Laboratory Safety Practices Associated with Potential Agents of Biocrime or Bioterrorism

The anthrax incident of 2001 in the United States clearly documented the threat posed by the intentional release of an infectious agent in a susceptible population. It also demonstrated that clinicians and clinical microbiology laboratories are key to the early detection of disease, identification of the putative agent, and notification of appropriate authorities. To be effective in this role, laboratories must be prepared for a possible biocrime or bioterrorism event. Preparation requires that laboratories have an awareness of the potential agents that may be used, laboratory techniques for the early identification of these agents, procedures for the management of the event, and knowledge of the safety precautions necessary to safely handle these infectious agents (7). Once prepared, laboratory personnel must constantly be alert for the possible isolation of these agents during the routine manipulations of cultures at the bench (10). With the exception of smallpox virus and viral hemorrhagic fever (VHF) agents, most of the biothreat agents are occasionally isolated from patients who have been naturally infected. To ensure a safe work environment, the laboratory must implement and strictly adhere to the routine safety practices that minimize risk to laboratory personnel (8, 9).

LABORATORY PREPAREDNESS

On a national level the Centers for Disease Control and Prevention (CDC) have developed a Bioterrorism Preparedness and Response Program that addresses a unified public health response to bioterrorism events (http://www.bt.cdc.gov). The program addresses disease surveillance; rapid laboratory diagnosis of biological agents; epidemiologic investigations; communication among local, state, and federal public health authorities; preparedness planning; and readiness assessment. Part of the program includes the development of the Laboratory Response Network to integrate multilevel laboratories (local, state, federal, military, veterinary, and environmental). The Laboratory Response Network provides for the rapid identification of biological agents and a capacity to respond to bioterrorism and other public health emergencies. The classification of laboratories is based on their presumed role in a bioterrorism event that is related to their safety and technical capability (11). Originally, this model contained four levels of laboratories (levels A through D) but these have evolved to essentially three levels: (i) Sentinel Laboratories (formerly level A), whose role is to recognize, rule out, and refer organisms, (ii) Reference Laboratories (formerly levels B and C), whose role is confirmatory testing, and (iii) two National Laboratories (formerly level D), i.e., the CDC and U.S. Army Medical Research Institute of Infectious Diseases. Most commercial and clinical laboratories are classified as Sentinel Laboratories.

ROLE OF THE SENTINEL LABORATORIES

The early recognition of a biocrime or bioterrorism event by the laboratory rests on microbiologists rapidly recognizing potential agents of bioterrorism (11). The agents that are likelier to be used in these incidents are not commonly encountered in most clinical microbiology laboratories (Table 1) (http://www.bt.cdc.gov). Because many of these agents grow in conventional culture media and are not easily recognized by laboratory personnel, the ordering physician or autopsy pathologist should notify the laboratory if the diagnosis is suspected. These potential agents of bioterrorism have been placed into three categories based upon (i) the ability of the agent to be easily disseminated, (ii) the ability to be transmitted from person to person, (iii) the ability to cause significant morbidity and mortality, and (iv) the ability to cause public panic and social disruption ( ttp://www.bt.cdc.gov/Agent/agentlist.asp). Category A agents pose the greatest threat because they can be easily disseminated or transmitted from person to person, are highly infectious, have the potential for major public health impact, and require special action for public health preparedness (1). The characteristics of category A agents and Brucella spp. and their associated disease states are listed in Table 2 (http://www.bt.cdc.gov). Category B agents are moderately easy to disseminate and have moderate morbidity and low mortality. Category C agents consist of emerging pathogens that could be engineered for mass dissemination in the future. Because most of these agents are rarely isolated and are often difficult to rapidly identify, clinical microbiology laboratories must employ diagnostic test protocols or algorithms for ruling out suspicious organisms encountered during the routine manipulation of cultures. These protocols are available on the CDC (http://www.bt.cdc.gov) and American Society for Microbiology (http://www.asmusa.org/pcsrc/biodetection.htm) websites and appear in other publications (12). To be effective, these protocols should be incorporated directly into the bench procedure to ensure rapid recognition and referral of any suspicious isolate to a Reference Laboratory (levels B and C) and, most importantly, to ensure safe handling of the agent (10). Protocols for identification of smallpox virus and botulism toxin are not listed because the Sentinel Laboratories (level A) should not process these specimens but should rather send the smallpox virus specimens to the CDC and the botulism toxin specimens to the Reference Laboratory. The CDC maintains an emergency telephone number ([770] 488-7100) with instructions for the collection and shipment of specimens. Sentinel Laboratories (level A) should not accept or process environmental or animal specimens for biocrime or bioterrorism agents. These specimens, after consultation with the Reference Laboratory and the Federal Bureau of Investigation, should be forwarded directly to the Reference Laboratory (levels B and C) that has the safety and technical capability to process them.

