Distinguishing Respiratory Features of Category A/B Potential Bioterrorism Agents from Community-Acquired Pneumonia

Abstract

Differentiating between illness caused by community-acquired respiratory pathogens versus infection by biothreat agents is a challenge. This review highlights respiratory and clinical features of category A and B potential biothreat agents that have respiratory features as their primary presenting signs and symptoms. Recent world events make such a reminder that the possibility of rare diseases and unlikely events can occur timely for clinicians, policymakers, and public health authorities. Despite some distinguishing features, nothing can replace good clinical acumen and a strong index of suspicion in the diagnosis of uncommon infectious diseases.

Differentiating between potential biothreat agents and community-acquired pneumonia (CAP) at the bedside can be challenging. Some diseases caused by potential biothreat agents have characteristic features in their latter stages but present as nonspecific febrile illnesses earlier. Awareness of potential “red flags” and a healthy index of suspicion, along with confirmatory diagnostics, may aid healthcare workers in making the correct diagnosis.

Twenty-sixty percent of patients with community-acquired pneumonia are hospitalized, with 10% to 20% requiring admission to the intensive care unit.¹ With US treatment guidelines for community-acquired pneumonia requiring early empirical treatment, there has been a decreased emphasis on determining the microbial cause; a specific pathogen was identified in only 38% of patients hospitalized with community-acquired pneumonia² and 50% of patients with severe community-acquired pneumonia.¹ In addition to influenza, other common causes of community-acquired pneumonia include Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, and rhinovirus. Rapid multiplex diagnostics may provide presumptive diagnoses, but clinicians should remain vigilant with broad differential diagnoses. A routine patient with presumed community-acquired pneumonia could be the index patient in a biothreat attack but might be missed without a larger cluster of patients.

Recent world events, including the use of chemical agents in Syria and an attack in the United Kingdom with a Novichok nerve agent, demonstrate the ongoing potential use of weapons of mass destruction on civilian populations. Countries that have developed chemical weapons often also included biological weapons in their arsenals. A 2017 report by Harvard's Belfer Center on North Korea's bioweapon activities provides a case in point.³ Using unclassified reports, defector testimonies, and interviews with subject matter experts, the report's authors argue that preparation for the threat is “urgent and necessary.” Moreover, North Korea is presumed to have assassinated Kim Jong-Un's half-brother with the nerve agent VX.

Therefore, we believe a reminder of potential biological weapons threats is timely. In 2002, the Centers for Disease Control and Prevention (CDC) assigned potential bioweapons threats to 3 categories, A, B, and C, with category A having the greatest potential to cause disruption and for which countermeasures were most needed. We focused our review on the agents in categories A and B that are known to cause respiratory manifestations as their primary means of presentation.⁴ Other category A and B agents that may have respiratory manifestations, but in which these symptoms generally occur as later disease complications rather than as their primary presenting features, are listed in a separate section with more succinct descriptions. Both sections list the agents in alphabetical order.

Agents that Present Primarily with Respiratory Manifestations

Anthrax

Anthrax is a zoonosis of herbivores worldwide, caused by Bacillus anthracis, a Gram-positive spore-forming bacillus. Humans are infected by inoculation, ingestion, or inhalation of spores from infected animals or animal products. The 2001 outbreak of inhalational and cutaneous anthrax arising from mailed letters containing B. anthracis spores highlighted the risk of B. anthracis as a terrorist weapon.

Pulmonary Manifestations

Inhalational anthrax occurs after spores are inhaled. As the spores germinate in the tracheobronchial and mediastinal lymph nodes, they secrete edema toxin and lethal toxin, which interfere with host innate immune response and impair lymphocyte, neutrophil, and macrophage function. Patients develop fever, chills, fatigue, malaise, nonproductive cough, and gastrointestinal symptoms (nausea, vomiting). As the organisms proliferate, they cause a hemorrhagic lymphadenitis of the subcarinal, hilar, and paratracheal lymph nodes, yielding the characteristic “widened mediastinum” on chest x-ray or chest computed tomography (CT).⁵ Although pulmonary infiltrates can occur, extensive consolidation is absent. Large pleural effusions may require thoracentesis. Prompt recognition and treatment is critical to prevent death, because organisms can cause bacteremia and systemic toxemia, in addition to hemorrhagic meningitis once the meninges are seeded.

A review of presenting symptoms from the 2001 outbreak⁶ showed that the majority of patients had dyspnea, drenching sweats, and pleuritic chest pain; half had myalgias and headache; only 20% (2/10) had pharyngitis; and only 1 had rhinorrhea. The absence of those latter symptoms might help to distinguish anthrax from respiratory viruses, especially during cold and influenza season. An analytic approach for distinguishing influenzalike illness from anthrax was published,⁷ in addition to an algorithm to discriminate anthrax from community-acquired pneumonia in cases and controls.⁸ Using the presence or absence of mediastinal widening, altered mental status, and an elevated hematocrit, the authors discriminated inhalational anthrax from community-acquired pneumonia with high sensitivity and specificity.

Without an approved rapid diagnostic for inhalational anthrax, diagnosis requires a high index of suspicion. The preferred treatment for inhalational anthrax is ciprofloxacin plus a protein synthesis inhibitor like clindamycin or linezolid, along with antitoxin therapy (raxibacumab, obiltoxaximab, or anthrax immunoglobulin).

