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Journal of Clinical Microbiology, July 2003, p. 2801-2809, Vol. 41, No. 7
0095-1137/03/$08.00+0 DOI: 10.1128/JCM.41.7.2801-2809.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Laboratory Safety Practices Associated with Potential Agents of Biocrime or Bioterrorism
David L. Sewell*
Pathology and Laboratory Medicine Service, Veterans Affairs Medical Center, and Department of Pathology, Oregon Health and Sciences University, Portland, Oregon 97239
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INTRODUCTION
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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).
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LABORATORY PREPAREDNESS
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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.
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ROLE OF THE SENTINEL LABORATORIES
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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.
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LABORATORY SAFETY MEASURES
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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.
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.
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LABORATORY SAFETY PRACTICES
WITH SPECIFIC AGENTS
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(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.
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IMMUNIZATION OF LABORATORY PERSONNEL
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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.
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DECONTAMINATION
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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.
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SECURITY
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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.
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FOOTNOTES
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* Mailing address: Pathology and Laboratory Medicine Service P5PATH, Veterans Affairs Medical Center, 3710 SW Veterans Rd., Portland, OR 97239. Phone: (503) 273-5363. Fax: (503) 721-7823. E-mail: david.sewell{at}med.va.gov. 
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REFERENCES
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- Centers for Disease Control and Prevention. 2000. Biological and chemical terrorism: strategic plan for preparedness and response. Recommendations of the CDC Strategic Planning Workgroup. Morb. Mortal. Wkly. Rep. 49(RR-4):1-14.[Medline]
- Centers for Disease Control and Prevention. 2001. Update: investigation of anthrax associated with intentional exposure and interim public health guidelines, October 2001. Morb. Mortal. Wkly. Rep. 50:889-893.[Medline]
- Centers for Disease Control and Prevention. 2002. Laboratory-acquired meningococcal disease—United States, 2000. Morb. Mortal. Wkly. Rep. 51:141-144.[Medline]
- Centers for Disease Control and Prevention. 2002. Update: cutaneous anthrax in a laboratory worker—Texas, 2002. Morb. Mortal. Wkly. Rep. 51:482.[Medline]
- Federal Register. 2002. Possession, use, and transfer of select agents and toxins; interim final rule. Fed. Regist. 240:76885-76905.
- Garner, J. S., et al. 1996. Guideline for isolation precautions in hospitals. Infect. Control Hosp. Epidemiol. 17:53-80.[Medline]
- Gilchrist, M. J. R., W. P. McKinney, J. M. Miller, and A. S. Weissfeld. 2000. Cumitech 33, Laboratory safety, management and diagnosis of biological agents associated with bioterrorism. Coordinating ed., J. W. Snyder. ASM Press, Washington, D.C.
- Hawley, R. J., and E. M. Eitzen, Jr. 2000. Bioterrorism and biological safety, p. 567-578. In D. O. Fleming and D. L. Hunt (ed.), Biological safety: principles and practices, 3rd ed. ASM Press, Washington, D.C.
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- Snyder, J. W., and A. S. Weissfeld. 2003. Laboratory detection of potential agents of bioterrorism, p. 121-128. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology, 8th ed. ASM Press, Washington, D.C.
- Voss, A., and E. Nulens. 2003. Prevention and control of laboratory-acquired infections, p. 109-120. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology, 8th ed. ASM Press, Washington, D.C.
Journal of Clinical Microbiology, July 2003, p. 2801-2809, Vol. 41, No. 7
0095-1137/03/$08.00+0 DOI: 10.1128/JCM.41.7.2801-2809.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
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