Biological weapons: Preparing for the worst.

Bioterrorism has been given significant attention during the past 2 years by the laboratory industry press and has been the subject of numerous seminars at national meetings of laboratory organizations. The likely precursors to this coverage were the discovery of a huge biological weapons program in Iraq after the Gulf War of 1991, the discovery of a major biological weapons program in the former Soviet Union called "Biopreparat," and the known recruitment of scientists from other rogue nations who worked on Biopreparat. [1,2] Terrorist attacks, such as the dispersal of sarin nerve gas in a Tokyo subway in 1995 by the Japanese cult Aum Shinrikyo, may also have contributed to the added attention bioterrorism has received. [3,4]

Many countries have biological weapons programs with virtually no safeguards against the transfer of weapons or the technology used to manufacture them to potential terrorist groups. These countries (many classified by the US State Department as supporting terrorism) include Syria, North Korea, Russia, Iran, Iraq, Libya, and China. [5]

The threat of a terrorist action against the US that involves lethal biological weapons is not only possible but also probable, according to experts. In his congressional testimony before the Subcommittee on National Security, Veterans Affairs, and International Relations for the US House of Representatives on Oct. 20, 1999, Raymond Zalinskas, PhD, the Senior Scientist-in-Residence for the Chemical and Biological Weapons Nonproliferation Project at the Center for Nonproliferation Studies in Washington, DC, said that the most likely attacks would be on food or water supplies. He also predicted a substantial increase in the possibility of biological attacks using airborne pathogens between 2006 and 2010. [6]

Three major trends have been cited as responsible for the increased threat of biological weapons: the expertise needed to develop biological agents is rapidly proliferating, the technology needed to manufacture these agents is readily available, and the will to use these agents may be increasing among terrorist organizations. [1-4] Many toxins can be readily produced with minimal scientific knowledge, facilities, and financial support. Biological weapons are appealing to terrorists because they are both cheap to produce and capable of causing cause massive casualties. [2,7] Any "basement laboratory" can produce significant quantities of toxic substances. Advances in genetic engineering also mean that a microbiologist can engineer new organisms that are more lethal and resistant to known antibiotics. Because of this, the threat for bioterrorism is the greatest it has ever been. Terrorist groups with specific political goals may be unwilling to use biological agents out of fear of backlash, but the more fanati c ethnic and religious organizations whose motives cannot be understood in political terms may be more prone to experiment with agents of biological warfare.

The use of biological weapons is not new. Diseased corpses were catapulted into enemy cities in medieval times; and in the Eighteenth century, blankets infested with smallpox were distributed to certain Native American tribes, [8,9]

Attempts to control the use of biological weapons are almost as old as the weapons themselves. Most of the members of the League of Nations signed the 1925 Geneva Protocol renouncing the use of chemical or biological weapons. Later, the 1972 Biological Weapons Convention was signed by 118 countries, including China, Japan, all NATO members, and the former Warsaw Pact countries. [2] The BWC defined both biological and chemical weapons as instruments of biological warfare and prohibits the development, production, and stockpiling of biological and chemical weapons. Because no verification method exists, however, this pact remains largely unenforceable. [1]

Domestic defense

In 1996, the US government developed the Biological Warfare Defense Program. The purpose of the program was to develop broad-spectrum approaches to neutralizing biological agents (bacteria, viruses, bioengineered organisms, and toxins) used in biological attacks. [10] This program partners with universities and other organizations to perform research that creates fast, simple methods for detecting and identifying biological agents. It also develops protective gear and mechanisms to destroy organisms before they enter the body. Even though this program provides expertise for the Department of Defense in biological attacks, many innovations will be transferred to the civilian sector for use in a bioterrorism attack.

That same year, Congress passed the Defense Against Weapons of Mass Destruction Act, which mandated that the Secretary of Defense create a program to improve the responses of state and local agencies to emergencies involving biological and chemical weapons. As a result, the Department of Defense established the Biological Weapons Improved Response Program, which uses the resources of he US Army's Chemical and Biologic Defense Command, the Department of Health and Human Services, the Department of Energy, the Department of Agriculture, the Federal Emergency Management Agency, the Federal Bureau of Investigation, the Environmental Protection Agency, and the Centers for Disease Control and Prevention to develop the Biological Weapons Response Template, which will help city and state governments prepare their defenses for an attack. [11,12]

Most published government and military reports state that the nation's medical and government structures would be unable to effectively respond to such a catastrophic event. [13] The US, local, state, and federal public health infrastructure is already overburdened with other pressing public health issues. [13] A simulated biological attack staged in New York, Chicago, and Los Angeles in 1996 revealed just how vulnerable the US is to bioterrorism. [14] Firemen were reported to have rushed into the "contaminated" area without protective clothing, and hospitals reported they would have been overwhelmed in the event of an actual attack. The US government responded in several ways, including appropriating $800 million for chemical and biological weapons defense in fiscal year 1997. [2] These and other government actions indicate that the detection, deactivation, and containment of biological weapons are becoming priorities for the US.

The lab's response

The execution of 4 critical steps will determine the success of the medical community's response to a bioterrorist attack [15]:

* prompt lab identification of agents of biological warfare,

* notification of local, state, and federal health officials,

* notification of appropriate law enforcement agencies, and

* support of healthcare providers who may be faced with caring for large numbers of infected patients.