TABLE 1.

Categories of potential agents and diseases

Category	Agent(s)	Disease
A	Bacillus anthracis	Anthrax
	Clostridium botulinum toxin	Botulism
	Yersinia pestis	Plague
	Variola major virus	Smallpox
	Francisella tularensis	Tularemia
	Filoviruses (Ebola and Marburg viruses)	VHF
	Arenaviruses (Lassa and Machupo viruses)
B	Brucella species	Brucellosis
	Burkholderia mallei	Glanders
	Burkholderia pseudomallei	Melioidosis
	Chlamydia psittaci	Psittacosis
	Coxiella burnetii	Q fever
	Rickettsia prowazekii	Typhus fever
	Food safety threats (e.g. Salmonella spp., Shigella spp., Escherichia coli O157)
	Water safety threats (Cryptosporidium parvum and Vibrio cholerae)
	Alphaviruses (VEE, EEE, and WEE)a	Encephalomyelitis
	Toxins (e.g., ricin and staphylococcal enterotoxin B)
C	Bunyaviruses (hantavirus and Crimean-Congo virus)	Hemorrhagic fever
	Flaviviruses (yellow fever and dengue viruses)	Yellow fever and dengue
	Nipah virus	Encephalomyelitis
	Tickborne viruses	Hemorrhagic fever or encephalitis

^aVEE, Venezuelan equine encephalomyelitis virus; EEE, Eastern equine encephalomyelitis virus; and WEE, Western equine encephalomyelitis virus.

TABLE 2.

Summary of diseases resulting from bioterrorism agents

Disease	Virulence factor(s)	Infective dose	Incubation period	Duration of illness	Mortality rate(s)	Person-to-person transmission (risk)	Isolation precautions for hospitalized patientsg	Persistence or stability of organism
Inhalation anthrax	Exotoxina and capsule	8,000 to 50,000 spores	1 to 6 days	3 to 5 days	≈100% if untreated; ≈99% if treated	No	Standard	In soil, for ≈40 yr
Brucellosis	LPS and PMNb	10 to 100 organisms	5 to 60 days	Weeks to months	≈5% if untreated; <1% if treatede	No	Standard	In water or soil, for ≈10 wks
Botulism	Neurotoxin	0.001 μg/kgd	6 h to 10 days	24 to 72 h	25% for first patient of outbreak; 4% for subsequent cases; 5 to 10% overall	No	Standard	In food or water, weeks
Tularemia	Intracellular survival	10 to 50 organisms	1 to 21 days	≈2 weeks	33% if untreated; <4% if untreated	No	Standard	In moist soil, for months
Pneumonic plague	V and W antigens LPS endotoxin, and F1 antigenc	<100 organisms	2 to 3 days	1 to 6 days	40 to 70% if untreated; 5% if treated	Yes (high)	Dropleth	In soil, for ≥1 yr
Smallpox		10 to 100 particles	7 to 17 days	≈4 weeks	<1% for variola minor; 20 to 50% for variola major	Yes (high)	Airborneh	Very stable
VHF		1 to 10 particles	4 to 21 days	7 to 16 days	53 to 88%	Yes (moderate)	Airborne and contacth	Unstable

^aB. anthracis exotoxin(s) consists of 3 components: the edema factor and lethal factor exert their effect within cells by interacting with a common transport protein, designated protective antigen (so named because, when modified, it contributes to vaccine efficacy). Expression of toxic factors is mediated by one plasmid and that of the capsule (d-glutamic acid polypeptide) is mediated by a second plasmid. Strains repeatedly subcultured at 42°C become avirulent as a result of losing virulence-determining plasmids; this loss is thought to be the basis for Pasteur's attenuated anthrax vaccine used at Pouilly-le-Fort, France, in 1881.