Glanders

Glanders, an exceedingly rare disease in humans, is caused by Burkholderia mallei, a nonmotile, facultative intracellular Gram-negative bacillus. The last naturally acquired case of glanders in the United States was reported in 1934, so any contemporary case should prompt consideration of an intentional release, a laboratory accident,⁹ or close contact with an equine host in foreign enzootic locales. Given the significant mortality of the disease occurring in animal aerosol challenge studies, stability in aerosol, and infection by the respiratory route, it is regarded as a potential biothreat. Glanders occurs as an acute or chronic disease. The chronic form (“farcy” in humans) typically manifests as generalized lymphadenopathy, with multiple ulcerating, draining skin nodules and induration, thickening, and regional lymphatic nodularity.

Pulmonary Manifestations

Pulmonary involvement occurs after direct inhalation or via hematogenous spread. Infected individuals typically present with fever, rigors, myalgia, profound malaise, adenopathy, mucopurulent rhinitis with copious sputum, and pleuritic chest pain. Radiographic findings include segmental or lobar consolidation or nodular opacities.¹⁰ Glanders can cause pulmonary cavitation, similar to tuberculosis, or abscesses in other organs, such as the liver and spleen. Systemic dissemination and septicemic glanders can occur regardless of entry portal and can be rapidly progressive, with additional manifestations of jaundice, diarrhea, and granulomatous and necrotizing lesions in any organ, especially the liver, spleen, and lungs. Its high mortality (up to 40% treated and 90% in untreated) requires prompt diagnosis.¹⁰ Given that human cases are rare, there is a limited amount of data with respect to antibiotic treatment in humans. Sulfadiazine has been shown to be effective in animal studies and human cases. Proposed treatment of glanders consists of ceftazidime or meropenem for initial parenteral therapy, followed by oral therapy with trimethoprim-sulfamethoxazole or doxycycline.

Melioidosis

Melioidosis is caused by the motile, facultative intracellular Gram-negative bacillus Burkholderia pseudomallei; which is endemic to Southeast Asia and northern Australia. In northeastern Thailand, B. pseudomallei causes 20% of community-acquired septicemia cases.¹¹ Individuals are usually infected by inhaling aerosols from contaminated soil, water, or mud or through percutaneous inoculation. Individuals with diseases that impair neutrophil function, such as diabetics and alcoholics, are at risk of severe manifestations. The potential ease of access to the organism, environmental stability, potential for antimicrobial resistance, direct infection by the respiratory route, and the significant morbidity associated with pulmonary infection make meliodosis a significant threat as a bioweapon.

Pulmonary Manifestations

Pneumonia is the most common clinical manifestation, with over 50% of patients presenting with primary pneumonia and a substantial proportion developing secondary pneumonia.¹² The pneumonia generally occurs acutely and is accompanied by septicemia and high mortality. Upper and lower lobes are affected with similar frequency, and multi-lobar involvement occurs in 30% of cases.¹² Chronic melioidosis pneumonia can mimic tuberculosis, with a predilection for the upper lobes or reactivation from a latent focus.¹² Similar to glanders, melioidosis can produce cavities in the lungs or other major organs. Bloody sputum occurs commonly in melioidosis and may help distinguish it from influenza or community-acquired pneumonia. First-line treatment of melioidosis consists of ceftazidime or meropenem for initial parenteral therapy, followed by eradication therapy with trimethoprim-sulfamethoxazole or doxycycline.

Plague

Plague, caused by the Gram-negative organism Yersinia pestis, remains endemic around the world, including in the United States, where a handful of cases occur every year. A large epidemic of plague, including pneumonic cases, recently occurred in Madagascar, which provides a reminder of this disease's epidemic potential. Infection usually occurs via an infected flea bite, which typically leads to bubonic plague. Symptoms include acute onset of fever, malaise, chills, headache, nausea, vomiting, and abdominal pain. Within 24 hours, an extremely painful, swollen lymph node (a bubo) develops proximal to the flea bite. Lymph nodes involved include femoral, inguinal, axillary, and cervical, in descending order of frequency. Some patients present with a tender palpable liver and/or spleen or an acute abdomen.^13,14

Most individuals develop bacteremia, which can be self-limited or lead to septicemic plague and subsequent spread to the meninges (causing plague meningitis) or the lungs (causing pneumonic plague).¹⁵

Pulmonary Manifestations

Pneumonic plague can occur “secondary” to bacteremia or primarily from person-to-person spread via respiratory droplets or by intentionally released aerosol. Patients may develop chest pain, cough with tachypnea, dyspnea, and hypoxia. Initial purulent sputum may become blood-tinged or grossly hemorrhagic;¹⁴ the presence of hemoptysis helps differentiate plague from other more common causes of pneumonia.

Chest x-ray findings in plague are variable, with patchy consolidated bronchopneumonia, frequently bilateral, and cavities or confluent consolidation and may appear more concerning than the exam might indicate initially.⁵ Gentamicin or streptomycin is used for the treatment of pneumonic plague. Doxycycline or ciprofloxacin are used for postexposure prophylaxis.

Psittacosis

Psittacosis is a zoonotic disease caused by the bacterium Chlamydia psittaci. Numerous species of domesticated and wild birds are susceptible to the disease. Individuals at highest risk include those with direct contact with birds: bird owners, pet store and aviary employees, poultry workers, and veterinarians.¹⁶ Psittacosis was introduced to the United States secondary to a pandemic in 1929. Fewer than 10 cases of the disease have been reported per year since 2010, mostly in the western United States; however, experts believe the disease is potentially underdiagnosed and underreported.¹⁷ Outbreaks of the disease have been identified among workers on poultry farms. The United States studied psittacosis as a potential biological weapon in the offensive biological warfare program that existed until it was closed in 1969.

