramatic reduction in livestock and human cases of anthrax in the United States since 1900 because of livestock and at-risk human immunization, proper disposal of infected animals, and restriction of imported wool, hides, and other products (13; 14).

Because of the complexities of the development and dispersal of an anthrax bioweapon, it had been anticipated that small-scale sabotage or attempts at personal murder were more likely than large-scale attempts at inflicting mass casualties with anthrax and that crude dispersal in a close area was the most likely mode of attack. In the 2001 United States outbreak of anthrax acquired through intentional contamination of mail, most cases involved media, government, and postal service employees; several civilian cases remain unexplained (24).

Epidemiologic clues to bioweapon use include (1) an outbreak or multiple, simultaneous outbreaks with large numbers of patients complaining of respiratory symptoms; (2) high case fatality numbers; (3) sick or dead animals of different species; and (4) multidrug-resistant pathogens (41). A World Health Organization report estimated that the release of 50 kg of anthrax spores upwind of a city of 500,000 population would result in 125,000 infections and nearly 95,000 deaths (132). A report by the United States Congressional Office of Technology Assessment estimated between 130,000 and 3 million deaths following the aerosolized release of 100 kg of anthrax spores upwind of Washington, D.C. (93).

Botulism. An average of 110 cases of botulism are reported annually in the United States, about 25% of which are foodborne botulism, whereas 75% are infant botulism (21). Five western states account for more than half of reported foodborne outbreaks since 1950: California, Washington, Colorado, Oregon, and Alaska.

Foodborne botulism is a significant public health problem among Alaska Natives and is associated with improper preparation and storage of traditional foods. At least two thirds of foodborne outbreaks are due to home-processed foods, most of the remainder are from unknown sources, and only 7% are due to commercially processed foods (21).

Although infant botulism has been the most common form of botulism in the U.S. since 1980, infant botulism is a rare sporadic disease that occurs in isolated cases, has no epidemic potential, and generally does not pose a major public health threat.

Infant botulism is epidemiologically distinct from foodborne botulism because it results from colonization (infection) of the intestine by spores of C. botulinum with subsequent in vivo toxin production rather than from ingestion of toxin preformed in contaminated foods. Although infant botulism was first described in 1976 (89; 98), earlier cases have been identified retrospectively (21).

Prevention

Much work needs to be done internationally to develop capabilities to manage victims of biological or chemical terrorism (74). At present, most physicians and hospital emergency departments in the United States are ill-prepared to treat victims of such terrorism (73; 129; 115).

Anthrax. Prevention of anthrax infection in civilians due to bioterrorist activity is limited to prevention of exposure and postexposure prophylaxis (postexposure prophylaxis is considered under management). In military personnel and selected at-risk populations, prevention has also included vaccination (37); however, legal challenges resulted in a temporary halt of military vaccinations as of December 2003.

Although previous CDC guidance (22) indicated that an anthrax vaccine could be requested through the CDC, such a vaccine was not provided to civilians during the 2001 outbreak in the United States (24; 80). Subsequently, civilians deemed at high risk of exposure who had completed a 60-day course of antibiotics were offered postexposure anthrax vaccination (30; 68).

Anthrax vaccine adsorbed, a cell-free, noninfectious anthrax vaccine prepared from a noncapsulating, nonproteolytic anthrax strain, was licensed by the U.S. Food and Drug Administration in 1970 for soldiers, at-risk scientists and laboratory workers (ie, those working with anthrax), at-risk veterinarians and livestock handlers, and at-risk wool mill workers (125). AVA was reapproved for licensure by the Food and Drug Administration in 1985 and remains the only licensed human anthrax vaccine in the United States (133). AVA, now marketed as BioThrax® (Emergent BioSolutions, Lansing, Michigan), is licensed for use in nonpregnant adults aged 18 to 65 years who are at high risk of exposure.