For their part, laboratory professionals must be aware of potential agents of biological warfare, know how to isolate and identify them, and know what precautions should be taken when handling such agents. Also important is having a procedure for notifying authorities of a suspected bioterrorist attack.

What are biological weapons?

Biological weapons include both biological agents, such as bacteria, protozoa, rickettsia, viruses, and fungi, as well as toxins, such as poison gas and chemicals. This article addresses the most commonly used biological weapons, most of which are biological agents.

Biological and chemical weapons are extremely pathogenic to both humans and animals. Their effects can include acute respiratory paralysis, central nervous system disorders, and organ failure, with death as the final outcome.

According to Zalinskas, the major biological threats to the US include emerging pathogens, reemerging pathogens, and transported infectious diseases. [11] Terrorist groups could use many different biological agents in a bioterrorism attack. The following biological agents are those put forth by the US Army Medical Research Institute of Infectious Diseases (USAMRIID) as those most likely to be used: Bacillus anthracis, Brucella sp., Burkbolderia mallei, Vibrio cholerae, Yersinia pestis, Francisella tularensis, Coxiella burnetii, Venezuelan equine encephalitis (VEE), viral hemorrhagic fevers, smallpox, botulinum toxin, staphylococcal enterotoxin B, ricin, and T-2 toxins. [8,16,17]

Anthrax

An aerobic, spore-forming, gram-positive rod called Bacillus anthracis causes anthrax. Anthrax is usually found in cattle, sheep, and horses, although other animals can also be infected. The infectious form of the organism is the spore, which remains viable for years in soil, water, and direct sunlight. The incubation period is 1-6 days.

Diagnosis. Initial symptoms include fever, malaise, fatigue, cough, and mild chest pain, which quickly progresses to severe respiratory distress, then shock and death within 24-36 hours after the initial symptoms. The gastrointestinal and cutaneous forms of anthrax are not lethal. A vaccine is available and is now required for all military members. Diagnosis usually comes late in the disease by blood culture or Gram stain of peripheral blood.

Treatment. Penicillin is effective before the symptoms appear, but high-dose antibiotic treatment with penicillin, ciprofloxicin, or doxycycline is recommended. [8,16,17]

Brucellosis 

Brucellosis can be caused by Brucella abortus, Brucella melitensis, and Brucella suis, which are gram-negative coccobacilli. These organisms are highly infectious, and as few as 10-100 bacteria can produce disease after being inhaled. The incubation period is from 5-60 days, but large aerosol doses can shorten the incubation period. Brucellosis is an incapacitating disease, but the mortality rate is less than 5% of infected individuals. Brucellosis also occurs as a percutaneous or enteric infection, which is contracted by consuming unpasteurized dairy products (especially goat's milk and cheese).

Diagnosis. Brucellosis presents as a nonspecific febrile illness with headache, fever, myalgia, arthralgia, back pain, sweats, chills, and malaise. The bacteria may cause meningitis in 5% of the cases, and the organism can be cultured from cerebrospinal fluid. Patients may present with anemia and thrombocytopenia, and blood and bone marrow cultures may be positive during the acute febrile phase. Enzyme immunoassays are under development as is a polymerase chain reaction test using a protein from B. abortus.

Treatment. Treatment is doxycycline plus rifampin for 6 weeks. Ofloxacin plus rifampin can also be given. For patients with meningitis or endocarditis, rifampin, tetracycline, and an aminoglycoside are administered. [8,16,17]

Cholera

Cholera is caused by a short, curved, motile, anaerobic, gram-negative rod called Vibrio cholera. Vi cholerae produces an enterotoxin that inhibits water absorption in the small intestine. The organism is transmitted by contaminated water, food, flies, or soiled utensils and can be killed by drying, chlorinating, steaming, and boiling water. This organism can withstand freezing for 3-4 days.

Diagnosis. Diagnosis is performed clinically by observing "rice water" diarrhea and dehydration. Laboratory diagnosis is made by identifying the motile vibrio under phase-contrast microscopy in the field or by culture in clinical laboratories.

Treatment. Treatment with antibiotics will reduce shedding of the organism so that the infected person does not pass as many organisms into his or her body fluids. However, antibiotics will not kill the organism [8,16,17]

Glanders 

Glanders is caused by a small, gram-negative rod called Burkholderia mallei (formerly known as Pseudomonas mallei). This organism usually produces disease in horses, mules, and donkeys, but may also infect humans. Infection occurs through inhalation or through cracks or sores in the skin. Laboratory cultures are considered extremely infectious and require biosafety level 3 practices.

Infections in patients can take 4 forms: acute localized, septicemia, acute pulmonary, or chronic cutaneous. The acute localized form can be either cutaneous or mucosal. In the cutaneous form, patients present with nodules and ulcerations. If the cutaneous form becomes systemic, a papular or pustular rash accompanies systemic invasion, and it can be mistaken for smallpox.

The septic form of this infection presents with a sudden onset fever, rigors, sweats, myalgia, chest pain, phototrophia, lacrimation, and diarrhea. The blood cultures are usually negative, and this form causes death quickly after onset.

