AUTHOR INFORMATION	Section 1 of 8
Author Information Historic Aspects Of Biological Warfare Agents Delivery, Dissemination, And Detection Of Biological Warfare Agents Bacterial Agents Viral Agents Biological Toxins Pictures Bibliography

Author: Daniel J Dire, MD, FACEP, FAAP, FAAEM, Clinical Associate Professor, Department of Emergency Medicine, University of Texas-Houston

Daniel J Dire, MD, FACEP, FAAP, FAAEM, is a member of the following medical societies: American Academy of Clinical Toxicology, American Academy of Emergency Medicine, American Academy of Pediatrics, American College of Emergency Physicians, and Association of Military Surgeons of the US

Editor(s): Edmond Hooker, MD, Assistant Clinical Professor, Department of Emergency Medicine, University of Louisville, Wright State University; John T VanDeVoort, PharmD, Clinical Assistant Professor, College of Pharmacy, University of Minnesota; Michael J Burns, MD, Instructor, Department of Emergency Medicine, Harvard University Medical School, Beth Israel Deaconess Medical Center; John Halamka, MD, Chief Information Officer, CareGroup Healthcare System, Assistant Professor of Medicine, Department of Emergency Medicine, Beth Israel Deaconess Medical Center; Assistant Professor of Medicine, Harvard Medical School; and Robert G Darling, MD, FACEP, Assistant Professor of Military and Emergency Medicine, Uniformed Services University of the Health Sciences, F Edward Hebert School of Medicine; Director, Center for Disaster and Humanitarian Assistance Medicine

Disclosure

	HISTORIC ASPECTS OF BIOLOGICAL WARFARE AGENTS	Section 2 of 8
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Biological weapons include any organism or toxin found in nature that can be used to incapacitate, kill, or otherwise impede an adversary. Biological weapons are characterized by low visibility, high potency, substantial accessibility, and relatively easy delivery.

The potential spectrum of bioterrorism ranges from hoaxes and use of non–mass casualty agents by individuals or small groups to state-sponsored terrorism that employs classic biological warfare (BW) agents and can produce mass casualties. Such scenarios would present serious challenges for patient treatment and for prophylaxis of exposed persons. Environmental contamination could pose continuing threats.

The use of biological agents is not a new concept, and history is replete with examples of biological weapon use. Before the 20th century, biological warfare took on 3 main forms: (1) deliberate poisoning of food and water with infectious material, (2) use of microorganisms or toxins in some form of weapon system, and (3) use of biologically inoculated fabrics.

Attempts to use BW date back to antiquity. Scythian archers infected their arrows by dipping them in decomposing bodies or in blood mixed with manure as far back as 400 BC. Persian, Greek, and Roman literature from 300 BC quote examples of the use of animal cadavers to contaminate wells and other sources of water. In 190 BC, at the Battle of Eurymedon, Hannibal won a naval victory over King Eumenes II of Pergamon by firing earthen vessels full of venomous snakes into the enemy ships.

In the 12th century AD, during the battle of Tortona, Barbarossa used the bodies of dead soldiers to poison wells. In the 14th century AD during the siege of Kaffa, the attacking Tarter force hurled the corpses of those who died of plague into the city to attempt to inflict a plague epidemic upon the enemy. This was repeated in 1710 when the Russians besieging Swedish forces at Reval in Estonia catapulted plague cadavers.

In the 18th century AD during the French and Indian War, British forces in North America gave blankets from smallpox patients to the Native Americans to create a transmission of the disease to the immunologically naïve tribes. In 1863, a confederate surgeon was arrested and charged with attempting to import yellow fever–infected clothes into the northern parts of the United States during the Civil War.

Biological warfare became more sophisticated against both animals and humans during the 1900s. During World War I, the Germans developed anthrax, glanders, cholera, and a wheat fungus for use as biological weapons. They allegedly spread plague in St Petersburg, infected mules with glanders in Mesopotamia, and attempted to do the same with the horses of the French Calvary.

In 1925, the Geneva Protocol was signed by 108 nations, including the 5 permanent members of the UN Security Council. This was the first multilateral agreement that extended prohibition of chemical agents to biological agents. No method for verification of compliance was addressed.

During World War II, the Japanese operated a secret BW research facility in Manchuria and carried out human experiments on Chinese prisoners. They exposed more than 3000 victims to plague, anthrax, syphilis, and other agents. Victims were observed for development of disease, and autopsies were performed.

In 1942, the United States formed the War Research Service. Anthrax and botulinum toxin initially were investigated for use as weapons, and sufficient quantities of botulinum toxin and anthrax cattle cakes were stockpiled by June 1944 to allow limited retaliation if the Germans first used biological agents. The British tested anthrax bombs on Gruinard Island off the northwest coast of Scotland in 1942 and 1943 and then prepared and stockpiled anthrax-laced cattle cakes.

The United States continued research on various offensive biological weapons during the 1950s and 1960s. From 1951-1954, simulants (Bacillus globigii, Serratia marcescens) were released off both coasts of the United States to demonstrate the vulnerability of American cities to biological agent attacks. This vulnerability was tested again in 1966 when the simulant B globigii was released in the New York subway system.

In 1957, the British government decided to end its offensive BW capabilities and destroy its weapon stockpiles.

The United States terminated its offensive biological weapons program in 1969 for microorganisms and in 1970 for toxins. The United States is a signatory nation of the Biological Toxin Weapons Convention of 1972. This convention addressed the prohibition of the development, production, stockpiling, and destruction of bacteriologic and toxin weapons. Signatories to this agreement are required to submit information annually to the United Nations concerning facilities where biological defense research is being conducted, scientific conferences that are held at specified facilities, exchanges of scientists or information, and disease outbreaks. American stockpiles of biological weapons were destroyed completely by 1973.

During the Vietnam War, Vietcong guerrillas used punji stakes dipped in feces to increase the morbidity from wounding by these stakes.