AVA is a considerable improvement over previously available vaccines, including the uncharacterized whole-cell vaccines developed initially in 1881 (109). Originally it was administered as a series of six subcutaneous exposures over 18 months, followed by annual boosters for persons with continuing potential exposure risk (41; 125). In 2008, the CDC published the results of a four-arm randomized controlled trial comparing the original regimen administered by subcutaneous administration with the original regimen administered by intramuscular injection, a reduced dose schedule administered by intramuscular injection, and a placebo that received saline injections at the same intervals. Intramuscular injection significantly reduced the occurrence of injection-side reactions, and immunological priming of reduced-dose regimens proved noninferior to the original licensed regimen (86). For pre-event or pre-exposure prophylaxis, the United States Advisory Committee on Immunization Practices now recommends five 0.5-ml doses of AVA administered intramuscularly at 0 and 4 weeks and 6, 12, and 18 months, followed by annual 0.5-ml booster injections after the 18-month dose (133). A booster dose of AVA for preexposure prophylaxis can be given every 3 years instead of annually to persons not at high risk for exposure to Bacillus anthracis who have previously received the initial AVA three-dose priming and two-dose booster series and want to maintain protection (10; 10). People with contraindications to intramuscular injections (eg, coagulopathies) may continue to receive AVA by subcutaneous administration (133). In contrast to the revised vaccine administration schedule and route for pre-event or preexposure prophylaxis, the FDA decided in June 2009 to continue the preexisting postexposure prophylaxis protocol until new data become available; therefore, for postexposure prophylaxis, AVA should only be administered under an Investigative New Drug protocol or Emergency Use Authorization in a three-dose series (at 0, 2, and 4 weeks) by the subcutaneous route in conjunction with antimicrobial therapy for a minimum of 60 days (133).

The Department of Defense Anthrax Vaccine Immunization Program, announced in 1997 and instituted in 1998, administered 8.4 million doses to approximately 2.1 million military personnel from March 1998 through December 2008 (133). Although previously mandatory for military servicemen and women, a United States District Court ruled on October 27, 2004, that the Department of Defense can no longer inoculate troops without their consent. The vaccine is associated with frequent local reactions but only rare serious side effects sufficient to require hospitalization (approximately once per 200,000 doses) (125). In fact, in a study employing a historical cohort before the inception of the Department of Defense Anthrax Vaccine Immunization Program in 1998, hospitalization following anthrax vaccination was significantly less likely from any cause, diseases of the blood and blood-forming organs, and diseases of the respiratory system among 170,723 active-duty U.S. service members who were vaccinated with the anthrax vaccine (128).

The Vaccine Adverse Event Reporting System has monitored adverse events reported following vaccination with AVA. The most common adverse events that occurred after AVA administration (either alone or concurrently with other vaccines) were arthralgia (17%), headache (16%), pruritus (15%), pain (14%), injection-site erythema (13%), fever (11%), erythema (10%), pain at the injection site (10%), rash (10%), and myalgia (10%) (133). There is no convincing evidence of long-term adverse health effects associated with AVA administration (133). Vaccine postmarketing surveillance has identified no association between optic neuritis and receipt of the anthrax vaccine by members of the U.S. military (97).

AVA is neither recommended nor licensed for use during pregnancy, and the Department of Defense continues a policy of avoiding anthrax vaccination in pregnant women (37). AVA is now available under an investigational new drug application in combination with extended antibiotic treatment for civilians exposed to inhalational anthrax who have completed a 60-day course of antibiotics (30; 68).

Botulism. Vaccination with a pentavalent toxoid of Clostridium botulinum toxin types A, B, C, D, and E is available as an investigational new drug for preexposure prophylaxis. This product has been administered to several thousand volunteers and occupationally at-risk workers and induces serum antitoxin levels that correspond to protective levels in experimental animals. Adequate antibody levels are transiently induced after three injections but decline prior to the 1-year booster.

