Role of the Hospital-Based Microbiology Laboratory in Preparation for and Response to a Bioterrorism Event

Many hospital-based clinical microbiology laboratories were called upon by local first responders (law enforcement, hazardous-material teams, and emergency medicine service personnel) to perform testing on powdery and miscellaneous environmental substances during the anthrax outbreak of September and October 2001. The post-September 11 anthrax outbreak was the first definitive challenge to the integrated laboratory system known as the Laboratory Response Network (LRN). The LRN strongly recommends that hospital-based laboratories restrict testing to human specimens and use only diagnostic test protocols developed in partnership with subject matter experts from the American Society for Microbiology (ASM), Centers for Disease Control and Prevention (CDC), and the Association of Public Health Laboratories. Only public health laboratories should analyze environmental and animal specimens. During the initial anthrax scare, the submission of powdery substances and other suspicious nonhuman specimens to hospital-based clinical microbiology laboratories was prompted by public panic, pressure from law enforcement to obtain an immediate answer regarding the presence or absence of anthrax spores, the unfamiliarity of first responders with the LRN, and pressure placed on laboratories to provide a public service. However, many laboratories were unfamiliar with or did not understand their role in the LRN. As a result of the anthrax experience, one of the major questions being asked by microbiologists is “what is or what should be the role of the hospital-based clinical microbiology laboratory when confronted with biothreats, biocrimes, or bioterrorism?” The laboratory's standard roles of detection, recovery, characterization, identification, and determining the etiological agent's susceptibility to antibiotics will continue, with additional emphasis being given to biothreat-associated agents. This role also involves an active role in surveillance and epidemiology. The main role of the hospital-based clinical microbiology laboratory in support of a biothreat, biocrime, or act of bioterrorism is to “raise suspicion” when a targeted agent is suspected in a human specimen. To be successful in this role, laboratory personnel, including the laboratory director, in conjunction with infection control and administrative personnel, should develop both laboratory and institution-wide response plans. These plans must include the following: (i) criteria for distinguishing the type of bioterrorism event; (ii) information regarding access to and utilization of the LRN, including diagnostic testing protocols; (iii) safety guidelines; (iv) communication and notification protocols; (v) criteria for the safe packaging and transport of infectious substances; and (vi) measures to increase laboratory security.

DISTINGUISHING TYPES OF BIOTERRORISM EVENTS

A bioterrorism event is either overt or covert in nature. The former is self-evident and usually announced. The most notable examples of an overt event were the anthrax spore-containing letters that were sent to Senators Daschle and Leahy and to Tom Brokaw with a written note announcing that they had been exposed to Bacillus anthracis spores. The package that was sent to the National Enquirer, from which the first case of anthrax originated, is an example of a covert event. There was no indication and it was not announced that the package had been deliberately contaminated with anthrax spores. As a consequence, there was a delay in making the diagnosis of anthrax, contributing to the eventual death of the index case patient. Because an overt event involves the use of environmental substances, the hospital-based clinical microbiology laboratory should not accept these samples for testing but rather, inform the first responders that such specimens are to be tested by the local or state public health laboratories. A similar policy also applies to the testing of animal, food, and water specimens.

The hospital-based clinical microbiology laboratory should be prepared to recognize and respond to a covert event involving the collection, preservation, transport, and testing of human specimens. This type of event does not have an immediate impact because of the delay between exposure and onset of illness, i.e., incubation period. Consequently, emergency medicine physicians and other primary healthcare providers, including the clinical microbiology laboratory staff, are the sentinels or medical first responders and will most likely identify the initial cases (1). Key indicators of a possible act of bioterrorism in raising suspicion follow: (i) a disease entity that is unusual or that does not occur naturally in a given geographical area, or combinations of unusual disease entities in the same population; (ii) multiple disease entities in one patient, indicating that mixed agents have been used in the event; (iii) increased rates of morbidity and mortality relative to the number of personnel at risk or within a population that inhabits the same area; (iv) epidemiological data suggesting a massive point source outbreak; (v) apparent aerosol route of infection; (vi) illness limited to localized or circumscribed geographical areas; (vii) low attack rates in personnel who work in areas with filtered air supplies or closed ventilation systems; (viii) sentinel dead animals of multiple species; and (ix) absence of a competent natural vector in the area of the outbreak for those biological agents that are vector-borne in nature (United States Army Research Institute for Infectious Diseases [USAMRIID] Biological Warfare and Terrorism Medical Issues and Response Satellite Broadcast, 26 to 28 September 2000). Detection and recognition of a suspicious event is also dependent on a vigilant laboratory staff comprised of well-trained personnel who are capable of recognizing unusual circumstances and possess a high index of suspicion. An additional component of the recognition process includes an active surveillance and monitoring program (2). Microbiological and epidemiological clues that may signal a potential biothreat or act of bioterrorism are summarized in Table 1 (USAMRIID Satellite Broadcast).