^bThe major virulence factor for brucellosis appears to be an endotoxic lipopolysaccharide (LPS) among smooth strains. Pathogenicity is related to an LPS containing poly N-formyl perosamine O chain, Cu-Zn, superoxide dismutase, erythrulose phosphate dehydrogenase, intracellular survival stress-induced proteins, and adenine- and guanine-monophosphate inhibitors of phagocyte functions. PMN, polymorphonuclear leukocyte.

^cThe V and W antigens and the F1 capsular antigens are expressed only at 37°C and not at the lower (body) temperature of the flea (20 to 25°C).

^dDose applies for type a botulism.

^eEndocarditis accounts for the majority of brucellosis-related deaths.

^fPeriods of communicability are as follows: for inhalation anthrax, brucellosis, botulism, or tularemia, none (no evidence of person-to-person transmission); for pneumonic plague, 72 h following initiation of appropriate antimicrobial therapy (or until sputum culture is negative); for smallpox, approximately 3 weeks (usually corresponds with the initial appearance of skin lesions to their final disappearance; most infectious during the first week of rash via inhalation of virus released from oropharyngeal lesion secretions of the index case); for VHF, at least for the duration of illness (varies with virus); and for Ebola or Marburg virus, up to 7 weeks after clinical recovery (transmission through semen).

^gGuideline for isolation precautions in hospitals (4b) (http://www.cdc.gov/ncidod/hip/isolat/isolat.htm).

^hIn addition to standard precautions, which apply to all patients.

LABORATORY SAFETY MEASURES

The category A and B agents have all been associated with laboratory-acquired infections (9, 13). However, most occupationally acquired infections caused by these microorganisms occurred because laboratory personnel did not recognize the risk associated with these infectious agents and failed to follow routine biosafety level 2 (BSL2) safety practices, especially practices for containment of aerosols. To minimize risk, management must implement a safety program that assesses risk associated with laboratory practices, provides appropriate containment facilities and personal protective equipment (PPE), writes safety procedures, and supports exposed individuals with medical care. It is the individual's responsibility to understand the risk associated with handling infectious agents and to employ safe practices to minimize exposure to other laboratory workers and to prevent contamination of the laboratory. Laboratories should institute the safety recommendations specified in the CDC and National Institutes of Health's publication “Biosafety in Microbiology and Biomedical Laboratories” (9), which can be accessed on the CDC's website (http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm).

It is likely that the first patients seen in a hospital following release of a bioterrorism agent will be routinely evaluated. These early cultures pose the greatest risk to clinical microbiologists because the agent may not be initially recognized. Once an outbreak is identified, Sentinel Laboratories without adequate staffing or safety facilities to handle the volume of specimens may choose to restrict receipt of specimens (http://www.asmusa.org/pasrc/intro.htm). The agents that pose the greatest risk are transmitted by aerosols produced by routine laboratory practices, such as pipetting, flaming inoculation loops, subculturing blood bottles, streaking agar plates, sonication, homogenization, mixing bacterial suspensions, expelling bubbles from syringes, and centrifugation (8). Specimens should be handled in a class II biological safety cabinet (BSC). Laboratory personnel should wear protective eyewear, closed laboratory coats with cuffed sleeves, and gloves stretched over the cuffs; avoid touching mucosal surfaces with their hands; and remove gloves and coat and wash hands prior to leaving the laboratory. Benches should be decontaminated after use, and waste material should be placed in appropriate biohazard containers (2, 8, 9).

To categorize the risk associated with an infectious agent and define the appropriate safety practices for handling the agent, the CDC and National Institutes of Health have proposed four BSLs (9). The BSLs imply increased occupational risk from exposure to an agent and need for additional containment for work with that agent. The guidelines for assigning microorganisms to a BSL are as follows: BSL1, well-characterized agents that are not known to consistently cause disease in healthy adult humans and are considered a minimal potential hazard to laboratory personnel and the environment; BSL2, agents of moderate potential hazard to personnel and the environment; BSL3, indigenous or exotic agents that cause serious or potentially lethal disease as a result of exposure by the inhalation route; and BSL4, dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease.

