Comprehensive Review of Bioterrorism

Function

The Centers for Disease Control and Prevention (CDC) have classified biological weapons into three categories based on various factors, including the morbidity and mortality caused by the disease in humans:[2]

• Category A: Highest priority. Pose a risk to national security. They are easily transmitted from person to person and have high morbidity and mortality. They would have a major public health impact, cause panic, and result in special public health preparedness requirements. 
• Category B: Second highest priority. These include diseases with lower morbidity and mortality as compared to category A. They are also more difficult to disseminate. 
• Category C: Third highest priority. They have the potential to cause significant morbidity and mortality but consist mostly of emerging pathogens that could potentially be engineered for mass dispersion in the future.

For the purpose of this review, the biological weapons and agents which can be used in bioterrorism have been broadly classified as bacterial, viral, fungal, protozoal, and toxins. A brief overview of specific agents which have been used in prior attacks as well as have the potential to weaponized are discussed in this review. The agents being discussed are summarized below:[1][4][5]

• Bacterial: Bacillus anthracis (anthrax), Brucella species (brucellosis), Burkholderia mallei (glanders), Burkholderia pseudomallei (melioidosis), Franciscella tularensis (tularemia), Salmonella typhi (typhoid fever), and other Salmonella species (Salmonellosis), Shigella species (shigellosis), Vibrio cholerae (cholera), Yersinia pestis (plague), Rickettsial agents including Coxiella burnetii (Q fever), Rickettsia prowazekii (typhus fever), Rickettsia rickettsii (Rocky Mountain spotted fever), and Chlamydia psittaci (Psittacosis).
• Viral: Variola major (Smallpox), viral hemorrhagic fevers, viral encephalitis.
• Fungal: Coccidiodes immitis (coccidioidomycosis), Histoplasma capsulatum (histoplasmosis).
• Protozoal: Cryptosporidium parvum (Cryptosporidiosis).
• Toxins: ricin, abrin, Clostridium perfringens toxins, Clostridium botulinum toxins, tetrodotoxin, nerve agents

Issues of Concern

There have been numerous incidents in the past where bioweapons were used in biowarfare. The intentional use of biological weapons, including infectious agents during the war, led to a new and yet unknown threat. The initial and early attempts at using biological weapons in warfare date to the middle ages and included crude methods such as using cadavers and carcasses of humans and animals to contaminate water sources of enemy armies and enemy civilians during warfare.[1]

One of the first reported instances of biological weapon use was as early as 600 BC. Solon used a purgative herb called hellebore during the siege of Krissa.[4] The ingestion of white hellebore (Veratrum Album L.) has been reported to cause nausea, vomiting, abdominal pain, bradycardia, and hypotension, with complete atrioventricular block reports in one patient who had accidental ingestion.[6] 

In 1155 AD, in Tortona, Italy, Emperor Barbarossa poisoned wells with human bodies. In 1346, the Tatar forces who were laying siege to the city of Kaffa (presently Feodosia, Ukraine) engaged in biological warfare by employing the use of catapulting people suffering from the bubonic plague over the walls of the city to initiate a bubonic plague epidemic in its inhabitants and weaken them.[1][4][7] This led to a bubonic plague outbreak followed by a retreat of the defending army and the subsequent conquest of Kaffa by the Tatars. Following this, ships carrying possibly bubonic plague-infected people and vectors (rats) sailed to Genoa, Constantinople, Venice, and other Mediterranean ports. This is thought to have directly contributed to the second plague pandemic, outlining the sheer destructive and unpredictable nature of biological weapons leading to unintended widespread disease.[8] In 1495, in Naples, Italy, the Spaniards mixed wine with blood from leprosy patients to sell to their French foes.[4] In 1710, similar to the events of 1346, Russian troops catapulted the bodies of bubonic plague victims into Swedish cities.[4]

In the 18th century, smallpox was a popular choice for biological weapons.[7] During the French and Indian War between 1754 to 1767, the commander of British forces in North America, Sir Jeffrey Amherst, employed the deliberate use of smallpox to "reduce" the populations of the Native American tribes who were hostile to the British.[7] A naturally occurring smallpox outbreak at Fort Pitt helped the British troops execute Amherst's plan by virtue of the generation of smallpox-laden fomites. In 1763, one of Amherst's subordinates, Captain Ecuyer, gave the Native Americans a handkerchief and a few blankets taken from the smallpox hospital. This resulted in an epidemic among the Native American tribes.[7] In 1797, Napoleon flooded the plains around Mantua in Italy to increase the spread of malaria. In the United States, in 1863, the Confederates sold clothing obtained from patients suffering from yellow fever and smallpox to Union troops during the Civil War.[4]

More recently, in the 20th century, biological weapons were reportedly used to a limited extent. Some evidence suggests that during World War I, Germany had developed a biological warfare program that planned on covert operations to infect the livestock and contaminate the animal feed, which was to be exported to the Allied forces from neutral countries. Reports circulated of attempts to ship cattle and horses inoculated with Bacillus anthracis (causing anthrax) and Burkholderia mallei (causing glanders) to the United States and other countries.[1][4] These same organisms were also used to infect Romanian sheep, which were planned to be exported to Russia. There were also other allegations of attempts to spread the plague in Russia and cholera in Italy. Germany denied all such allegations of indulging in biological warfare. However, following these allegations, keeping in mind the horrific and unpredictable nature of these agents, the Geneva Protocol was signed in 1925, which prohibited the development, production, and use of Biological weapons in war.[3]

During World War II, there were once again attempts made by various nation-states to indulge in the use of biological weapons.[1] Japan engaged in research related to biological weapons from 1932 until the end of World War II. The agents of interest to the Japanese biological weapons program included Bacillus anthracis, Vibrio cholerae, Shigella spp, Neisseria meningitidis, and Yersinia pestis. Between 1932 and 1945, more than 10,000 prisoners died due to experimental infection as part of this biological weapons research. A majority of these prisoners’ deaths were a direct consequence of experimental inoculation of biological weapons and pathogens, which caused anthrax, cholera, meningococcal infection, dysentery, plague, and so on. Research on tetrodotoxin (an extremely potent toxin derived from the ‘fugu’ fish) was conducted.[4][5]

Prisoners in Nazi concentration camps in Germany were deliberately infected with Rickettsia prowazekii, Plasmodium species, and hepatitis A virus to be treated with experimental drugs and vaccines.[7] In England, experiments involving weaponized spores of Bacillus anthracis were conducted off Scotland's coast, which resulted in heavy contamination. Still, viable anthrax spores remained on the island until it was decontaminated with formaldehyde and seawater in 1986.

In 1942, the United States initiated a biological warfare program. The program included research on B. anthracis and Brucella suis in various research facilities, including a development facility at Camp Detrick in Maryland, known today as the US Army Medical Research Institute of Infectious Diseases (USAMRIID). About 5000 bombs containing B. anthracis spores were developed at Camp Detrick, but since the facility lacked adequate safety measures, further production during World War II was stopped.[4][7]

Post World War II, various other nation-states and organizations dabbled in developing biological weapons. By the 1960s, the United States military developed a large biological weapons arsenal that consisted of various biological pathogens, toxins, and fungal plant pathogens that could induce crop failure and result in subsequent famines. In 1972, a United States-based extremist group called themselves ‘Order of the Rising Sun’ was found to possess typhoid bacteria cultures intended to disseminate the water supplies of numerous major mid-western cities.

In 1978, a Bulgarian exile, Georgi Markov, was assassinated in London in what later came to be known as the “umbrella killing” due to the murder weapon being a device hidden inside an umbrella. A tiny pellet was discharged into Markov’s leg at a bus stop in London. The next day, he became severely ill and died 3 days after the incident. 10 days before Markov’s assassination, an attempted assassination had occurred in Paris, France. Another Bulgarian exile, Vladimir Kostov, felt a sharp pain in his back in a metro stop in Paris. He reported seeing a man carrying an umbrella fleeing the scene. Two weeks later, after learning of Markov's assassination, Kostov was examined by a French medical team, and they extracted a similar pellet. It was made out of an alloy of platinum and iridium and contained the plant toxin ricin (made from castor beans).

In 1979, there was an outbreak of anthrax in the Russian city of Sverdlovsk (now Yekaterinburg). The outbreak occurred in people close to a Soviet military microbiology facility (called Compound 19). As well as humans, livestock in the area also died of anthrax. The unintentional release of anthrax spores was thought to have resulted in a total of 66.[9] In 1980, the Baader-Meinhof group (also called the Red army faction) in Germany was found to have access to Clostridium botulinum cultures as well as to a biological laboratory.[1] In 1986, the Rajneesh cult in Dalles, United States, contaminated salad bars in local restaurants with a resulting 751 cases. The cult was attempting to prevent citizens from voting in an upcoming election.[1][10]

From 1990 to 1994, a Japanese religious cult calling themselves Aum Shinrikyo (presently called Aleph) made nine failed attempts to release anthrax spores as well as an aerosol containing botulinum toxin in Tokyo with the intent to murder innocent civilians.[1] However, in 1995, they succeeded in releasing a nerve gas called sarin in Tokyo’s subway system, which resulted in the death of 12 people and injury to approximately 3,800 people.[9]

In 2001 in the United States, a series of letters containing anthrax spores were mailed to senators, journalists, and media buildings. There were 5 casualties and 22 people who were seriously injured. A large-scale investigation finally implicated a former United States scientist as the perpetrator.[3] Using letters in the post as a mode of delivery of biological weapons remains a popular choice among bioterrorists. There were more than 20 attacks involving ricin between 1990 to 2011. Due to the highly destructive nature of biological weapons and the relative ease with which they may be produced, they remain a big threat. 

Specific Biological Weapons

Bacterial Agents

Bacillus anthracis (Anthrax)

Bacillus anthracis is an aerobic or facultatively-anaerobic, encapsulated, gram-positive or gram-variable, spore-forming bacilli that grow well on blood agar in the form of large, irregular-shaped colonies. The word anthrax originates from "anthrakis" in Greek, meaning black, which refers to the necrotic lesions which are encountered in cutaneous anthrax. B. anthracis is categorized as a ‘category A’ priority organism by the Centers for Disease Control and Prevention due to its potential capability to be disseminated as a bioweapon.[2] 

The pathogenesis of anthrax infections depend on the route of inoculation, with three routes reported in humans: Inhalational anthrax by the accumulation of B. anthracis spores initially in the lung alveoli followed by transport to the regional lymph nodes where it germinates, multiplies, and begins toxin production with subsequent systemic illness, bloodstream infection and septic shock. Cutaneous anthrax occurs by the inoculation of anthrax spores through a break in the skin into the subcutaneous tissues. B. anthracis germinates and multiplies locally along with toxin production, which causes the characteristic edema as well as cutaneous ulceration. Gastrointestinal anthrax occurs due to ingestion of meat contaminated with anthrax spores, leading to mucosal ulceration and bleeding. Another fourth form of anthrax has been reported recently in northern European intravenous drug users due to the use of contaminated needles, producing lesions at the injection site clinically similar to cutaneous anthrax but can also present with a deeper infection including myositis.[11]

Symptoms and Signs[11]

Inhalational anthrax has an incubation period of around 1 to 6 days following exposure. It presents with a non-specific prodromal phase, including fever, malaise, nausea, vomiting, chest pain, and cough. The second stage of bacterial replication follows this in the mediastinal lymph nodes, which causes hemorrhagic lymphadenitis and mediastinitis, with subsequent progression to bacteremia. Meningitis can occur in up to 50% of cases. Death can result, ranging from 1 to 10 days after symptom onset. 