TABLE 1.

Epidemiological and microbiological clues that may signal a biological act of terrorism

Epidemiological or microbiological clue
Large numbers of ill persons with a similar disease or syndrome
Large numbers of cases of unexplained diseases or deaths
Unusual illness in a population (e.g., unexplained respiratory infection)
Single case of disease caused by an uncommon agent (e.g., pulmonary anthrax, Burkholderia mallei or Burkholderia pseudomallei, tularemia, viral hemorrhagic fever)
Several unusual or unexplained diseases coexisting in one patient without any other explanation
Higher morbidity and mortality in association with a common disease or syndrome or failure of such patients to respond to usual therapy
Disease with an unusual geographical or seasonal distribution (e.g., tularemia in an area where it is not endemic influenza in the summer)
Illness that is unusual (or atypical) for a given population or age group (e.g., outbreak of measles-like rash in adults)
Unusual disease presentation (e.g., pulmonary instead of cutaneous anthrax)
Similar genetic type among agents isolated from the distinct sources at different times or locations
Unusual, atypical, genetically engineered, or antiquated strain of an agent (or antibiotic resistance pattern)
Stable native disease with an unexplained increase in incidence (e.g., tularemia, plague)
Simultaneous clusters of similar illness in noncontiguous areas, domestic or foreign
Atypical disease transmission through aerosols, food, or water, which suggests deliberate sabotage
Ill persons who seek treatment at about the same time (point source with compressed epidemic curve)
No illness in persons who are not exposed to common ventilation systems (have separate closed ventilation systems) when illness is seen in persons in close proximity who have a common ventilation system
Unusual pattern of death or illness among animals (which may be unexplained or attributed to an agent of bioterrorism), which precedes or accompanies illness of death in humans

ACCESSING AND INTEGRATION WITH THE LRN

The LRN is a four-tier system in which private and hospital-based laboratories, along with some local public health and military laboratories, are categorized as level A with the major responsibility being to rule out and refer suspicious isolates to the next higher level (level B, C, or D) for confirmatory testing (1, 3, 6). Laboratories designated as level B (local or state public health laboratory), level C (state health laboratories with advanced testing capacity), or level D (CDC and USAMRIID) are responsible for confirming the identification of a suspicious isolate. The level D laboratory primarily performs advanced genetic analysis, archiving, and direct analysis of specimens suspected of harboring agents requiring biosafety level 4 (BSL-4) precautions, e.g., smallpox and agents of viral hemorrhagic fever (1). Although level A laboratories have access to but are not official members of the LRN, they are regarded by public health officials as being critical to the success of the LRN. It is important that level A laboratories develop a relationship with their local and state health laboratories, many of whom provide formal training in laboratory safety, testing procedures for ruling out targeted biological agents, clinical characteristics of diseases produced by these agents, and packaging and shipping of infectious substances. The hospital-based clinical microbiology laboratory should have a bioterrorism response plan and a standing operating procedure, which include the telephone numbers of the local and state health department laboratories and the CDC. The laboratory should have a copy of the current Targeted (Critical) Agent list developed by the CDC and its affiliated partners (Table 2). Laboratory personnel should be familiar with the biothreat categories (A, B, and C) of the different agents. Because many of these agents are uncommon, especially in the United States, or are limited to specific geographical areas, the hospital-based clinical microbiology laboratory must be familiar with the general morphological, cultural, and Gram stain characteristics of these agents and perform common conventional diagnostic tests, currently listed in the LRN level A testing protocols for the bacterial agents of anthrax, brucellosis, plague, and tularemia, and rule out or refer suspicious isolates to the next higher level for confirmation of identification. Level A laboratory testing protocols are available at the ASM (www.asmusa.org/pcsrc/biodetection.htm) and CDC (www.bt.cdc.gov) websites. Laboratory preparedness is also dependent on making improvements in the clinical and laboratory surveillance systems in addition to the training of clinicians and laboratory personnel (1, 7). To facilitate the recognition of biothreat-related microbial agents, the hospital-based clinical microbiology laboratory must attract and retain qualified personnel for conducting tests and raising awareness. Laboratory personnel should receive specialized training in the recognition of targeted agents, the use of the LRN, and the application of the level A standardized testing protocols for ruling out and referring suspicious isolates (3).