Most clinical microbiology laboratories (Sentinel Laboratories or level A laboratories) function at BSL2 and employ BSL2 laboratory practices (Table 3) (http://www.bt.cdc.gov) (5, 8, 11). In general, clinical specimens containing these agents can be handled at BSL2 because few organisms are present. The exposure risk increases when the infectious agent is amplified in culture or when laboratory practices that may generate aerosols are used to manipulate the culture. In these situations, BSL3 practices should be employed (Table 4) (http://www.bt.cdc.gov). BSL2 practices require that all manipulations that produce an aerosol be performed in a class II BSC, whereas BSL3 practices specify that all manipulations should be performed in a BSC. Routine manipulations of cultures (e.g., preparation of bacterial suspension in saline) may potentially produce aerosols. Aerosol production during the routine manipulation of cultures of Neisseria meningitidis may have caused laboratory-acquired infections that resulted in death (3). Laboratories should assess the risk of aerosol production for all procedures, including automated instrumentation (8). Certain procedures such as sonication or vortexing of bacterial suspensions of unidentified organisms are likely to produce an aerosol and should be performed in a BSC (http://www.asmusa.org/pasrc/intro.htm). All procedures that produce aerosols should be performed in a BSC or discontinued. Once an agent is identified as a BSL3 pathogen or when a specimen is submitted to rule out a BSL3 agent, the material should be labeled as BSL3 and all subsequent manipulations should be performed in a BSC or in a BSL3 laboratory where available. After laboratory practices that produce aerosols are identified and contained or discontinued, the risk associated with handling BSL3 agents will be minimized.

TABLE 3.

Summary of BSL1 and 2 precautions containment^a of infectious agents

BSLd	Description of agent(s)	Primary containmentb		Secondary containment facilitiesc (secondary barriers)
		Practices or techniques to follow	Safety equipment (primary barriers)
1	Well-characterized agents (e.g. Bacillus subtilis) not known to consistently cause disease in healthy adults and of minimal potential hazard to laboratory personnel and environmente	BSL1 practices: Laboratory personnel specifically trained to perform any procedure conducted in the laboratory Supervision by a scientist with general training in microbiology Limited access to laboratory while experiments are in progress Washing of hands after handling of cultures and before exit from laboratory Eating, drinking, insertion of contact lenses or application of cosmetics, and storage of food in the laboratory prohibited Mouth pipetting prohibited “Sharps” policy instituted Creation of aerosols minimized during all procedures Work surfaces decontaminated after spills and at end of day Waste disposal policy instituted Biohazard sign (with name of agent(s) and name and phone number of supervisor) posted at entrance when infectious agents are present Insect and rodent control program in effect	For BSL1, none required but the following are recommended: Work performed on open bench top Lab coats, gowns, or uniforms worn to protect street clothes Gloves worn if skin on hands is broken or if a rash is present (alternatives to powdered latex gloves should be available) Protective eyewear worn for procedures in which splashes are anticipated Persons wearing contact lenses also wear goggles or face shields	At least a sink for washing hands but also the following: Laboratory is not necessarily separated from the general traffic patterns of the building Laboratory should have doors for access control Laboratory designed to be cleaned easily (carpets and rugs are not appropriate) Bench tops impervious to water and resistant to moderate heat, organic solvents, acids, alkalis or chemicals used to decontaminate the work surfaces Furniture can support anticipated weight load and uses, with spacing between cabinets, benches, and equipment accessible for cleaning Windows fitted with screens
2	Agents associated with human disease (e.g., Bacillus anthracis, Shigella spp., and Yersinia pestis)f	BSL1 practices plus the following: Laboratory personnel are specifically trained to handle pathogenic agents and are directed by competent scientists Policies and procedures allow only persons meeting specific entry and/or training requirements to enter laboratory Individuals at increased risk of acquiring infection are allowed limited access to or are restricted from entering the laboratory area Posted biohazard sign (as above) lists biosafety level, required immunization, required personal protective equipment, and any procedures required for exiting laboratory Immunizations or tests for agents in laboratory (e.g., hepatitis B virus vaccine or TB skin testing) are provided Personnel receive appropriate training in safety precautions, exposure prevention, and “sharps” precautions and receive annual updates for procedure/policy changes Biosafety manual defining infectious waste handling or decontamination and medical surveillance policies is available Decontamination policy for work surfaces, spills, and contaminated equipment is established An accident policy involving an accidental or overt exposure to infectious materials is established that requires immediate reporting to the laboratory director for documentation; medical evaluation, surveillance, and any necessary treatment	For BSL2: Properly maintained BSCg (Class I or II but preferably Class II) for all manipulations involving splashes or aerosols of infectious materials Personal protective equipment, including the following: Protective laboratory clothing (this clothing is removed and left in the laboratory area prior to exit, and it is either disposable or laundered by the institution; it should never be taken home) Gloves are worn when hands may contact potentially infectious materials, surfaces, or equipment; disposable gloves are not to be reused, washed, or used to touch “clean surfaces” (telephones, etc.); hands are washed following glove removal Face protection (goggles, mask, face shield, or splatter guard) is used for anticipated splashes or sprays of hazardous materials for manipulations outside the BSC	BLS1 requirements plus the following: Autoclave available Doors for facilities that house restricted agents can be locked Laboratory separated from general traffic patterns and away from public areas Sinks for handwashing equipped with foot, knee, or automatic faucet operation BSC optimally located for containment Eyewash station readily available