Differential diagnosis

Confusing conditions

History of exposure to neurotoxic biological warfare agents may narrow down the differential diagnosis to agents involved. In febrile patients with acute neurologic deterioration after suspected exposure to biological or chemical agents, anthrax should be considered in the differential diagnosis if there are the following findings: (1) dark necrotic pustules on the extremities, (2) Gram-positive rods in the CSF, and (3) multifocal areas of unexplained intracerebral hemorrhage on CT scans.

Botulism should be differentiated from other neurologic conditions, including Guillain-Barré syndrome, myasthenia gravis, diphtheria, tick paralysis, viral encephalitis, basilar artery stroke, toxic effects of drugs (eg, atropine and aminoglycoside antibiotics), and multiple cranial nerve palsies due to lesions involving the skull base. Once botulism is considered, it is important to differentiate the pulmonary inhalation form from the food-borne form. If bioterrorism is suspected, differentiation should be made between the chemical nerve agents, such as sarin and botulinum toxin exposure effects, as shown in Table 2 (104).

Table 2. Characteristics of Poisoning with Botulinum Toxin and that Distinguish it from Sarin Exposure

Diagnostic workup

Neurologic workup. Workup includes lumbar puncture with examination of CSF and brain imaging (CT or MRI). Normal CSF helps to differentiate botulism from other neurologic disorders, such as Guillain-Barré syndrome. Electrophysiological studies can provide presumptive proof of botulism in patients with the clinical signs of botulism; this is especially helpful when bioassay studies are negative. The most consistent electrophysiological abnormality is a small evoked muscle action potential in response to a single supramaximal nerve stimulus in a clinically affected muscle done pre- and postexercise (131).

If a biological attack occurs, clinical diagnosis should be supplemented by rapid and accurate detection methods. Laboratory diagnostics assume greater importance in the absence of clinical manifestations immediately following exposure.

Biological agents. Combinations of diagnostic technologies, such as culture, immunoassay, and molecular assay can provide rapid diagnostic response capabilities to microbial threats with antimicrobial-resistant organisms, new emerging infectious disease agents, and possible agents of bioterrorism. Conventional microbiology protocols are used at lower-level laboratories, whereas molecular diagnostic assays are used at higher-level laboratories. Polymerase chain reaction-based methods with freeze-dried reagents can be used for quick field detection of microorganisms and bacterial toxins. An ELISA detects circulating toxins. In addition, commercial biosensors are now available for rapid diagnosis of biological agents (eg, Bacillus anthracis and botulinum toxin) (102). Electrochemical biochips enable detection of chemical and biological warfare agents simultaneously.

The United States Army Medical Research Institute of Infectious Diseases (USAMRIID, Fort Detrick, Maryland) has the molecular diagnostic facilities for biological warfare agents. Microarrays using genomic and genetic technologies can reduce the time required for serogroup or strain identification of pathogens affecting the nervous system from days and weeks of cultures to less than a few hours. Although the Institute's primary focus is on protecting military service members, it has collaborations with the CDC, the World Health Organization, and academic centers of excellence worldwide.

Anthrax. Treatment should not be delayed pending a diagnostic workup for possible anthrax. Detailed interim guidelines for investigation of, and response to, Bacillus anthracis exposures have been published (31; 32).

No reliable rapid screening test is available to diagnose inhalational anthrax in the early stages (31).

With inhalational anthrax, a chest x-ray may demonstrate hilar or mediastinal adenopathy, a widened mediastinum, pleural effusions, or infiltrates (41; 23; 31). Anthrax should be diagnosed if a chest x-ray demonstrates a widened mediastinum in the setting of fever and other constitutional symptoms, in the absence of another obvious cause (eg, severe blunt chest trauma or postsurgical infection).

Diagnostic recommendations for anthrax meningitis vary by setting (11).