The mucosal form involves infection of oral, nasal, and/or conjunctival mucosa, which causes mucopurulent, bloodstreaked discharged from the nose. Turbinate nodules and ulcerations are also present in the nose. Systemic invasion can occur from here.

The chronic form presents with cutaneous and intramuscular abscesses on the legs and arms. There is also enlargement and induration of the lymph glands. Patients with this form of the disease may still erupt into acute septicemia.

Diagnosis. Diagnosis is achieved through a Gram stain of exudates and routine culture. B. mallei takes 48 hours to grow on nutrient agar, but colony growth can be enhanced by the addition of meat infusion media. Antibody detection tests are not positive for 7-10 days and are difficult to interpret because of a high background titer. Complement fixation tests are more specific and are considered positive at a titer of 1:20.

Treatment. Most antibiotics have been tested only on animals, so each organism encountered must be tested to determine susceptibility to antibiotic agents. Experts expect high mortality despite antibiotic use. [8,16,17]

Smallpox

Smallpox is an orthopoxvirus. It has two forms: variola major and variola minor. Variola major produces more severe disease. The virus is highly infectious when transported by aerosols, is easy to make, and the world population does not have immunity to it. In 1980, the World Health Organization declared that smallpox was globally eradicated and approved CDC and the Institute for Viral Preparedness in Moscow to hold live cultures of smallpox. In the 1980s, children in the US stopped receiving smallpox vaccinations, and the military stopped vaccination programs in 1989. This means that a good part of the US population and its military force are now susceptible to variola major. This fact makes smallpox a choice biological agent because even though smallpox was "globally eradicated" and a vaccine exists, countries with live cultures can still use it in biological weapons. The incubation period for smallpox is around 12 days, but because it is extremely infectious as pustules, patients and contacts are quarantin ed for about 16 days after exposure.

Symptoms. The patient infected with variola major presents with malaise, fevers, rigors, vomiting, headache, and backache. After 48-72 hours, patients develop a rash on the face, hands, and forearms, which then extends to the legs and trunk over the next week. Lesions are plentiful on the face and extremities--this is called centrifugal distribution and is diagnostic. Approximately 2 weeks after onset of nonspecific symptoms, pustules form scabs, which then become depressed and depigmented. After healing, scars are left on the skin. Patients are considered infectious until all scabs disappear. Variola major can cause up to 30% mortality among the unvaccinated population.

The early stages of smallpox are indistinguishable from other diseases such as chicken pox or allergic-contact dermatitis. Particularly troubling is the fact that many vaccinated people could contract mild cases of the disease and spread it before they are quarantined.

Diagnosis. Smallpox is usually diagnosed by appearance of virions on electron microscopy of vesicular scrapings. Guarnieri's bodies in vesicular scrapings can be seen with Gispen's modified silver stain, but the inclusions do not differentiate between cowpox, monkeypox, and smallpox.

The problem with smallpox is that it is highly contagious. If a vesicular exanthem (eg, skin eruptions or vesicles on the lips, nose, or tongue) is discovered on a patient, physicians must suspect a biological attack and undertake appropriate quarantine measures. A confirmed case of smallpox is an international emergency and needs to be reported to public health officials immediately. One sobering thought: only persons vaccinated within the past 3 years are considered immune; however, if vaccination is given within 7 days of exposure to smallpox, the disease can be prevented.

Treatment. There are no effective antiviral drugs at this time. Variola immune globulin (VIG) does exist and can provide protection for people who cannot take the smallpox vaccine. [8,16,17]

Plague

A gram-negative, nonmotile, nonsporulating, aerobic bacteria named Yersinia pestis is responsible for the plague. Plague is a zoonotic infection that is passed onto humans by fleas. If a human is bitten by an infected flea, this person develops the bubonic form of plague, but the pneumonic form of plague is better suited for a biological weapon because all people are susceptible. The organism can remain alive in water, meals, and grains for weeks; but at near freezing, it can remain alive from months to years. It is killed when exposed to temperatures of 72[degrees]C for 15 minutes and several hours of sunlight exposure.

Diagnosis. The pneumonic plague presents with malaise, high fever, chills, headache, myalgia, cough with a bloody sputum, and toxemia. Patients have bronchopneumonia, which progresses rapidly to dyspnea, stridor, and cyanosis. They die from respiratory failure, circulatory collapse, and a bleeding diathesis. The typical incubation period for pneumonic plague is 2-3 days.

Diagnostic laboratory tests include CBC with a WBC of as high as 20,000/L with increased bands and [greater than] 80% polymorphonuclear cells on the differential. Patients experience a lowgrade disseminated intravascular coagulation (DIC) with a positive fibrin degradation product (FDP) and elevated alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin levels. Presumptive diagnosis consists of finding gram-negative coccobacillus in lymph node aspirate, sputum, or CSF. The gram-negative coccobacillus exhibits safety-pin bipolar staining with Giemsa stain. Definitive diagnosis is made from culture of blood, sputum, bubo aspirates, and CSF. Yersinia pestis will grow on blood and MacConkey agars.