^aContainment refers to safe methods for managing infectious materials in the laboratory environment; its purpose is to reduce or eliminate exposure of laboratory workers, other persons, and the outside environment to potentially hazardous agents.

^bPrimary containment is the protection of personnel and the immediate laboratory environment from exposure to infectious agents. The most important element of containment is strict adherence to standard microbiological practices and techniques.

^cSecondary containment is the protection of the environment external to the laboratory from exposure to infectious materials, provided by facility design and operational practices.

^dRisk assessment factors, such as pathogenicity, route of transmission, agent stability, infectious dose, organism concentration, specimen origin, animal study data, availability of prophylaxis, medical surveillance, and technical proficiency, are but a number of elements that contribute to the establishment of a given biosafety level.

^eAppropriate for undergraduate and secondary educational training and teaching laboratories.

^fBSL2 recommendations and Occupational Safety and Health Administration requirements focus on the prevention of percutaneous, ingestive, and mucous membrane exposure(s) to clinical materials.

^gBSC, Biological Safety Cabinet.

TABLE 4.

Microbiology biosafety

Agent	BSL for:		Specimen types posing risk in case of exposure	Recommended precautions for level A laboratories
	Specimen handling	Culture handling		BSL2	BSL3	BSL4
Bacillus anthracis	2	2	Blood, skin lesion exudates, CSF, pleural fluid, sputum, and, rarely, urine and feces	Activities may include collection of clinical material and handling of only diagnostic quantities of infectious cultures	Activities may include those with high potential for aerosol or droplet production
Brucella spp.a	2	3	Blood, bone marrow, CSF, tissue, semen, and occasionally urine	Activities limited to collection, transport and plating of clinical material	All activities involving manipulation of cultures
Clostridium botulinumb	2	2	Food specimens, clinical material (serum, gastric, and fecal specimens), and environmental samples (soil and surface water)	Activities with materials known or potentially containing toxin allowed but must be handled in a Biological Safety Cabinet (Class II) with a lab coat, disposable surgical gloves, and a face shield (as needed)	Activities with high potential for acrosol or droplet production
Francisella tularensisc	2	3	Skin lesion exudates, respiratory secretions, CSF, blood, and urine, as well as tissues from infected animals and fluids from infected arthropods	Activities limited to collection, transport, and plating of clinical material	All activities involving manipulations of cultures
Yersinia pestisd	2	2	Bubo fluid, blood, sputum, CSF, feces, and urine	Activities may include collection of clinical material and handling of only diagnostic quantities of infectious cultures	Activities with high potential for aerosol or droplet production
Smallpoxe	4	4	Lesion fluid or crusts, respiratory secretions, and tissue			Specimen collection and/or transport
VHFf	4	4	Blood, urine, respiratory and throat secretions, semen, and tissue			Specimen collection and/or transport

^aLaboratory-acquired brucellosis has occurred via sniffing of cultures; aerosols generated by centrifugation; mouth pipetting; accidental parenteral inoculations; and sprays into eyes, nose, and mouth; and, finally, by direct contact with clinical specimens.