An evidence-based assessment tool has been developed for screening patients for meningitis during an anthrax mass casualty incident (72). Severe headache, altered mental status, meningeal signs, and other neurologic signs at presentation independently predicted meningitis in a derivation cohort. The presence of a single one of these signs and symptoms on admission had a sensitivity of 89% for finding anthrax meningitis in the adult validation cohort and 83% in the pediatric validation cohort. Anthrax meningitis was unlikely in the absence of any of these signs or symptoms, whereas anthrax meningitis was very likely in the presence of two or more of these signs or symptoms.

A diagnosis of hemorrhagic meningitis should raise strong suspicion of anthrax infection (66; 67; 80). In the index case in the 2001 bioterrorist outbreak in the United States, the initial diagnosis was made from CSF (17). Findings are fairly consistent across reported cases (54; 120; 103; 101; 119; 52; 57; 17; 80). CSF in anthrax may appear cloudy, with yellowish or pinkish coloration, or may be grossly bloody; evolution across this spectrum has been reported in serial lumbar punctures in an individual patient. The supernatant is frequently xanthochromic. Opening pressure may be normal or increased (30 to 50 cm of CSF). Hypoglycorrhachia is common, with CSF glucose frequently ranging from 20 to 40 mg/dl. CSF protein is generally elevated, with initial CSF protein values from 120 to 1150 mg/dl (median 400 mg/dl) and with maximum reported values of 1333 mg/dl. CSF is frequently hemorrhagic, but in the early stages, there may be no red cells. There is a polymorphonuclear CSF pleocytosis with a range of 560 to 4750 white cells per cubic mm on initial lumbar puncture (median 2500); the percentage of polymorphonuclear cells ranges from 67% to 95% (median 81%). CSF cultures are usually positive, but CSF may be sterile after several days of antibiotics. Gram stain generally shows numerous polymorphonuclear leukocytes as well as many large gram-positive rods, either singly or in short or long chains; indeed, virtually all case reports of anthrax meningitis that give gram stain results report these findings. Serial lumbar punctures may document an evolution from cloudy to grossly bloody CSF, with increasing protein concentration, numbers of red cells and polymorphonuclear leukocytes, and proportion of polymorphonuclear leukocytes among white cells.

Other laboratory findings in patients with anthrax meningoencephalitis include elevated white blood cell counts (up to 80,000) with a left shift, positive blood cultures in approximately 75%, positive skin biopsy cultures in patients with skin lesions, and frequently abnormal chest x-rays in patients with respiratory symptoms.

Computed tomography or magnetic resonance imaging of the head in patients with anthrax meningoencephalitis may demonstrate focal intracerebral hemorrhage, subarachnoid hemorrhage, intraventricular hemorrhage, diffuse cerebral edema, and prominent leptomeningeal enhancement (52; 76; 58). Parenchymal cerebral enhancement has not been reported (76). Findings may progress rapidly on serial brain imaging studies (52).

Botulism. Like with anthrax, diagnostic recommendations for botulism vary by setting and specifically differ in conventional and contingency settings and crisis settings (105).

Laboratory diagnosis of botulism is typically based on the detection of toxin in the patient's serum, stool, or wound. However, because of the minute amount of toxin needed to produce botulism, it may not be possible to recover toxin from tissues or body fluids. Both ELISA and PCR can be used to detect botulinum toxins A and B within 2.5 hours.

Glyco-quantitative PCR enables the detection of active botulinum neurotoxin with high sensitivity (78). Eight known types of botulinum toxin (A through H) can be elaborated by Clostridium botulinum, and knowing which type is present can provide epidemiologic clues regarding the source of exposure (eg, toxin type G does not cause natural disease in humans) (08).

Immunological strategies to screen for botulinum neurotoxin-containing samples include: (1) sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses; (2) a bead-based suspension array is used for screening followed by conventional ELISA for quantification; and (3) an ELISA plate format assay for serotype-specific immunodetection of botulin neurotoxin-cleaved substrates to detect the activity of the light chain rather than the toxin protein (112).

Management

Most physicians are not adequately prepared to diagnose and manage potential bioterrorism agents including anthrax (42), and not much has changed since the U.S. bioterrorism outbreak in 2001 (115).