Gastro-intestinal can have oropharyngeal and/or intestinal involvement. In oropharyngeal anthrax, ulcers may develop on the posterior oropharynx, which can cause dysphagia and regional lymphadenopathy. In intestinal anthrax, patients may have fever, nausea, vomiting, and diarrhea. They can also have acute abdomen-like features with associated hematemesis, bloody diarrhea, and massive ascites. Untreated patients can progress to septicemia with a mortality range of 25% to 60%. 

Cutaneous anthrax, which is also called hide-porter's disease, can present one to 10 days following exposure with a pruritic and papular lesion which can progress over days into a painless ulcer. The primary lesion can have associated satellite vesicles that can progress to a necrotic center with non-pitting edema surrounding it. A painlessness lesion is considered characteristic of cutaneous anthrax. The eschar may dry and slough off in about 1 to 2 weeks, but the mortality rate can approach nearly 20% without treatment. 

Investigations and Management [11]

The CDC recommends using PCR, gram stain, and bacterial cultures depending on clinical features from blood, pleural fluid, ulcer, cerebrospinal fluid, or stool. Routine diagnostic tests, including a complete blood count and chest x-ray, are also recommended. Chest X-ray in inhalational anthrax can show an enlarged mediastinum. A CT scan shows enlarged hilar lymph nodes, evidence of mediastinal hemorrhage or pleural effusions. Cutaneous anthrax may be diagnosed using a methylene blue stain that can show a non-motile gram-positive bacillus. 

The laboratory personnel must be adequately warned about the possibility of anthrax. All inhalational anthrax cases should be considered as a bioterrorism event, and decontamination is done appropriately. Anthrax is reportable and, if identified, should be immediately reported to local authorities and the CDC.  

Treatment for inhalational anthrax involves a regimen using one bactericidal agent + one protein-synthesis inhibitor drug. Intravenous ciprofloxacin + clindamycin/linezolid is the preferred regimen. In meningitis, a three-drug regimen is preferred with the addition of a bactericidal agent from a different drug class, such as a beta-lactam. In cutaneous lesions, oral ciprofloxacin/doxycycline is effective, however with extensive edema or in cases of head and neck involvement, an intravenous multi-drug regimen is preferred. An antitoxin may be recommended along with the multi-drug regimen. Treatment with Anthrax immune globulin is added on for systemic treatment. Anthrax vaccine may be considered for exposed people following a bioterrorism event. In the event of inhalation exposure, should undergo prophylactic treatment for 60 days, regardless of vaccination. A combination of doxycycline and ciprofloxacin is the recommended first-line therapy in post-exposure prophylaxis.

Importance in Bioterrorism[3]

Anthrax has been used in bioterrorist attacks in the past, with the most prolific example being the ‘Anthrax letter’ attacks in the United States in 2001. It is a category A priority pathogen as per the CDC. It is highly stable in aerosolized form, making it one of the most popular biological weapons choices. 

Brucella species (Brucellosis)

In humans, Brucellosis may be caused by four different species, including B. suis, B. abortus, B. melitensis, and B. canis. Brucella species are gram-negative, non-motile Cocco-bacilli, which are facultatively intracellular and do not form spores or toxins.[12]

Symptoms and Signs[12]

Brucellosis can present with clinical features based on the underlying clinical syndrome, potentially including features of meningoencephalitis, myelitis, transaminitis, orchitis, epididymitis, endocarditis, sacroiliitis, spondylodiscitis, osteomyelitis, septic arthritis, epidural abscesses, and hepatic abscesses. Respiratory symptoms can include a cough, breathlessness, and pleurisy, with the presence of focal lung abscesses and pleural effusions having been reported. Guillain-Barre syndrome and Subarachnoid hemorrhage have been reported in acute neurobrucellosis. Dermatological manifestations include maculopapular rashes, erythema nodosum, cutaneous abscesses, and panniculitis. Endocarditis and aortic fistulas can rarely occur. Although lymphadenopathy, hepatomegaly, splenomegaly, and clinical features of an underlying clinical syndrome may be found, the physical examination may be normal. Brucellosis frequently presents as a fever of unknown origin, prompting significant workup before a diagnosis is reached. The mortality rate ranges from two to five percent. 

Investigations and Management[12]

Patients may have anemia, leukopenia, or even pancytopenia along with elevated inflammatory markers, serum lactate dehydrogenase, transaminases, and alkaline phosphatase. In spondylitis, disc space narrowing, sclerosis, and bone destruction may be visible on imaging. On liver biopsy, granulomas may be observed. Blood cultures on tryptose medium may yield growth of Brucella, but due to its slow-growing nature, it may take more than a week. Bone marrow cultures have a higher yield when compared to blood cultures. In endemic areas, standard agglutination tests are commonly used. ELISA or Rose Bengal plate agglutination test also may be used. Doxycycline, along with another agent, which may be streptomycin, gentamicin, rifampin, or co-trimoxazole, is the mainstay of treatment. As Brucella is an intracellular organism, several weeks of treatment may be necessary. Monotherapy should be avoided due to high relapse rates. A regimen of co-trimoxazole plus rifampin for four to six weeks may be used in the pediatric population. Rifampin is used during pregnancy, with co-trimoxazole added postpartum. Surgical debridement may be required in certain cases, especially in fistulas and bone involvement.

Importance in Bioterrorism

Brucella has been successfully engineered as a biological weapon by the United States and several other countries, although it has never been used during the war. Brucella can be easily aerosolized, and it survives well in the aerosol form. In a bioterrorist event using Brucella, treatment remains the same as for naturally occurring Brucella infections as detailed above. The relatively lower mortality rate of Brucellosis has led to it falling out of favor as a potential bioweapon and has more historical significance.[12][13]

Burkholderia mallei (glanders) and Burkholderia pseudomallei (melioidosis)

Glanders is caused by Burkholderia mallei which is a gram-negative, aerobic, non-motile bacillus. Melioidosis is caused by Burkholderia pseudomallei, which is also a gram-negative, aerobic bacillus but is motile. These two bacteria are related closely and can both present with the disease in humans.[14]

Symptoms and Signs

The incubation period for glanders ranges between one to 21 days but can even be months to years. Glanders usually starts as fever, followed by pustules, abscesses, and pneumonia. Acute glanders is usually fatal within seven to ten days of onset. Chronic glanders causes death within months, and the survivors become carriers.[14]

Burkholderia pseudomallei can enter the human host through three modes: ingestion, inhalation, or direct inoculation. The incubation period of melioidosis is highly variable. It can range from two days to several years. Acute melioidosis presents with fever, cough, pleurisy, myalgia, arthralgia, headache, night sweats, and anorexia. Liver, spleen, prostate, and parotid abscesses are common. In about 10% of cases, symptoms last more than 2 months, which constitutes Chronic melioidosis. Direct inoculation through wounds in the body and the organism's ability to use an axoplasmic transport mechanism to invade the central nervous system using the peripheral nervous system results in neuromelioidosis, which is notoriously difficult to diagnose as well as a treat.[14][15]

Investigations and Management

Cultures can diagnose both organisms. In melioidosis, blood, sputum, urine, and throat swab cultures may be indicated. It is recommended to perform the laboratory work on these organisms under BSL-3 precautions. Latex agglutination, indirect hemagglutination, and direct immunofluorescence tests may be available based on region. Patients may have non-specific anemia, leukopenia or leukocytosis, and elevated inflammatory markers. Imaging of appropriate body sections based on clinical presentation may reveal abscesses which can point towards melioidosis. Histopathology of affected tissue may show granulomas.[14][15]

In both diseases, patients with significant lungs' significant involvement can progress to respiratory failure and require mechanical ventilation. Sepsis may also occur. In glanders, the recommended treatment regimen includes imipenem and doxycycline for two weeks, which should be followed by doxycycline and azithromycin for six months. A post-treatment CT can show improvement of underlying abscesses. Glanders tends to be fatal in 95% of cases without appropriate treatment, and death can occur within seven to ten days of onset. Mortality may still be as high as 50%, even with appropriate antibiotic treatment.[14]

The mortality in melioidosis ranges between 20% to 50%.[16] This can exceed 90% in sepsis, but it may decrease to 10% in uncomplicated cases with appropriate antibiotic therapy.[14] The recommended treatment regimen in acute melioidosis includes intravenous ceftazidime. Carbapenems, including meropenem and imipenem, are also effective. Like glanders, patients undergo treatment with intravenous antibiotics for two weeks, followed by doxycycline and co-trimoxazole for up to 20 weeks to eradicate the disease. Most abscesses often resolve with antibiotic therapy, but some may require surgical debridement. There may be a recurrence in 20% of cases, but this is reduced to less than five percent with co-trimoxazole eradication therapy. Due to the risk of relapse, lifelong follow-up may be required. No vaccines or approved antibiotic prophylaxis regimens are currently available for melioidosis or glanders.[14]

Importance in Bioterrorism

As per the CDC, both B. pseudomallei and mallei are category B bioweapons. During World War I, German sympathizers in numerous countries infected horses meant for dispatch to conflict areas with B. mallei to induce glanders. This led to the combat operations being affected due to the infection of humans and horses. Japan and the Soviet Union researched the use of B. mallei before World War II. The Japanese deliberately infected Chinese prisoners using B. mallei. Numerous countries, including the United States, studied B. pseudomallei for its potential use as a bioweapon. However, there have not been any reports of the malicious use of B. pseudomallei or B. mallei in recent years.[17]

Franciscella tularensis (tularemia)[18]

Francisella tularensis is a highly infectious gram-negative coccobacillus. Infection can occur through various entry points, including inhalation, direct contact with a break in the skin or mucous membranes, ingestion, or through ticks or fly vectors. 

Symptoms and Signs[19]

F. tularensis infections can cause distinct clinical syndromes based on the mode of exposure. Percutaneous inoculation usually causes ulceroglandular tularemia, which is characterized by a cutaneous ulcer at the inoculation site as well as tender regional lymphadenopathy. Inhalation can result in primary pneumonia. Ingestion can cause oropharyngeal disease, which consists of tonsillitis or pharyngitis with associated cervical lymphadenopathy. Other presentations of tularemia can include oculoglandular and typhoidal (pyrexia without a localizing sign). Mortality is ranges between 2 to 24% depending on the strain, with certain strains such as the type A strain being more lethal. 

