Laboratory Diagnosis and Biosafety Issues of Biological Warfare Agents E

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Laboratory diagnosis and biosafety issues of biological warfare agents E. Nulens and A. Voss

University Medical Center St Radboud, Department of Medical Microbiology (440 MMB), Nijmegen, The Netherlands Bioterrorism events have been rare until recently. Many clinical laboratories may not be familiar with handling specimens from a possible bioterrorism attack. Therefore, they should be aware of their own responsibilities and limitations in the handling and treatment of such specimens, and what to do if they are requested to process clinical samples. The Centers for Disease Control and Prevention has developed the Laboratory Response Network to provide an organized response system for the detection and diagnosis of biological warfare agents based on laboratory testing abilities and facilities. There are potentially many biological warfare agents, but probably a limited number of agents would be encountered in case of an attack, and their identiﬁcation and laboratory safety will be discussed. Keywords biosafety, bioterrorism, biological warfare, Bacillus anthracis, Francisella tularensis, Clostridium botulinum Clin Microbiol Infect 2002; 8: 455–466

laboratory personnel are not familiar with the BIOSAFETY appearance and characteristics of these agents, A great variety of biological agents could poten- or with the reception, handling and laboratory tially be used for biological warfare, but fortu- treatment of clinical specimens. Owing to the pre- nately only a few agents can be efficiently sent events, clinical laboratories may be requested disseminated in the community. As well as being to process clinical or environmental specimens easily dispersed (1–5-mm particles), the ‘ideal’ bio- (from real or hoax attacks). Therefore, all micro- logical agents should be highly lethal, easily pro- biological laboratories should be familiar with the duced in large quantities, stable, preferably methods and precaution measures involved in transmitted by the aerosol route or from person handling these agents. to person, resistant to standard antibiotics, and not The Centers for Disease Control and Prevention preventable by vaccination [1]. This limits the list (CDC) has created the Laboratory Response Net- of possible agents to certain bacteria (Bacillus work to provide an organized response system for anthracis, Brucella spp., Clostridium botulinum, Yer- the detection and diagnosis of biological warfare sinia pestis, and Francisella tularensis) and viruses agents based on laboratory testing abilities and (smallpox and viral hemorrhagic fevers) [1,2]. facilities [3,4]. There are four levels of laboratory Among these, Bacillus anthracis and smallpox are capacity (A–D), and each level has designated the two agents with the most potential to fulfill the core-testing capacities. Level A laboratories have criteria for mass casualties [1]. the minimum core capacity, and they would rule Until recently, bioterrorism was a very rare out suspected isolates by simple testing and refer event, and the agents involved in it were hardly them to a higher-level laboratory. Level D labora- ever encountered in (clinical) laboratories; hence tories, like the CDC, are biological safety level 4 (BSL-4) facilities, and they have the highest capacity and most advanced techniques (Figure 1). Corresponding author and reprint requests: A. Voss, UMC Most clinical, hospital and private laboratories St Radboud, Medical Microbiology, 440 MMB, PO Box 9101, are classified as level A, operating at BSL-2. Level 6500 B Nijmegen, The Netherlands Tel: þ31 24 3617575 A laboratories are most likely to be the first con- Fax: þ31 24 3540216 tacted and questioned with regard to specimen E-mail: [email protected] collection and shipment, culture, and treatment.

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specimens. As a result, strict adherence to standard microbiological practices and techniques is the most important factor in containment, and each laboratory should develop a biosafety manual. To minimize and/or eliminate exposure to certain agents, laboratory personnel must be con- tinually trained to ensure awareness of diagnostic and preventive measures. Safety equipment comprises primary barriers against biological materials, including biological safety cabinets (BSCs), enclosed containers, and