Treatment. Patients must be isolated for 72 hours after starting antibiotics. Antibiotics of choice include streptomycin, tetracycline, chloramphenicol, gentamicin, and ceftriaxone. A vaccine is available, but it is a 3-dose vaccine that requires boosters at 6, 12, and 18 months; then every 1-2 years after vaccination. People who need prophylaxis can take doxycycline to prevent infection in face-to-face encounters with infected people. [8,16,17]

Tularemia 

Tularemia is caused by a small, nonmotile, aerobic, gramnegative coccobacillus called Francisella tularensis. This organism can survive in water, carcasses, hides, and for years in frozen rabbit meat. It also survives in soil or water at freezing temperatures and below. It is susceptible to heat and disinfectants.

Tularemia manifests itself in several forms in man: ulceroglandular, septicemic (typhoidal), and pneumonic. The most probable form of the disease caused by a bioterrorism attack would be septicemic tularemia. Septicemic tularemia occurs after intradermal, respiratory, or gastric inoculation of the organism. This is the presenting form of tularemia in 5-15% of cases. Pneumonic tularemia can be primary (inhaled bacteria) or secondary (produced after patient is septic).

Diagnosis. Acute onset of disease usually occurs from 2-10 days after exposure. Diagnosis of tularemia is difficult because signs and symptoms are nonspecific. The organism grows poorly on conventional media or is overgrown by normal flora. Direct Gram stains from ulcer fluids or sputums are usually not helpful.

Treatment. Tularemia is treated with intramuscular streptomycin for 10-14 days or gentamicin. Tetracycline and chloramphenicol are also used, but there are more relapses associated with these drugs. A new investigational drug was recently developed. [8,16,17]

Q fever

Q fever is caused by a rickettsia called Coxiella burnetii that usually infects sheep, cattle, and goats. Coxiella burnetii is extremely infective via the aerosol route. As few as 1-10 organisms can produce clinical disease when inhaled by a human. The incubation period for this disease is 10-20 days, after which the illness is usually self-limiting--lasting from 2 days to 2 weeks.

Diagnosis. The disease usually presents with headache, myalgia, and fatigue. About half of the patients develop pneumonia, but only 25% of those patients have a productive cough. About one-third of the patients have an elevated WBC, and most patients have an elevated ALT and AST.

Treatment. Tetracycline or doxycycline treatment will shorten the length of the illness, and the fever will disappear after 2 days of antibiotic treatment. A vaccine is available and provides at least 5 years of immunity against Q fever. Prophylaxis with doxycycline or tetracycline can prevent the disease if given 8-12 days after exposure and continued for 10 days. [8,16,17]

Venezuelan equine encephalitis

Venezuelan equine encephalitis (VEE) is caused by an alphavirus of the same name. This virus usually affects horses, mules, and donkeys and is usually carried by mosquitoes. Wet and dry forms are stable and can be released by aerosol or through water and food contamination. The virus can be inactivated with heat and disinfectants.

This virus causes an inflammation of the meninges and the brain, with fatality occurring in less than 1% of infected patients. The disease is usually acute and short-lived. Recovery from this disease produces lifelong immunity. The incubation period is usually 1-5 days before the onset of symptoms.

Diagnosis. Symptoms include malaise, fever, rigors, severe headache, photophobia, and myalgia of the legs and lower back. Secondary symptoms include nausea, vomiting, diarrhea, cough, and sore throat. The WBC differential shows leukopenia and lymphopenia. CSF may contain as many as 1,000 leukocytes/[mm.sup.3] and an elevated protein level. Serological tests such as IgM ELISA, IFA, and complement-fixation tests are available for this virus.

Treatment. Treatment of the symptoms is the only relief for patients infected with this virus. Recovery usually takes 1-2 weeks. There is no commercially available vaccine. [8,16,17]

Viral hemorrhagic fevers

This group of diseases is caused by RNA viruses from the Filoviridae (Ebola and Marburg), Arenaviridae (Lassa fever, Argentine and Bolivian hemorrhagic fever), and Bunyaviridae (Hantavirus, Congo-Crimean hemorrhagic fever, Rift Valley fever, and Yellow fever) families; the Dengue hemorrhagic fever virus; and others (see Table 1). Thankfully, aerosol biological weapons do not exist for some of these viruses, but the symptoms may confuse physicians treating infected people.

Viral hemorrhagic fevers target the vascular system and cause changes in vascular permeability, which then leads to microvascular damage. The initial symptoms include fever, myalgia, mild hypotension, flushing, and petechiae. These symptoms quickly evolve to shock with generalized mucous membrane hemorrhage. Renal failure accompanies the cardiovascular decline, and mortality can be 5-20% or higher. The fatality rates for Ebola are 50-90%.

Diagnosis. A detailed history is important because many of these viral hemorrhagic fevers are spread by mosquitoes or other arthropod vectors. Therefore, if a patient has visited a geographic location where the disease is endemic, a terrorist attack can probably be ruled out. When large numbers of cases are diagnosed in a nonendemic area, however, a biological attack should be suspected. A viral hemorrhagic fever should not be ruled out if the patient presents with hypotension, petechiae, and flushing of the face and chest. Other diseases that may be confused with viral hemorrhagic fevers include typhoid fever, rickettsial disease, relapsing fever, and leptospiral disease. Diseases that lead to DIC such as leukemias, lupus erythromatosus, idiopathic or thrombotic thrombocytopenic purpura, or hemolytic ureniic syndrome can also be confused with viral hemorrhagic fevers and must be ruled out.