The Soviet Union (USSR) continued to develop biological weapons from 1950-1980. In the 1970s, the USSR and its allies were suspected of having used "yellow rain" (trichothecene mycotoxins) during campaigns in Loas, Cambodia, and Afghanistan. In 1979, an accidental release of anthrax from a weapons facility in Sverdlovsk, USSR, killed at least 66 people. The Russians denied this accident until 1992.

Since the 1980s, terrorist organizations have become users of biological agents. The most frequent bioterrorism episodes have involved contamination of food and water. In September and October of 1984, 751 persons were infected with Salmonella typhimurium after an intentional contamination of restaurant salad bars in Oregon by followers of the Bhagwan Shree Rajneesh.

In 1985, Iraq began an offensive biological weapons program producing anthrax, botulinum toxin, and aflatoxin. During Operation Desert Shield, the coalition of allied forces faced the threat of chemical and biological agents. Following the Persian Gulf War, Iraq disclosed that it had bombs, Scud missiles, 122-mm rockets, and artillery shells armed with botulinum toxin, anthrax, and aflatoxin. They also had spray tanks fitted to aircraft that could distribute 2000 L over a target.

Currently, 17 countries are suspected of having an offensive BW program. In 1992, 20 people were administered chemoprophylaxis after a Virginia man sprayed his roommates with a substance that he claimed was anthrax. In 1994, a Japanese sect of the Aum Shinrikyo cult attempted an aerosolized release of anthrax from the tops of buildings in Tokyo. In 1995, 2 members of a Minnesota militia group were convicted of possession of ricin, which they had produced themselves for use in retaliation against local government officials. In 1996, an Ohio man was able to obtain bubonic plague cultures through the mail.

In 1997, the Defense Against Weapons of Mass Destruction Act directed the Department of Defense to establish a domestic preparedness program to improve the ability of local, state, and federal agencies to respond to biological incidents. During 1998 and 1999, multiple hoaxes occurred involving the threatened release of anthrax in the United States that resulted in decontamination and antibiotic prophylaxis for the intended victims. Nearly 6000 persons across the United States have been affected by these threats. According to a study by the Centers for Disease Control and Prevention (CDC), an intentional release of anthrax by a bioterrorist in a major US city would result in an economic impact of $477.8 million to $26.2 billion per 100,000 persons exposed.

From September to November 2001, a total of 23 confirmed or suspected cases of bioterrorism-related anthrax (10 inhalation, 13 cutaneous) occurred in the United States. Most cases involved postal workers in New Jersey and Washington DC, and the rest occurred at media companies in New York and Florida, where letters contaminated with anthrax were handled or opened. As a result of these cases, approximately 32,000 persons with potential exposures initiated antibiotic prophylaxis to prevent anthrax infections.

The threat that biological agents will be used on military forces and civilian populations is now more likely than at any point in all of history.

	DELIVERY, DISSEMINATION, AND DETECTION OF BIOLOGICAL WARFARE AGENTS	Section 3 of 8
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Biological agents are easy to acquire, synthesize, and use. The small amount of agents necessary to kill hundreds of thousands of people in a metropolitan area make the concealment, transportation, and dissemination of biological agents relatively easy. In addition, BW agents are difficult to detect or protect against; they are invisible, odorless, and tasteless, and their dispersal can be performed silently.

Dissemination of BW agents may occur by aerosol sprays, explosives (artillery, missiles, detonated bombs), or food or water contamination. Variables that can alter the effectiveness of a delivery system include particle size of the agent, stability of the agent under desiccating conditions, UV light, wind speed, wind direction, and atmospheric stability.

The use of an explosive device to deliver and disseminate biological agents is not very effective, since such agents tend to be inactivated by the blast. Contamination of municipal water supplies requires an unrealistically large amount of agent and introduction into the water after it passes through a regional treatment facility.

To be an effective biological weapon, airborne pathogens must be dispersed as fine particles less than 5 mm in size. Infection with an aerosolized agent usually requires deep inspiration of an infectious dose. Advanced weapons systems (eg, warheads, missiles) are not required for the aerosolized delivery of biological agents. Low-technology aerosolization methods including agricultural crop-dusters; aerosol generators on small boats, trucks, or cars; backpack sprayers; and even purse-size perfume atomizers suffice. Aerosolized dispersal of biological agents is the mode most likely to be used by terrorists and military groups.

Detection of biological agents involves either finding the agent in the environment or medical diagnosis of the agent's effect on human or animal victims. Early detection of a biological agent in the environment allows for early specific treatment and time during which prophylaxis would be effective. Unfortunately, currently no reliable detection systems exist for BW agents. The US Department of Defense has placed a high priority on research and development of a detector system. Methods are being developed and tested to detect a biological aerosol cloud using an airborne pulsed laser system to scan the lower altitudes upwind from a possible target area. A detection system mounted on a vehicle also is being developed. This system will analyze air samples to provide a plot of particle sizes, detect and classify bacterial cells, and measure DNA content, ATP content, and identify agents using immunoassays.

A BW agent attack is likely to be covert. Thus, detection of such an attack requires recognition of the clinical syndromes associated with various BW agents. Physicians must be able to identify early victims and recognize patterns of disease. This requires integrated epidemiologic surveillance systems performing real-time monitoring with information shared at many levels of the health care system (eg, ED to ED, ED to public health officials). Preliminary criteria for suggestive outbreaks of disease that could provide indications of a possible biological weapons event include the following:

• Disease (or strain) not endemic

• Unusual antibiotic resistance patterns

• Atypical clinical presentation

• Case distribution geographically and/or temporally inconsistent (eg, compressed time course)

• Other inconstant elements (eg, number of cases, mortality and morbidity rates, deviations from disease occurrence baseline)

For excellent patient education resources, visit eMedicine's Bioterrorism and Warfare Center. Also, see eMedicine's patient education articles Biological Warfare and Personal Protective Equipment.

	BACTERIAL AGENTS	Section 4 of 8
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For excellent patient education resources, visit eMedicine's Bioterrorism and Warfare Center. Also, see eMedicine's patient education article Anthrax.