TABLE 2.

Critical biological agents for bioterrorism and civilian preparedness^a

Organism(s)	Disease, common name, or toxin	Biothreat levelb
Bacteria
Bacillus anthracisc	Anthrax	A
Yersinia pestisc	Plague	A
Francisella tularensisc	Tularemia	A
Coxiella burnettic	Q fever	B
Brucella speciesc	Brucellosis	B
Burkholderia mallei	Glanders	B
Salmonella species	Salmonellosis	B
Shigella dysenteriae	Shigellosis	B
Escherichia coli O157:H7	Hemorrhagic colitis	B
Vibrio cholerae	Cholera	B
Mycobacterium tuberculosisd	Tuberculosis	C
Viruses
Variola major virusc	Smallpox	A
Ebola virusc	Hemorrhagic fever	A
Marburg virusc	Hemorrhagic fever	A
Lassa virusc	Lassa fever	A
Junin virusc	Argentine hemorrhagic fever	A
VEE,c WEE, EEEe	Encephalomyelitis	B
Nipah virus	Encephalomelitis	C
Hantavirus	Hemorrhagic fever, hantavirus pulmonary syndrome	C
Tick-borne viruses	Hemorrhagic fever and/or encephalitis	C
Yellow fever virus	Yellow fever	C
Protozoa
Cryptosporidium parvum	Cryptosporidiosis	C
Toxin sources
Clostridium botulinuma	Botulinum toxin	A
Staphylococcus aureusc	Entertoxin B	B
Clostridium perfringens	Epsilon toxin	B
Ricinus communisa (castor bean)	Ricin	B
Some marine dinoflagellates	Saxitoxin
Various species of fungi	Trichothene mycotoxins

^aAdapted from reference 7 with permission of the publisher.

^bBiothreat level A, agents that pose the greatest threat due to their infectiousness, relative ease of transmission, or a high rate of mortality; biothreat level B, agents having a moderate ease of transmission and morbidity with a low rate of mortality; biothreat level C, emerging pathogens and potential risks for the future.

^cOrganisms categorized as critical agents for bioterrorism or biocrime.

^dMultidrug-resistant strains.

^eVEE, Venezuelan equine encephalomyelitis; WEE, western equine encephalomyelitis; EEE, eastern equine encephalomyelitis.

SAFETY

Knowledge of the BSLs is critical for the handling and management of specimens suspected of containing any of the targeted agents. Most hospital-based clinical microbiology laboratories function at BSL-2, which includes the availability of a class II biosafety cabinet (4). Once a culture or preliminary test result raises suspicion of a biothreat-related agent, all further manipulations should be performed in a biosafety cabinet. Although Bacillus anthracis is classified as a BSL-2 agent, the CDC and LRN recommend that all manipulations be performed in a biological safety cabinet. Therefore, all microbiology laboratory personnel should understand and be trained in the levels of biosafety (BSL-1, -2, -3, and -4).

A major reason for restricting level A laboratories to the testing of human specimens is to maintain and promote the safety of the institution, personnel, and patients. Major concerns for testing environmental samples in a hospital-based microbiology laboratory are the unknown nature of the sample. The sample could be explosive or a volatile, toxic chemical or contain a radioactive substance. Therefore, risk assessment becomes the responsibility of the clinical microbiologist, infection control personnel, hospital risk management office, and infectious disease physicians. Furthermore, a postexposure plan that describes the policy for managing laboratory or other personnel following exposure to a biothreat-related agent must be developed. Decisions must be made concerning the use of prophylactic antibiotics, administration of available vaccines, or simply instituting a fever watch policy and observing for the onset of symptoms in exposed individuals.

Of equal concern is the possibility that a specimen containing smallpox virus, a BSL-4 agent, is submitted to the virology laboratory. This virus can easily grow and amplify in most routine cell lines that are used for the cultivation of herpesviruses, e.g., varicella-zoster virus and herpes simplex virus. The technologist may neither suspect nor recognize a hazard. Failing to obtain confirmation of the more common viruses via direct fluorescent-antibody staining, the technologist may pass or manipulate the culture multiple times before terminating efforts to confirm the source of the cytopathic effects. The result is increased risk of exposure should the specimen contain smallpox virus. How to recognize and safely manage such situations is a critical challenge to the laboratory. In the event of such a circumstance, the laboratory director should call both the state health laboratory and the CDC for guidance.