Factors that could potentially mitigate the consequences of an anthrax attack include the ability to promptly recognize and manage the illness and its public health consequences, limitation of secondary contamination through appropriate decontamination, and development and implementation of genotyping methods that can facilitate investigations and thereby influence the governmental response to an attack (47). In addition, though, the circumstances of a significant outbreak of anthrax (eg, as in an accidental or intentional release of anthrax spores) could easily overwhelm treatment resources, forcing triage protocols and the use of alternative treatment regimens.

Bacterial biothreat agents. Combining an earlier-generation antibiotic and a broad-spectrum antimicrobial peptide is a useful approach for combating diverse pathogens, including five bacterial biothreats—the etiologic agents of glanders (Burkholderia mallei), melioidosis (Burkholderia pseudomallei), plague (Yersinia pestis), tularemia (Francisella tularensis), and anthrax (Bacillus anthracis) (43). Of these, only anthrax is particularly prone to involve the nervous system.

Anthrax. Naturally occurring Bacillus anthracis is generally susceptible to many antibiotics (24; 80).

For postexposure prophylaxis of inhalational anthrax following intentional exposure to Bacillus anthracis, the CDC recommends initial treatment with ciprofloxacin or doxycycline until antibiotic susceptibility is determined, with total therapy for at least 60 days, in combination with a three-dose series of anthrax vaccine adsorbed administered at time zero, 2 weeks, and 4 weeks (27; 33; 68; 116). Both ciprofloxacin and doxycycline are approved by the U.S. Food and Drug Administration for postexposure prophylaxis of inhalational anthrax in all age groups; ciprofloxacin and doxycycline are considered equivalent first-line agents for postexposure prophylaxis and have similar susceptibility profiles for naturally occurring isolates, similar safety profiles, and a low rate of anaphylactic reactions (116). Long-term use of these antibiotics appears to be relatively safe to treat a large-scale exposure to anthrax in a bioterrorism attack (87). Alternative antimicrobial drugs that can be considered for postexposure prophylaxis if first-line agents are not tolerated or are unavailable include levofloxacin, moxifloxacin, amoxicillin, penicillin VK (if the isolate is penicillin-susceptible), and clindamycin (62).

Anthrax vaccine is now available under an investigational new drug application in combination with extended antibiotic treatment for civilians exposed to inhalational anthrax who have completed a 60-day course of antibiotics (30; 68; 116). A short course of postexposure antibiotic prophylaxis combined with vaccination (ie, initiated simultaneously) has been shown to improve protection against inhalational anthrax in animal models and may shorten the duration of antibiotic prophylaxis needed to protect against inhalational anthrax (126).

There are no controlled trials in humans of potential treatments for inhalational anthrax. Based on studies in nonhuman primates, other animal and in vitro data, and limited clinical experience in people, intravenous ciprofloxacin or doxycycline should be included as essential components of initial therapy until antimicrobial susceptibility is established (24; 27). Ciprofloxacin is the drug of choice in pregnant women (34). Because of the high mortality associated with inhalational anthrax, and because both ciprofloxacin and doxycycline have poor penetration into the CNS, the CDC now recommends combination therapy with at least two drugs that are predicted to be effective. Thus, initial therapy for inhalational (as well as gastrointestinal or oropharyngeal) anthrax should include ciprofloxacin or doxycycline and one or two additional antibiotics with in vitro activity against Bacillus anthracis (eg, ciprofloxacin, rifampin, and vancomycin; ciprofloxacin, rifampin, and clindamycin) (24). The initial dosing of ciprofloxacin in adults is 400 mg intravenously every 12 hours, whereas in children, dosing is 10 to 15 mg/kg every 12 hours (not to exceed 1 gm/d). Initial dosing of doxycycline in adults (or children over 8 years of age and over 45 kg in weight) is 100 mg intravenously every 12 hours, whereas in younger or smaller children, dosing is 2.2 mg/kg intravenously every 12 hours. Initial therapy may be altered based on the clinical course and antimicrobial susceptibility; one or two antibiotics administered orally (eg, ciprofloxacin or doxycycline) may be sufficient if the patient improves (24). Because of the presence of constitutive or inducible beta-lactamases in Bacillus anthracis isolates from the 2001 U.S. outbreak, penicillin alone (eg, penicillin G or amoxicillin) is not recommended as therapy (24; 27; 2001l). Aztreonam, trimethoprim-sulfamethoxazole, or third-generation cephalosporins should not be used. Corticosteroids should be considered as adjunctive therapy for inhalational anthrax associated with extensive edema, respiratory compromise, or meningitis (24). Three doses of the anthrax vaccine can be administered at 0, 2, and 4 weeks, and antibiotics should be continued throughout this period (41; 30; 68). Because the disease is not spread by respiratory secretions, standard precautions are adequate.