Plague is a zoonotic infection caused by Yersinia pestis, a gram-negative coccobacillus, which has been the cause of 3 great human pandemics in the Common Era, in the 6th, 14th, and 20th centuries. Throughout history, the oriental rat flea (Xenopsylla cheopis) has been largely responsible for spreading bubonic plague. After the flea ingests a blood meal on a bacteremic animal, bacilli can multiply and essentially block the flea's foregut with a fibrinoid mass of bacteria. When an infected flea with a blocked foregut attempts to feed again, it regurgitates clotted blood and bacteria into the victim's blood stream and so passes the infection onto the next victim, whether rat or human. As many as 24,000 organisms may be inoculated into the host.

Although the largest outbreaks of plague have been associated with X cheopis, all fleas should be considered dangerous in plague-endemic areas. The most important vector in the United States is Diamanus montanus, the most common flea of rock squirrels and California ground squirrels. The black rat, Rattus rattus, has been most responsible worldwide for the persistence and spread of plague in urban epidemics.

Plague is characterized by the abrupt onset of high fevers, painful lymphadenopathy, and bacteremia. Septicemic plague sometimes can ensue from untreated bubonic plague or, de novo, after a fleabite. Patients with the bubonic form of the disease may develop secondary pneumonic plague. This complication can lead to human-to-human spread by the respiratory route and cause primary pneumonic plague. Pneumonic plague is the most severe form of disease and, untreated, has a mortality rate approaching 100%.

Mortality from endemic plague continues at low rates throughout the world despite the availability of effective antibiotics. People continue to die of plague, not because the bacilli have become resistant but, most often, because physicians do not include plague in their differential diagnosis, and treatment is delayed.

PATHOPHYSIOLOGY - PLAGUE

Y pestis is a gram-negative, nonacid-fast, nonmotile, nonsporulating coccobacillus. Its bipolar appearance is best appreciated when Wright-Giemsa, Wayson, or Gram stains are used. Y pestis grows optimally at 28°C. Biochemically, the plague bacillus produces no hemolysins, is positive for catalase, and is negative for hydrogen sulfide, oxidase, and urease.

The known virulence factors of Y pestis are encoded on the chromosomes of its 3 plasmids. The pH6 antigen, a protein located on the surface of the bacterium, is necessary for complete virulence. It is induced in vivo at sites of inflammation and cellular necrosis and within phagocytic cells. The low calcium response (LCR) plasmid, which is homologous in Y pestis and the other 2 Yersinia pathogens, Yersinia pseudotuberculosis and Yersinia enterocolitica, codes for several secreted proteins and is also necessary for virulence.

As few as 1-10 organisms of Y pestis are sufficient to infect rodents and primates via the oral, intradermal, subcutaneous, or intravenous routes. After being introduced into the mammalian host by a flea, the organism is thought to be susceptible initially to phagocytosis and killing by neutrophils. However, some of the bacteria may grow and proliferate within tissue macrophages. Within the human host, several environmental signals (temperature of 37°C, contact with eukaryotic cells, location within mononuclear cells, pH) are thought to induce the synthesis and activity of a multitude of factors that contribute to virulence. Bacteria become resistant to phagocytosis and proliferate unimpeded extracellularly.

During the incubation phase, the bacilli most commonly spread to regional lymph nodes, where supportive lymphadenitis develops, producing the characteristic bubo. Dissemination from the local site is thought to be related to the action of both plasminogen activator and Yop M. Infection progresses if untreated; septicemia develops, and the infection spreads to other organs. The endotoxin probably contributes to the development of septic shock, which is similar to the shock states observed with other causes of gram-negative sepsis.

Tissues most commonly infected include the spleen, liver, lungs, skin, and mucous membranes. Late infection of the meninges also occurs, especially if suboptimal antibiotic therapy has been administered.

Primary pneumonic plague, the most severe form of the disease, arises from inhalation of an infectious aerosol. Primary pneumonic plague is more rapidly fatal than the secondary form, because the inhaled droplets already contain phagocytosis-resistant bacilli, which have arisen from their growth in the vertebrate host.

Primary septicemia plague can arise from direct inoculation of bacilli into the bloodstream, bypassing initial multiplication in the lymph nodes.

CLINICAL FEATURES - PLAGUE

In the United States, most patients (85-90%) with human plague present clinically with the bubonic form, 10-15% with the primary septicemia form, and 1% with the pneumonic form. Secondary septicemic plague occurs in 23% of patients who present with bubonic plague, and secondary pneumonic plague occurs in 9%. If Y pestis were used as a BW agent, it most likely would be inhaled as an infectious aerosol and result in primary pneumonic plague (epidemic pneumonia). If fleas were used as carriers of disease, bubonic or septicemic plague would result.

Bubonic plague: Buboes manifest after a 1- to 8-day incubation period. Their appearance is associated with the onset of sudden fever, chills, and headache, which often are followed by nausea and vomiting several hours later. Presenting symptoms include severe malaise (75%), headache (20-85%), vomiting (25-49%), chills (40%), altered mentation (26-38%), cough (25%), abdominal pain (19%), and chest pain (13%). Buboes occur in the groin (90% femoral, more frequent femoral than inguinal), axillary, or cervical regions, depending on the site of inoculation, 6-8 hours after the onset of symptoms. Buboes become visible within 24 hours and are characterized by severe pain. Untreated, septicemia develops in 2-6 days. Approximately 5-15% of bubonic plague patients develop secondary pneumonic plague and thus the ability to spread illness from person to person by respiratory droplets.

Septicemia plague: Septicemia plague may occur primarily or secondarily as a result of hematogenous dissemination of bubonic plague. Presenting signs and symptoms of primary septicemic plague are essentially the same as those for any gram-negative septicemia and include fever, chills, nausea, vomiting, and diarrhea; later, purpura, disseminated intravascular coagulation (DIC), and acrocyanosis and necrosis occur.