Laboratory results include thrombocytopenia (except Lassa), leukopenia (except Lassa, hantaviral, and severe Crimean-Congo hemorrhagic fever), and proteinuria and/or hematuria (found in Argentine hemorrhagic fever, Bolivian hemorrhagic fever, and hantaviral infections). Serological tests for antibodies to Lassa, Argentine hemorrhagic fever, Rift Valley fever, Crimean-Congo hemorrhagic fever, yellow fever, and hantaviral disease may also be used to confirm viral hemorrhagic fevers. These viruses all require biosafety level 4 containment facilities.

Treatment. Supportive therapy is given to patients with viral hemorrhagic fevers. If DIC is present, heparin therapy should be given. Dengue and hantaviral infections should be managed differently than the rest of the viral hemorrhagic fevers because of the severe consequences of Dengue fever and the renal involvement of hantaviral infections. The antiviral drug ribavirin can reduce mortality in Lassa fever. Dengue fever, Yellow fever, Ebola, and Marburg fever are not responsive to ribavirin therapy. [8,6,7]

Botulinum toxin

Clostridium botulinum produces 7 neurotoxins, types A through G. All neurotoxins produce symptoms of botulism. These toxins are considered some of the most toxic substances in the world. (The botulinum toxins are 100,000 times more toxic than sarin nerve gas.) The toxins can be delivered as an aerosol, and people who inhale the toxins will become ill with symptoms of botulism. The toxins can produce paralysis in infected persons because they bind to the presynaptic nerve terminal at neuromuscular junctions and prevent release of acetyicholine and ultimately, neurotransmission.

Diagnosis. Patients can become ill in as little as one day after inhaling the toxins. The initial symptoms include blurred vision, diplopia, photophobia, dysarthria, dysphagia, and dysphonia. Next, skeletal muscles are affected, which causes a symmetrical, descending, progressive weakness that can result in respiratory failure. Patients may also experience dry and crusted mucous membranes--especially in the mouth--and can experience difficulty speaking and lose their gag reflex.

If many patients present with no fever, but exhibit a descending paralysis, botulism should be expected. This condition can be confused with Guillain-Barre syndrome, myasthenia gravis, or tick paralysis. Laboratory testing is not helpful in diagnosing this condition.

Treatment. Antitoxin is available from the CDC, but because it is equine-based, side effects may include anaphylaxis and serum sickness from the horse proteins. A toxoid made from Clostridium botulinum toxin types A, B, C, D, and E has been used on groups at high risk for inhalation of botulinum toxins. The toxoid induces antitoxin production that will protect against the adverse effects of the toxins. [8,6,7]

Staphylococcal enterotoxin B

Staphylococcal enterotoxin B is a pyrogenic toxin that is produced by Staphylococcus aureus and that causes food poisoning. In bioterrorism, this toxin would probably be introduced using an aerosol. It produces a different disease when inhaled as compared with ingestion. Although it would not cause high mortality if used in an attack, it would cause a great number of people to be incapacitated and to require medical treatment. This toxin causes disease by activating a nonspecific immune response from a person, and it induces T-cell proliferation that leads to increased interleukin-2 production, which produces severe nausea and vomiting. Inhalation of the toxin also causes production of tumor necrosis factor and interferon gamma.

Diagnosis. Symptoms begin 3-12 hours after inhalation. There is a sudden onset of fever, headache, chills, myalgias, and a nonproductive cough. Soon after the initial symptoms, nausea, vomiting, and diarrhea occur and lead to heavy fluid loss. The fever ranges from 103 to 106[degrees]F and may last up to 5 days. Severe cases can develop pulmonary edema and acute respiratory distress syndrome.

Laboratory tests are not useful in diagnosing staphylococcal enterotoxin B inhalation, so it must be diagnosed clinically and epidemiologically. This disease presentation is similar to anthrax, tularemia, Q fever, plague, adenovirus, influenzae, and mycoplasma infections. Epidemiologists should be concerned about a bioterrorist attack if large numbers of patients present with the initial symptoms in a short period of time.

Treatment. Treatment is limited to supportive care, no antitoxin or other specific treatment is available. There is also no vaccine on the market to prevent reaction to this toxin. [8,16,17]

Ricin

Ricin is a toxin that is derived from castor beans. After the oil is extracted from the beans, the waste product contains about 5% ricin. Ricin is easy and inexpensive to produce in large quantities and is toxic to cells because it inhibits protein synthesis. The amount of ricin inhaled determines the severity of disease produced. Sublethal doses will produce fever, chest tightness, cough, dyspnea, nausea, and arthralgias within 4-8 hours of exposure. If enough toxin is inhaled, severe damage occurs in the airways and alveoli, causing death in approximately 18-72 hours. If ricin is ingested, gastrointestinal hemorrhage and hepatic, splenic, and renal necrosis occurs.

Diagnosis. Because of the nonspecific symptoms, an aerosol attack would be diagnosed by clinical and epidemiological data. Ricin is immunogenic, so survivors will have antibody protection for a short period of time.