Ã other engineering controls. Safety equipment may Figure 1 Laboratory Response Network laboratory levels also include items for personal protection, such as [5]. gloves, face shields, and safety glasses, which are often used in combination with a BSC. There are Although these laboratories are not intended to currently three types of BSC: open-fronted class I actually handle critical biological agents, they and II BSCs offer protection to laboratory person- might receive clinical samples initially not sus- nel and to the environment; class II BSCs addi- pected to contain these agents. Consequently, even tionally provide protection from external level A laboratories should have the necessary contamination; and the gas-tight class III BSC facilities, equipment and competence to process provides the highest attainable level of protection critical biological agents [3]. to personnel and environment [5]. In the present situation, all laboratories hand- The secondary barriers will depend on the ling clinical and environmental specimens should risk of transmission of specific agents (Table 1) reach a basic standard: [5]. The design and the construction of the 1. Knowledge of the current biosafety level within laboratory facility form part of these secondary the laboratory. barriers. They should contribute to the labora- 2. Development and availability of protocols tory workers’ protection, as well as protect the related to the chain of guardianship. community environment from infectious agents 3. Collection, preservation and shipment of speci- which may be accidentally released from the mens, as well as culture and identification of laboratory. targeted agents. A detailed description of biosafety level prac- 4. Knowledge of the location of the nearest higher- tices and classification of BSCs can be found in the level reference laboratory, and of current guide- 4th edition of the Centers for Disease Control and lines to ensure the safe handling and shipment Prevention and National Institutes of Health pub- of biological agents. lication Biosafety in Microbiological and Biomedical 5. Knowledge of the basic characteristics of the Laboratories, which is also available on the internet current targeted agents [3,4]. (http://www.cdc.gov) [5]. BSL-1 represents a Containment involves the use of safe methods basic level of containment with no recommenda- for managing infectious materials in the laboratory tions for special primary or secondary barrier environment. Its purpose is to reduce or eliminate precautions. In BSL-1 laboratories, work on an exposure of laboratory workers and the environ- open bench is performed with microorganisms ment to potentially hazardous agents [5]. Primary that cause no disease in healthy adults, but may containment is the protection of personnel and the behave as opportunistic pathogens in very young immediate laboratory environment; secondary or older patients and immunocompromised indi- containment is the protection of the environment viduals. Moderate-risk organisms, such as hepati- external to the laboratory [5]. Hence, the three tis B virus and salmonellae, which may cause most important topics in laboratory safety are: disease of varying severity, should be treated in (1) laboratory practice and techniques; (2) safety a BSL-2 laboratory. The primary hazards relate to equipment; and (3) design of facilities. percutaneous or mucous membrane exposure, or Laboratory personnel must always be aware of ingestion of infectious materials. Procedures with the potential hazards when working with clinical aerosol or splash potential that may increase the

ß 2002 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 8, 455–466 Nulens and Voss Laboratory diagnosis and biosafety issues of biological warfare agents 457

Table 1 Biosafety levels

BSL [5] Agents Practices Safety equipment Facilities

1 Not known to Standard microbiological None required Open benchtop sink consistently cause practices required disease in healthy adults 2 Associated with BSL-1 practice plus: Primary barriers ¼ BSL-1 plus: human disease, limited access class I or II BSCs autoclave available hazard ¼percutaneous biohazard warning signs or other physical injury ingestion ‘sharps’ precautions containment mucous membrane biosafety manual defining devices used for all exposure any needed waste manipulations of agents decontamination or medical that cause splashes or surveillance policies aerosols of infectious materials PPEs: laboratory coats; gloves; face protection as needed 3 Indigenous or exotic BSL-2 practice plus: Primary barriers ¼ BSL-2 plus: agents: with potential controlled access class I or II BCSs physical separation for aerosol transmission decontamination of all or other physical from access corridors disease may have serious waste decontamination of containment devices self-closing, double- or lethal consequences laboratory clothing used for all open door exhausted air before laundering manipulation of agents not recirculated baseline serum PPEs: protective negative airflow into laboratory clothing; laboratory gloves; respiratory protection as needed 4 Dangerous/exotic BSL-3 practices plus: Primary barriers ¼ BSL-3 plus: agents: which pose clothing change before all procedures separate building or high risk of life- entering conducted in isolated zone threatening disease shower on exit class III BSCs dedicated supply and aerosol transmitted all material or class I or II BSCs in exhaust, vacuum, and laboratory infections decontaminated on combination with decontamination or related agents with exit from facility full-body, air-supplied, systems other unknown risk of positive-pressure requirements outlined transmission personnel suit in the text

BSL, biosafety level; BSC, biological safety cabinet; PPE, personal protective equipment.

risk of exposure must be conducted in a BSC, and the primary hazards. The laboratory worker must other primary and secondary barriers must be be completely isolated from aerosolized infectious available. In a BSL-3 facility, all laboratory manip- materials by working in a class III BSC or in a full- ulations are performed in a BSC, and more empha- body, air-supplied positive-pressure personnel sis is placed on primary and secondary barriers. suit (Table 2). Mainly, viral agents are processed Exposure to infectious aerosols, ingestion and in a BSL-4 laboratory [5]. autoinoculation are the primary hazards to the For bacterial warfare agents, activities concern- personnel in contiguous areas, the community, ing specimen handling and diagnosis of suspected and the environment. Examples are Mycobacterium cultures should be forwarded to at least a BSL-2 tuberculosis and Coxiella burnetii. Exotic agents that facility (Table 3). This implies that all specimens, pose the risk of life-threatening disease, which after reception and labeling, should be further may be transmitted via aerosol, and for which handled in a class I or II BSC. Manipulation and there is no available vaccine or therapy, must be production of large quantities of bacterial cultures, handled in a BSL-4 facility. Manipulations that and other activities which might lead to aerosol may lead to exposure of the mucous membrane formation, should be avoided, and should be or broken skin, including autoinoculation, pose referred to a BSL-3 laboratory.