Pulmonary Manifestations

Clinical symptoms develop within 5 to 14 days and include fever, chills, headache, muscle aches, and productive cough. In the majority of cases, respiratory manifestations of psittacosis are limited to cough; consequently, it is most likely a disease that is underdiagnosed, as people with milder cases may not seek medical attention. A minority of patients develop pneumonia or, alternatively, respiratory failure secondary to pulmonary edema. Despite variability in patient presentation, approximately 80% of cases will require hospital admission. Chest x-ray findings are initially normal and remain normal in approximately a third of patients; in more severe cases, air space consolidation and ground-glass attenuation of the lung is denoted bilaterally on chest x-ray.¹⁸ In patients with abnormal chest x-rays, their imaging will return to normal baseline after a mean of 7.2 weeks. Prompt antibiotic therapy is key to mitigating disease morbidity and mortality: Tetracycline and doxycycline are the antibiotics of choice. Treating patients for 2 to 3 weeks prevents relapse.

Q Fever

Q (for Query) fever is caused by the rickettsialike, Gram-negative organism Coxiella burnetii. Q fever occurs worldwide, except in New Zealand. Unlike brucellosis, there is an extensive wildlife reservoir, although the primary reservoirs are sheep, cattle, and goats. Cats, dogs, and rabbits can also become infected. Ticks can serve as both reservoirs and vectors, but they are primarily involved in transmission among animal species.^19,20

Infected animals are usually asymptomatic, but Coxiella localizes in the uterus and mammary glands and is known for causing spontaneous abortion. Massive amounts of organisms (up to 10⁹ guinea pig infective doses per gram of tissue) in placental tissues put animal handlers at risk.¹⁹ Others who work closely with animals or animal carcasses are at occupational risk, including abattoir workers, veterinarians, and farmers. Vehicles kicking up dust on roads around farms have been known to spread organisms.^21,22 Rarer modes of infection include skinning of infected animals, ingestion of raw milk, and inhalational lab exposure.^23-25

Like Bacillus anthracis, Coxiella burnetii has a hardy, spore-like stage. Unlike anthrax, it has a very low infective dose of approximately 1 to 10 organisms, which makes Coxiella a potential bioweapon agent. The incubation period can range from a few days to several weeks, with the severity of infection varying related to the infectious dose.²⁶

Acute Coxiella infection can present as pneumonia, hepatitis, bradycardia, osteomyelitis, meningoencephalitis, Guillain-Barré syndrome, mononeuropathy, optic neuritis, pericarditis, myocarditis, anemia, bone marrow necrosis, thrombocytosis, thrombocytopenia, splenic rupture, or erythema nodosum. Approximately 1% to 2% of untreated individuals can progress over months to years to chronic Q fever disease, with endocarditis as the most common long-term complication. Because of the difficulty in growing this organism, these cases often present as “culture-negative” endocarditis. Having preexisting valvular abnormalities is a risk factor. Osteomyelitis may also occur, especially with preexisting bone disease or prosthetic hardware. Pregnancy can be complicated by fetal death, prematurity, or low birth weight if infection occurs during the first or second trimester.^19,20

Pulmonary Manifestations

Q fever pneumonia typically presents with high fever lasting 1 to 2 weeks, chills, sweats, headache, myalgias, shortness of breath, cough (productive and nonproductive), and chest pain. Occasionally, respiratory symptoms are accompanied by GI symptoms: anorexia, abdominal pain, nausea, vomiting, and diarrhea. Physical findings may include hypoxemia, bradycardia, tachypnea, decreased breath sounds, crackles, and/or rhonchi. Chest imaging either by x-ray or CT is most often nonspecific and may reveal unilateral or bilateral lobar, segmental, or patchy infiltrates. Hilar lymphadenopathy and pleural effusions also can be seen. “Round pneumonia” infiltrates (5-10 cm) in the lower lobes have been associated with Q fever in outbreak settings.^19,20,27,28 Generally, 2% to 5% of acute Q fever cases result in disease severe enough to require hospitalization, although that increased to 21% in a recent large outbreak in the Netherlands (2007-2009). Of those hospitalized, 29% were characterized as having severe Q fever pneumonia. Median duration of hospitalization for the Q fever pneumonia cohort was 5 days (IQR 3-7), while direct mortality related to Q fever pneumonia was 1.1%; all-cause 2-year mortality related to Q fever was 6%.²⁹ Q fever pneumonia is treated with doxycycline. Moxifloxacin, trimethoprim-sulfamethoxazole, and clarithromycin or azithromycin are alternative agents.

Ricin

Ricin is a potent cellular toxin derived from the castor bean.^30,31 In fact, the “waste mash” resulting from the production of castor oil contains 3% to 5% ricin by weight. This fact, combined with the worldwide availability of castor beans and the low degree of technological sophistication necessary to extract the toxin, has led to the inclusion of ricin among the CDC's category B agents of bioterrorism. A potent inhibitor of protein synthesis, the bi-chain ricin molecule is toxic via multiple routes of exposure, including inhalation.

Pulmonary Manifestations

The pulmonary effects of inhalational ricin exposure are dose-dependent. Data derived from accidental sublethal exposures in humans document the development, within 4 to 8 hours, of fever, nausea, cough, dyspnea, chest tightness, and arthralgia. Based on experimental animal data, exposure to higher doses is likely to result in the necrosis of respiratory epithelium, with resultant tracheitis, bronchitis, interstitial pneumonia, perivascular edema, and alveolar flooding. Death in these animals typically occurred within 36 to 72 hours after exposure. Chest radiographs would likely demonstrate the nonspecific findings associated with bilateral pneumonitis and severe pulmonary edema.³² Diagnosis depends on a high index of suspicion in the proper epidemiologic context. Lack of mediastinal involvement may help differentiate ricin intoxication from anthrax, and a failure to respond to antibiotics further supports a nonbacterial etiology. Ricin intoxication acquired via ingestion or percutaneous exposure is not likely to produce significant pulmonary symptoms. There is no specific antitoxin for ricin, and treatment is largely supportive.