Treatment. Treatment is only supportive because no antitoxin is available. There is also no vaccine available, but gas masks can prevent inhalation of the toxin. [8,16,17]

T-2 toxins

Filamentous fungi, especially those of the Fusarium, Myrotecium, Trichoderma, and Stachybotrys genera, produce trichothecene mycotoxins. These mycotoxins are extremely heat stable and resist ultraviolet light inactivation. If the mycotoxins are ingested, they produce a lethal illness called alimentary toxic aleukia (ATA) with the following initial symptoms: abdominal pain, diarrhea, vomiting, and prostration. These progress into fever, chills, myalgias, and bone marrow depression causing granulocytopenia and sepsis. If the patient survives these initial stages, the next set of symptoms are painful pharyngeal/laryngeal ulceration and diffuse bleeding into the skin, bloody diarrhea, hematuria, hematemesis, epistaxis, and vaginal bleeding. Some believe that these mycotoxins were used in Southeast Asia and Afghanistan in the form of "yellow rain" to produce casualties and deaths among the civilian populations. [8]

Mycotoxins can enter the body through the skin, stomach, or lungs and inhibit protein and nucleic acid synthesis. The first cells attacked are the rapidly dividing cells such as bone marrow, skin, mucosal epithelia, and germ cells. When skin is exposed to mycotoxins, burning, redness, blistering, and skin necrosis occur. When nasal mucosa is exposed to mycotoxins, this produces nasal pain, sneezing, rhinorrhea, dyspnea, wheezing, cough, and blood tinged saliva and sputum. Exposure of the eyes to mycotoxins produces eye pain, tearing, redness, and blurred vision. Once the mycotoxins enter the system, symptoms include weakness, prostration, dizziness, ataxia, loss of coordination, and in fatal cases, tachycardia, hypothermia, and hypotension. Death may occur in minutes, hours, or days.

Diagnosis. Laboratory tests are not available to diagnosis exposure to T-2 toxins. Toxic exposure can only be confirmed when tissue samples taken at autopsy are tested using a mass spectrometer.

Treatment. Again, exposure can be prevented with a gas mask and protective chemical gear. All treatment is supportive because no antitoxins or antifungals are presently available. [8,16,17]

Bioterrorism readiness plan

In 1999, the Association of Professionals in Infection Control and Epidemiology's Bioterrorism Task Force and the CDC's Hospital Infections Program Bioterrorism Working Group collaborated to produce a reference document to help healthcare facilities develop readiness plans in case a bioterrorism attack occurs in their city. [18] This plan includes reporting requirements; detection of outbreaks; infection control practices for patient management; postexposure management; patient, visitor, and public information; and laboratory support and confirmation. When these agencies developed this initial template, only 4 diseases were considered potential biological agents: anthrax, botulinum toxin, plague, and smallpox. More recently, other agents have been added to the list by USAMRIID.

Reporting requirements. Because healthcare facilities will probably be the first to recognize a bioterrorism attack, they will be responsible for notifying local infection control personnel, administrators, public health facilities, FBI field offices, local police, the CDC, and emergency medical services. Every facility should have these phone numbers available.

Detection of outbreaks. Terrorist attacks using biological agents can occur as announced or unannounced events. Healthcare facilities must be able to deal effectively with both types of attacks. To do this, facilities should determine syndrome-based criteria and epidemiological features of the outbreak to determine if terrorists have used a biological agent.

Infection control practices for patient management. Healthcare facilities must determine what isolation procedures should be used for individual biological agents because some agents are not transmitted via person-to-person contact and others are. Patient placement and patient transport is important to avoid both exposure of the infected patient to other agents and exposure of unaffected patients to toxic agents. In addition, cleaning, disinfection, and sterilization of equipment and the environment are very important to prevent the spread of the agent to other healthy patients. Postexposure plans should include discharge management of patients to ensure reinfection does not occur. Clinical laboratories and pathology departments need to be informed of suspected agents so that extra biosafety precautions can be taken with bodies and bodily substances after postmortem examinations.

Postexposure management. Patients and the environment need to be decontaminated after release of a biological agent. People who were exposed or thought to have been exposed should be (1) given medication to prevent onset of the disease and its symptoms and (2) immunized against a particular biological agent. Healthcare facilities must develop plans for triage and management of large-scale exposure and suspected exposures without shutting down the hospital. Masses of people can overwhelm healthcare facilities, so this possibility should be dealt with up-front so that plans can be made to keep the system flowing. Finally, psychological aspects of bioterrorism must be taken into account and planned for. Mental health professionals need to be an integral patt of the response team.

Patient, visitor, and public information. All 3 audiences must be kept adequately informed to prevent widespread panic. The information given must be clear, concise, and understandable. Fact sheets can be prepared ahead of time and distributed to interested parties. All lines of communication with the outside world need to be coordinated in advance so that misunderstanding and anxiety can be minimized.

Laboratory support and confirmation. Clinical microbiology laboratories routinely identify infectious agents that cause disease in humans and animals. Laboratories provide 80% of objective data used to make a diagnosis; and in a bioterrorism attack, providers will expect the laboratory to identify the bioterrorism agent and its antimicrobial susceptibility pattern. Bioterrorism agents used can be categorized as biosafety level 2 pathogens such as salmonella, biosafety level 3 pathogens such as Venezuelan equine encephalitis, or biosafety level 4 pathogens such as smallpox (see Table 2). Most clinical laboratories routinely identify biosafety level 2 pathogens, and larger laboratories may identify biosafety level 3 pathogens; but only 2 labs in the US have the capability to identify biosafety level 4 pathogens--the CDC and USAMRIID. If an attack with a biosafety level 4 agent occurred anywhere in the country, clinical laboratories would be unable to identify the agent.