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Table 2 Microbiology biosafety for biological warfare agents

BSL

Specimen Culture Recommended precautions for Agent handling handling level A laboratories

Bacillus anthracis 2 2 BSL-2: BSL-3: activities involving clinical activities with high material collection and potential for aerosol or diagnostic quantities of droplet production infectious cultures Brucella spp.a 2 3 BSL-2: BSL-3: activities limited to all activities involving collection, transport and manipulations of plating of clinical cultures material Clostridium botulinumb 2 2 BSL-2: BSL-3: activities with materials activities with high known to contain toxin or potential for aerosol or potentially containing toxin droplet production must be handled in a biological safety cabinet (class II) with a laboratory coat, disposable surgical gloves, and a face shield (as needed) Francisella tularensisc 2 3 BLS-2: BSL-3: activities limited to all activities involving collection, transport and manipulations plating of clinical material of cultures Yersinia pestisd 2 2 BSL-2: BSL-3: activities involving clinical activities with high material collection and potential for aerosol or diagnostic droplet production quantities of infectious cultures Smallpoxe 4 4 BSK-4: specimen collection/transport Hemorrhagic fever 4 4 BSL-4: virusesf specimen collection/transport aLaboratory-acquired brucellosis has occurred through: ‘sniffing’ cultures; aerosols generated by centrifugation; mouth pipetting; accidental parenteral inoculations; sprays into eyes, nose, and mouth; and by direct contact with clinical specimens. bExposure to toxin is the primary laboratory hazard, since absorption can occur through direct contact with skin, eyes, or mucous membranes, including the respiratory tract. The toxin can be neutralized by 0. 1 M sodium hydroxide. Clostridium botulinum is inactivated by a 1 : 10 dilution of household bleach. The contact time is 20 min. If the material contains both toxin and organisms, the spill must be sequentially treated with bleach and sodium hydroxide for a total contact time of 40 min. cLaboratory-acquired tularemia infection has been more commonly associated with cultures than with clinical materials/ animals. Direct skin/mucous membrane contact with cultures, parenteral inoculation, ingestion and aerosol exposure have resulted in infection. dSpecial care should be taken to avoid the generation of aerosols. eIngestion, parenteral inoculation and droplet or aerosol exposure of mucous membranes or broken skin to infectious fluids or tissues are the primary hazards for laboratory workers. fRespiratory exposure to infectious aerosols, mucous membrane exposure to infectious droplets and accidental parenteral inoculation are the primary hazards for laboratory workers.

Bacillus anthracis, Y. pestis and specimens con- Smallpox and the hemorrhagic fever viruses are taining botulism toxins can be safely handled extremely infectious, and specimen collection, using BSL-2 practices, but BSL-2–3 practices are handling and further treatment should be referred recommended for F. tularensis and Brucella spp. to a BSL-4 facility (Table 2) [4].

Table 3 Specimens, specimen processing and diagnostic tests for biological warfare agents

Agent Form Specimen Media Techniques

Bacillus anthracis Respiratory Blood SBA Culture Sputum CA Serology Pleural fluid MAC Amplification Cutaneous Blood Toxin detection Vesicular swabs Antigen detection with electrochemiluminescence Gastrointestinal Blood Gastric aspirate Ascites Stool PLET Meningitis Blood Liquor F. tularensis Respiratory Blood SBA Culture Wounds Sputum CA Serology Bronchial/tracheal CHA Amplification wash Pleural fluid TMA Lymph node BCYE Conjunctival scraping Biopsy Gastric aspirate Brucella spp. Blood SBA Culture Sputum CA Serology Throat MAC Bronchial/tracheal wash TMA Biopsy BCYE Abscess Serum Y. pestis Pneumonic Blood SBA Culture Bubonic Sputum MAC F1 antigen detection Septicemic Throat BHIB Amplification Bronchial/tracheal wash Lymph node Botulism Food-borne Enema fluid Mouse bioassay (toxin detection) Wound Food Toxin gene amplification Infant Gastric fluid Culture Intestinal fluid Nasal swab Serum Stool Vomitus Wound/tissue Environmental sample Smallpox Rash Vesicular fluid Chorioallantoic Electron microscopy membranes of chicken embryos Lesions Human cell cultures Giemsa stain Saliva Cell culture Biopsy Serology Serum Amplification VHF Blood Human cell cultures Culture Serum Serology Throat swab/washings Immunohistochemical Liver biopsy Amplification Cerebrospinal fluid

SBA, sheep blood agar; CA, chocolate agar; MAC, McConkey agar; PLET, polymyxin–lysozyme–EDTA–thallous acetate agar; TMA, Thayer–Martin agar; CHA, cysteine heart agar; BCYE, buffered charcoal yeast extract; BHI, brain–heart infusion broth; VHF, viral hemorrhagic fever.