Staphylococcal Enterotoxin B

The bacteria Staphylococcus aureus produces several toxins, one of which is staphylococcal enterotoxin type B (SEB). The toxin is referred to as an enterotoxin because of its primary effect on the intestine. This toxin is one of several that causes food poisoning in humans who ingest foods that have been stored or handled improperly. Alternatively, this agent can be aerosolized, which produces minimal mortality at low doses but can cause severe incapacitation. During the US offensive biological weapons program, SEB was studied extensively as an incapacitating weapon by the inhalational route. The incapacitating dose in 50% of humans was determined to be 0.0004 ug/kg, and the estimated lethal dose in 50% (LD₅₀) was at 0.02 uk/kg.³³ Latent period depends on route of exposure: 3 to 12 hours for inhalation versus 4 to 12 hours for ingestion. An increased index of clinical suspicion in conjunction with a correlating epidemiologic history is necessary for diagnosis.³⁴

Pulmonary Manifestations

Cases exposed to inhaled toxin present with fever, chills, headache, myalgia, and cough. Inhalational exposure to SEB results in persistent fever (103°F to 106°F), which can last up to 5 days, along with accompanying prodromal symptoms. Cough may persist for up to 4 weeks. Most cases are self-limited, so chest x-ray is usually unremarkable; however, in patients with more severe illness, atelectasis or corresponding increase in lung markings may be present. A limited number of cases progress to pulmonary edema or acute respiratory distress syndrome (ARDS). Antibiotics have not shown efficacy in the treatment of SEB; supportive care is the mainstay of clinical treatment.³⁵

Tularemia

Tularemia is caused by the pleomorphic, Gram-negative coccobacillus Francisella tularensis.³⁶ The low infectious dose and the organism's hardiness in the environment justify concerns for its use as a potential bioweapon.

The disease primarily occurs in the northern hemisphere, and approximately 100 to 200 cases are reported in the United States each year.³⁷ Infection occurs after direct contact with infected animals, inhalation or ingestion of the organism, arthropod bites, or laboratory exposure. There is no human-to-human transmission. Clinical presentations include localized (ulceroglandular) and systemic (typhoidal and pneumonic) forms. Symptoms include fever, headache, myalgias (typhoidal), lymphadenopathy and skin lesions (glandular and ulceroglandular), conjunctivitis (oculoglandular), cough and dyspnea (pneumonic), and nausea, vomiting, diarrhea, and abdominal pain (oropharyngeal/gastrointestinal).³⁸

Pulmonary Manifestations

Pneumonic tularemia typically occurs after inhalational exposure, but it can occur secondarily via hematogenous dissemination after gastrointestinal or cutaneous inoculation. With pneumonia, initial symptoms are followed by the development of cough, dyspnea, hemoptysis, and chest pain. Chest x-ray findings are nonspecific and may range from normal to patchy bilateral or unilateral infiltrates that may cavitate, hilar lymphadenopathy, pleural effusions, and granulomas.³⁹ Early recognition is important, as reported case fatality rates in untreated patients are as high as 60% for pneumonic and other severe forms of the disease, but drop to 2% with treatment in the United States.⁴⁰ The drugs of choice for tularemia are streptomycin or gentamicin, although the former is difficult to obtain and the latter is used with less and less frequency in the United States. Patients with mild to moderate disease may be treated with oral agents, including ciprofloxacin or doxycycline.

Agents with Limited Respiratory Manifestations or Other Distinguishing Features

Botulism

Eight serotypes of Clostridium botulinum produce neurotoxins that cause botulism, which usually occurs after foodborne toxin ingestion. Serotypes A, B, and E (rarely F) cause naturally occurring disease in humans. Botulism does not cause cough or sputum production, and victims are generally afebrile. Pulmonary symptoms would be related to the paralytic manifestations, including difficulty maintaining an open airway, and potentially progressing to respiratory failure from diaphragmatic paralysis. Antitoxin therapy is the main treatment option for botulism.

Brucellosis

Although inhalation of contaminated aerosols is the primary route of exposure for brucellosis in laboratory or abattoir workers, frank pulmonary signs and symptoms or pneumonia occur rarely.^41-43 Bacteremia may seed the lungs following exposure via other routes.^41,44 Pulmonary brucellosis, accounting for 0.6% to 7% of cases, may manifest solely as a chest x-ray abnormality or as part of a syndromic illness.^41,43,45 Pulmonary manifestations include lymphadenopathy (hilar and paratracheal), granuloma, pleural effusion, empyema, bronchopneumonia, and lung abscess.^41,43,46-48 Most cases of pulmonary brucellosis are not life threatening and respond rapidly to treatment.⁴⁵

Multiple imaging modalities may be used to assess for abscesses or granulomas, including chest x-ray, plain films of areas of concern, CT/MRI, ultrasound, or technetium/gallium scans. Brucellosis is treated with doxycycline plus streptomycin or gentamicin, or with doxycycline plus rifampin.

Smallpox

Smallpox presents with a high fever, headache, backache, and other systemic complaints. After a few days, patients develop a synchronous pustular rash that is more prominent on the face and extremities than the trunk. Although patients may develop pneumonia as a complication, the major manifestations are due to the exanthem and enanthem in the respiratory and gastrointestinal systems, respectively. Smallpox virus is stable in the environment and highly infectious via the aerosol route. Smallpox (Variola) virus was eradicated by the World Health Organization's global eradication campaign, which ended in 1980.⁴⁹ Minimal use of vaccination for decades has left a significant portion of the global population immunologically naïve and highly vulnerable, which creates the potential for significant devastation and risk of spread from a bioweapon attack.