Investigations and Management:[19]

Tularemia requires a high index of clinical suspicion as laboratory identification is difficult. Patients may have raised inflammatory markers or leukocytosis. The cornerstone of laboratory diagnosis relies on the serologic diagnosis. An initial titer of more than 1:160 or a four-fold increase in titers between the initial and convalescent samples indicates a diagnosis of tularemia. Early testing may yield negative results as antibodies take time to form. Hence, a negative serology early on does not rule out Tularemia. Cultures of blood, CSF, lymphatic tissue, and ulcer swabs may yield the growth of F. tularensis. However, it is important to note that culturing should be attempted only in highly controlled settings as lab workers' accidental inhalation can potentially cause pneumonic tularemia. The culture of F. tularensis also requires specialized culture media and longer incubation. On other laboratory evaluations. 

The recommended treatment regimen for tularemia consists of intravenous gentamicin for seven to 14 days. Fluoroquinolones such as ciprofloxacin also have a role in mild disease, but data of its use in the more virulent type A infections is limited. Bacteriostatic agents like tetracyclines should be avoided due to the high risk of relapse. Incision and drainage of the affected lymph nodes are indicated in select cases. 

Importance in Bioterrorism[19]

F. tularensis has been designated as a category A agent due to a low infectious dose, its ability to aerosolize, and its history of being developed as a biological weapon.

Salmonella typhi (typhoid fever) and other Salmonella species (Salmonellosis)

Salmonella typhi is a gram-negative, flagellated bacillus that causes typhoid fever. It is usually contracted by ingesting contaminated water or food with an infectious dose ranging between 1000 and 1 million bacteria. Salmonella typhi can enter the small intestine submucosal layer by direct penetration into the epithelial tissue, following which it causes hypertrophy of the Peyer’s patches. It then disseminates through the lymphatics and the bloodstream.[20]

Salmonella genus is motile, gram-negative, produces hydrogen sulfide, acid-labile, and facultative intracellular bacteria that belong to the Enterobacteriaceae family.[21]

Symptoms and Signs

In Typhoid fever, the incubation period ranges between seven to 14 days after initial inoculation. Following this, patients can present with fever and abdominal symptoms, including abdominal pain, nausea, vomiting, diarrhea, or constipation. A stepladder pattern of fever and relative bradycardia is classically associated with typhoid and enteric fever.[20][22] Hepatomegaly and splenomegaly can develop during disease progression. Rose spots, which are blanching erythematous maculopapular rashes consisting of lesions that are two to four mm in diameter, can develop on the abdomen and chest.[20]

Other Salmonella infections can present with bacteremia or as focal infections, including gastroenteritis, meningitis, osteomyelitis, and urinary tract infections.[21]

Investigations and Management

In Typhoid fever, a blood count can show either leukopenia or leukocytosis with a left shift. Relative anemia could be seen. Blood and stool cultures are recommended in the workup. Blood cultures may be positive in 40 to 80% of patients, while stool cultures could be positive in 30% to 40%. The most sensitive test remains a bone marrow aspirate for culture, with more than 90% being positive for Salmonella typhi. Widal test is a measure of agglutinating antibodies against the flagellar H and lipopolysaccharide O antigens. A positive Widal constitutes a four-fold increase in the antibody titers when taken 10 days apart. Currently, ciprofloxacin or ofloxacin is the mainstay of treatment in non-endemic regions. In endemic areas, and when resistance to quinolones is suspected, an extended-spectrum cephalosporin like ceftriaxone should be used with or without azithromycin based on local guidelines and resistance patterns. Approximately around 1% to 5% of patients can become chronic carriers of Salmonella typhi despite appropriate antimicrobial therapy.[20]

For other Salmonella infections, the gold standard test for diagnosis is bacterial culture. Stool, blood, urine, bile, CSF, and bone marrow may be cultured based on the clinical syndrome. Due to the production of hydrogen sulfide, Salmonella forms black colonies on Hektoen Agar. PCR for specific Salmonella species is also commercially available and is being increasingly used in clinical medicine.[21]

Importance in Bioterrorism[23]

Salmonella is a category B bioweapon as per the CDC. It has been used in biowarfare by the Germans during World War I and in the infamous attack by the Rajneesh cult in Dallas, TX in the United States in 1986, where they contaminated salad bars in local restaurants with Salmonella to influence an upcoming election. 

Shigella species (shigellosis) and Escherichia coli O157:H7

Shigella is a gram-negative, non-motile, facultatively anaerobic, and non-spore-forming bacillus which has 4 serotypes, including serotype A (Shigella dysenteriae with 12 serotypes), serotype B (Shigella flexneri with 6 serotypes), serotype C (Shigella boydii with 23 serotypes), and serotype D (Shigella soneii with 1 serotype). Transmission occurs mainly via the fecal-oral route and maybe water or food-borne. The number of organisms required to cause illness is usually only 10 to 200 bacteria. It produces enterotoxin one and two, which causes Shigella-associated diarrhea and is responsible for cytotoxicity and complications such as hemolytic uremic syndrome.[24]

Escherichia coli O157: H7 is a Shiga-like toxin-producing strain that is a food and waterborne pathogen. It is a gram-negative bacillus and belongs to the Enterobacteriaceae family. Naturally occurring infections occur through the fecal-oral route by consumption of contaminated food and water. Only a relatively low inoculum (102 CFU) is required to cause infection.[25]

Symptoms and Signs

Shigellosis can present with abdominal discomfort or severe diffuse colicky abdominal pain. There can be mucoid diarrhea that can precede dysentery. Fever, nausea, vomiting, lethargy, anorexia, and tenesmus are also common. Physical examination may indicate lethargic or toxic patients with fever and altered vital signs. An abdominal examination can show a distended abdomen with tenderness in the lower abdomen because of the sigmoid colon and rectum's involvement.[24]

In E. coli O157: H7 infections, patients present with acute onset bloody diarrhea and abdominal cramping with or without fever. There may also be nausea, vomiting, and profuse diarrhea resulting in dehydration and decreased urine output. Abdominal tenderness may be elicited by virtue of Shiga-like toxin-induced intestinal inflammation. Systemic signs of dehydration may be present.[26]

Investigations and Management

In shigellosis, a complete blood count can show leukocytosis with a shift to the left or leukopenia. Anemia and/or thrombocytopenia may be present. Inflammatory markers may be raised. Stool analysis can show fecal leukocytes and red blood cells. A stool culture can yield the growth of Shigella. Blood cultures may be positive in complicated cases. There may be a mild elevation of bilirubin and creatinine. Electrolytes may be deranged with hyponatremia and hypokalemia. ELISA can be used to detect S. dysenteriae type-1 toxin in the stool. PCR can be used to identify virulent genes such as ipaH, virF, and virA genes. The mainstay shigellosis treatment involves hydration and electrolyte management. In adults, empiric antibiotic therapy is based on resistance patterns. Fluoroquinolones are recommended when there are no risk factors for resistance. A third-generation cephalosporin is recommended when resistance is suspected or in high-risk cases. Second-generation cephalosporins, ampicillin, and co-trimoxazole may also be used if susceptibility is documented. In children, the preferred first-line agent is azithromycin. Cefixime or ceftibuten may be used in case of resistant strains. Intravenous antibiotics are indicated with suspected or proven severe shigellosis with signs of bacteremia, potentially including lethargy, fever > 102.2 F, underlying immune deficiency, and in children unable to take oral drugs.[24]

In E. coli O157: H7 infections, complete blood count can show leukocytosis, anemia due to hemolysis, and thrombocytopenia. Metabolic profile is important, especially in dehydration, which can result in electrolyte disturbances and uremia. A stool culture may be positive for E. coli 0157:H7. Culturing the stool with sorbitol MacConkey agar can differentiate non-pathogenic E. coli from the pathogenic E. coli O157:H7 as the O157:H7 strain cannot metabolize sorbitol. PCR for the presence of O157: H7 antigens or toxin genes in the stool may be useful. Treatment of E. coli O157 is based on supportive care and hydration of the patient. Most patients recover within ten days with supportive care. Antibiotic therapy does not improve outcomes and may even worsen prognosis by increasing the chances of developing hemolytic uremic syndrome (HUS). In the setting of HUS, patients may require hemodialysis.[26]

Importance in Bioterrorism

During World War II, the Japanese bioweapon program studies the use of Shigella species. Many prisoners died due to experimental inoculation causing dysentery.[4]

E. coli O157:H7 strain is considered a Category B priority pathogen by the CDC as it is a potential food safety threat.[27]

Vibrio cholerae (cholera)[28]

Toxin-producing strains of Vibrio cholerae cause cholera. V. cholerae is a motile, comma-shaped, gram-negative rod that has a single polar flagellum. Cholera is transmitted through the fecal-oral route by contaminated water or food. 

Symptoms and Signs

Cholera presents with profuse painless diarrhea, abdominal discomfort, and vomiting but without fever. Severe cases can lead to hypovolemic shock as a result of massive fluid and electrolyte loss. Classical diarrhea consists of watery and foul-smelling mucous, which is described as "rice-water" stools. The rate of fluid loss can be up to 1 liter per hour. In the absence of adequate treatment, mortality rates may be as high as 70%. Cholera sicca is a variant of cholera where the fluid accumulates inside the intestinal lumen, followed by circulatory collapse and resulting death before diarrhea presents.[28]

Investigations and Management

Laboratory tests usually reveal hypokalemia, hypocalcemia, and metabolic acidosis as a result of massive fluid loss, but hyponatremia may not be evident as salt is lost too. Confirmatory diagnosis of V. cholerae is by the isolation of bacteria in stool cultures, PCR, and other rapid tests. Stool cultures remain the gold standard in the diagnosis of cholera.[28]

Oral rehydration therapy (ORT) remains the mainstay in the treatment of acute cholera. The degree of fluid replacement may be determined by the degree of hypovolemia ascertained by a physical exam. Rehydration must be started as soon as cholera is suspected. In patients with severe hypovolemia, intravenous replacement with the appropriate replacement of electrolytes and glucose is key. Oral rehydration may begin as soon as the patient is able to drink. It is also paramount to assess the ongoing fluid losses and replace them appropriately with periodic reassessment of the volume status. Antibiotics are added as an adjunctive treatment in cholera once the volume deficit is corrected. The recommended agents include tetracyclines, macrolides, and fluoroquinolones, with tetracyclines being the most used agents.

Importance in Bioterrorism[4]

Similar to Shigella, the Japanese bioweapon program during World War II experimented with cholera. Numerous prisoners died due to experimental inoculation using Vibrio cholerae. 