ß 2002 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 8, 455–466 460 Clinical Microbiology and Infection, Volume 8 Number 8, 2002

other Bacillus species are relatively easy to culture, BACILLUS ANTHRACIS even on media routinely used in bacteriology Infections due to Bacillus anthracis may lead to (Table 1). Usually, a sheep blood agar (SBA) or a three clinical manifestations. Cutaneous anthrax chocolate agar (CA) is used. Polymyxin–lyso- is seen in 95% of the cases, while inhalation zyme–EDTA–thallous acetate agar (PLET) is a anthrax is found in only 5% of cases. Gastrointest- selective medium that is used for culturing Bacillus inal anthrax is very rare and is encountered in less anthracis from heavily contaminated specimens than 1% of cases [4]. All three manifestations of such as stool [6,8]. All media are incubated under anthrax may lead to bacteremia, which is often ambient conditions at 35 8C and are examined after fatal. Meningitis may occur as a complication of 18–24 h. Bacillus anthracis bacteremia [6,7]. For the majority of strains, Bacillus anthracis is The Bacillus genus comprises more than 60 dif- easily identified and differentiated from other ferent species. Bacillus species are Gram-positive Bacillus species [8,10]. On nutrient agar, colonies aerobic or facultative anaerobic bacilli that pro- are flat to slightly convex, 2–5 mm in size, similar duce endospores [7]. In clinical specimens, the to Bacillus cereus, white or gray–white with irre- bacteria often appear in short chains, but in vitro, gular borders and curly tailing at the edges longer chains of bacilli are formed [6,8]. The bac- (Medusa head appearance). The colonies often teria grow well on sheep blood agar and are have a firm consistency. Bacillus anthracis is non- usually catalase positive [7]. Most Bacillus species hemolytic, but slight hemolysis may be noticed in can be divided into three groups, depending on areas of heavy growth [11]. The organism is non- the morphology of the spore and the sporangium. motile, and a motility test by the hanging drop Bacillus anthracis belongs to the Bacillus cereus method is one of the most useful and reliable group, which is composed of at least seven closely procedures for screening isolates [11]. Bacillus related species [7,9,10]. Bacillus species can be anthracis does not grow on McConkey agar differentiated from other spore-forming bacteria, (MAC), and capsule formation is not seen if the such as Clostridium species, by the formation of bacteria are cultured on artificial media [8]. In the ellipsoidal spores without swelling of the sporan- presence of 0.7% sodium carbonate in the medium gium, in the presence of oxygen [7,8,11]. Spores are and during incubation in 5–20% CO2, capsule rarely seen in clinical specimens, since the CO2 synthesis by Bacillus anthracis is enhanced and content of the body prevents spore formation [6]. mucoid colonies are produced [6–8]. If a colony Toxin production, which is thought to inhibit the from a nutrient agar is suspended in defibrinated immune response, and capsule formation, which blood and incubated for 5 h, a smear of the suspen- inhibits phagocytosis of vegetative anthrax bacilli, sion may show encapsulated bacteria [8]. In con- are the two major virulence factors of Bacillus trast to Bacillus cereus and other Bacillus species, anthracis. Smears stained with the M’fadyean stain Bacillus anthracis is usually susceptible to penicil- (polychrome methylene blue stain) show dark blue lin, although resistant strains have been reported bacilli, surrounded by a pink capsule [8,11]. In an [9]. The most important biochemical features are India ink wet preparation, the capsule is not stained, summarized in Table 4, although, in the majority butclearhalosaroundBacillusanthracisbacillicanbe of strains, additional testing is rarely necessary seen[8].TheGramstainisnotsuitableforvisualizing [8,10]. the capsule of Bacillus anthracis, but can show typical Serology is useful for the diagnosis of a Bacillus Gram-positive square-ended rods (box cars) [8]. anthracis infection, but is only retrospective, and Depending on the clinical manifestations, different should mainly be used as an epidemiologic tool specimens can be obtained for culture of Bacillus [8]. Acute and convalescent serum samples, taken anthracis(Table 3). Antibiotic treatment mayquickly 2–4 weeks apart, are necessary to confirm infec- sterilize blood and tissues. Although residual forms tion. Capsular antigens and exotoxin components may still be seen in clinical smears, specimens are the major immunogenic proteins [2,9]. An should be obtained before antibiotic therapy is immunochromatographic protective antigen assay initiated [8]. is a very reliable and sensitive test for the detection To obtain a suspension of cells, clinical speci- of the Bacillus anthracis toxin in sera of patients, mens should be mixed with sterile nutrient broth even if the bacteria are no longer viable [8]. (cave aerosol formation) [11]. Bacillus anthracis and An immunomagnetic electrochemiluminescence

Table 4 Key characteristics to dif- ferentiate Bacillus anthracis and other Bacillus cereus and Bacillus species [7,11] Characteristics Bacillus anthracis other Bacillus species Hemolysis (sheep blood agar) Àþ Motility Àþ(usually) Penicillin susceptibility þÀ Gelatin hydrolysis (7 days) Àþ Salicin fermentation Àþ Enhanced hydrolysis of þÀ p-nitrophenyl-a-D glucoside in the presence of 1% Triton X-100 Growth on phenylethyl Àþ alcohol blood agar