^bThe toxin may be present in the specimen types listed and is extremely poisonous. Exposure to toxin is the primary laboratory hazard since absorption can occur with direct contact with skin, eyes, or mucous membranes, including the respiratory tract. The toxin can be neutralized by 0.1 M sodium hydroxide. C. botulinum is inactivated by a 1:10 dilution of household bleach. Contact time is 20 min. If 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 or animals. Direct contact of skin or mucous membranes 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 exposure of mucous membranes to droplet or aerosol form, and exposure of broken skin to infectious fluids or tissues are the primary hazards to laboratory workers.

^fExposure of respiratory passages to infectious aerosols, exposure of mucous membranes to infectious droplets, and accidental parenteral inoculation are the primary hazards to laboratory workers.

The potential release of BSL4 agents, such as smallpox virus or VHF agents, poses a critical risk to health care and laboratory personnel. When infection by these agents is suspected, the CDC and the state health department should be notified. Instructions for the collection and shipment of specimens directly to the CDC can be obtained during this communication. Many state public health care practitioners have received training in the recognition of infection caused by these agents. The BSL4 agents are extremely hazardous and when amplified should be contained in a BSL4 facility at the CDC or the U.S. Army Medical Research Institute of Infectious Diseases. The unsuspected amplification of a BSL4 agent in a routine virology laboratory is a potential risk. Virology laboratories should assess this potential, and laboratory workers should become familiar with the cell lines that support the growth of BSL4 agents and the cytopathic effect produced in each cell line (http://www.bt.cdc.gov). All virology laboratories should have a BSC, and their members should be trained in the correct use of a BSC (8) (http://www.cdc.gov/od/ohs/biosfty/bsc/bsc.htm). In the case of smallpox, the laboratory and facility should implement a clinical pathway for patients with vesicular or pustular rash illness (http://www.idsociety.org). Because most clinicians have little or no clinical experience diagnosing patients infected with VHF agents, a high degree of suspicion is also required. Adherence to standard precautions and handling clinical specimens in a BSC should minimize exposure risk to laboratory workers from smallpox virus and VHF agents.

LABORATORY SAFETY PRACTICES WITH SPECIFIC AGENTS

(i) Bacillus anthracis.

The agent may be present in a variety of clinical specimens, including blood, cerebrospinal fluid (CSF), pleural fluid, sputum, wound exudates, and, rarely, urine and feces. In clinical specimens, the B. anthracis cells are primarily vegetative, and not easily transmitted. The primary hazards to laboratory personnel are direct and indirect contact of intact and broken skin with cultures and accidental parenteral inoculation. The recent case of cutaneous anthrax in a laboratory worker manipulating an environmental sample illustrates this potential risk (4). Exposure to infectious aerosols is an additional hazard associated with the screening of environmental samples. Most Sentinel Laboratories (level A) are not trained or equipped for work with environmental samples. Samples should be transported to a Reference Laboratory (levels B and C) for testing. Clinical specimens and subsequent cultures can be handled under BSL2 practices, containment equipment, and facilities. BSL3 conditions are required for work involving production quantities, concentrations of cultures, or activities with a high potential for aerosols, including manipulation of suspect powders.

(ii) Francisella tularensis.

F. tularensis is a common cause of laboratory-acquired infection from aerosol exposure. However, most cases occur in research facilities that handle large quantities of liquid cultures. The agent may be present in nearly all clinical specimens. Laboratory hazards include direct contact of skin or mucous membranes with infectious material, accidental parenteral inoculation or ingestion, and exposure to infectious aerosols or droplets through manipulation of cultures. The greatest risk to laboratory personnel is associated with manipulation of cultures. The recommended laboratory precautions for handling clinical specimens are BSL2 practices, containment equipment, and facilities. BSL3 conditions are recommended for all activities involving cultures.

(iii) Brucella spp.