Viral Hemorrhagic Fevers

Four families of single-stranded RNA viruses—arenaviruses, bunyaviruses, flaviviruses, and filoviruses—cause viral hemorrhagic fever. Among these, the arenaviruses (Lassa, new world arenaviruses) and filoviruses (Ebola, Marburg) are considered potential bioweapons threats. Patients present with fever, myalgias, maculopapular rash, and conjunctival injection, followed by gastrointestinal manifestations. As the illness progresses, patients may develop multi-organ failure, vascular leak, or bleeding manifestations. Pulmonary manifestations may occur but usually from pulmonary edema during volume resuscitation. Hantavirus pulmonary syndrome, caused by new world hantaviruses, has characteristic pulmonary features, but these viruses are generally not considered viable bioweapon threats because of the difficulty of growing these agents in large concentrations.

Diagnostics

Although some of the diseases discussed in this review have potential distinguishing features, the nonspecific nature of many, especially early in the illness, makes the use of optimal diagnostic tools imperative. Rapid diagnostic assays for influenza (rapid influenza diagnostic tests, or RIDTs) are important for distinguishing this common cause of respiratory illness from other diseases discussed in this review, and these continue to evolve, but their sensitivity and specificity remain suboptimal. The type of sample collected and time from illness onset dramatically affect test accuracy. Moreover, some rapid influenza diagnostic tests are specific for influenza A or B, but not both. However, their rapidity (often as short as 20 minutes) compared with viral culture (which often requires 3 to 10 days) makes rapid influenza diagnostic tests appealing.

Diagnostics for the other infectious agents listed in this review usually include blood culture, Gram stain or other special stains, and PCR of blood, sputum, or specific lesions. With some exceptions, these approaches are useful only while the pathogen is present in the bodily fluid or tissue; therefore, the diagnostic window is short for some infections. Additionally, PCR-based diagnostics are pathogen-specific, requiring a reasonable pretest hypothesis.

Newer exploratory diagnostic assays focus on pathogen-agnostic—or at least multiplex—approaches to identifying infecting pathogens. Multiple platforms using multiplex PCR aim to enable pathogen identification in the absence of a clear presumptive diagnosis,^50-52 and the potential applications of CRISPR-based diagnostics might enable development of a new class of nucleic acid–based diagnostics that combine diagnosis with treatment.⁵³ The only truly pathogen-agnostic approach to pathogen identification and potentially to pathogen discovery, however, is next-generation sequencing (NGS)–based approaches. Advances in portable sequencing technology are raising the possibility of one day enabling sequencing-based diagnostics in the field, as well as in outer space.^54-56 However, the technology supporting that end is still in development, and issues with both front-end sample processing and back-end data analysis need to be considered.^57-59 A thorough review of culture-independent approaches has recently been published.⁶⁰ As with influenza diagnostics, however, negative diagnostic results are often inconclusive.

Serologic assays are available for most agents discussed in this review, but they are beneficial only weeks after acute infection. Laboratory diagnostics to consider are listed in Table 1. If not available locally, diagnostics for biothreat agents can be accessed through local or state health departments, through the laboratory response network, or through the CDC.

Table 1.