assay may be used to detect antigens, in concen- amino acids cysteine and cystine for growth trations as low as 0.1–1.0 pg/mL [2]. [13,14]. It is weakly catalase positive, as well as PCR techniques for the detection of specific oxidase and urease negative. Cysteine heart agar plasmid-borne virulence genes of Bacillus anthra- (CHA), SBA, CA, Thayer–Martin agar (TM), cis, such as the toxin genes and enzymes leading to enriched chocolate agar with cysteine with 9% capsule formation, exist or are under investigation. sheep blood (CHAB) and buffered charcoal yeast Many of these techniques are useful in the extract (BCYE) may be used for clinical specimens detection and identification of Bacillus anthracis, (Table 3) [13]. Although growth of F. tularensis in but are more likely to be used in non-level A supplemented broth (trypticase soy broth, brain– laboratories [8–10]. heart infusion, thioglycolate) is poor, its recovery from clinical specimens is enhanced [12,13]. Broth cultures should be incubated for 10 days. F. tular- FRANCISELLA TULARENSIS ensis may grow initially on SBA, CA and TMA, but The taxonomy of F. tularensis is still unresolved, subcultures on SBA usually fail to grow [13]. Blood but at present F. tularensis has two biovars: F. cultures may become positive after 3–7 days with tularensis biovar tularensis, which is the most the BACTEC system [14]. CO2 may stimulate pathogenic biovar, and F. tularensis biovar palaearc- growth [12,13]. Viscous materials and skin and tica, which is less virulent to humans [12]. wound scrapings should first be suspended in Transmission of F. tularensis is possible through broth before being inoculated on plates [12,13]. the skin, by inhalation or via ingestion [12]. In After 18 h of incubation, pinpoint colonies may laboratory workers not strictly adhering to safety appear, if there is a high colony count in the clinical protocols, infections via damaged skin have been specimens [12]. After 2 days of inoculation, the reported [4,13]. In the case of bioterrorism, inhala- smooth, shiny and flat colonies reach a size of 1– tion and consequent pneumonia would probably 1.5 mm. The medium immediately surrounding be the primary mode of transmission. colonies is characteristically green in appearance F. tularensis is an obligate aerobe bacterium, [4,12–14]. On CA and TMA, colonies are 1–2mm non-motile, non-encapsulated, and non-spore- indiameter,blue–whitetogray;onCHAandCHAB, forming. The bacteria are small (0.5–0.2 mm), coloniesare2–4 mmindiameterandgreenish–white pale-staining, Gram-negative uniform coccoba- [4,13]. Culture platesshould be incubated for at least cilli, usually pleomorph in older cultures [12,14]. 10 days [13]. The inability of F. tularensis to grow on The organism can be distinguished from other MAC or at room temperature can be used for differ- bacteria by its small size, faint bipolar staining entiation from Y. pestis [13,14]. F. tularensis is bio- with aniline dyes, fluorescent antibody reaction, chemically inert, and testing is thus of little value. agglutination with a specific antiserum, and lack of Usually, the diagnosis is based on clinical findings, a growth or poor growth on ordinary media [12]. F. compatible Gram stain, poor growth on SBA and at tularensis is a fastidious organism that needs the room temperature, and a typical metallic sheen of