Yersinia pestis (plague)

Yersinia pestis is a gram-negative, non-motile bacillus with a bipolar staining pattern with Giemsa, Wright, or Wayson staining.[29] As a result of the lymph nodes' pathophysiologic involvement, more than 80 to 95% of Y. pestis infections usually present with suppurative adenitis, known as the bubonic plague. Other presentations include septicemic plague and pneumonic plague.[30]

Symptoms and Signs

The most common presentation is the bubonic plague which has a two to eight-day incubation period. Symptoms include sudden fever, chills, headache, and malaise. A bubo develops within a day or so, starting as intense pain and swelling in the regional lymph node area, commonly inguinal, followed by axillary or cervical nodes' involvement. The masses are usually non-fluctuant with overlying warmth. There may be tachycardia and hypotension, indicating progression to shock. There may also be hepatosplenomegaly. Septicemic plague is similar to bubonic plague in most signs, except that there is no associated bubo.[30]

Pneumonic plague commonly occurs following the hematogenous spread of the organism from the bubo and can present with fever, cough, chest pain, and hemoptysis. It can also occur without buboes. Primary pneumonic plague can occur following inhalational exposure to another patient having a cough. Rarely, patients may present with meningitis and pharyngitis.[30]

Investigations and Management

Suspected patients should be immediately isolated along with droplet precautions for at least 48 hours following initiation of antibiotic therapy. Laboratory personnel should also be informed to allow for precautions while handling samples. In all presentations, a high degree of clinical suspicion is required. In the setting of clinical suspicions, an aspirate taken from the bubo is stained and cultured to demonstrate the organism. Complete blood counts can show significant leukocytosis as well as thrombocytopenia. Neutrophils may demonstrate Dohle bodies, but this may not be specific. Other tests include PCR, immunofluorescence, and ELISA. In the US, confirmation of the diagnosis is possible by sending samples to the CDC for culturing.[30]

Rapid initiation of antibiotics is necessary for effective treatment due to the rapid progression. Aminoglycosides such as streptomycin or gentamicin are considered first-line treatment for seven to ten days. The alternative agents include tetracycline or doxycycline and tetracycline for 14 days. Co-trimoxazole has decreased efficacy as compared to the first-line antibiotics. Chloramphenicol is preferred in case of meningitis. Levofloxacin is also licensed for use in the plague. As Y. pestis is a potential bioweapon, vaccines exist with unconfirmed efficacy, and potentially better vaccines are under production.

Importance in Bioterrorism[29]

Plague is a category A bioweapon as per the CDC. Epidemiology of plague in the event of a bioterrorist attack would be significantly different from natural infections. It would likely be an aerosol leading to a pneumonic plague outbreak. Patients would initially present with symptoms similar to other severe respiratory infections. The incubation period may range between one to six days, and the mortality rate may be substantially based on the strain used. An outbreak of the plague in areas not previously reported to have enzootic infections coupled with an absence of a large number of dead rats would indicate a deliberate bioterrorist attack.

Rickettsial Infections 

The CDC has included four rickettsial organisms, including Coxiella burnetii (Q fever), Rickettsia prowazekii (typhus fever), Rickettsia rickettsii (Rocky Mountain spotted fever), and Chlamydia psittaci (Psittacosis), as potential biological weapons.[31][32]

Coxiella burnetii is an obligate intracellular, gram-negative, pleomorphic bacteria that causes Q fever. C. burnetii exhibits a form of antigenic shift, namely, phase variation, where it exists as a highly infectious phase I form in animals and as a non-infectious phase II form when it is subcultured.[33]

Rickettsia prowazekii is an obligate intracellular, gram-negative bacteria that causes typhus.[34]

Rickettsia rickettsii is an obligate intracellular, Cocco-bacillary organism that causes Rocky Mountain spotted fever.[35]

Chlamydia psittaci is an obligate intracellular, gram-negative bacteria that can infect both mammals and avians, having multiple genotypes. Birds are the major epidemiological reservoir, while human-to-human transmission is rare.[36]

Symptoms and Signs

The severity of Q fever may range from being completely asymptomatic to serious illness. The incubation period ranges between two to six weeks. The usual spectrum involves a febrile illness which is usually associated with a headache. The illness plateaus in two to four days, and the patient returns to normal in five to 14 days. The fever may last longer if untreated. There may be atypical pneumonia characterized by non-productive cough, minimal auscultatory findings, and very non-specific findings on chest radiograph. There may also be hepatitis which may be without clinical manifestations, with hepatomegaly or hepatitis with evidence of granulomas on biopsy, which may present as a fever of unknown origin. There can be cardiac involvement in the form of myocarditis or pericarditis, which is a major cause of death. Dermatological manifestations include a pink macular or papular rash on the trunk, seen in about five to 21% of cases. Neurological involvement involves lymphocytic meningitis, encephalitis/meningoencephalitis, or peripheral neuropathy.[33]

For Rickettsia prowazekii, the incubation period ranges between one to two weeks. Symptoms include high fever (105 to 106 F), severe headache, myalgias, delirium, dry cough, stupor, and an erythematous rash which begins on the trunk and spreads peripherally with sparing of the palms and soles. The disease can progress to hypotension, shock, and death. Recrudescent cases can occur even decades after initial infection, presenting as severe headache, high fever, chills, and cough.[34]

Rocky Mountain spotted fever occurs four to ten days following exposure to the Rickettsia rickettsii. The symptoms classically include a triad of fever, headache, and a maculopapular or petechial rash. The rash begins as a maculopapular rash on the wrists and ankles, which can later progress to petechia. Other symptoms and signs include lymphadenopathy, confusion or neck rigidity, vomiting, myalgia, arthralgia, and cardiac involvement.[35]

Psittacosis has an average incubation period of five to 14 days. Symptoms are mainly respiratory, but the system involvement can vary tremendously. The organism can spread hematogenously to other organ systems after initial respiratory replication. The symptoms initially include fever, chills, headache, and cough. Signs include altered mental status, photophobia, neck stiffness, pharyngitis, and hepatosplenomegaly. Other symptoms and signs vary based on the systems involved in the disease.[36]

Investigations and Management

C. burnetii can be isolated on cell culture media, but as there is a risk of lab transmission, culture should only be attempted in BSL 3 labs. Serology remains the mainstay in the diagnosis of Q fever, with indirect immunofluorescence being the reference test. Patients with acute Q fever can have normal leukocyte counts, thrombocytopenia, elevated transaminases, the presence of smooth muscle, and anti-phospholipase antibodies. In chronic Q fever, there may be anemia, leukocytosis or leukopenia, thrombocytopenia, elevated transaminases, raised serum creatinine, and the presence of smooth muscle autoantibodies, antinuclear antibodies, and rheumatoid factor. Treatment is most effective when initiated within three days of symptom onset. In acute Q fever, the preferred treatment regimen includes doxycycline 100mg/day for 14 days or fluoroquinolones, minocycline, co-trimoxazole. In chronic Q fever, a regimen of doxycycline plus hydroxychloroquine for at least 18 months is preferred. A vaccine is available which may be useful against a bioterrorist event using C. burnetii as the bioweapon.[33]

Serology is the mainstay in diagnosing Rickettsia prowazekii infections. A four-fold increase between the acute and convalescent titers is considered diagnostic. Indirect fluorescence antibody tests, agglutination tests, and enzyme immunoassays are commonly employed. Patients can have an initial IgM response followed by the production of IgG antibodies. Primary treatment of Rickettsia prowazekii infections includes doxycycline 100 mg twice daily for seven to ten days. The alternative regimen includes chloramphenicol 500 mg four times daily for seven to ten days.[34]

The diagnosis of R. rickettsiae infection depends on IgM and IgG serologic responses in the setting of clinical suspicion. Serology can be negative early in the course of the disease, and repeat tests may be warranted. Rickettsia can be cultured but is difficult and has a high BSL requirement due to exposure risk. PCR is an alternative means of diagnosis. The patient can have thrombocytopenia, hyponatremia, and CSF pleocytosis. Doxycycline is the antibiotic of choice for treatment, including in children. Defervescence usually occurs within three days of starting therapy, and treatment is usually continued for seven to ten days or at least three days following defervescence. Mortality rates may be as high as 20% to 30% without appropriate treatment. Prompt initiation of treatment on the grounds of clinical suspicion is recommended.[35]

In psittacosis, workup may show mild leukopenia during the acute phase of the disease, which can later progress to profound leukopenia. There may be anemia likely secondary to hemolysis. Liver transaminase, as well as gamma-glutamyl transpeptidase, may be variably elevated. There may also be raised inflammatory markers. A chest X-ray is abnormal in about 80% to 90% of patients who require hospitalization. Findings on the X-ray include unilateral or bilateral consolidation, miliary lesions, interstitial infiltrates, or nodular infiltrates. The CDC diagnostic criteria for psittacosis include any one of the following in the setting of clinical suspicion: isolation of C. psittaci from respiratory secretions, a four-fold increase in the serum antibody titers between in samples collected two weeks apart, a single IgM antibody titer of 1:16 or higher. The treatment regimen of choice for psittacosis is doxycycline 100 mg twice daily for 10 to 14 days. Alternative regimens including in pregnancy include macrolides such as azithromycin and erythromycin for seven days. Third-line agents include fluoroquinolones, but these are less effective than first and second-line agents.[36]

Importance in Bioterrorism

C. burnetii is a category B biological weapon as per the CDC. Even though it can be disseminated on a large scale, its relatively low mortality rates make it less dangerous than category A agents. However, it might still be more suitable as a biological weapon due to its widespread availability, environmental stability, and potential for aerosolized use. Aerosolised C. burnetii is extremely infectious, with a single bacterium being enough to produce disease.[31]

Concerning other rickettsial organisms, the CDC classifies Rickettsia prowazekii, Rickettsia rickettsii, and Chlamydia psittaci as category B biological weapon agents as well. The minuscule infectious dose required (less than 10 bacteria) coupled with the ability of the organism to aerosolize efficiently and the resulting poor clinical outcomes in patients make them potential bioterror agents.[32]

VIRAL  

Variola Major (Smallpox)[37]

Variola major is a DNA virus that belongs to the orthopoxviridae genus and the Poxviridae family, consisting of other poxviruses such as parapoxvirus, suipoxvirus, capripoxvirus, and molluscipoxvirus. Only variola and molluscum contagiosum are specific human viruses. However, the other orthopoxviruses, including vaccinia, monkeypox, and cowpox, can also cause significant illness in humans, but only variola major has human to human transmission. All orthopoxviruses are large, brick-shaped virions with a complex structure and a diameter of about 200 nm. The viruses replicate in the host cell cytoplasm by utilizing a DNA-dependent RNA polymerase. 