Pneumonic plague: Pneumonic plague may occur primarily from inhalation of aerosols or secondarily from hematogenous dissemination. Patients typically have a productive cough with blood-tinged sputum within 24 hours of symptom onset. The findings on chest x-ray are variable, but bilateral alveolar infiltrates appear to be the most common findings in pneumonic plague.

Plague meningitis: This is observed in 6-7% of patients. The condition manifests itself most often in children after 9-14 days of ineffective treatment. Symptoms are similar to those of other forms of acute bacterial meningitis.

DIAGNOSIS - PLAGUE

The diagnosis of bubonic plague should be made readily on clinical grounds if a patient presents with a painful bubo, fever, prostration, and history of exposure to rodents or fleas in an endemic area. However, if the patient presents in a nonendemic area or without a bubo, then the diagnosis can be difficult to make. When a bubo is present, the differential diagnosis should include tularemia, cat scratch disease, lymphogranuloma venereum, chancroid, TB, streptococcal adenitis, and scrub typhus.

The differential diagnosis of septicemic plague also includes meningococcemia, gram-negative sepsis, and rickettsioses. A presentation of systemic toxicity, a productive cough, and bloody sputum suggests a large differential diagnosis. However, demonstration of gram-negative coccobacilli in the sputum readily should suggest the correct diagnosis, because Y pestis is perhaps the only gram-negative bacterium that can cause extensive, fulminant pneumonia with bloody sputum in an otherwise healthy, immunocompetent host. In addition, Y pestis has unique bipolar, safety-pin morphology.

In patients with lymphadenopathy, perform a bubo aspiration. Air-dry the aspirate on a slide for Gram, Wright-Giemsa, or Wayson stain. If available, obtain a DFA stain of the aspirate for the presence of Y pestis capsular antigen. A positive DFA is more specific for Y pestis than the other stains listed.

Perform cultures of blood, bubo aspirate, sputum, and cerebrospinal fluid (CSF). Tiny 1- to 3-mm beaten copper colonies appear on blood agar in 48 hours. It is important to remember that colonies may be negative at 24 hours.

Complete blood counts (CBCs) often reveal leukocytosis with a left shift. Platelet counts may be normal or low, and activated partial thromboplastin times (aPTTs) may be increased. When DIC is present, fibrin degradation products are elevated. Because of liver involvement, alanine aminotransferase, aspartate aminotransferase, and bilirubin levels may be increased.

Most naturally occurring strains of Y pestis produce an F1-antigen in vivo, which can be detected in serum samples by immunoassay. Because fractional antigen and antibody do not occur early in the infection, perform titers for both on several sequential blood specimens. A 4-fold rise in antibody titer in patient serum is retrospectively diagnostic.

TREATMENT - PLAGUE

Isolate patients with plague for the first 48 hours after treatment initiation. If pneumonic plague is present, continue isolation for 4 days.

Since l948, streptomycin has been the treatment of choice for bubonic, septicemic, and pneumonic plague. Administer it in a dose of 30 mg/kg/d IM divided bid. In patients with meningitis or hemodynamic instability, add intravenous chloramphenicol (50-75 mg/kg/d) divided qid. Gentamicin has had much less clinical usage but can be used as an alternative to streptomycin. Continue treatment for a minimum of 10 days or 3-4 days after clinical recovery. In patients with very mild bubonic plague who are not septic, tetracycline can be used orally at a dose of 2 g/d divided qid for 10 days. Doxycycline, ofloxacin, and ceftriaxone have been demonstrated to be effective in animal models.

In pregnant women, use streptomycin or gentamicin unless chloramphenicol specifically is indicated. Streptomycin is also the treatment of choice for newborns.

If treated with antibiotics, buboes typically recede in 10-14 days and do not require drainage. Patients are unlikely to survive primary pneumonic plague if antibiotic therapy is not initiated within 18 hours of symptom onset. Without treatment, the mortality rate is 60% for bubonic plague and 100% for the pneumonic and septicemic forms.

PREVENTION/PROPHYLAXIS - PLAGUE

All plague control measures must include insecticide use, public education, and reduction of rodent populations with chemicals such as cholecalciferol. Fleas always must be targeted before rodents, because killing rodents may release massive amounts of infected fleas.

Treat contacts of patients with pneumonic plague and individuals who have been exposed to aerosols with tetracycline 15-30 mg/kg/d divided qid for 6 days. If tetracycline is not available, doxycycline 100 mg bid is an effective alternative. Pregnant women and children younger than 8 years should receive trimethoprim/sulfamethoxazole (40 mg sulfa/kg/d) divided bid for 6 days.

Contacts of patients with bubonic plague do not require prophylactic therapy. However, administer prophylaxis to people who were in the same environment and potentially exposed to the same source of infection. In addition, previously vaccinated individuals should receive prophylactic antibiotics if they have been exposed to a plague aerosol.

Only individuals at high risk for plague should be immunized with a licensed, killed, whole cell vaccine. Vaccinate military troops and personnel working in endemic areas, lab personnel working with Y pestis, and people who reside in enzootic or epidemic areas. While epidemiologic evidence supports the efficacy of the current vaccine against bubonic plague, its efficacy against aerosolized Y pestis is believed to be poor.

For excellent patient education resources, visit eMedicine's Bioterrorism and Warfare Center. Also, see eMedicine's patient education article Plague.

Cholera is an acute and potentially severe GI disease caused by Vibrio cholerae. V cholerae is a short, curved, motile, gram-negative, nonsporulating rod. Two serogroups (01, 0139) have been associated with cholera in humans. The 01 serotype exists as 2 biotypes, classical and El Tor. The organisms are strongly anaerobic, preferring alkaline and high-salt environments. They do not invade the intestinal mucosa but rather adhere to it. Cholera is the prototype toxigenic diarrhea, which is secretory in nature.

This agent has been investigated in the past as a biological weapon. Cholera does not spread easily from human to human; therefore, it appears that major drinking water supplies would have to be contaminated heavily for this agent to be effective as a biological weapon. The rate of symptomatic-to-asymptomatic cases during exposures is 1:400.