ß 2002 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 8, 455–466 462 Clinical Microbiology and Infection, Volume 8 Number 8, 2002 colonies on cysteine heart agar with chocolatized Brucella grows slowly, forming small, non- blood [12,14]. Cultures suspected of growing F. hemolytic,glistening,convex,bluish–whitecolonies tularensis should be sent to a reference laboratory on SBA. A presumptive identification of Brucella can for confirmation by agglutination or ELISA tests be made on the basis of the Gram stain; non-hemo- [2,14]. Furthermore, a TaqMan 50-nuclease assay lytic colonies that do not ferment glucose or lactose directed against the F. tularensis outer-membrane are obligate aerobes, and are oxidase and urease protein gene, and a PCR enzyme immunoassay positive [12,16]. Urease production can easily be directed against the tul4 gene, may be applied [2]. detected on Christensen urea agar [12,14,16]. The definitive identification of Brucella species is based on CO requirement, H S production, sensitivity to BRUCELLA 2 2 fuchsin, thionin and thionin blue dyes, and agglu- Brucella species are small, faintly staining, Gram- tination with monospecific sera [4,14,16]. Clinical negative, single-appearing coccobacilli or short laboratories that are not able to identify Brucella rods, arranged in pairs and short chains. They species definitively should try a presumptive iden- are non-motile, non-sporulating strict aerobes, tification/differentiation with other coccobacilli, usually oxidase positive, catalase positive, and leaving the definitive identification to a reference urease variable [4,14,16]. The metabolism of Bru- laboratory (Table 5) [12,14]. cella is mainly oxidative on carbohydrates in con- Serology is helpful in the diagnosis of infection ventional media [16]. Thiamine, niacin and biotin due to Brucella. Acute-phase serum should be are required for growth; some strains require the obtained at the onset of the infection, followed addition of serum [16]. The optimal growth tem- by convalescent-phase serum collected after 14– perature is 37 8C, with a range between 10 8C and 21 days [16]. 40 8C [16]. The genus Brucella has six species— Brucella abortus, Brucella melitensis, Brucella suis, YERSINIA PESTIS Brucella canis, Brucella ovis, and Brucella neoto- mae—each including different biotypes [16]. Of Human plague is caused by Y. pestis, and there are those species, the first four are associated with three clinical forms: bubonic plague, pneumonic human disease, Brucella melitensis being consid- plague, usually secondary to bubonic plague, and ered the most virulent [12,14]. Reports have been a septicemic form. Y. pestis is a Gram-negative, published of Brucella as an occupational hazard to facultative anaerobe, and is a member of the Enter- laboratory workers [12,14]. obacteriaceae [18,19]. In a smear, fat, single or Since Brucella infects the reticuloendothelial sys- short-chained, sometimes bipolar, bacilli can be tem, blood and bone marrow are crucial clinical seen [20]. Even though the bipolar staining, e.g. in specimens (Table 3). Depending on the infectious a Wright–Giemsa stain, is typical of Yersinia,itis complications, other specimens (cerebrospinal not enough to identify the organism [19,20]. Like fluid, biopsies, abscess, etc.) should be submitted other Yersinia species, Y. pestis does not react, or for culture [14,16]. Specimens are refrigerated if reacts unreliably, in commonly used biochemical immediate inoculation is not possible [12,16]. tests [18]. It is a fastidious organism that does not Blood and other fluids can be cultured in a Cas- form spores and is non-motile [18,20]. Lymph taneda bottle, incubated with 5–10% CO2 [16]. node aspirates, spleen or liver biopsies and blood Other blood culture systems may be used for and sputum specimens are inoculated on SBA and detection of Brucella, but for optimal detection MAC and in brain–heart infusion broth [4,20]. subcultures are needed [12,14,17]. Tissues, fibrous Bacteremia is characteristically intermittent, so clots and exudates should be inoculated onto SBA, multiple blood cultures should be done to increase CA, MAC, and, if available, BCYE, after being sensitivity [4]. The optimal growth temperature of aseptically crushed [4,14,16]. When contam- Y. pestis is between 25 8C and 30 8C [4,19,20]. There- ination is likely, a selective medium should be fore, for rapid recovery of the organism, the speci- used, such as Farrell’s medium, or modified mens should be incubated at 28 8C. Y. pestis Thayer–Martin medium [16]. All cultures should expresses a temperature-regulated antigen (F1) be incubated for 21 days in 5–10% CO2 at 35–37 8C; when incubated at 37 8C that can be used for for subcultures, 7 days of incubation is sufficient identification [20]. Colonies grow slowly on SBA [4,12,14,16]. and have the appearance of fried eggs when

viewed under the stereomicroscope [19,20]. After incubation for 48 h, the colonies are non-hemolytic, 1–2 mm in diameter, gray–white to slightly yellow, and opaque [20]. Y. pestis grows as lactose-nega-

Francisella tularensis Various À À tive colonies on MAC [4]. After 24–48 h in brain– heart infusion broth, Y. pestis gives characteristic growth of ﬂocculent or crumbly clumps at the sides and bottom of the tube, while the rest of the medium remains clear [20]. The clumps stay visible even in a cloudy, contaminated broth culture [20]. Although the clumping growth in brain– heart infusion broth is very suggestive of Y. pestis, Haemophilus influenzae Various V Y. pseudotuberculosis and Streptococcus pneumoniae may exhibit the same type of clumping growth [20]. Y. pseudotuberculosis and Y. pestis can be differentiated by urease production, being positive and negative, respectively [18]. spp. The F1 antigen of Y. pestis is detected in serum samples by an immunoassay [2,18,20]. Molecular 0

Urinary tract detection methods include a 5 -nuclease PCR, for the detection of the plasminogen activator gene (pla) in blood and oropharyngeal swabs, and a microchip PCR array. The assays have detection thresholds of 2.1 105 copies of the pla target and of 105 cells/L, respectively [2]. þþ V Psychobacter phenylpyruvicusVarious Oligella CLOSTRIDIUM BOTULINUM Clostridium botulinum is a slightly curved Gram- positive, sporulating motile rod [4], producing spp. seven distinct (A–G) neurotoxins, which are the most toxic compounds known. Three forms of botulism are known: food-borne botulism (intox- ication) is the classical form; wound botulism V Acinetobacter Various (infection and toxin production) is the rarest form; and infant botulism (colonization of the intestines,

species [15] and toxin production) is the most common form [4,21]. As biological weapons, botulinum toxins may be aerosolized or used to sabotage food sup- spp. Brucella plies [2]. Inhalation causes the same symptoms as ingestion, but the toxin is less likely to be found in þÀ þþ þ ÀÀÀÀþÀ Brucella Blood serum and stool [4]. In general, clinical laboratories are not involved in the identiﬁcation of Clostridium botulinum or in detection of the neurotoxins [4]. A mouse bioassay is classically used to detect the presence of toxin in stools, vomitus, gastric contents and serum [2,21]. Competitive reverse tran- scription PCR measures the level of toxin- encoding messenger RNA in Clostridium botuli- Presumptive differentiation of num, and is more rapid and sensitive than the bioassay [2]. Other PCR assays are used to detect Gram stain morphologyOxidase Urea hydrolysis X or Tiny V coccobacilli factor requirement Broad coccobacilli Broad coccobacilli Tiny coccobacilli Small coccobacilli Tiny coccobacill Table 5 Test Specimen source the C1 toxin gene in sediment and the type B gene

