Cumitech 33: Biological Agents Associated with Bioterrorism

33 Laboratory Safety, Management, and Diagnosis of Biological Agents Associated with Bioterrorism MARY J. R. GILCHRIST, W. PAUL MCKINNEY, J. MICHAEL MILLER, AND ALICE S. WEISSFELD COORDINATING EDITOR

JAMES W. SNYDER

Cumitech CUMULATIVE TECHNIQUES AND PROCEDURES IN CLINICAL MICROBIOLOGY

Cumitech 1B

Blood Cultures III

Cumitech 2B

Laboratory Diagnosis of Urinary Tract Infections

Cumitech 3A

Quality Control and Quality Assurance Practices in Clinical Microbiology

Cumitech 5A

Practical Anaerobic Bacteriology

Cumitech 6A

New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide

Cumitech 7B

Lower Respiratory Tract Infections

Cumitech 12A

Laboratory Diagnosis of Bacterial Diarrhea

Cumitech 13A

Laboratory Diagnosis of Ocular Infections

Cumitech 16A

Laboratory Diagnosis of the Mycobacterioses

Cumitech 18A

Laboratory Diagnosis of Hepatitis Viruses

Cumitech 19A

Laboratory Diagnosis of Chlamydia trachomatis Infections

Cumitech 21

Laboratory Diagnosis of Viral Respiratory Disease

Cumitech 23

Infections of the Skin and Subcutaneous Tissues

Cumitech 24

Rapid Detection of Viruses by Immunofluorescence

Cumitech 25

Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing

Cumitech 26

Laboratory Diagnosis of Viral Infections Producing Enteritis

Cumitech 27

Laboratory Diagnosis of Zoonotic Infections: Bacterial Infections Obtained from Companion and Laboratory Animals

Cumitech 28

Laboratory Diagnosis of Zoonotic Infections: Chlamydial, Fungal, Viral, and Parasitic Infections Obtained from Companion and Laboratory Animals

Cumitech 29

Laboratory Safety in Clinical Microbiology

Cumitech 30A

Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections of the Gastrointestinal Tract

Cumitech 31

Verification and Validation of Procedures in the Clinical Microbiology Laboratory

Cumitech 32

Laboratory Diagnosis of Zoonotic Infections: Viral, Rickettsial, and Parasitic Infections Obtained from Food Animals and Wildlife

Cumitech 33

Laboratory Safety, Management, and Diagnosis of Biological Agents Associated with Bioterrorism

Cumitech 34

Laboratory Diagnosis of Mycoplasmal Infections

Cumitech 35

Postmortem Microbiology

Cumitech 36

Biosafety Considerations for Large-Scale Production of Microorganisms

Cumitech 37

Laboratory Diagnosis of Bacterial and Fungal Infections Common to Humans, Livestock, and Wildlife

Cumitech 38

Human Cytomegalovirus

Cumitech 39

Competency Assessment in the Clinical Microbiology Laboratory

Cumitech 40

Packing and Shipping of Diagnostic Specimens and Infectious Substances

Cumitechs should be cited as follows, e.g.: Gilchrist, M. J. R., W. P. McKinney, J. M. Miller, and J. W. Snyder. 2000. Cumitech 33, Laboratory safety, management, and diagnosis of biological agents associated with bioterrorism. Coordinating ed., J. W. Snyder. ASM Press, Washington, D.C. Editorial Board for ASM Cumitechs: Alice S. Weissfeld, Chair ; Vickie Baselski, B. Kay Buchanan, Mitchell I. Burken, Roberta Carey, Linda Cook, Lynne Garcia, Richard M. Jamison, Karen Krisher, Michael Saubolle, David L. Sewell, James W. Snyder, Allan Truant. Effective as of January 2000, the purpose of the Cumitech series is to provide consensus recommendations regarding the judicious use of clinical microbiology and immunology laboratories and their role in patient care. Each Cumitech is written by a team of clinicians, laboratorians, and other interested stakeholders to provide a broad overview of various aspects of infectious disease testing. These aspects include a discussion of relevant clinical considerations; collection, transport, processing, and interpretive guidelines; the clinical utility of culture-based and non-culture-based methods and emerging technologies; and issues surrounding coding, medical necessity, frequency limits, and reimbursement. The recommendations in Cumitechs do not represent the official views or policies of any third-party payer. Copyright © 2000 ASM Press American Society for Microbiology 1752 N Street NW Washington, DC 20036-2904 All Rights Reserved 10 9 8 7 6 5 4 3 2

Laboratory Safety, Management, and Diagnosis of Biological Agents Associated with Bioterrorism Mary J. R. Gilchrist University of Iowa Hygienics Laboratory, Iowa City, Iowa 52242