Brucellosis is the most commonly reported laboratory-acquired infection, owing in part to the low infectious dose. The agent is present in blood, CSF, semen, and, rarely, urine. Laboratory hazards include direct contact of skin or mucous membranes, accidental parenteral inoculation or ingestion, and exposure to aerosols generated during manipulation of cultures. As with F. tularensis, BSL2 conditions suffice for handling clinical specimens and BSL3 conditions are recommended for all activities involving cultures.

(iv) Yersinia pestis.

Y. pestis is a rare cause of infection in laboratory personnel. The agent may be present in bubo fluid, blood, sputum, CSF, feces, and urine. The primary hazards to laboratory personnel are direct contact with cultures and infectious materials, autoinoculation or ingestion, and exposure to aerosols or droplets produced by manipulation of cultures. BSL2 conditions are recommended for specimen and culture handling, while BSL3 practices are recommended for activities with a high potential for droplet or aerosol production.

(v) Smallpox virus.

Smallpox virus is highly transmissible and presents a significant risk to laboratory personnel. The agent is present in lesion fluid or crusts, respiratory secretions, and tissue. The primary hazards to laboratory personnel are ingestion, parenteral inoculation, and droplet or aerosol exposure of mucous membranes or broken skin with infectious material. If a patient is identified as high or moderate risk for smallpox, physicians should immediately contact the state health department before collection of specimens (http://www.idsociety.org). Only recently vaccinated personnel wearing appropriate protective equipment should collect specimens. All testing of specimens from patients at high risk for smallpox should be performed by a National Laboratory (level D). Consult the CDC [phone, (770) 488-7100] for specific instructions on the shipment of specimens. Once smallpox is confirmed in a geographical area, additional cases can be diagnosed clinically. Testing specimens on patients not at moderate or high risk for smallpox can be performed under BSL2 conditions. If smallpox cannot be ruled out through testing, contact the local or state health department.

(vi) VHF agents.

The hemorrhagic fever viruses also pose a significant risk to laboratory personnel, and laboratory-acquired infections from handling these agents are documented. The agents may be present in blood, urine, respiratory and throat secretions, semen, and urine. The primary hazards in the laboratory setting include accidental parenteral inoculation, mucous membrane exposure to infectious droplets, and exposure to infectious aerosols. Specimens from patients with suspected VHF should be referred, after consultation, to a National Laboratory for testing. At a minimum, laboratory staff including autopsy personnel should be alerted to the potential diagnosis and only designated personnel should handle specimens. When possible, point-of-care analyzers should be used at the bedside. Specimens transported to the laboratory should be identified, double bagged, and hand carried at predetermined times. No specimens should be sent in pneumatic tubes. Serum samples should be pretreated with Triton X-100 or heated at 60°C for 1 h before testing to inactivate the virus. When handling specimens, laboratory personnel should wear appropriate PPE and process specimens in a BSC following BSL3 practices (http://www.idsociety.org).

(vii) Botulism.

Botulinum toxin may be present in food, serum, gastric contents, feces, and environmental samples. Toxin can be absorbed after direct contact with skin or mucous membranes, including the respiratory tract. Materials should be handled in a BSC while using BSL2 practices, including gloves and a face shield. Manipulations producing aerosols require BSL3 conditions. Sentinel Laboratories (level A) should collect appropriate specimens and, after consultation, ship the specimens to a Reference Laboratory.

IMMUNIZATION OF LABORATORY PERSONNEL

The availability of vaccines for biocrime or bioterrorism agents and CDC's recommendations for immunization of laboratory personnel are shown in Table 5 (http://www.bt.cdc.gov). There are no generally available vaccines against brucellosis or VHF at this time, but some investigational vaccines are being evaluated for VHF (http://www.idsociety.org). Immunization of laboratory personnel against the other agents is not recommended.

TABLE 5.