Symptoms and Signs[37]

After transmission of variola, primary viral replication (primary viremia) occurs at the infection site. Secondary viremia happens around the eighth day after infection, which presents with sudden onset of a fever. After a 12 to 14 day incubation, there can be high-grade fever, malaise, and a headache. There can be an associated maculopapular rash on the oral mucosa, pharynx, and face, spreading to the trunk and limbs. The typical rash is centrifugal and is most prominently visible on the face, limbs, palms, and soles. Smallpox lesions appear over one to two days. Smallpox is infectious during the initial week of the rash. Patients become non-infectious once the scabs separate. 

In addition to the ordinary type, three other distinct forms exist. Hemorrhagic type is associated with skin petechiae and mucosal or conjunctival bleeding. The mortality rates associated with hemorrhagic type are high. The flat-type is associated with toxemia and slow onset of skin lesions. This type also has a high mortality rate. The modified type is seen in previously vaccinated patients where the skin lesions evolve rapidly and are variable. This type has a low mortality rate. 

Investigations and Management[37]

Smallpox may be clinically diagnosed, but lab confirmation is important during the initial stages of an outbreak. Pustular fluid or scabs from suspected patients should be collected by recently vaccinated personnel using personal protection equipment. The specimen must be collected in an evacuated tube, sealed with tape, and transported in a second water-tight container. The relevant health department labs must immediately be notified. Specimen examination must be performed only in a BSL-4 facility. Identification of the species-specific DNA sequences is the preferred investigation of choice. Electron microscopy, immunohistochemistry, PCR, and serology are also useful. The definitive diagnosis and species identification are based on viral culture and subsequent characterization by PCR.

Before 1972, routine vaccination against smallpox was common in the United States and Europe. This was stopped following the completed eradication of the disease. Protective immunity from vaccination has never been evaluated satisfactorily. Hence, the present population globally is considered immunologically naive and unprotected against smallpox if the disease is reintroduced. The CDC and the WHO maintain a small stockpile of conventional vaccines. There are recent efforts to produce a newer vaccine based on live cell culture. Since only very few doses of the vaccine exist, an Advisory Committee on Immunization Practices in 2001 recommended that preventive vaccination be started in first-line responders, including emergency and other first-line health care and law enforcement personnel, with a booster dose every 10 years. However, at present, widespread vaccination is administered only in case of an epidemic that can potentially occur due to a lab error or deliberate acts of bioterrorism. 

No antiviral drugs have been found to be effective against human smallpox infections. Hence, surveillance and containment, which was the strategy used in smallpox eradication in the past, are the strategies currently in place in the event of an outbreak. Contact tracing involving the identification and surveillance of the patients’ contacts are the cornerstones of these techniques. Administration of vaccines within four days of exposure is considered to be effective in preventing smallpox. Vaccinated contacts do not transmit the disease and do not require isolation. Environmental decontamination after a bioterrorist attack using aerosolized smallpox is paramount to control the spread. 

Importance in Bioterrorism[37]

Even though smallpox was eradicated completely in 1980, it is still a potentially dangerous bioweapon. The CDC categorizes it as a category A organism due to its ability to easily transmit from human to human. Smallpox has a high mortality rate with the potential to cause panic and subsequent social disruption in the event of a bioterrorist attack. The release of aerosolized smallpox is a looming threat, with numerous models developed for emergency response. 

Viral Hemorrhagic Fevers[38]

Viral hemorrhagic fevers include a group of severe illnesses characterized by bleeding manifestations of varying degrees, caused by four virus families, including Arenaviridae, Bunyaviridae, Filoviridae, and Flaviviridae. 

• The Arenaviridae family includes:
  • Chapare virus (CHPV), which causes Chapare hemorrhagic fever
  • Guanarito virus (GTOV), which causes Venezuelan hemorrhagic fever
  • Junin virus (JUNV) causes Argentine hemorrhagic fever 
  • Lassa virus (LASV), which causes Lassa fever 
  • Lujo virus (LUJV), which causes Lujo hemorrhagic fever
  • Lymphocytic choriomeningitis virus (LCMV) which causes Lymphocytic choriomeningitis 
  • Machupo virus (MACV) which causes Bolivian hemorrhagic fever
  • Sabia virus (SABV) causes Brazilian hemorrhagic fever 

• The Bunyaviridae family includes: 
  • Crimean-Congo hemorrhagic virus (CCHFV), which causes Crimean-Congo hemorrhagic fever 
  • Dobrava-Belgrade virus (DOBV), which causes hemorrhagic fever with renal syndrome 
  • Hantaan virus (HTNV), which causes hemorrhagic fever with renal syndrome 
  • Puumalavirus (PUUV), which causes hemorrhagic fever with renal syndrome 
  • Rift Valley fever virus (RVFV), which causes Rift Valley fever
  • Saaremaa virus (SAAV), which causes Hemorrhagic fever with renal syndrome 
  • Seoul virus (SEOV), which causes hemorrhagic fever with renal syndrome 
  • Sin Nombre virus (SNV), which causes Hantavirus pulmonary syndrome 
  • Severe fever and thrombocytopenia syndrome virus (SFTSV), which causes severe fever, and thrombocytopenia syndrome 
  • Tula virus (TULV), which causes hemorrhagic fever with renal syndrome 

• The Filoviridae family includes:
  • Bundibugyo ebolavirus (BDBV), which causes Ebola virus disease
  • Marburg marburgvirus (MARV), which causes Marburg hemorrhagic fever 
  • Sudan ebolavirus (SUDV), which causes Ebola virus disease 
  • Tai Forest ebolavirus (TAFV), which causes Ebola virus disease 
  • Zaire ebolavirus (EBOV), which causes Ebola virus disease 

• The Flaviviridae family includes:
  • Dengue virus (DENV-1-4), which causes Dengue fever 
  • Kyasanur forest disease virus (KFDV), which causes Kyasanur forest disease 
  • Omsk hemorrhagic fever virus (OHFV), which causes Omsk hemorrhagic fever 
  • Yellow fever virus (YFV), which causes Yellow fever 

Arenaviridae is associated with rodent vectors and is sub-divided into two groups: the New World and Old World groups. Infection can occur via direct contact with rodent droppings or urine or via aerosol transmission. There can be human-to-human as well as nosocomial infections with high mortality rates. For instance, the Lassa virus has a case fatality rate as high as 50%. 

Bunyaviruses are associated with arthropods and rodents. These viruses can present with mild to moderate illness or severe illness with high mortality. For instance, in Crimean-Congo hemorrhagic fever, human-to-human transmission can occur through exposure to blood and bodily fluids with subsequent high mortality.  

Filoviruses can cause Ebola and Marburg hemorrhagic fever and are associated with African bats. Human to human transmission is reported with extremely high case fatality rates. Ebola outbreaks tend to have case fatality rates of more than 80% to 90%. Case fatality rates in Marburg hemorrhagic fever have been reported to be around 82% in a previous outbreak. 

Flaviviruses are transmitted by arthropods and include the Dengue virus, which is transmitted by the Aedes aegypti mosquito. Dengue fever has a relatively low mortality rate of around 0.8% to 2.5%, but this can increase in more severe forms of the disease, namely dengue hemorrhagic fever. 

Symptoms and Signs[38]

Patients with viral hemorrhagic fevers can present with very non-specific symptoms, including fever, headache, and malaise. Common clinical features across the different viral hemorrhagic fevers include arthralgia, retro-orbital pain, eye redness, vomiting, abdominal pain, and diarrhea. There may also be hemorrhagic manifestations, including bleeding gums, epistaxis, petechiae, and other major bleeding episodes. 

Investigations and Management

The lab tests in viral hemorrhagic fevers should include complete and differential blood counts, blood type and cross, coagulation studies, liver function tests, kidney function tests, chest x-ray, urinalysis, urine culture, and blood cultures to rule out other more common differentials.[38] Leukopenia, thrombocytopenia, and transaminitis are commonly encountered.[39] Serological testing for the specific IgM and IgG is useful, but molecular-based testing, including PCR, is the most sensitive test. Virus isolation by cell culture may also be used in diagnostic testing.[38] 

Management of viral hemorrhagic fevers relies on early diagnosis to increase the chances of survival and prevent secondary bacterial infections. For a large number of viral hemorrhagic fevers, patients should be isolated with the treating staff using personal protective equipment except in certain viruses such as Dengue. The cornerstone of currently available treatment options is supportive care. A few examples of specific infections include: the Lassa virus and ribavirin improve treatment outcomes when administered early in the course of the infection. In Crimean-Congo hemorrhagic fever, treatment is supportive care. In Ebola virus disease and Marburg hemorrhagic fever, the treatment again involves supportive care. However, there is currently an approved Ebola vaccine against Ebola Zaire. There are no effective antiviral agents available in dengue fever, and management is based on supportive care. A vaccine is currently available in South America and South-East Asia.[38]

Importance in Bioterrorism[40]

Viral hemorrhagic fevers are considered category A bioweapons by the CDC. These viruses are potential candidates as biological weapons as they are stable when aerosolized, can cause severe disease, and are difficult to treat. The Soviet Union and other countries have performed research regarding their use in warfare in the past.

Viral Encephalitis

A number of viruses that can cause encephalitis are considered potential bioweapons. These include tick-borne encephalitis virus (TBEV), Japanese encephalitis, West Nile virus, and Nipah virus. 

The tick-borne encephalitis virus (TBEV) is a spherical and lipid-enveloped RNA virus that belongs to the genus of Flavivirus in the Flaviviridae family. Humans usually contract the disease by the bite of an infected tick. Following the bite, the virus replicates locally. Dendritic skin cells serve as the site for viral replication followed by transport to regional lymph nodes. The virus disseminates into the spleen, liver, and bone marrow from the nodes, where it continues to replicate. Following this, TBEV infects the central nervous system and produces typical clinical manifestations.[41]

Japanese encephalitis is the most common naturally occurring cause of viral encephalitis, and it is caused by a flavivirus and is transmitted by Culex mosquitos. 