For anthrax meningitis, ciprofloxacin may be more effective than doxycycline because of better CNS penetration (27; 110), and intravenous ciprofloxacin is, therefore, specifically recommended over doxycycline for treatment of severe systemic or life-threatening anthrax infection (116; 62; 11). Because both ciprofloxacin and doxycycline penetrate poorly into the CSF of patients with meningitis, anthrax meningoencephalitis should be treated with a polydrug antibiotic regimen, utilizing agents that have good CNS penetration in meningitis and provide good antibacterial coverage for Bacillus anthracis for at least 2 to 3 weeks, or until the patient is clinically stable, whichever is longer. Guidelines suggest that empiric treatment for anthrax in which anthrax meningitis is suspected, or cannot be ruled out, should include at least three antimicrobial drugs (62; 11; 99). The preferred regimen includes a fluoroquinolone (ciprofloxacin), a carbapenem (meropenem), and a protein-synthesis inhibitor (linezolid). Intravenous ciprofloxacin is the preferred primary bactericidal component of the multidrug regimen, although levofloxacin and moxifloxacin are considered equivalent alternatives; these fluoroquinolones have adequate CNS penetration and are not known to be susceptible to natural resistance. The carbapenem drugs are highly resistant to beta-lactamases and provide good CNS penetration; meropenem is preferred as the second drug of the combination regimen, although doripenem and imipenem/cilastatin are considered equivalent alternatives. At least one antimicrobial drug that inhibits protein synthesis should also be used to reduce exotoxin production: linezolid is preferred as the first-line protein synthesis inhibitor and is likely to provide better CNS penetration than clindamycin, although toxicity issues must be carefully considered and weighed with linezolid. Doxycycline should not be used if meningitis is suspected because it does not adequately penetrate the CNS.

Although the CDC recommends the use of doxycycline, ciprofloxacin, penicillin G, and amoxicillin for the treatment of human cases and for postexposure prophylaxis to anthrax spores, B anthracis shows a high degree of in vitro susceptibility to many other antimicrobials, suggesting that alternative choices for prophylaxis and therapy may be possible (85). Isolates collected over more than 3 decades were susceptible to gentamicin, streptomycin, penicillin G, clindamycin, chloramphenicol, vancomycin, linezolid, tetracycline, erythromycin, rifampin, amoxicillin, ciprofloxacin, and doxycycline; the isolates showed intermediate susceptibility to ceftriaxone and cefotaxime but high minimal inhibitory concentration (MIC) values only for trimethoprim.

Corticosteroids have been recommended as an adjunct to the treatment of meningitis in children and are thought to (1) reduce neutrophil migration across the blood-CSF barrier, (2) decrease neutrophil lysosome release in the subarachnoid space, and (3) facilitate reconstitution of the blood-CSF barrier. Steroids have been recommended for consideration as adjunctive agents without any age restriction in anthrax meningitis (24; 62); nevertheless, they should generally be avoided if the antibiotic regimen includes vancomycin because CSF vancomycin levels may be decreased by corticosteroids (49).