COMMUNICATION

The laboratory, preferably the laboratory director, must establish and include in the laboratory bioterrorism response plan a notification policy that is enacted when a suspicious isolate cannot be ruled out and must be referred to the next higher level laboratory for confirmation of the organism's identity. The policy must also include the point-of-contact individual within the hospital who is responsible for notifying other key personnel and the local health department officials. Event recognition within the hospital resembles an epidemiological investigation. Case definitions must be made and then followed by aggressive case analysis. In the event that a suspicious agent is detected, the first priority of the laboratory, as always, should be to notify the treating clinician of test results so that they can initiate appropriate management of patients (5, 8). To facilitate evaluation of any potential threat to the local community, the laboratory should then promptly alert the institutional infection control personnel, who in turn should notify the institutional leadership. It should be the responsibility of the infection control personnel to notify city or county public health officials, who will then notify state public health officials that an unusual illness has occurred. It is the public health authorities who will determine whether a threat to the general public health exists and who will recommend whether patient specimens or isolates should be forwarded to higher-level laboratories for further analysis and, if warranted, notify law enforcement officials. Under no circumstances should the laboratory notify local, state, or federal law enforcement officials or make the decision that a bioterrorism event has occurred. The identification of an isolate as an organism that is on the list of potential agents of bioterrorism is not evidence that a bioterrorism event has occurred (8). For example, Francisella tularensis, Brucella spp., and Yersinia pestis are naturally found in certain geographical areas of the United States and are associated with low rates of sporadic infections. Public health officials and law enforcement authorities determine if an event is creditable. The Federal Bureau of Investigation (FBI) has ultimate responsibility, under Presidential Decision Directive 39, for declaring that a bioterrorism event has occurred. During the process of determining if a threat exists to the general public health, the clinical microbiologist should notify the nearest LRN level B laboratory, obtain guidance concerning the referral of the organism for confirmatory testing, and be available to serve in an advisory capacity.

PACKAGING AND SHIPPING OF INFECTIOUS SUBSTANCES

Specimens and agents must be packaged and shipped in accordance with guidelines specified by the International Air Transport Association and United Nations 6.2 packaging regulations. All specimens, including pure cultures, are classified as infectious substances and must be packaged and transported in the same manner, whether its destination is intra- or interstate. Laboratory personnel must be trained and certified to package and ship infectious substances using approved packaging materials and containers (9). The laboratory director should know which commercial carriers will accept infectious substances, most of which will be transported to an LRN level B laboratory. Currently, the only commercial carriers that will accept and transport infectious substances are the United States Postal Service and Federal Express. Although not normally a major issue for the level A clinical microbiology laboratory, the FBI may require that a chain-of-custody protocol be developed for potential litigation. It is also recommended that the laboratory retain a stock culture of any suspicious isolates that have been referred to the next higher-level LRN laboratory. Isolates being saved for litigation purposes must be stored in a secure area to prevent unauthorized personnel (e.g., engineering staff, housekeeping personnel) from gaining access.

LABORATORY SECURITY

The issue and importance of laboratory security have become major concerns of officials and agencies involved with the planning of national security. In addition to maintaining the security of the aforementioned suspicious isolates, microbiologists should give strong consideration to implementing a laboratory-wide limited access policy (7). Only authorized personnel should have access to the laboratory. Installing an electronic access system and requiring all members of the laboratory staff to possess a scanning card for entry into the laboratory can accomplish this. Unauthorized individuals would be required to report to a central greeting area and be screened for appropriate identification and the nature of their business. Additional security measures include locking all storage cabinets, refrigerators, incubators, and doors to sensitive areas (e.g., tuberculosis section and virology laboratory). In these matters, security takes precedence over convenience.

SUMMARY

In conjunction with local, state, and federal health agencies, the hospital-based clinical microbiology laboratory is expected to play a primary role in determining if a biocrime or bioterrorism event has occurred. While this role will not compromise the routine operations of the clinical laboratory, it will enhance their capabilities and awareness. The primary focus is to raise the index of suspicion related to a biocrime or bioterrorism event. In the event that the laboratory encounters a suspected biothreat agent, the bioterrorism response plan is enacted. This plan includes event recognition, access to and interaction with the various LRN level laboratories, communication protocols, safety guidelines, training of personnel to ensure competence and awareness, packaging and shipment of infectious substances, and laboratory security.