The West Nile virus is a single-stranded, enveloped RNA virus that belongs to the Flaviviridae family. In natural infections, Culex mosquitos are the most common vector.[42]

Nipah virus is an RNA virus that belongs to the Paramyxoviridae family and Henipavirus genus. Nipah virus is a BSL 4 category pathogen and is featured on the WHO's priority list of organisms that are likely to cause outbreaks.[43]

Symptoms and Signs

In TBEV, between 70% to 98% of infections are asymptomatic. The incubation period of TBEV infections ranges from two to 28 days. The illness may take on a monophasic or biphasic clinical picture. Patients with monophasic courses usually have central nervous system involvement in the form of meningitis or meningoencephalitis. A small number of patients present with a febrile illness and headache but do not develop meningitis which is termed the abortive form. The first phase correlates with the viremia and manifests as fever, headache, myalgia, arthralgia, malaise, anorexia, nausea, etc., which lasts for two to seven days, followed by an asymptomatic interval for about one week. The second phase presents as meningitis in approximately 50% of patients, meningoencephalitis in about 40%, and meningoencephalomyelitis in about 10% of cases.[41]

In Japanese encephalitis, the incubation period ranges between four to 15 days. A prodrome of non-specific symptoms, including fever, headache, vomiting, diarrhea, and myalgia, is common. Encephalitis develops following this, presenting as altered mental status, confusion, and overt psychosis. Meningism and seizures may develop. Rarely mutism and flaccid paralysis can occur. In the later course of the illness, patients can develop dystonia and choreo-athetoid movements.[44]

In West Nile virus infections, the incubation period varies from four days to two weeks. Symptoms include fever, myalgia, malaise, headache, vomiting, anorexia, and a maculopapular rash on the trunk. In some cases, there may be encephalitis or meningitis and other neurologic presentations, including seizures, muscle weakness, altered mental status, or flaccid paralysis. West Nile infection can also cause myelitis, resulting in a polio-like presentation. In West Nile infections involving the nervous system, mortality is high.[42][45]

Nipah virus has an incubation period ranging from four to 21 days. It can cause distinct syndromes in the form of acute encephalitis or respiratory illness, or a combination of both, depending on the strain. The mortality rate, too, varies with the strain but is generally high. Some patients may remain asymptomatic or may have a sub-clinical course. The symptomatic disease begins with prodromal symptoms, including fever, headache, and myalgia. Encephalitis can develop within a week, presenting with altered mental status, hypotonia, areflexia, myoclonus, gaze palsy, weakness, and a myriad of other neurological symptoms and signs. Rapid deterioration into a coma and subsequent mortality are reported in some outbreaks. In about 20% of survivors, the patients may have residual neurological deficits. Nipah virus infections also may relapse or present with late-onset encephalitis. The respiratory presentation can present with cough, breathing difficulties, and atypical pneumonia.[43] 

Investigations and Management

A case of TBEV infection is diagnosed by the following criteria: symptoms or signs of meningitis or meningoencephalitis, an elevated CSF cell count, and microbiologic evidence by the identification of specific IgM and IgG. Other than the specific criteria, ESR and CRP may be normal or elevated. Detection of viral RNA by RT-PCR in blood or CSF is limited to the initial phase of the illness, following which it may become negative. TBEV serum IgM antibodies can remain detectable for many months following acute infection, and TBEV IgG antibodies persist lifelong and prevent symptomatic reinfection. No specific antiviral treatment exists for the treatment of TBEV infections. Supportive care remains the mainstay of treatment. Dexamethasone is found to be useful in the reduction of cerebral edema in acute encephalitis. Two vaccines are approved for use in Europe.[41]

In Japanese encephalitis, workup may show leukocytosis or hyponatremia. MRI or CT can show bilateral thalamic lesions or hemorrhage. ACSF study may show significant opening pressure elevation, increased protein, and normal glucose. Japanese encephalitis virus-specific serum or CSF IgM using ELISA is the most useful test. There are no effective antiviral agents licensed for Japanese encephalitis. The cornerstone of management is supportive care. Anti-convulsants are useful for seizure control. In around 30% to 50% of survivors, there may be residual neurological deficits and psychiatric symptoms post-recovery of the acute illness. An effective vaccine in a short-course regimen is currently available against Japanese encephalitis. The CDC recommends the vaccine in people traveling to endemic areas for a long period and travelers to a place with a known outbreak.[44]

In West Nile virus infections, labs can reveal non-specific leukocytosis and raised inflammatory markers. Hyponatremia is common if the nervous system is involved. The definitive diagnosis depends on detecting West Nile virus serology using ELISA for the IgM in serum or CSF samples. A CSF study is usually typical of a viral meningitis picture with elevated protein, lymphocytes, and normal glucose levels. CT brain may not show any features in acute disease, but MRI may be useful to detect CNS involvement after several weeks. Treatment of West Nile virus infection is supportive care. Mild cases may be managed symptomatically with an excellent prognosis. Cases with CNS involvement will usually require rehabilitation with physical and occupational therapy. Some patients have persistent neurological defects, including cognitive, gross, and fine motor abnormalities, even after recovery from the infection.[42]

In Nipah virus infections, throat swabs, blood, urine, and CSF for PCR are the mainstay of diagnosis in a patient suspected of having the disease. These must be done only in a BSL 4 laboratory. Virus isolation using a Vero cell line followed by definitive identification of the virus using PCR is also useful for diagnosis. Still, it may be of limited utility in an outbreak scenario. Serum or CSF IgM by ELISA is also used for diagnosis but may be useful only relatively late in the course of the disease. The serum neutralization test is considered the gold standard but is time-consuming. The management of Nipah virus infection depends on good supportive care. Ribavirin has been reported to decrease mortality, but reports are conflicting. Neutralizing human monoclonal antibodies is approved in an outbreak setting in India based on reports of efficacy.[43] The virus is highly infectious, with human-to-human transmission occurring through the respiratory route and body fluids, thereby necessitating isolation of the patients and strict contact tracing.[46]

Importance in Bioterrorism

Tickborne encephalitis viruses are category C biological weapons as per the CDC. They are emerging viruses that can potentially be engineered for mass dissemination and have the potential for high morbidity and mortality.[47] Japanese encephalitis and West Nile virus are potentially transmissible by aerosolization, which makes them a potential bioweapon.[45]

Due to its high mortality rates, the respiratory route of human to human transmission, and its potential to be weaponized as an aerosol, the Nipah virus is considered a Category C priority organism by the CDC. Prasad et al. developed a lethal model using the Bangladesh strain of the Nipah virus and infected African green monkeys through aerosol exposure and demonstrated a lethality suggesting that the Nipah virus has a high potential for weaponization.[48]

Fungal Agents

Coccidiodes immitis (coccidioidomycosis) and Histoplasma capsulatum (histoplasmosis)

Coccidioides are dimorphic fungi that can exist as mycelia or as spherules. It is endemic in certain states in the United States. In naturally occurring infections, the infectious particles of Coccidioides called arthroconidia are inhaled into the lung by the patient causing coccidioidomycosis or San Joaquin Valley fever.[49] 

Histoplasma capsulatum is a soil-dwelling dimorphic fungus present in pockets worldwide and is endemic in certain states of the United States.[50]

Symptoms and Signs

In coccidioidomycosis, around 60% of cases are asymptomatic. The incubation period can range between seven to 21 days. The symptoms include fever, cough, breathlessness, and chest pain. Headache, loss of weight, and a rash in the form of a faint maculopapular rash, erythema nodosum, or erythema multiforme may be seen. The combination of fever, erythema nodosum, and arthralgia is called desert rheumatism. Other than the common pulmonary presentation in the form of pneumonia, the patient may present with signs of pulmonary cavities, meningitis, abscesses, or disseminated infection involving multiple systems.[49]

In histoplasmosis, primary infections may be asymptomatic or may present with mild flu-like symptoms. The incubation period ranges between seven to 21 days. Symptoms in acute histoplasmosis include fever, headache, cough, and chest pain. The symptoms usually resolve in 10 days. Arthralgias, erythema nodosum, or erythema multiforme can develop in a small proportion of patients, but this is less common in histoplasmosis than coccidioidomycosis. Some patients may present as chronic pulmonary histoplasmosis in the form of cavitary or non-cavitary illness. In others, there may be disseminated histoplasmosis with uncontrolled growth and proliferation of the fungus in multiple organs, and the patient presents with fever, weight loss, hepatomegaly, and splenomegaly.[50]

Investigations and Management

In coccidioidomycosis, isolation of coccidioides provides definite evidence of infection, and this can be done through sputum examination in patients suspected to have lung involvement. However, patients may not produce sputum, and fungal cultures may not be feasible or of no growth, and serology may be used to diagnose the disease in these cases. Tube precipitin antibodies may be detected in about 90% of coccidioidomycosis patients in the initial few weeks following exposure. Complement-fixing antibodies can be detected in the body fluids, including in CSF, which helps diagnose coccidial meningitis. An enzyme immunoassay (EIA) based Coccidiosis IgM, and IgG test is available. PCR to detect Coccidioides DNA in clinical specimens are not commercially available but are reported to be 98% sensitive and 100% specific. The treatment of coccidioidomycosis uses fluconazole 400 mg to 1200 mg daily or itraconazole for three months. In fibro-cavitary disease, treatment may be extended to a year. Therapy with azoles is lifelong in case of meningitis. Surgical resection has a role in lung lesions and cavitary lesions amenable to surgery.[49]

In histoplasmosis, workup includes imaging which may reveal healed granulomas in the lungs, liver, or spleen. CBC may reveal bone marrow suppression in the form of a non-specific reduction in any of the cell lines. A bronchoscopic alveolar lavage may sometimes be positive, especially in the setting of cavitary lesions. Complement-fixing antibodies appear three to six weeks after infection in 95% of the patients and can persist for years. A single titer of 1:32 or a fourfold increase is diagnostic of an acute infection. Histoplasmin is the antigen extract of Histoplasma mycelial form. Antibodies to histoplasmin, including C, H, and M, may be detected. ELISA IgM and IgG are also useful in diagnosis. Detection of urinary antigen is useful in acute disease and disseminated histoplasmosis. The urinary antigen levels may be used for diagnosis as well as to assess the response to therapy. Regarding treatment, acute pulmonary infections lasting less than four weeks do not require treatment. In case symptoms persist, itraconazole for three months is the treatment of choice. In chronic disease and non-cavitary disease, therapy is extended to six months, while in cavitary disease, treatment may be required for a year. In disseminated disease, amphotericin-B induction therapy for two to four weeks followed by maintenance therapy with itraconazole for one year is the preferred regimen.[50]

Importance in Bioterrorism

Most of the fungi that are human pathogens tend to produce spores that are naturally designed for airborne spread. There have been reports of the ability of fungal spores that have spread across countries and continents through the airborne route. Specifically, wind storms across California have led to the outbreak of coccidioidomycosis in other non-endemic regions of the United States. The opening of Petri dishes in laboratories has resulted in the dispersal of C. immitis spores and subsequent infections. Lumbering has resulted in histoplasmosis amongst onlookers, which suggests that spore dispersal and subsequent infection can occur. Due to the aerosolization potential and the relatively low inoculum requirement, C. immitis has been included in a select agent list of over 80 organisms with bioweapon potential by the CDC. H. capsulatum exhibits similar properties and hence is an organism of interest in bioterrorism even though not included in this list.[51]

Protozoan Agents

Cryptosporidium parvum (Cryptosporidiosis)

Cryptosporidium belongs to the coccidia protozoan group. More than 15 species of Cryptosporidium are implicated in human infections, with Cryptosporidium hominis and Cryptosporidium parvum being the most commonly encountered organisms. Naturally occurring infections are transmitted through the consumption of contaminated water and the fecal-oral route.[52]

Symptoms and Signs

Cryptosporidiosis presents with profuse and watery diarrhea along with features of malabsorption. Symptoms may be cyclical, with alternating periods of worsening and improvement lasting one to two weeks. In a majority of cases, the symptoms resolve without treatment within seven to 14 days. Other than diarrhea, patients may have a fever, nausea, vomiting, and abdominal pain. Immunosuppressed patients may have chronic diarrhea that can last for months to years, or they may also develop complications with other system involvement.[52]

Investigations and Management

Diagnosis of Cryptosporidium infections relies on identifying Cryptosporidium parasite in stool samples using special stains, antigen detection assays, or PCR tests. Modified acid-fast stains may identify mature oocysts. However, PCR can distinguish the species. Serology is of limited use and may be of value in epidemiological studies. In immunocompetent patients, the treatment of choice is nitazoxanide. Paromomycin or azithromycin are alternatives. In immunocompromised patients, correction of immunodeficiency is paramount in treating the disease, along with addressing the infection.[52]

Importance in Bioterrorism

Cryptosporidium is a category B priority pathogen as per the CDC as it is a potential water safety threat.[37]

Toxins

Ricin

Ricin is a highly potent plant toxin derived from the castor bean plant Ricinus communis, grown worldwide for industrial production of castor oil. The plant itself has a storied past with several uses being detailed in various contexts spread throughout history, such as wound healing, as an emetic/purgative, as well as a potential treatment for a host of other medical conditions around the world. Ricin constitutes up to 5% of the press cake's protein content left behind after castor oil extraction. It is toxic by ingestion, inhalation, and injection. Ricin consists of 2 subunits; a catalytic ricin toxin A chain (RTA) and a galactose binding B chain (RTB).