All the recommended antibiotic agents for postexposure prophylaxis or treatment of anthrax may be associated with allergic reactions, and all are likely to cause side effects when administered for 60 days, although the penicillins are likely to be the best tolerated (35). Fluoroquinolones, including ciprofloxacin, can cause various side effects (eg, nausea, vomiting, abdominal pain, diarrhea, anorexia, dizziness, headache, insomnia, mood changes, tremor, restlessness, confusion, and rarely psychosis, hallucinations, seizures, and Achilles tendon rupture). In children, they may cause permanent cartilage damage and arthropathy. Tetracyclines, including doxycycline, frequently cause gastrointestinal disturbances, can cause photosensitivity reactions, and may cause staining and deformity of the teeth in young children (up to age 8 years) or when administered in utero after the fourth month of pregnancy. In pregnant women, doxycycline should be reserved for cases where there are clear contraindications to other appropriate antimicrobial drugs (34). Amoxicillin can cause diarrhea. In a small survey of 490 persons taking postexposure prophylaxis antibiotics in Florida, approximately 20% reported adverse events attributed to the antibiotics after only 1 to 2 weeks, including symptoms of itching, breathing problems, and swelling of the face, neck, or throat (36). Vancomycin requires dosage adjustment and close monitoring in patients with renal failure.

Three anthrax antitoxin antibody preparations were developed with biodefense funding and have received approval from the FDA as adjunctive therapies for inhalational anthrax: raxibacumab, Anthrasil anthrax immune globulin intravenous (AIGIV), and ETI-204 (44). Antitoxin therapy treats the toxemia component of anthrax infection that antibiotics cannot address and that is, in fact, responsible for most of the morbidity and mortality associated with anthrax infection. Despite a lack of human data on these new antitoxins, limited experimental data from animal studies suggest that adjunctive antitoxin therapy may improve survival (65; 44).

Botulism. Rapid diagnosis and multidisciplinary supportive care are the most important components for managing a victim of toxic exposure to botulinum toxin. There is no known cure for the toxic paralysis of botulism. Respiratory support may be required for up to several weeks. In an epidemic of food-borne botulism with respiratory failure and autonomic nervous system involvement, patients receiving botulinum antitoxin within a few days of exposure had a shorter period of ventilator dependency. Unlike the situation with nerve agent intoxication, where there is too much acetylcholine due to inhibition of acetylcholinesterase, the problem in botulism is the lack of the neurotransmitter in the synapse.

Early administration of botulinum antitoxin to neutralize the circulating toxin is important in patients with symptoms that continue to progress. When symptom progression ceases, no circulating toxin remains, and the antitoxin has no effect. Antitoxin may be particularly effective in food-borne cases, where presumably, toxin continues to be absorbed through the gut wall. Treatment of botulism involves the administration of equine-derived heptavalent (A to G) antitoxin, which has been approved by the FDA and is available from the CDC. There is no vaccine against botulinum toxin, although the antitoxin may induce host immunity to the toxin, and, therefore, the antitoxin may be efficacious when used as a vaccine (02).

The effectiveness of Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G), or BAT, is based solely on efficacy studies conducted in animal models of botulism. BAT is made from equine plasma, and hypersensitivity reactions are possible, including infusion reactions, anaphylaxis, and delayed allergic reactions (serum sickness). In clinical trials with healthy volunteers, the most common adverse reactions observed in at least 5% were headache, nausea, pruritus, and urticaria. In a clinical study, the most common adverse reactions reported in at least 1% of patients were pyrexia, rash, chills, nausea, and edema, and only one serious adverse reaction of hemodynamic instability was observed in a single patient.

A systematic review of randomized controlled trials found low- and moderate-certainty evidence supporting the use of intravenous botulinum immune globulin for infant intestinal botulism (39).

Special considerations

Pregnancy

Botulin toxin. Botulinum toxin does not cross the placental barrier. Anecdotal reports indicate that women suffering from botulism have borne healthy infants.