Vaccines

Disease or agent	Immunity by natural exposure	Vaccine type	Vaccine efficacy in case of aerosol exposure	Comments
Anthrax	Yesa	For humans, cell-free culture filtrate of an avirulent, nonencapsulated derivative of a bovine isolate designated V770; for animals, a spore suspension of an avirulent, nonencapsulated live strain	Two-dose efficacy (against 200 to 500 LD50e in monkeys)	Required for level A Laboratory: No Laboratory-acquired cases: None reported since the late 1950s, at which time the human vaccine was introduced Immunity: Combined vaccine efficacy against both forms of anthrax (inhalational and cutaneous) = 93%
Brucellosis	Yes	For humans, no vaccine available in the United States; for animals, in 1996, RB51, a live, attenuated strain of B. abortus replaced the S19 strain, which was also a live, attenuated vaccine	No vaccine	Required for level A Laboratory: None available Laboratory-acquired cases: Brucellosis is the most commonly reported bacterial infection acquired in laboratories; one of the largest reported incidences involved 45 cases with 1 death; protection is based on adherence to BSL3 precautions Immunity: Studies of humans demonstrate that immunity is acquired after active infection; both cellular and humoral responses are required
Botulism	No	Pentavalent (ABCDE) toxoidc	Three-dose efficacy, 100% (against 25 to 250 LD50 in primates)	Required for level A Laboratory: No Laboratory-acquired cases: There has been one report of laboratory-associated botulism Immunity: In cases of foodborne exposure, immunity does not develop even with severe disease, and its repeated occurrence has been reported
Tularemia	Partial	Live, attenuated vaccine	80% protection against 1 to 10 LD50	Required for level A Laboratory: No Laboratory-acquired cases: Over the past 50 years, tuleremia has been the third-most-common bacterial infection acquired in laboratories, mostly in research laboratories; Immunity: Multiple episodes of reinfection have been documented among vaccinated laboratory personnel and in unimmunized individuals
Plague	Partial	Suspension of killed (formalin-inactivated) Y. pestis organisms	Has yet to be measured precisely in controlled studies. At least two vaccinated persons contracted pneumonic plague following exposure to Y. pestis	Required for level A Laboratory: No Laboratory-acquired cases: Few laboratory-associated cases have been reported; since 1936, only three cases of pneumonic plague have been documented Immunity: Indirect evidence, mainly from the military, indicates that the plague vaccine is effective for preventing flea-borne transmission of disease
Smallpox	Yes	Vaccinia (smallpox) vaccined (grown in the skin of vaccinated bovine calves)	Vaccine protects against large doses in primates	Required for level A Laboratory: No Laboratory-acquired cases: none Immunity: If a smallpox sample is handled at a level A laboratory, vaccination within 3 days postexposure is considered effective in preventing serious infection and death; vaccinia immune globulin may also be considered but may compromise postexposure vaccination efficacy
VHF	?b	None available	No vaccine	Required for level A Laboratory: No
				Laboratory-acquired cases: Exposure of skin and/or mucous membrane to virus-laden material, i.e., blood, cell cultures, body fluid/secretions, has been responsible for most recognized cases among humans
				Immunity: To be determined

^aSome degree of immunity is evident following cutaneous anthrax, i.e., the dose generally considered to be lethal is too low to produce an immune response.

^bImmunity to Lassa fever reinfection occurs following infection, but the length of protection is unknown.

^cDistributed by the CDC under an investigational new drug protocol and used to protect laboratory workers at high risk, i.e., those actively working with C. botulinum or the toxins.

^dDistributed by the CDC.

^eLD₅₀, 50% lethal dose.

DECONTAMINATION

Standard disinfectants (e.g., 0.5% household bleach) are considered adequate for cleaning surfaces potentially contaminated by bioterrorism agents (6). As with all biological spills, personnel should wear gloves, protective eyewear, and PPE during the cleanup process. Respiratory protection should be worn when aerosolization is suspected. Following direct exposure, clothing and body surfaces should be washed with soap and water. All laundry that is potentially contaminated with smallpox virus or VHF agents should be incinerated or autoclaved prior to washing in hot water containing bleach.

SECURITY

All of these agents are classified as select agents and are regulated by the federal government (4a). The regulation is available at http://www.cdc.gov/od/sap. Laboratories that handle select agents must meet the regulations that went into effect on 7 February 2003. For most Sentinel Laboratories, that means that none of the select agents may be kept in the laboratory unless the regulations are met. As a minimum, the CDC must be informed whenever a select agent is recovered from a clinical specimen and the destruction of the agent must be documented.