The CDC, USAMRIID, and the Association for Public Health Laboratories identified the need for a laboratory network for responding to bioterrorism and emerging infectious diseases. [19] This network is being developed and would consist of hospital clinical laboratories and physician office laboratories (level A), commercial reference laboratories (level B), public health and military laboratories (level C), and CDC and USAMRIID (level D). Hospital clinical and physician office laboratories would be first-response labs because patients would seek medical care at local facilities as soon as they became ill. (After a covert bioterrorism attack; the disease incubation period could be as short as 1 day or as long as 1 month before victims became symptomatic.) This period of time between exposure and developing symptoms would increase the difficulty of identifying a bioterrorism attack. The level A labs would be responsible for ruling out bioterrorism agents, referring suspected agents to level C labs, and evaluati ng specimens from patients.

The responsibilities of level B labs would be to confirm laboratory specimens referred from level A labs, to isolate and identify bioterrorism agents, to train and educate level A lab personnel, and to test for antimicrobial susceptibility of isolates. If an agent of biological warfare was suspected, commercial reference labs would refer the specimen to level C labs (public health and military labs).

Level C labs would have particular expertise in working with particular organisms, and specimens would be referred to those labs specializing in the characterization or molecular fingerprinting of that organism. This would be necessary to compare and identify isolates from other suspected victims of bioterrorism. These labs would also be responsible for evaluating and distributing devices or new rapid identification methods for identifying biological agents.

Level D labs would be responsible for developing new rapid identification tests for bioterrorism agents, testing all agents for chimeras (genetically altered organism that may not be detectable by current methods), and for culturing and performing antimicrobial susceptibility tests on all biosafety level 3 and 4 bioterrorism agents.

This laboratory network would rely heavily on information technology to provide timely identification and antimicrobial susceptibility of bioterrorism agents. The current vision shows level C and D labs developing new methods and procedures, then passing these newly developed tests to the level A and B labs. A central Web-based repository would be available to laboratories through a secure Internet site. Level D labs would also develop algorithms to aid level A and B labs in ruling out and identifying bioterrorism agents. CDC and APHL envision 2 parallel networks (civilian and military) that can be cross-linked so that both can retrieve relevant data. [20]

Even though some experts do not anticipate bioterrorism attacks in the next 5 years, the medical community must expect the unexpected. Plans should be developed that have criteria for determining exposure to biological agents, triaging thousands of patients, reducing risk for both unexposed and infected patients, and keeping the public from panicking. As one looks at all the potential biological agents and the similarities between the syndromes produced, detecting and identifying biological agents is a monumental task. The civilian community can look to the military for some answers, but the military detection and identification techniques for biological agents are still infantile--if they exist. When bioterrorist attacks occur in the US, we must be prepared or pay dearly for a lesson learned.

Donna Leach is associate professor and chair of the Clinical Laboratory Science Department and Denny G. Ryman is assistant professor in the Clinical Laboratory Science Department at Winston-Salem State University, Winston-Salem, NC.

References

(1.) Alibek K Biohazard. New York: Random House.

(2.) Seldon Z. Assessing the biological weapons threat. Business Executives for National Security Special Report. www.bus.org/pubs/bwc.html. Accessed July 17, 2000.

(3.) Carus SW. The threat of bioterrorism. From the National Defense University's Institute for National Strategic Studies, Strategic Forum. Sept 1997. www.ndu.edu/niss/strforum/forum 127.html. Accessed July 21, 2000.

(4.) Tucker JB. Historical Trends Related to Bioterrorism: An Empirical Analysis. Emerging and Infectious Diseases. Vol. 5, no. 4, July-August, 1999. www.cdc.gov/ncidod/eid/vol5no4/tucker.html. Accessed on April 5, 2000.

(5.) Anderson JH. Microbes and Mass Casualties: Defending America Against Bioterrorism, The Backgrounder. The Heritage Foundation, www.heritage.org/library/backgrounder/bgl182es.html. Accessed on August 17, 2000.

(6.) Zalinskas R. (20 October 1999). Assessing the Threat of Bioterrorism: Congressional Testimony by Raymond Zalinskas. Center for Nonproliferation Studies, Monteray Institute of International Studies, National Defense University.

(7.) MacKenzie D. (September 19, 1998). Bioarmageddon. New Scientist 2000, Online conference reports, www.newscientist.com/nsplus/insight/ bioterrorism/bioarmageddon.html. Accessed on 4/5/00.

(8.) US Army Medical Research Institute of Infection Diseases. Medical Management of Biological Casualties. September 1999. www.nbcmed.org/SiteContent/HomePage/What'sNew/MedManual/Sep99/Current /Handbook.html. Accessed on April 15, 2000.

(9.) Kupperman RH, Smith DM. Coping with biological terrorism. In: Roberts B, ed. Biological Weapons: Weapons of the Future? Washington, DC: Center for Strategic and International Studies; 1993.