PATHOPHYSIOLOGY- CHOLERA

All strains of V cholerae elaborate the same enterotoxin, a protein molecule with a molecular weight of 84,000 daltons. The entire clinical syndrome is caused by the action of the toxin on the intestinal epithelial cell. Cholera toxin causes active secretion of chloride and blocks sodium absorption in the small intestine, with the colon relatively insensitive to the toxin. The large volume of fluid produced in the upper intestine overwhelms the capacity of the lower intestine to absorb. The diarrhea is classically thin, grayish brown, and mucoid and may reach a rate of 1 L/h.

Transmission is made through direct or indirect fecal contamination of water or foods and by heavily soiled hands or utensils. All populations are susceptible, while natural resistance to infection varies.

Drying easily kills the organism. It is not viable in pure water but survives up to 24 hours in sewage and as long as 6 weeks in certain types of relatively impure water containing organic matter. It can withstand freezing for 3-4 days. It is killed readily by dry heat at 117°C, steam and boiling, short-term exposure to ordinary disinfectants, and chlorination of water.

CLINICAL FEATURES - CHOLERA

Infection generally occurs within a week of exposure and is classically of abrupt onset following a brief nonspecific prodrome. Fever is rare. The syndrome is characterized by sudden onset of nausea and vomiting and profuse diarrhea with a classic rice water appearance. If untreated, the disease generally lasts 1-7 days. The clinical manifestations of cholera are related to the profound fluid and electrolyte depletion that occurs. Acute treatment consists of rapid, aggressive fluid resuscitation with isotonic solutions and potassium.

Children may experience seizures caused by hypoglycemia and hypernatremia and may have potassium depletion severe enough to cause an arrhythmia. The rapid loss of body fluids often leads to toxemia and frequent cardiovascular collapse. The mortality rate can range as high as 50% in untreated patients.

DIAGNOSIS - CHOLERA

The incubation period ranges from 12-72 hours and depends on the dose of ingested organisms. Onset of illness usually is sudden. Initially, the disease presents with intestinal cramping and painless diarrhea. Vomiting, malaise, and headache often accompany the diarrhea, especially early in the illness.

On microscopic examination of the stool, few or no red cells, white cells, and almost no protein are found. The absence of inflammatory cells and erythrocytes reflects the noninvasive character of V cholerae infection of the intestinal lumen. The organism can be identified in liquid stool or enrichment broths by darkfield or phase contrast microscopy and by identifying darting motile Vibrio species. Bacteriologic diagnosis is not necessary for treatment, as it can be diagnosed clinically.

TREATMENT - CHOLERA

Treatment depends on replacement of fluids and electrolyte losses. This is best accomplished using oral rehydration therapy, but intravenous fluid replacement is occasionally necessary for persistent vomiting or high rates of stool loss (10 mL/kg/h). Antibiotics shorten the duration of diarrhea and reduce fluid losses. Tetracycline (500 mg q6h for 3 d) or doxycycline (300 mg once or 100 mg bid for 3 d) is an acceptable alternative. However, due to resistance, ciprofloxacin (500 mg q6h for 3 d) or erythromycin (40 mg/kg/d divided qid for 3 d) also has been accepted.

PREVENTION/PROPHYLAXIS - CHOLERA

A licensed, killed vaccine is available for use in those considered to be at risk for exposure. The vaccine is protective for only approximately 50% of those immunized, and protection lasts for no more than 6 months. The vaccination schedule is an initial dose followed by another dose 4 weeks later, with booster doses every 6 months.

An inactivated oral vaccine (WC/rBs) is safe and provides rapid short-term protection. WC/rBs requires 2 doses and has approximately 85% efficacy lasting 2-3 years for both El Tor and classic biotypes.

Tularemia is a zoonosis caused by the gram-negative, facultative intracellular bacterium Francisella tularensis. The disease is characterized by fever, localized skin or mucous membrane ulceration, regional lymphadenopathy, and occasionally pneumonia. GW McCay discovered the disease in Tulare County, California, in 1911. The first confirmed case of human disease was reported in 1914. Edward Francis, who described transmission by deer flies via infected blood, coined the term tularemia in 1921. F tularensis has been considered an important BW agent because of its high infectivity after aerosolization.

F tularensis is a nonmotile, obligate aerobic, gram-negative coccobacillus with 2 subspecies. F tularensis subsp tularensis is the most common in the United States. F tularensis subsp palearctica is more common outside the United States. The subspecies are indistinguishable serologically, although they may be distinguished by 169 ribosomal ribonucleic acid (rRNA) analysis. A capsule has been reported to contribute to virulence. No known toxins are produced.

The principal reservoir in North America is the tick. In North America, the rabbit is the most common vertebrate associated with transmission of tularemia. In other areas of the world, tularemia is maintained in water rats and other aquatic animals.

PATHOPHYSIOLOGY - TULAREMIA

F tularensis usually is introduced into the host through breaks in the skin or through the mucous membranes of the eye, respiratory tract, or GI tract. Ten virulent organisms injected subcutaneously and 10-50 organisms given by aerosol can cause infection in humans. After inoculation, F tularensis is ingested by and multiplies within macrophages. The host defense against F tularensis is mediated by a T cell-independent mechanism, which appears early after infection (<3 d), and a T cell-dependent mechanism, which appears later (>3 d) after infection. The role of humoral-medicated immunity and neutrophils in the host defense against F tularensis remains unclear.

CLINICAL FEATURES - TULAREMIA

Tularemia can be divided into the ulceroglandular (75% of patients) and typhoidal (25% of patients) forms based on clinical findings. Patients with ulceroglandular tularemia have lesions of the skin or mucous membranes, lymph nodes greater than 1 cm in diameter, or both. Patients with typhoidal tularemia present with lymph nodes less than 1 cm in diameter and without skin or mucous membrane lesions.

After an incubation period of 3-6 days, patients with the ulceroglandular form of the disease develop a constellation of symptoms consisting of fever (85%), chills (57%), headache (45%), cough (38%), and myalgia (31%). Patients also may complain of chest pain, vomiting, arthralgia, sore throat, abdominal pain, diarrhea, dyspnea, back pain, or neck stiffness.