ß 2002 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 8, 455–466 464 Clinical Microbiology and Infection, Volume 8 Number 8, 2002 in feces [2]. These techniques are used when asses- the lungs, liver, spleen, and kidneys. Specimens sing the infectious forms of botulism, in which the can be stored at À20 8C or, even better, at À70 8C toxin is produced by ingested organisms, but [22]. Electron microscopy is commonly used, and would be ineffective in classical food-borne botu- is very reliable for the preliminary identification of lism, in which the causative agent is primarily characteristic brick-shaped virions, although no toxin and not Clostridium botulinum itself [2]. Clos- differentiation between smallpox and monkeypox tridium botulinum may be cultured from speci- is possible [2,4,22,23]. Guarnieri bodies, which are mens, and isolates are subsequently tested for eosinophilic aggregations of variola particles, can toxogenicity [4]. Commercial identification sys- be seen in Giemsa-stained smears of clinical speci- tems are not very reliable, and may misidentify mens, under a light microscope [4,23]. Different Clostridium botulinum. human cell cultures are also used for culture of smallpox, and detection of Guarnieri bodies. Var- iola may produce a cytopathogenic effect within 1– SMALLPOX 3 days, depending on the inoculum (Table 6). Pox Smallpox (variola) is a double-stranded DNA lesions in chorioallantoic membranes of chicken virus that belongs to the genus orthopoxvirus. embryos will develop if clinical specimens Dissemination from person to person occurs via infected with smallpox are inoculated. Soluble aerosols from oropharyngeal secretions, by direct viral antigen is found in blood, vesicular/pustule contact, or via contaminated clothing [2,22]. contents and extracts from scrapings. It can be Laboratory confirmation of the diagnosis of detected with complement fixation, immunofluor- smallpox is only performed in high-containment escence, or Ouchterlony techniques [4]. The ser- facilities (BSL-4). Clinical specimens that contain ologic diagnosis is established by a four-fold rise in smallpox viruses remain infectious at room tem- titer in complement fixation or hemagglutination perature for 1 year, for several months at 4 8C, and inhibition [4,22]. The ultimate identification and for years at À20 8CtoÀ70 8C [4]. Smallpox virus characterization of smallpox is accomplished using produces a soluble hemagglutinin [22]. The virus PCR and restriction length polymorphism [2]. is detected in vesicular or pustular fluid collected from scraped lesions. Vesicular fluid is collected in VIRAL HEMORRHAGIC FEVERS capillary tubes, on swabs, or on glass slides as a thick smear, but without spreading the smear. The viral hemorrhagic fever syndrome may be caus- Suitable autopsy specimens include sections of ed by several RNA viruses from the Filoviridae,

Table 6 Diagnosis of variola by laboratory tests [4]a

Detection or Antigen Detection of Microscopic isolation of virusb detectionc antibodiesd Stage Material used examination (1 h) (1–3 days) (3–24 h) (3 h to 3 days)

Pre-eruptive Blood þ/Àþ/ÀÀ Maculopapular Blood þ/Àþ/Àþ/À Skin lesions þþ þ Saliva þ Vesicular Blood þ/Àþ Skin lesions þþ þ Pustular Blood þ Pustular fluid þ/Àþ þ Crusting Blood þ Crusts Àþ þ aThe time required for completion of each test is given. Results: þ, usually positive; À, usually negative; þ/À, positive or negative. bCulture on chorioallantoic membrane of chicken embryos or in tissue culture. cComplement fixation, agar gel precipitation, or immunofluorescence. dHemagglutination inhibition, complement fixation, or neutralization. Antibodies may appear earlier in previously vaccinated patients. þ, rise in antibody titer.