Ricin enters the cell by a multi-step pathway which includes receptor-mediated endocytosis. It is then transported by vesicular transport to the Golgi apparatus and subsequently to the endoplasmic reticulum by a chaperone protein named calreticulin. Ricin is a type two ribosome-inactivating protein. RTA chain inactivates the ribosome by hydrolyzing the N-glycosidic bond of an adenosine residue in the 28 S ribosomal RNA of eukaryotic cells. Ribosome binding is essential for ribosome depurination, inhibition of translation, and subsequent toxicity of RTA in mammalian cells.[53][54][55][56]

Symptoms and Signs

Ricin toxicity can occur through ingestion, inhalation, or injection. Most cases of ricin toxicity occur following voluntary or accidental ingestion of castor seeds. The lethal oral dose is estimated to be around one to 20 milligrams per kilogram. About five to six seeds of the castor bean plant are considered lethal in children, while in adults, it about 20 seeds. The clinical presentation usually depends on the route of inhalation. Symptoms usually set in within 12 hours of ingestion and initially may include nausea, vomiting, diarrhea, and abdominal pain. Massive gastrointestinal fluid and electrolyte loss have been described and are complicated by hematemesis and melena and progressing to hepatic failure, renal dysfunction, and death due to multiorgan failure or cardiovascular collapse. Inhalation of aerosol particles between the size of 1 to 5-micrometer diameter can penetrate deep in the lung and cause toxicity. Post inhalation symptoms usually begin immediately or within the first 8 hours and may present as cough, dyspnoea, fever, pneumonia, and pulmonary edema leading to respiratory failure and death. Post injection symptoms may include erythema, induration, the formation of blisters, capillary leak, as well as localized necrosis.[57][58][59][60]

Investigations and Management

Initial investigations may reveal elevated liver transaminases, amylase, creatinine kinase, electrolyte abnormalities, myoglobinuria, and altered renal function tests. Laboratory detection of the ricin protein depends on immunological assays, liquid chromatography-mass spectrometry, or functional activity assays. Field-based diagnostic tests, including lateral flow assays, provide an effective immunoassay technique for detection.[59][60]

At present, no antidote or specific therapy exists for ricin poisoning or prevention of the illness after exposure. Hence the treatment is largely supportive and involves airway management and positive pressure ventilation in inhalation toxicity cases. Activated charcoal can be considered in patients who present early without emetic symptoms once the airway is secured. Coagulopathy and dyselectrolytemia should be corrected. Other laboratory parameters such as liver function tests and renal function tests should also be closely monitored.[60][61][62]

Importance in Bioterrorism

Ricin is a category B biological weapon as per the CDC. Various studies have been carried out testing the use of ricin as an agent of bioterrorism. In 1978, the Bulgarian dissident Georgi Markov was killed with ricin placed in the tip of an umbrella, which was poked into the back of his leg. In 2003, a package that contained ricin and a letter that threatened the deliberate contamination of water supplies was found in a post office in South Carolina, United States. This became the first potential chemical terrorism event involving ricin in the United States. It is important to develop a multi-disciplinary approach using effective countermeasures with the help of trained healthcare workers and first responders to enable rapid epidemiological and laboratory investigation, disease surveillance, and efficient medical management.[60][62][63]

Abrin

Abrin is a toxic toxalbumin isolated from Abrus precatorius. It is also known as rosary pea or jequirity bean. It can be weaponized for purposes of biowarfare by aerosolization as dry powder/liquid droplets or by adding to food and water as a contaminant. Abrin is a heterodimer consisting of 2 polypeptide chains A and B, linked by a disulfide bond. The mechanism of action is similar to ricin. It enters the cell by endocytosis and inhibits protein synthesis. The A chain acts as an RNA N -glycosidase and cleaves the C-N bond in adenine. The resultant adenine depurination prevents ribosomal binding to elongation factors and thereby prevents protein synthesis.[62][64]

Symptoms and Signs

Abrin is significantly more toxic than ricin. The estimated fatal dose is 0.1 to 1 microgram per kilogram body weight. Poisoning occurs following ingestion of seeds or contamination of food or water. It presents as abdominal pain, vomiting, diarrhea, hemorrhagic gastritis, and renal failure. Following inhalation, pulmonary edema can occur. Cerebral edema, convulsions, and CNS depression can also occur. Death can occur within 72 hours of exposure.

Investigations and Management

In cases of suspected exposure, monitor laboratory parameters such as CBC, liver transaminases, renal function tests, and electrolytes. Treatment is supportive in the form of fluid resuscitation, ventilatory support, and decontamination measures. No vaccine or antidote has been identified to date.[62][64]

Importance in Bioterrorism

Abrin is among the strongest plant toxins known and hence can be exploited for use in bioterrorism. Owing to its stability, toxicity, and ease of purification, it has been classified as a category B agent, with potential for use in bioterrorism by the CDC. No cases have been reported so far, but it is important to be vigilant to the possibility of future use.[62][64]

Botulinum Neurotoxins

Botulinum neurotoxin (BoNT) is among the most toxic substances known to man in its purified form. Its extremely low lethal dose poses a grave biological threat, with high morbidity and mortality. Botulinum neurotoxins are produced by the Gram-positive, spore-forming, anaerobic bacteria of the genus clostridium. The toxin is a double chain protein with a molecular weight of 150 kDa and exists in 7 different serotypes (A - G) with over 40 subtypes. In the past few years, with the help of advanced DNA sequencing techniques, newer serotypes and subtypes have been discovered. In 2013, a new toxin (BoNT/H), produced by clostridium botulinum, was isolated from a case of human infant botulism with lower potency and slower progression of symptoms compared to the other BoNTs.

Several other BoNT like toxins produced by non-clostridial bacterial species such as Enterococcus faecium and Chryseobacterium piperi have also been discovered. BoNTs are zinc endopeptidases and can be produced by several genus clostridium members, most commonly C. botulinum. The active form is made up of a C terminal heavy chain, which has the binding and translocation domains and a catalytic light chain forming the N terminal. A single disulfide bond connects the two chains. The light chain has proteolytic activity and is considered the active part. BoNTs bind selectively and irreversibly to the nerve terminals and cleave SNARE (“Soluble NSF Attachment Protein Receptor”) proteins such as VAMP/Synaptobrevin, Syntaxin, and SNAP-25. The SNARE proteins are responsible for the synaptic vesicle's fusion containing the neurotransmitter acutely choline with the presynaptic plasma membrane. This effectively blocks the release of acetylcholine, leading to flaccid paralysis.[62][65]

Symptoms and Signs

In human beings, botulism is usually caused by serotypes A, B, E, and F. There are six recognized forms of botulism: foodborne, infant, intestinal, wound, iatrogenic, and inhalational, characterized by different routes of exposure and incubations periods. The lethal dose for an adult is 0.7 to 0.9 micrograms of inhaled toxin to 70 micrograms of ingested toxin. Food-borne botulism is usually caused by ingestion of home-preserved food and presents typically within the first 4 hours. Infant botulism occurs by the ingestion of spores in infants one to six months in age and has an incubation period ranging from five to 30 days. Intestinal botulism is caused by the ingestion of spores by children over the age of years and adults and has a variable incubation period. Wound botulism is caused by the germination of spores in a wound and has an incubation period of 1 to 2 weeks.

Iatrogenic botulism is caused by the injection of commercial or non-approved preparations of BoNTs. Inhalation of BoNTs leading to botulism can occur. However, it has not been reported in humans. The clinical presentation is usually not dependent on the route of exposure. It usually presents as a descending, symmetric paralysis. Initially, there is diplopia, dysphagia, dysarthria, followed by dyspnoea due to intercostal respiratory muscle and diaphragm paralysis. Death usually occurs due to respiratory failure.[62][65]

Investigations and Management

The diagnosis is difficult and depends heavily on the clinical presentation. Laboratory detection of BoNTs from clinical specimens such as vomitus, gastric aspirate, nasal swabs, or stool samples via mouse bioassay is considered the gold standard. ELISA may be performed on samples such as bronchoalveolar lavage in select cases. Treatment is supportive care in the form of assisted ventilation, hydration, and prevention of secondary infections. Administration of the anti-toxin, preferably within the first 24 hours and not later than 72 hours, to neutralize the circulating BoNTs is a critical step in the management. In the EU, a trivalent (A, B, E; equine) antitoxin is used. In the United States in 2013, a human-derived BoNT antitoxin (BIG-IV) was approved by the FDA to treat infant botulism.[62][65]

Importance in Bioterrorism

Botulinum neurotoxin is the deadliest toxin known to man. It is categorized as a category A biological agent that can be used for bioterrorism. BoNTs are lethal at low doses (LD 50 is lower than any other known substance) and can be deployed through multiple routes, including aerosols or contaminated food and water. It is also colorless and odorless, making it ideal for silent attacks. However, production, purification, storage, and transportation can prove to be challenging. Vigilance and continued research into BoNTS and BoNT like toxins are necessary to understand and prevent public health catastrophe due to the possible use of such agents for bioterrorism activities in the future.[62][65]