A cutaneous chancrelike ulcer occurs in approximately 60% of patients and is the most common sign of tularemia. Ulcers are generally single lesions with heaped up borders 0.4-3 cm in diameter. Lesions associated with infection acquired from mammalian vectors usually are located on the upper extremities, whereas lesions associated with infection from arthropod vectors usually are located on the lower extremities.

Enlarged lymph nodes are observed in approximately 85% of patients and may be the initial or the only sign of infection. Although enlarged lymph nodes usually occur as single lesions, they may appear in groups. The appearance of enlarged lymph nodes in upper or lower extremities and the correlation with the vector is the same as for ulcerative lesions. Enlarged lymph nodes may become fluctuant, drain spontaneously, or persist for as long as 3 years. When fluctuant, they may be confused with buboes of bubonic plague. A minority of patients with typhoidal disease develop a morbilliform eruption.

Pharyngitis may occur in as many as 25% of patients with tularemia. On occasion, patients with pharyngitis also may develop a retropharyngeal abscess or suppuration of regional lymph nodes. Pharyngeal ulcers may be found in patients with aerosol-induced disease.

The lower respiratory tract is involved in 47-94% of patients. Approximately 30% of patients with ulceroglandular and 80% of patients with typhoidal tularemia have pneumonia. Patients present with productive or nonproductive cough and less commonly with pleuritic chest pain, shortness of breath, or hemoptysis. Fifty percent of patients have radiographic evidence of pneumonia, and 1% or fewer have hilar adenopathy. Pleural effusions are observed in 15% of patients with pneumonia.

DIAGNOSIS - TULAREMIA

Tularemia can be diagnosed by recovery of F tularensis in culture. Although difficult to culture, it can be recovered from blood, ulcers, sputum, conjunctival exudate, pharyngeal exudates, and gastric washings. On media containing cysteine, F tularensis appears as small, smooth, opaque colonies after 24-48 hours of incubation at 37°C. Identification of the organism is made on the basis of its growth characteristics and bacterial agglutination or fluorescent stain using antiserum specific for F tularensis.

Most diagnoses of tularemia are made serologically using bacterial agglutination or ELISA. The serologic response may be blunted by the use of antibiotics and may not appear for more than 2 weeks.

Patients usually do not have abnormalities in the hemoglobin, hematocrit, or platelet count. The peripheral white blood cell count usually is elevated only mildly and often shows a lymphocytosis late in the disease. Patients may have microscopic pyuria, which may lead to erroneous diagnosis of urinary tract infection. Some patients demonstrate mild elevations in levels of lactic dehydrogenase, serum transaminases, and alkaline phosphatase. CSF is usually normal.

TREATMENT - TULAREMIA

Patients with tularemia who do not receive appropriate antibiotic therapy may have a prolonged illness characterized by malaise, weakness, and weight loss. With appropriate therapy, tularemia has a mortality rate of only 1-2.5%.

Streptomycin (30 mg/kg/d IM divided bid for 10-14 d) is the drug of choice for tularemia. Gentamicin (3-5 mg/kg/d parenterally for 10-14 d) is also effective. Tetracycline and chloramphenicol are effective as well but have been associated with significant relapse rates. Although laboratory-related infections with this organism are common, human-to-human spread is unusual, and respiratory isolation is not required.

PROPHYLAXIS/PREVENTION - TULAREMIA

Antibiotic prophylaxis after exposure to tularemia is difficult, because the ideal drug, streptomycin, must be administered parenterally. Tetracycline is effective after exposure to an aerosol of tularemia if administered within 24 hours of the exposure at an oral dose of 2 g/d for 14 days.

A live attenuated vaccine has been developed and used in humans since 1940. In the 1960s, a further purified derivative was introduced and called live vaccine strain (LVS). Extensive studies have demonstrated that the LVS vaccine protected humans against an aerosol challenge with virulent F tularensis. Evidence indicates that immunization with the LVS vaccine prevents the typhoidal and ameliorates the ulceroglandular forms of tularemia.

Brucellosis is a zoonotic infection of domesticated and wild animals caused by an organism of the genus Brucella. The organism infects mainly cattle, sheep, goats, and other ruminants, causing abortion, fetal death, and genital infection. Humans, who usually are infected incidentally by contact with infected animals, may develop numerous symptoms in addition to the usual ones of fever, malaise, and muscle pain. The disease often becomes chronic and may relapse, even with appropriate treatment. The ease of transmission by aerosol suggests that Brucella species may be useful as a BW agent.

PATHOPHYSIOLOGY - BRUCELLOSIS

Brucella species are small, nonmotile, nonsporulating, aerobic, gram-negative coccobacilli that may represent a single species. However, they are classified into 6 species. Each species has a characteristic predilection to infect certain animal species. Only Brucella melitensis, Brucella suis, Brucella abortus, and Brucella canis cause disease in humans. Infection of humans with Brucella ovis and Brucella neotomae has not been described.

Animals may transmit Brucella organisms during septic abortion, at the time of slaughter, and in their milk. Brucellosis rarely, if ever, is transmitted from human to human. Brucella species can enter mammalian hosts through skin abrasions or cuts, the conjunctiva, the respiratory tract, and the GI tract. Organisms are ingested rapidly by polymorphonuclear leukocytes, which generally fail to kill them. Organisms also are phagocytized by macrophages, which traffic to lymphoid tissue and eventually localize in lymph nodes, liver, spleen, joints, kidneys, and bone marrow.

Brucellosis also can replicate extracellularly in host tissue. The host cellular response may range from abscess formation to granuloma formation with caseous necrosis.