W. Paul McKinney Department of Medicine, Division of Internal Medicine and Geriatrics, University of Louisville, and Louisville Veterans Administration Medical Center, Louisville, Kentucky 40202

J. Michael Miller Hospital Environment Laboratory Branch, Hospital Infections Program, Centers for Disease Control and Prevention, Atlanta, Georgia 30333

Alice S. Weissfeld Microbiology Specialists Inc. and Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77054

COORDINATING EDITOR: James W. Snyder Department of Pathology, Division of Laboratory Medicine, University of Louisville School of Medicine and Hospital, Louisville, Kentucky 40292

Introduction ......................................................................................... General Principles of Laboratory Safety in Counterbioterrorism ................ Specific Guidelines for Specimen Safety ................................................. Clinical Features and Laboratory Diagnosis of Biological Agents Targeted for Use in Acts of Bioterrorism ...........................................................

1 2 6 9

Anthrax ..................................................................................................................... 9 Plague .................................................................................................................... 12 Botulism ................................................................................................................. 13 Tularemia ................................................................................................................ 14 Brucellosis ............................................................................................................... 15 Smallpox ................................................................................................................. 16

Additional Information ........................................................................ 17 References ......................................................................................... 17

crobiology laboratories of the late 1800s and those of today are the number and types of microorganisms that have been detected in the 20th century as distinct pathogens. Many of the infectious diseases that challenged the capacity of the early laboratories (e.g., malaria, intestinal parasitosis, tuberculosis, syphilis, gonorrhea, and diphtheria) continue to challenge the modern-day laboratory in addition to new and evolving infectious diseases that have been discovered during the 20th century, such as Legionnaires’ disease, human immunodeficiency virus (HIV) disease, and

INTRODUCTION

T

he clinical microbiology laboratory is a sentinel that will play a vital and pivotal role in the event of a terrorist action involving the use of biological agents. Its major role will continue to be that which was first practiced in the late 1800s with the advent of the first microbiology laboratories and continues to be practiced in the 20th century, the detection, cultivation, and confirmation of the etiological agent. The major differences between the clinical mi1

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CUMITECH 33

FIGURE 1.

Laboratory response network.

many other viral, fungal, bacterial, and parasitic infections. The current challenges in recognizing and detecting emerging and reemerging infectious agents along with the worldwide increase in antibiotic resistance are potentially superseded by the threat posed by biological agents and their potential use in terrorist activities. Biological terrorism is defined as the intentional or threatened use of viruses, bacteria, fungi, or toxins from living organisms to produce death or disease in humans, animals, or plants. The NATO Handbook on the Medical Aspects of NBC Defensive Operations lists 31 biological agents that have been targeted as most likely to be used in a bioterrorism attack (15). Of these, six have been designated as high-priority agents: Bacillus anthracis, the agent of anthrax; Yersinia pestis, the agent of plague; Francisella tularensis, the agent of tularemia; Brucella spp., the agents of brucellosis; botulinum toxin, produced by Clostridium botulinum; and variola major virus, the agent of smallpox. To serve as an effective agent of bioterrorism, the ideal biological agent must (i) be pathogenic to humans, plants, or animals; (ii) be environmentally stable; (iii) be effective at low doses; (iv) be transmissible via aerosol; (v) cause high rates of morbidity and/or mortality; and (vi) be difficult to diagnose and/or treat. In the event of a biological attack, whether an overt or covert act, the early recognition and detection of these agents will pose a formidable challenge to the microbiologist and the clinical microbiology laboratory staff. Not only will the clinical microbiology laboratory be expected to detect and identify the agent in a timely manner, but it will also be expected to provide information regard-

ing the selection, collection, safe handling, and transport of diagnostic specimens to an appropriate laboratory with a level of biosafety capability compatible with the perceived threat. This Cumitech is primarily directed to community and hospital clinical microbiology laboratories and focuses on the laboratory aspects of biosafety related to the likely agents of bioterrorist threats; specimen safety, including collection, transport, and management in the clinical microbiology laboratory; the clinical features of these selected agents; and their isolation and identification in a clinical and hospital microbiology laboratory. Terms such as “biocrime” and “biothreat” as well as “biothreat agents” and “bioterrorist agents” are used interchangeably throughout this document. Each is defined as follows: “biocrime”—a criminal act involving the use of biological agents as weapons; “biothreat”—the suspected but unconfirmed release of a biological agent(s); “biothreat agents” or “bioterrorist agents”—microbial pathogens and/or toxins which have been previously considered or used in biological warfare and recent terrorist events.