Arenaviridae, Bunyaviridae, and Flaviviridae [2]. 11. Doyle RJ, Keller KF, Ezzell JW. Bacillus. In: These viruses are highly infectious by the aerosol Lennette EH, Balows A, Hausler WJ Jr, Shadomy route. Only BSL-4 facilities are equipped for the HJ, eds. Manual of clinical microbiology, 4th edn. diagnosis of viral hemorrhagic fevers. Culture, Washington, DC: American Society for Microbiol- ogy, 1985: 211–15. serology, immunohistochemical techniques and 12. Weaver RE, Hollis DG, Bottone EJ. Gram-negative amplification methods are used for diagnosis fermentative bacteria and Francisella tularensis. In: [2,4,24]. Lennette EH, Balows A, Hausler WJ Jr, Shadomy Blood, urine and throat swabs or washings are HJ, eds. Manual of clinical microbiology, 4th edn. used for virus isolation; liver biopsies are used Washington, DC: American Society for Microbiol- postmortem. Virus isolation is usually successful ogy, 1985: 309–29. only if specimens are obtained within the first few 13. Centers for Disease Control and Prevention. Basic days of illness. For isolation, blood, cerebrospinal laboratory protocols for the presumptive identification of Francisella tularensis. Atlanta: Centers for Dis- fluid, brain or other tissues should be collected, ease Control and Prevention, 2001. http://www. 8 and refrigerated at 4 C. Heparinized whole blood bt.cdc.gov/Agent/Tularemia/ftu_la_cp_121301. or homogenized clots are satisfactory for virus pdf isolation [24]. 14. Koneman EW. Miscellaneous fastidious Gram- negative bacteria. In: Koneman EW, Allen SD, REFERENCES Janda WM, Schreckenberger PC, Winn WC Jr, eds. Color atlas and textbook of diagnostic micro- 1. Kortepeter MG, Parker GW. Potential biological biology. Philadelphia: Lippincott-Raven, 1997: weapons threats. Em Infect Dis 1999; 5: 523–7. 395–472. 2. Broussard LA. Biological agents: weapons of war- 15. Centers for Disease Control and Prevention. Basic fare and bioterrorism. Mol Diagn 2001; 6: 323–33. laboratory protocols for the presumptive identification of 3. Jortani SA, Snyder JW, Valdes R Jr. The role of the Brucella species. Atlanta: Centers for Disease Control clinical laboratory in managing chemical or biolo- and Prevention, 2001. http://www.bt.cdc.gov/ gical terrorism. Clin Chem 2000; 46: 1883–93. Agent/Brucellosis/bsp_cla_cp_121201.pdf 4. Klietmann WF, Ruoff KL. Bioterrorism: implica- 16. Hausler WJ, Moyer NP, Holcomb LA. Brucella. In: tions for the clinical microbiologist. Clin Microbiol Lennette EH, Balows A, Hausler WJ Jr, Shadomy Rev 2001; 14: 364–81. HJ, eds. Manual of clinical microbiology, 4th edn. 5. Centers for Disease Control and Prevention and Washington, DC: American Society for Microbiol- National Institutes of Health. Biosafety in microbio- ogy, 1985: 382–6. logical and biomedical laboratories, 4th edn. Washing- 17. Reimer LG, Wilson ML, Weinstein MP. Update on ton, DC: US Department of Health and Human detection of bacteremia and fungemia. Clin Microb Services, Public Health Service, 1999. Rev 1997; 10: 444–65. 6. Centers for Disease Control and Prevention. Basic 18. Kelly MT, Brenner DJ, Farmer JJ III. Enterobacter- laboratory protocols for the presumptive identification of iaceae. In: Lennette EH, Balows A, Hausler WJ Jr, Bacillus anthracis. Atlanta: Centers for Disease Shadomy HJ, eds. Manual of clinical microbiology, 4th Control and Prevention, 2001. http://www.bt. edn. Washington, DC: American Society for Micro- cdc.gov/Agent/Anthrax/LevelAProtocol/Anthra- biology, 1985: 263–81. cis20010417.pdf 19. Koneman EW. The Enterobacteriaceae. In: Kone- 7. Koneman EW. The aerobic Gram-positive bacilli. man EW, Allen SD, Janda WM, Schreckenberger In: Koneman EW, Allen SD, Janda WM, Schreck- PC, Winn WC Jr, eds. Color atlas and textbook of enberger PC, Winn WC Jr, eds. Color atlas and diagnostic microbiology. Philadelphia: Lippincott- textbook of diagnostic microbiology. Philadelphia: Raven, 1997: 171–252. Lippincott-Raven, 1997: 651–708. 20. Centers for Disease Control and Prevention. Basic 8. World Health Organization. Guidelines for the laboratory protocols for the presumptive identification of surveillance and control of anthrax in humans and Yersinia pestis. Atlanta: Centers for Disease Control animals. Emerging and other communicable diseases, and Prevention, 2001. http://www.bt.cdc.gov/ surveillance and control, 3rd edn. Geneva: WHO, 1998. Agent/Plague/ype_la_cp_121301.pdf http://www.who.int/emc-documents/zoonoses/ 21. Midura TF. Update: infant botulism. Clin Microb whoemczdi986c.html Rev 1996; 9: 119–25. 9. Dixon TC, Meselson MM, Guilemin JG, Hanna PC. 22. Nakano JH. Poxviruses. In: Lennette EH, Balows A, Anthrax. N Engl J Med 1999; 341(11): 815–26. Hausler WJ Jr, Shadomy HJ, eds. Manual of clinical 10. Turnbull PCB. Definitive identification of Bacillus microbiology, 4th edn. Washington, DC: American anthracis—a review. J Appl Microbiol 1999; 87: 237–40. Society for Microbiology, 1985: 733–41.

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