CLINICAL FEATURES - BRUCELLOSIS

Clinical manifestations of brucellosis are diverse, and the course of the disease varies. Patients may present with an acute, systemic, febrile illness; an insidious chronic infection; or a localized inflammatory process. The disease may be abrupt or insidious in onset, with an incubation period of 3 days to several weeks. Patients usually have nonspecific symptoms such as fever, sweats, fatigue, anorexia, and muscle or joint aches. Neuropsychiatric symptoms such as depression, headache, and irritability occur frequently. In addition, focal infection of bones, joints, or the genitourinary tract may cause local pain. Cough and pleuritic chest pain also may be noted.

Symptoms often last 3-6 months and occasionally for longer than a year. Brucellosis usually does not cause leukocytosis, and patients may be neutropenic. B melitensis tends to cause more severe, systemic illness than the other Brucella species. B suis is more likely to cause localized suppurative disease.

Infection with B melitensis leads to bone or joint disease in approximately 30% of patients. Sacroiliitis develops in 6-15%, particularly in young adults. Arthritis of large joints occurs with about the same frequency as sacroiliitis. In contrast to septic arthritis caused by pyogenic organisms, joint inflammation observed with B melitensis is mild, and erythema of overlying skin is uncommon. Synovial fluid is exudative, with cell counts in the low thousands, predominantly mononuclear. In both sacroiliitis and peripheral joint infections, destruction of bone is unusual. Organisms can be cultured from fluid in approximately 20% of patients. Spondylitis tends to affect middle-aged or elderly patients, causing back (usually lumbar) pain, local tenderness, and occasionally radicular symptoms.

Radiographic findings, similar to those of tuberculous infection, include disk space narrowing and epiphysitis. Paravertebral abscesses occur rarely. In contrast to frequent infection of the axial skeleton, osteomyelitis of long bones is rare.

Infection of the genitourinary tract, an important target in ruminant animals, also may lead to signs and symptoms of disease in humans. Pyelonephritis, cystitis, and, in males, epididymo-orchitis may occur. Both diseases may mimic their tuberculous counterparts with sterile pyuria on routine bacteriologic cultures.

Lung infections also have been described. Although as many as 25% of patients may complain of respiratory symptoms (mostly cough, dyspnea, or pleuritic pain), chest radiographic examinations usually are normal. Diffuse or focal infiltrates, pleural effusions, abscesses, and granulomas may be observed.

Hepatitis and, rarely, liver abscess also occur. Mild elevations of serum lactase dehydrogenate and alkaline phosphatase levels are common. Biopsy findings may show well-formed granulomas or nonspecific hepatitis with collections of mononuclear cells.

Other sites of infection include the heart, central nervous system (CNS), and skin. Brucella endocarditis, a rare but feared complication, accounts for 80% of deaths from brucellosis. CNS infection usually manifests itself as chronic meningoencephalitis, but subarachnoid hemorrhage and myelitis also occur. Cases of skin abscess also have been reported.

DIAGNOSIS - BRUCELLOSIS

A thorough history eliciting details of appropriate exposures (animals, animal products, environmental exposures) is the most important diagnostic tool. Strongly consider brucellosis in the differential diagnosis when military troops exposed to a biological attack have febrile illnesses. PCR and antibody-based antigen detection systems may demonstrate the presence of organisms in environmental samples collected from attack areas.

When the disease is considered, diagnosis usually is made based on serology. The tube agglutination test remains the criterion standard. This test reflects the presence of anti-O-polysaccharide antibody. Most patients already have high titers at the time of clinical presentation. Serum testing always should include dilution to at least 1:320. The tube agglutination test does not detect antibodies to B canis, because this organism does not have O-polysaccharide on its surface.

In addition to serologic testing, pursue diagnosis by microbiologic cultures of blood or body fluid samples. Hold cultures for at least 2 months. The reported frequency of isolation from blood varies widely, from less than 10% to 90%. B melitensis is said to be cultured more readily than B abortus. Culture of bone marrow may increase the yield.

TREATMENT - BRUCELLOSIS

Therapy with a single drug has resulted in a high relapse rate, so use combined antibiotic regimens whenever possible. A 6-week regimen of doxycycline 200 mg/d PO with the addition of streptomycin 1 g/d IM for the first 2 weeks is effective in most adults with most forms of brucellosis. Patients with spondylitis may require longer treatment. A 6-week oral regimen with both rifampin 900 mg/d and doxycycline 200 mg/d is effective. Several studies have demonstrated that treatment with a combination of streptomycin and doxycycline may result in less frequent relapse than treatment with the combination of rifampin and doxycycline.

Endocarditis likely is best treated with a combination of rifampin, streptomycin, and doxycycline for 6 weeks. Replace infected valves early. CNS disease responds to a combination of rifampin and trimethoprim/sulfamethoxazole but may need prolonged therapy. The latter combination is also effective for children younger than 8 years. Rifampin is recommended for pregnant women.

PREVENTION/PROPHYLAXIS - BRUCELLOSIS

Animal handlers should wear appropriate protective clothing when working with infected animals. Meat should be well cooked, and milk should be pasteurized. Laboratory workers should culture the organism only with appropriate Biosafety Level 2 or 3.

In the event of a biological attack, the standard gas mask should protect personnel adequately from airborne Brucella species. No commercially available vaccine exists for humans.

Q fever is a zoonotic disease caused by Coxiella burnetii, a rickettsialike organism of low virulence but remarkable infectivity. A single organism may initiate infection. In addition, despite the fact that C burnetii is unable to grow or replicate outside host cells, a sporelike form of the organism is extremely resistant to heat, pressure, and many antiseptic compounds. This allows C burnetii to persist in the environment for long periods under harsh conditions. In contrast to this high degree of inherent resilience and transmissibility, the acute clinical disease associated with Q fever is usually a benign, although temporarily incapacitating, illness in humans. Even without treatment, most patients recover.

The primary reservoir for natural human infection is livestock, particularly parturient females, and the distribution is worldwide. Humans who work in animal husbandry, especially those who assist during parturition, are at risk of acquiring Q fever.

The potential of C burnetii as a BW agent is related directly to its infectivity. It has been estimated that 50 kg of dried C burnetii would produce casualties at a rate equal to that of similar amounts of anthrax or tularemia organisms.