Clostridium Perfringens Toxins

Clostridium perfringens is a Gram-positive, anaerobic, spore-forming rod known to secrete more than 20 virulent toxins associated with disease in both humans and animals. Six toxins, namely the alpha (CPA), beta (CPB), enterotoxin (CPE), necrotic enteritis B like toxin (NetB), epsilon (ETX), and iota (ITX), have the potential for toxicity. The epsilon and iota toxins have been categorized as category B agents that can be used in biowarfare by the CDC. The epsilon toxin is the most potent of all the toxins produced by C. perfringens. It is the third most potent toxin behind C. botulinum and C. tetani neurotoxins. ETX shows relative resistance to the proteases found in the gastrointestinal tract of mammals. It causes pore formation in the cell membranes, resulting in degenerative and necrotic changes culminating in organ failure. ETX can also bind to myelin fibers in the CNS after crossing the blood-brain barrier resulting in central nervous system demyelination. The iota toxin causes cytoskeleton damage via enzymatic action on ADP ribosylation leading to cell death. Another important toxin is enterotoxin (CPE), which disrupts the tight junctions in the gastrointestinal epithelial cells, causing food poisoning and non-foodborne gastrointestinal diarrhea.[62][66]

Symptoms and Signs

A necrotic inflammatory bowel disease called Dambrand or enteritis necroticans was reported in West Germany after the second world war, facilitated by poor post-war sanitary conditions and malnutrition. The disease seemed to disappear a few years after it was first described. In 1966, a new form of enteritis necroticans, also known as Pigbel, an inflammatory gut disease described as spontaneous gangrene of the small intestine, was reported in Papua New Guinea. Presently C. perfringens is associated with acute watery diarrhea contributing to a significant number of food poisoning cases around the world. It presents as intestinal cramps, watery diarrhea without fever or vomiting about eight to 14 hours after consuming contaminated food. It is self-limiting with low mortality and usually settles within 24 hours. C. perfringens is also associated with non-foodborne diarrhea, such as antibiotic-associated and sporadic diarrhea. C. perfringens has also been associated with pre-term infant necrotizing enterocolitis. Perfringolysin O is a pore-forming toxin that has been implicated in gas gangrene by its synergistic effects with CPA.[62][66][67]

Investigations and Management

In suspected cases of C. perfringens, stool culture and ELISA testing for toxin can be considered. In cases of clostridial myonecrosis, laboratory evaluation with routine blood tests including blood culture, CK level, ABG, and lactic acid, as well as imaging of the affected area with an x-ray or CT scan. Treatment is generally supportive with fluid resuscitation. In clostridial sepsis, the patients may present with shock due to intravascular hemolysis, which occurs secondary to toxin-mediated destruction of red cells. Antibiotic therapy is with penicillin G, clindamycin, tetracycline, or metronidazole. Surgical debridement of necrotic tissue reduces mortality. In severe cases of clostridial myonecrosis, hyperbaric oxygen is shown to improve outcomes.[62][66][68]

Importance in Bioterrorism

The epsilon and iota toxins of C. perfringens have been categorized as category B agents that can be used in biowarfare by the CDC. The toxin can be used in an aerosolized form that can be used as a bioterrorist weapon or dispersed in food intended for human consumption. A multidisciplinary approach is necessary to control outbreaks and ensure that the necessary public health departments are aware to minimize spread.[62][66]

Tetrodotoxin

Tetrodotoxin (TTX) is a lethal neurotoxin found in marine animals. It is about 1200 times more toxic than cyanide and has no known antidote. It acts by inhibiting the transport of sodium ions through voltage-gated sodium channels found in the muscles and nerves, halting the propagation of action potentials and leading to paralysis. TTX was first isolated from pufferfish of the family Tetraodontidae. There is, to this date, no clarity about the origin, biosynthesis, or function of TTX. Pufferfish with other marine animals are believed to harbor bacteria, which produces TTX. The incidence of poisoning is rare and is reported mainly from countries such as Japan, Bangladesh, and Taiwan, where pufferfish is regularly consumed.[69][70][71]

Signs and Symptoms

The severity of symptoms is dose-dependent. In most cases, symptoms will start to develop between 30 minutes to 6 hours following ingestion. Symptoms may include headache, perioral numbness, loss of coordination, nausea, vomiting, abdominal pain, and in severe cases, hypotension, cardiac arrhythmia, respiratory failure, and death.[69][72][73]

Investigations and Management

Diagnosis is based on identifying the clinical presentation. No specific laboratory tests exist to confirm tetrodotoxin poisoning. Treatment is supportive. Provide ventilatory support and watch out for cardiac arrhythmia. If the patient presents early gastric lavage or activated charcoal can be considered. There is no antidote to date.[69]

Importance in Bioterrorism

The lethal dose of TTX for a human is about 0.5 to two mg when ingested. Due to its potency, it can be weaponized for biological warfare. Hence, it is important that adequate knowledge be imparted to healthcare professionals about the potential consequences of the same to prevent widespread mortality.[74]

Nerve Agents

Nerve agents are a subcategory of organophosphorus compounds. They are among the most toxic substances known. They are used extensively as insecticides and have contributed immensely to modern agricultural practices. The high toxicity profile, ease of synthesis, and widespread availability of these agents have lead to their use in chemical warfare over the years. The nerve agents are classified into 4 types; 1. the G-series, which include tabun (GA), sarin (GB), soman (GD), and cyclosarin (GF) 2) V-series, where V stands for venomous, which include VE, VG, VM, and VX, Chinese VX and Russian VX, 3) GV-series which have combined properties of both series G and V, for example, GV, 2-dimethylaminoethyl-(dimethylamido)-fluorophosphate and 4) Novichok series of compounds, such as Novichok-5, and Novichok-7. The organophosphorus compounds ( OPCs) cross the dermis's epithelial membrane and the respiratory tract with ease. They then undergo biotransformation into their active form. They bind to the acetylcholine esterase and form a complex, which inhibits the hydrolysis of acetylcholine, causing it to accumulate and leads to a cholinergic crisis. The removal of the functional group in nerve agents makes it more deadly, as the bond between the agent and the enzyme becomes permanent. The acetylcholine esterase is thus irreversibly inactivated, which is known as the aging of the enzyme.[75][76]

Symptoms and Signs

The clinical presentation is usually of cholinergic excess. The lungs and eyes absorb nerve agents the fastest. The presentation may include mitosis, bronchorrhoea, chest tightness, loss of bladder control, salivation, vomiting, sweating, abdominal pain, and cramps. In severe cases, they may present with stupor, convulsions, and respiratory compromise. The intermediate syndrome may occur before 24 hours or after 96 hours of exposure and may present as pneumonia, aspiration pneumonitis, and respiratory failure.[75][76][77]

Investigations and Management

Decontamination after donning appropriate personal protective equipment (PPE) is the most important step. Liquid agents may be absorbed through the skin and pose a threat to those around the suspected case, while inhaled nerve agents are systemically absorbed and metabolized. The first step is to disrobe the patient, rinse with soapy water, remove all jewelry and personal items and shift to the ICU if ventilatory support is required. Treatment includes 3 types of therapies. The first is to administer antimuscarinic agent atropine. The UK military and North Atlantic Treaty Organization (NATO) protocol recommends an initial dose of 5–10 mg intravenous (IV)/intraosseous (IO) atropine for severely poisoned patients, titrated to effect every 5 min until atropinization (reversal of the ‘3Bs’—bradycardia, bronchospasm, bronchorrhea) takes place. If given early, oxides can re-activate the acetylcholine esterase. The most commonly used is Pralidoxime, given as 2g intravenous or intraosseous slow infusion. The third step is appropriate supportive therapy, including ventilatory support and anticonvulsants.[76][77]

Importance in Bioterrorism

Nerve agents are amongst the most lethal agents used in chemical warfare. The first known nerve agent, Tabun, was developed by the German chemist Gerhard Schrader in the 1930s during his research into the development of new OP insecticides. Following this, many other nerve agents such as Sarin and Soman were developed for military use. In February 2017, Kim Jong Nam, the half-brother of Korean dictator, Kim Jong-un, was assassinated inside the Kuala Lumpur airport. An autopsy identified the nerve agent ethyl N-2-diisopropylaminoethyl methylphosphonothiolate (VX). In 1994, nerve agent sarin was released in the Tokyo subway system, resulting in the poisoning of 640 people. On 4 March 2018, Sergei Skripal, a former Russian military intelligence officer, and his daughter, Yulia Skripal, collapsed on a park bench in Salisbury, the United Kingdom, after eating dinner at a local restaurant. It was later confirmed that Novichok was present in biological sampling from the Skripals as well as from the site of suspected exposure. Chemical warfare with nerve agents poses a considerable threat to the health of the civilian population, military personnel, and peacekeeping forces. Healthcare providers should recognize symptoms of exposure, understand regional and international notification procedures for potential attacks, as well as the indications for and available supply of antidotal therapy.[78][79][80]

Clinical Significance

The ever-looming threat of a terrorist attack, in particular, a bioterrorist event, necessitates that healthcare professionals must prepare to manage victims of these catastrophic events. A broad understanding of potential biological weapons, including identifying the offending agent and the steps in the management of the patients, is paramount in limiting the morbidity and mortality associated with a bioterrorist attack. The healthcare professional's initial role in the event of a bioterrorist attack is to identify that an attack has taken place. Bioweapons, especially infectious agents, may simulate naturally occurring infections.

The healthcare professional should be able to differentiate between these by a thorough epidemiological analysis of the outbreak. Once this is identified, appropriate disease-specific interventions can be started to reduce the morbidity and mortality associated with the attack. The choice of the bioweapon may depend on the technical and economic capabilities possessed by the terrorist organization. Healthcare professionals should be familiar with both the epidemiology and the control measures in such an event to respond if an outbreak should occur. Research targeted at improving the diagnostic and therapeutic capabilities as well as the implementation of effective response plans is another step against bioterrorism. Training healthcare workers and other frontline workers in identifying and managing a bioterrorist attack with regular refresher sessions may also be useful.

Panic and fear among the public can be another aspect of a bioterrorist attack. Healthcare workers also have a role in minimizing panic by explaining the situation to the victims and starting appropriate and timely management to minimize the agent's impact. With prompt identification of the causative biological weapon along with rapid initiation of treatment and containment measures, healthcare professionals can play a direct role in preventing mass casualties and limiting large-scale damage to human life.

Enhancing Healthcare Team Outcomes

The management of a patient who is a victim of an act of bioterrorism is challenging and complex. This requires an interprofessional team consisting of healthcare professionals that include physicians in different specialties, nurses, laboratory technologists, pharmacists, and possibly governmental agencies. Without rapid identification of the causative agent followed by proper management, morbidity and mortality may be high. It may also lead to an outbreak if an agent with the human-to-human transmission is used. The internist, infectious diseases physician, may rapidly identify the offending pathogen or toxin or medical toxicologist.

The patient will require emergency care and possible intensive care, which requires a team effort. Cross consultation involving the various other medical subspecialties may also be required in the case of the disease's systemic involvement. The need for preparedness by healthcare professionals and an interdisciplinary approach in managing the patient is highly recommended to lower the morbidity and improve outcomes. [Level 5]