Laboratory Diagnostic Testing for Biothreat Agents

Disease/Agent	Specimen Type	Presumptive Diagnostic Test by Sentinel Laboratory (BSL-2)	Presumptive & Confirmatory Diagnostic Tests by LRN Reference Laboratory (BSL-3) & LRN National Laboratory (BSL-4)
Anthrax (Bacillus anthracis)	Blood, skin lesion exudates, CSF, pleural fluid, sputum, and rarely urine and feces	• Direct Gram stain • Culture—colony morphology • Biochemical tests: ○ Catalase ○ Motility ○ RedLine Alert Test	Presumptive (serum specimens): • Quick ELISA Anthrax-PA kit • Detection of lethal factor (LF) by LF mass spectrometry Confirmatory: • Isolation of Bacillus anthracis from a clinical specimen; or • Detection of B. anthracis antigens in tissues by immunohistochemistry using both B. anthracis cell wall and capsule monoclonal antibodies; or • Evidence of 4-fold rise in antibodies or antigen between acute and convalescent sera using CDC IgG ELISA testing; or • Documented anthrax environmental exposure AND evidence of B. anthracis DNA.
Botulism (botulinum toxin, Clostridium botulinum)	Toxin may be present in food specimens, clinical material (serum, gastric, and feces) TOXIN IS EXTREMELY POISONOUS!	• Due to the inherent dangers in working with C. botulinum, specimens suspected of containing botulinum toxin are sent to LRN reference lab for testing.	• Detection of toxin by mouse toxicity and toxin neutralization method • Isolation of Clostridium botulinum from stool
Brucellosis (Brucella melitensis, B. abortus, B. suis, B. canis)	Blood, bone marrow, CSF, tissue, semen, and occasionally urine	• Direct Gram stain • Culture—colony morphology • Biochemical tests: ○ Catalase ○ Oxidase ○ Urease • Enzyme immunoassay	Presumptive: • Brucella total antibody titer of ≥160 by standard tube agglutination test (SAT) or Brucella microagglutination test (BMAT) (not applicable to B. canis) in 1 or more serum specimens obtained after onset of symptoms • Detection of Brucella DNA in clinical specimen by PCR assay Confirmatory: • Isolation of Brucella spp. from a clinical specimen, or • 4-fold or greater rise in Brucella agglutination titer between acute- and convalescent-phase serum specimens obtained ≥2 weeks apart and studied at the same laboratory.
Melioidosis and glanders (Burkholderia pseudomallei and Burkholderia mallei)	Blood, sputum, CSF, tissue, abscesses, and urine	• Direct Gram stain • Culture—colony morphology • Biochemical tests: ○ Catalase ○ Oxidase ○ Spot indole ○ Polymyxin B or colistin disk ○ Susceptibility test to amoxicillin-clavulanic acid and penicillin (B. mallei) ○ Growth on B. cepacia selective agar (B. pseudomallei) ○ Motility ○ Growth at 42°C • API 20NE, API 20E or Vitek II (B. pseudomallei; to differentiate B. cepacia)	Presumptive: • Evidence of a 4-fold or greater rise in B. pseudomallei antibody titer by indirect hemagglutination assay (IHA) between acute and convalescent-phase serum specimens obtained ≥2 weeks apart, or • Complement fixation (CT) test for B. mallei, titer equal to or exceeds 1:20. • Immunofluorescent test or latex agglutination • Evidence of B. pseudomallei or B. mallei DNA by RT-qPCR Confirmatory: • Isolation of B. pseudomallei or B. mallei from clinical specimen
Plague (Yersinia pestis)	Bubo fluid, blood, sputum, CSF, feces, and urine	• Direct Gram stain; Wright or Giemsa stain • Culture—colony morphology • Biochemical tests: ○ Catalase ○ Oxidase ○ Urease ○ Indole	Presumptive: • Elevated serum antibody titer(s) to Y. pestis fraction 1 (F1) antigen (without documented 4-fold or greater change) in a patient with no history of plague vaccination, or • Detection of F1 antigen in a clinical specimen by fluorescent assay. Confirmatory: • Isolation of Yersinia pestis from a clinical specimen, or • 4-fold or greater change in serum antibody titer to Y. pestis F1 antigen.
Psittacosis (Chlamydia psittaci)	Serum, nasopharyngeal (NP), and/or oropharyngeal (OP) swabs, and any lower respiratory tract specimen including bronchoalveolar lavage (BAL) and sputum; tissue, CSF, isolates, and purified nucleic acid		• Serologic test—complement fixation (CF), microimmunofluorescent antibody (MIF) test—4-fold increase in antibody titer between acute and convalescent serum samples• Direct immunofluorescent antibody staining• Real-time PCR; touchdown enzyme time release (TETR)-PCR• Isolation of Chlamydia psittaci in culture
Q-fever(Coxiella burnetii)	Blood, tissue, body fluids, feces; manipulation of tissues from infected animals and tissue culture should be performed at BSL-3.	Acute Q fever: • A single positive IFA IgG titer ≥1:128 (CDC cutoff value) to phase II antigen (phase I titers may be elevated as well), or• Elevated (as defined by the testing laboratory) phase II IgG or IgM antibody reactive with C. burnetii antigen by ELISAChronic Q fever:IgG antibody titer to C. burnetii phase I antigen ≥1:128 and <1:800 by IFA	Acute Q fever: • 4-fold or greater changes in antibody titer between paired acute- and convalescent-phase serum samples by immunofluorescence antibody (IFA) testing (1 taken during the first week of illness and a second 3-6 weeks later), or• Detection of C. burnetii target of IS1111 in affected tissue by RT-PCR• Immunohistochemical (IHC) staining of biopsy material from affected organs, or• Isolation of C. burnetii in cell culture of biopsy or blood samplesChronic Q fever:• IFA titer to phase I antigen ≥1:800 with phase I titer being higher than the phase II titer, or• Detection of C. burnetii target of IS1111 in affected tissue by RT-PCR, or• Immunohistochemical detection of C. burnetii, or• Isolation of C. burnetii in cell culture.
Ricin toxin(Ricinus communis, toxin)	Urine, serum, environmental or food samples	• PCR	Confirmatory: • Time resolved fluorescence immuno assay (TRFIA) • Liquid chromatography/tandem mass spectrometry • Matrix assisted laser desorption/ionization time of flight mass (MALDI-TOF-MS)Spectrometry (MALDI-TOF-MS)
SEB(Staphylococcal enterotoxin type B)	Serum, urine, respiratory secretions, nasal swabs, stool/gastric aspirate, food specimens, environmental samples, isolates	• Culture—sent for toxin testing• Serology• Sentinel laboratories should not accept environmental (including food samples) or animal specimens for testing; such specimens should be forwarded directly to the next level LRN reference laboratory.	• Time-resolved fluorescence (TRF) immunoassay• A solid-phase, noncompetitive sandwich ELISA of capture antibody, antigen, and detection antibody• Gel diffusion assay• Reverse passive latex agglutination test (in food)• PCR
Smallpox(Variola virus)	Lesion fluid or crusts, respiratory secretions, or tissue	• There is no sentinel laboratory test to rule out smallpox.	• Generic tests for orthopox, including non-variola orthopox PCR, orthopox PCR, and electron microscopy• Variola-specific tests include variola culture with PCR confirmation and variola PCR from clinical specimen.• Additional tests may include Vaccinia PCR.
Tularemia(Francisella tularensis)	Skin lesion exudates, respiratory secretions, CSF, blood, urine, tissues from infected animals, and fluids from infected arthropods	• Direct Gram stain• Culture—colony morphology; satellite growth• Biochemical tests: ○ Catalase ○ Oxidase ○ B-lactamase	Presumptive:• Elevated serum antibody titer(s) to F. tularensis antigen (without documented 4-fold or greater change) in a case with no history of tularemia vaccination, or ○ Detection of F. tularensis in a clinical specimen by fluorescent assay.Confirmatory:• Isolation of F. tularensis in a clinical specimen (culture must be held for 21 days before specimen can be reported as negative), or• 4-fold or greater change in serum antibody titer between acute and convalescent sera to F. tularensis antigen. Antibodies usually appear in week 2 of disease.• PCR
Viral hemorrhagic fevers (VHF) (Ebola, Marburg, Lassa, Crimean-Congo hemorrhagic fever, Rift Valley fever, and others)	Blood, urine, respiratory, and throat secretions, semen, and tissue		• Detection of VHF viral antigens in blood by ELISA antigen detection• Isolation of VHF virus in cell culture for blood or tissues• Detection of VHF viral genes using reverse transcriptase with PCR amplification (RT-PCR) from blood or tissues• Detection of VHF viral antigens in tissues by immunohistochemistry

Note: CSF = cerebrospinal fluid; LRN = Laboratory Response Network.