The causative agent of Q fever was designated Coxiella burnetii to recognize the outstanding contribution of both Harold Cox and MacFarlane Burnet in the isolation and characterization of the pathogen. The disease now has been identified in at least 51 countries and on 5 continents.

PATHOPHYSIOLOGY - Q FEVER

The genus Coxiella has only 1 species. C burnetii is extremely infectious. Under experimental conditions, a single organism is capable of producing infection and disease in humans.

The host range of C burnetii is diverse and includes a large number of mammalian species and arthropods. Among these, the human is the only host identified that experiences an illness as a result of infection. A number of different strains of C burnetii have been identified worldwide, and different clinical manifestations and complications may be associated with the various strains.

Humans have been infected most commonly by contact with domestic livestock, particularly goats, cattle, and sheep. The risk of infection is increased substantially if humans are exposed to these animals at parturition. During gestation, the proliferation of C burnetii in the placenta facilitates aerosolization of large numbers of the pathogen during parturition. Survival of the organism on inanimate surfaces, such as straw, hay, or clothing, allows for transmission to individuals who are not in direct contact with infected animals.

Human infection with C burnetii is usually the result of inhalation of infected aerosols. Following this, host cells phagocytize the organisms. After phagocytosis by host cells, dissemination of the pathogen occurs as a result of circulation of organism free in the plasma, on the surface of the cells, and carried by circulatory macrophages.

Little host reaction occurs at the initial portal of entry, either in the lung following inhalation of aerosol or in the skin following a tick bite. Q fever develops without formation of a primary infectious focus in the area of the tick bite, and the organism does not infect the vascular endothelium, as do other rickettsial pathogens. The presence of a lipopolysaccharide on the cell surface of C burnetii protects the pathogen from host microbicidal activity.

CLINICAL FEATURES - Q FEVER

Humans are the only hosts that commonly develop an illness as a result of the infection. Incubation varies from 10-40 days. The duration of the incubation period is correlated inversely with the magnitude of the inoculum. A higher inoculum also increases the severity of the disease. Q fever in humans may be manifested by asymptomatic seroconversion, acute illness, or chronic disease. The frequency of chronic disease (usually endocarditis) is probably less than 1% of the total infected population.

No characteristic illness is described for acute Q fever, and manifestations may vary considerably between locations where the disease is acquired. The onset of symptomatic Q fever may be abrupt or insidious. Fever, chills, and headache are the most common signs and symptoms. Diaphoresis, malaise, myalgias, fatigue, and anorexia are also common. Arthralgias are relatively uncommon. Cough often occurs later in the illness. Chest pain occurs in a minority of patients. Although nonspecific, evanescent skin eruptions have been reported. No characteristic rash results.

Most patients appear mildly to moderately ill. The temperature tends to fluctuate, with peaks at 39-40°C, and is biphasic in approximately 25% of patients. The fever generally lasts less than 13 days but has been reported to last longer in older adults.

Encephalopathic symptoms, headache, hallucinations (visual, auditory), expressive dysphasia, facial pain resembling trigeminal neuralgia, diplopia, and dysarthria have been reported.

Physical findings in acute Q fever are as nonspecific as the clinical symptomatology. Rales are probably the most commonly observed physical finding; evidence of pleural effusion and consolidation also may be noted but not in most infections.

Reports of abnormalities on chest radiographic examination vary with locale, but abnormalities probably are observed 50-60% of the time. The most common abnormality reported is a unilateral homogenous infiltrate involving 1 or 2 lobes. Rounded opacities and hilar adenopathy are not uncommon. Consider the diagnosis of Q fever when these abnormalities are observed in the setting of acute pneumonia.

Patients with acute Q fever may present with a clinical picture of acute hepatitis with elevations of aminotransferases that are 2- to 3-fold higher than the upper limit of normal. The total bilirubin can be expected to be elevated in 10-15% of patients with acute Q fever. The white blood cell count is usually normal. The erythrocyte sedimentation rate is elevated in 33% of patients. Mild anemia or thrombocytopenia also may be observed.

Chronic infection with C burnetii usually is manifested by infective endocarditis, which also is the most severe complication of Q fever. In addition, hepatitis, infected vascular prostheses, aneurysms, osteomyelitis, pulmonary infection, cutaneous infection, and an asymptomatic form have been reported.

In Q fever endocarditis, fever has been recorded in 85% of patients, along with other systemic symptoms (eg, chills, headache, myalgias, weight loss). Other frequently reported clinical features of Q fever endocarditis include heart failure, splenomegaly, hepatomegaly, clubbing, and cutaneous signs. Routine blood cultures in patients with Q fever endocarditis are negative, and Q fever should be considered when culture-negative endocarditis is encountered. The diagnosis of infective endocarditis secondary to Q fever is confirmed by serologic testing.

DIAGNOSIS - Q FEVER

Diagnosis of Q fever usually is accomplished using serologic testing; the most common methods are complement fixation, indirect fluorescent antibody, and ELISA. Significant antibody titers usually are not identifiable until 2-3 weeks into the illness.

Of the methods currently used for the diagnosis of Q fever, ELISA is the most sensitive and easiest to perform. This assay can establish a diagnosis of Q fever from a single serum specimen with a sensitivity of 80-84% in early convalescence and 100% in intermediate and late convalescence.

TREATMENT - Q FEVER

Tetracycline has been the mainstay of therapy since the 1950s. When initiated within the first few days of the illness, treatment significantly shortens its course. Macrolide antibiotics, such as erythromycin and azithromycin, are also effective.

When chronic Q fever infection is manifested by infective endocarditis, the mortality is 24% even when patients receive appropriate treatment. At least 2 years of therapy are required, usually with a tetracycline combined with rifampin or a quinolone, although trimethoprim-sulfamethoxazole also has been used.

PREVENTION/PROPHYLAXIS - Q FEVER

Although an effective vaccine (Q-Vax) is licensed in Australia, all Q fever vaccines used in the United States are investigational. Q fever can be prevented by immunization.