(10.) Kozaryn L. (February 9, 2000). Biological Defenses on the Horizon. American Forces Information Service. www.defenselink.mil/news/Feb2000/n02092000_20002092.html. Accessed on 4/5/00.

(11.) Boyce N. Nowhere to hide. New Scientist, Online Conference Reports. www.newscientist.com/nsplus/insight/bioterrorism/nowhere.html. March 21, 1998.

(12.) Hutchinson R. Improving Local and State Agency Response to Terrorist Incidents Involving Biological Weapons. Response to Nunn-Lugar-Domenici Domestic Preparedness Program by Department of Defense. August 1, 2000. dp.sbccom.army.mil/fr/dp_bwirp_interim_planning_guide_download.html. Accessed on August 16, 2000.

(13.) CDC Strategic Planning Workgroup. Biological and chemical terrorism: Strategic plan for preparedness and response. MMWR. April 21, 2000;49(RR04);1-14.

(14.) "US Cities Prepare to Deal with Terror Attacks, But Drills Point to Weankess in Rescue Plans." Wall Street Journal. Monday, June 3, 1996: A16.

(15.) Wilson ML. Bioterrorism Q&A. Laboratory Medicine. 1999;30(9):568.

(16.) Zajtchuk R. Textbook of Military Medicine: Medical Aspects of Chemical and Biological Warfare. Published by the Office of the Surgeon General, Department of the Army; 1997.

(17.) Murray PR, Baron EJ, Pfsllor MA, et al, eds. Manual of Clinical Microbiology. 6th ed. Washington, DC: ASM Press; 1995.

(18.) APIC Bioterrorism Task Force and CDC Hospital Infections Program Bioterroism Working Group. Bioterrorism Readiness Plan: A Template for Healthcare Facilities. April 13, 1999. www.cdc.gov/ncidod/hip/bio/13apr99APIC-CDCBioterrorism.PDF. Accessed on April 5, 2000.

(19.) Gilchrist MJR. A national laboratory network for bioterrorism: Evolving from a prototype network of laboratories performing routine surveillance. Military Medicine. 2000;165(supplement 2:001):28-31.

(20.) Ascker MS. A civilian-military virtual public health laboratory network. Military Medicine. 2000;165(supplement 2:001):1-4.

CDC: Our national resource for responding to biological attacks

If the unthinkable happens and an attack on the US with biological weapons occurs, an immediate call would be placed to the Centers for Disease Control and Prevention, which has set up an office to coordinate a national response to bioterrorist attacks. The next step would depend on whether the event was a chemical or a biological episode, said Elaine Gunter, Chief, NHANES Laboratory (National Health and Nutrition Examination Survey), CDC. "With biological events, it may be 2 weeks before you know what people are exposed to. They are going to trickle into doctors, county health offices, and hospitals. When a chemical event occurs, people are going to drop like flies. You know something has happened right away," she explained.

"Seven of us in this laboratory are medical technologists. Two of us would be dispatched within 2 hours to help first responders with specimen collection. We would look primarily for certain groups of chemicals, such as organophosphate nerve agents, agents used in riot control, or ricin. We developed a rapid toxic screen that enables us to identify these chemicals with a very small amount of blood or urine." Within 24 hours, 75 chemical agents can be identified through the use of various tests conducted on very high level instruments such as mass spectrometers that are capable of measuring down to parts per trillion, noted Gunter. "In another year, we hope to be able to measure 150 chemical agents within 48 hours." The rapid toxic screen answers 3 critical questions: Which agents were used? Who was exposed? How much exposure occurred? "We're not only looking for the exotic agents. We're looking for agents that are commonly available," she said.

In addition to helping collect blood and urine from victims, the team helps first responders package and ship samples to the CDC. They establish a "really strict chain of custody in accordance with the FBI's guidance because these are forensic specimens that will be used in a criminal trial. We have to be extremely careful," added Gunter.

The CDC's Chemical Terrorism Laboratory Network is composed of the laboratory at CDC and five state laboratories in New York, California, Virginia, Michigan, and New Mexico. CDC provides training, technical assistance, and proficiency testing for the state labs. "We bring them on board basically one method at a time. Most of these methods didn't exist 18 months ago."

On the biological side, the network is a complex one that goes from level A (the physician or county hospital) to level D (the CDC and US Army Medical Research Institute of Infectious Diseases). On the chemical side, the division of laboratory sciences at the CDC is the reference laboratory and has close ties to US Army Medical Institute of Chemical Warfare Agents. The states applied for CDC grants in five areas: preparedness and prevention, detection and surveillance, diagnosis and characterization of biological and chemical agents, response, and communication systems.

The CDC received $178 million in fiscal year 1999 to prepare against bioterrorism and to establish this network of biological and chemical labs to assist with measurement. (In addition, the CDC received $52 million to establish a pharmaceutical stockpile, which will ensure the availability of drugs, vaccines, prophylactic medicines, chemical antidotes, medical supplies, and equipment that will be needed to support a medical response to a terrorist incident.)

The CDC is holding regional meetings with all the state labs--in Philadelphia, Atlanta, St. Louis, Denver, and San Francisco. "We are telling them who we are, what the CDC's response plan is, and how we can help them so that this becomes a partnership," Gunter explained.

Iris Rosendahl