Discussion

Recent world events prompt the need for a reminder to clinicians and public health practitioners of the potential for bioterrorism events and other unexpected disease activity. We elected to focus this review on potential biothreat agents that present primarily with respiratory features and on differentiating them from community-acquired pneumonia, a step critical to the selection of proper therapy. Often-recommended empiric outpatient treatment regimens for community-acquired pneumonia in adults without co-morbidities include azithromycin and clarithromycin, which have little efficacy against the majority of category A and B threat agents. Conversely, doxycycline, also recommended for empiric therapy of adult community-acquired pneumonia, does have efficacy against anthrax, plague, tularemia, glanders, melioidosis, psittacosis, and Q fever, although it would likely prove inadequate when given alone. In adults with community-acquired pneumonia and co-morbid conditions, fluoroquinolones are often recommended. Again, these have efficacy against anthrax, plague, and tularemia but not against other agents. Moreover, they, too, are often inadequate in many cases when given as monotherapy. Therefore, following current guidelines for treating community-acquired pneumonia, if either a quinolone or doxycycline is selected, there may be partial coverage by chance of anthrax, plague, and tularemia.

In children, the situation is even more problematic. Amoxicillin, Augmentin, and azithromycin, all used frequently for community-acquired pneumonia, are likely to prove inadequate when used alone against any category A or B threat agent.

The threats covered in this review may present initially as nonspecific febrile illnesses (Table 2). Later in the illness, without adequate treatment, some may develop characteristic features that could help distinguish them from community-acquired pneumonia (Table 3). In some cases, chest imaging may help (Table 4). Anthrax causes a widened mediastinum on chest x-ray. Pneumonic plague presents with bloody sputum and severe sepsis. Glanders and melioidosis can present with bloody sputum and multiple cavitary lesions in the lungs, as well as in other major organs. The more difficult infections to distinguish include Q fever, which may have minimal pulmonary findings or a chest x-ray more concerning than the clinical illness; tularemia; SEB exposure; and psittacosis, which may not have any specific distinguishing features.

Table 2.

Hallmark Clinical Symptoms of Category A/B Biothreat Agents, Early Clinical Symptoms that May Occur on Presentation for Care

Biothreat Agent	Clinical Presentation
Anthrax	fever, chills, fatigue, nonproductive cough, and mild chest discomfort
Botulism	difficulty swallowing or speaking, nausea, vomiting, dry mouth, facial weakness, blurred or double vision, drooping eyelids, trouble breathing, abdominal cramps
Brucella	fever, malaise, anorexia, headache, muscle/joint/back pain, chills, weight loss, nausea and vomiting
Glanders	fever, chills, sweating, localized infection, muscle aches, headache, nasal discharge, muscle tightness, light sensitivity
Melioidosis	fever, anorexia, headache, cough (productive), localized pain or swelling/ulceration/abscess
Plague	fever, chills, malaise, nausea, vomiting, diarrhea, cough (hemoptysis), dyspnea
Psittacosis	fever, chills, headache, myalgia, cough (nonproductive)
Q fever	fever, malaise, headache, nausea, vomiting, chills or night sweats, cough (nonproductive)
Ricin	fever, chest tightness, cough, dyspnea, nausea
Smallpox	fever, headache, malaise, chills, vomiting, abdominal pain, pain in back or specific muscular distributions prior to the onset of the rash, rash (centrifugal distribution)
SEB	fever, chills, headache, malaise, myalgia, and nonproductive cough
Tularemia	fever, chills, malaise, fatigue, headaches, myalgia, arthralgia, skin ulcer at the point of contact with an infected animal or at the site of a bite, swollen lymph nodes in the distribution of the skin ulcer (the axillae and the groin are the most commonly affected areas)
VHF	fever, fatigue, dizziness, muscle/bone/joint aches, vomiting, diarrhea

Table 3.

Hallmark Clinical Symptoms of Category A/B Biothreat Agents, Late Clinical Signs/Symptoms Without Adequate Treatment

Biothreat Agent	Clinical Presentation
Anthrax	fever, diaphoresis, pain in chest or specific muscular distributions, shortness of breath progressing to acute respiratory distress syndrome, circulatory shock
Botulism	cranial nerve findings (difficulty swallowing, difficulty speaking or change in intonation, diplopia), descending flaccid paralysis, respiratory paralysis
Brucella	joint/muscle/back pain, arthritis, spondylitis, sacroiliitis; specific organ involvement: epididymo-orchitis, abortion, endocarditis, respiratory and neurological signs, cutaneous changes
Glanders	abscess (multiple sites)
Melioidosis	weight loss, seizures, respiratory distress, abdominal/chest discomfort, urinary obstruction, bloody sputum
Plague	chest pain, severe bronchopneumonia, sepsis, bloody sputum
Psittacosis	chest pain (pleuritic)
Q fever	dyspnea, chest pain/abdominal pain, hepatitis, and weight loss
Ricin	abdominal pain, anuria, pupillary dilation, headache, arthralgia, airway necrosis, pulmonary edema
Smallpox	fever, chills, fatigue, headache, back pain (severe), pustular rash with centrifugal distribution
SEB	dyspnea, retrosternal chest pain, pulmonary edema, acute respiratory distress syndrome
Tularemia	renal failure, septic shock, acute respiratory distress syndrome
VHF	delirium/coma, renal failure, bleeding manifestations, hypotension, acidosis, pulmonary edema

Table 4.

Chest Imaging for Specific Biothreat Agents

Biothreat Agent	Imaging
Anthrax (Bacillus anthracis)	Chest x-ray: hilar prominence, pleural effusions and parenchymal infiltrates, peribronchial opacities, mediastinal lymphadenopathy, widening of the mediastinum (particularly in the right paratracheal area)CT chest without contrast: mediastinal lymphadenopathy, widened mediastinum, pleural effusions
Botulism (botulinum toxin, Clostridium botulinum)	N/A
Brucellosis (Brucella melitensis, B. abortus, B. suis, B. canis)	Chest x-ray: rare abnormalities, nonspecific findings, lung cavitation can be visualized in a minority of casesPET/CT chest: increased FDG activity in the pulmonary lesions and in the mediastinal and hilar nodes, no extrathoracic abnormalities
Glanders (Burkholderia mallei)	Chest x-ray: lung abscess (potentially multiple)CT chest: lung abscesses, liver abscesses
Melioidosis (Burkholderia pseudomallei)	Chest x-ray: lung abscess (potentially multiple, 30% multi-lobar involvement); chronic disease can mimic tuberculosis (predilection for the upper lobes)CT chest: small pleural nodule, pleural effusions with an associated lung lesion, or hydropneumothorax
Plague (Yersinia pestis)	Chest x-ray: bilateral alveolar and parenchymal infiltrates, bilateral pleural effusions; in severe cases, may progress to a clinical picture similar to acute respiratory distress syndromeCT chest: acute respiratory distress syndrome–like presentation, consolidation (focal or lobular)Nuclear imaging: lymphadenitis and meningeal inflammation
Psittacosis (Chlamydia psittaci)	Chest x-ray: variable presentation, normal vs multiple patterns of consolidation (patchy infiltrates to well-localized confluent presentations have been documented in clinical reviews)
Q fever (Coxiella burnetii)	Chest x-ray: nonspecific findings; features may appear worse than the clinical disease; unilateral or bilateral infiltrates (lobar, segmental, or patchy infiltrates). Hilar lymphadenopathy and pleural effusions; round pneumonia in the lower lobesCT chest: nonspecific findings; patchy infiltrates vs lobar consolidation with or without small pleural effusions, mild lymphadenopathy
Ricin (Ricinus communis toxin)	Chest x-ray: nonspecific findings associated with bilateral pneumonitis and pulmonary edema
Smallpox (Variola virus)	Chest x-ray: rare pneumonia, pulmonary edema in flat or hemorrhagic smallpox; upper lobe nodular lesions in previously vaccinated individuals
SEB (Staphylococcal enterotoxin type-B)	Chest x-ray: normal chest x-ray (mild clinical illness); interstitial edema (moderate to severe cases); discoid atelectasis (upon recovery)
Tularemia (Francisella tularensis)	Chest x-ray (ulceroglandular or typhoidal form): bilateral or lobar infiltrates, cavitations and pleural effusions may be visible (occurs in approximately 1/3 of cases, less marked in tularemia type B)Chest x-ray (pneumonic tularemia): may present with normal findings on chest x-ray. Alternatively, may present with multifocal segmental or lobar infiltrates, hilar adenopathy (in a minority of cases).CT chest: prominent hilar nodes, cavitary lesions
VHF (includes Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae, and Paramyxoviridae)	Chest x-ray (Arenaviridae): CXR findings are often normal; lobar opacities have been documented in a minority of cases (20%) of patients who have Argentinian hemorrhagic fever with bacterial superinfectionChest x-ray (Bunyaviridae): Only American hantaviruses are known to cause extensive change on chest radiograph; marked interstitial edema with prominent Kerley B lines (early manifestation); subpleural edema with significant fluid accumulation in the bases of the lungs (late manifestation, hantavirus pulmonary syndrome)Chest x-ray (Filoviridae): Changes on CXR are rare, unless secondary to fluid volume overload from aggressive fluid volume resuscitation

The absence of rhinorrhea or pharyngitis may aid in defining an appropriate differential diagnosis. These 2 features were not widely seen during the 2001 anthrax attacks, although some of the diseases mentioned here can also have upper respiratory findings. For example, oropharyngeal anthrax can cause tonsillar exudates and cervical adenopathy. Pharyngeal plague carriers can have asymptomatic colonization with organisms grown from the pharynx. Tularemia pharyngitis can occur in both ulceroglandular and typhoidal tularemia. Overall, 25% of cases present with acute exudative pharyngitis or tonsillitis, with or without mucosal ulceration or cervical adenopathy. Although not associated with pharyngitis, melioidosis is known for causing acute parotitis. Varieties of oronasal glanders also can occur.

Identifying any of these diseases first requires a thorough history and physical exam, as well as a basic knowledge about the natural epidemiology of the diseases, so that a more detailed exposure history can be obtained. Armed with this information, the frontline clinician needs to be a trained observer and be willing to consider the rare “zebra” when they hear hoof beats, because with all these diseases, nothing can replace a healthy index of suspicion for the unusual and the knowledge of how to obtain the appropriate diagnostics.

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