GENERAL PRINCIPLES OF LABORATORY SAFETY IN COUNTERBIOTERRORISM For laboratories that are enrolled in the Laboratory Response Network that has been created to provide an organized response system for the detection and diagnosis of biological agents, four levels of laboratory capacity have been identified based on the level of risk (Fig. 1). Level A laboratories, primarily those located in hospitals, clinics, and small public health

CUMITECH 33

Bioterrorism Agents

Table 1. Recommended BSLs for different activities with bacteria and their toxins BSL of practices (facility) for: Organism or agent

Clinical specimen manipulation

Clinical diagnostic culture

Aerosolizing and/or production

B. anthracis Brucella species Botulinum toxina F. tularensis Y. pestis

2 (2) 2 (2} 2 (2) 2 (2) 2 (2)

2 (2) 3 (3) NAb 3 (3) 2 (2)

3 (3) Not specified 3 (3) Not specified 3 (3)

a b

See instructions regarding toxoid vaccine. NA, not applicable.

laboratories, will function at biosafety level 2 (BSL-2). Level B laboratories will follow BSL-3 practices because they will work with larger numbers of organisms, but since they are limited in the scope of their work, they are only required to work in a BSL-2 facility. Level C laboratories, those working with greater numbers of organisms and employing developmental techniques that may entail greater aerosolization, will employ BSL-3 practices. Level D laboratories, federal facilities such as the Centers for Disease Control and Prevention (CDC) and the Department of Defense, will employ BSL-4 for agents meriting this level of containment. These are minimum requirements, and may be enhanced at the laboratory director’s discretion when circumstances suggest the need for additional safety practices. The recommendations and requirements for the various BSLs in clinical microbiology laboratories are detailed in the latest edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL) (51), which is also available on the Internet (www.cdc.gov/od /ohs/biosfty/bmbl4/bmbl4toc.htm). The levels of biosafety prescribed in BMBL escalate in a fashion that is based upon the extent of potential exposure. Thus, clinical specimens represent less risk than cultured microorganisms in the clinical microbiology laboratory, and large volumes of cultured organisms represent the greatest risk. For a given organism, the prescribed BSL increases in a stepwise fashion with perceived risk. The recommended BSLs for handling those agents of primary concern in a bioterrorist threat are summarized in Table 1. Although some of these agents are classified as BSL-3 agents when present at amplified levels in cultures, the paragraphs that follow present a practical guideline for application of good biosafety practices that will serve to prevent laboratory exposure to and/or infection with these agents when they are encountered either from natural transmission or from a bioterrorist attack. Most hospital clinical microbiology laboratories function and perform diagnostic testing at, or approaching, BSL-2. Since all hospitals and clinics are at

3

risk for encountering these agents without prior warning either from natural transmission or from unannounced bioterrorist attacks, these guidelines will reflect the intent of BMBL and provide a practical application that will allow the clinical microbiology laboratory to function safely and effectively by assuring strict adherence to BSL-2 practices unless a BSL-3 agent is suspected or known. The reader is referred to BMBL for specific details on the implementation of general biosafety practices related to the various BSLs. For the purposes of this section, it is assumed that the laboratory is currently practicing standard precautions when handling body specimens to provide a barrier to avoid exposure to blood and body fluids (22). Biological agents most likely to be used in an attack are characterized by their ability to survive in dried form in an aerosol and to be transmitted through inhalation of the aerosol, since this is one of the most efficient means of secret administration of an infective dose to a targeted population. Thus, protection of the laboratory staff from aerosol transmission is of vital importance. The applicable practice for BSL-2 specifies that all procedures which may produce aerosols should be conducted in a biological safety cabinet (BSC), whereas the specification for BSL-3 is that all manipulations of the (open) culture (vessel) must be conducted in the BSC. The distinction is semantic in nature, because virtually all manipulations create an aerosol of greater or lesser magnitude. Although we traditionally think of aerosolizing procedures as those of blending, shaking, and vortexing, it has been demonstrated that simple manipulations (e.g., preparation of a wet mount by emulsifying a colony in saline) create aerosols of lesser proportions. Thus, it is wise to think of aerosolization as a continuum and to attempt to contain the greater part of the continuum. Since personnel in most diagnostic laboratories do not know the identity of the agents they work with until they are nearly or completely finished working with them, the guidelines require thoughtful application. If there is no reason to suspect a BSL-3 agent, either acquired endemically or from terrorist transmission, the laboratory may function safely by fully implementing BSL-2 practices, e.g., by containing all aerosols. Sound BSL-2 practices will create a “near 3” level of safety. For example, instead of using a Bunsen burner for sterilizing an inoculating loop, all such practices should be conducted with a microincinerator to contain particles which may be expelled upon exposure to heating. There are other changes that may be indicated. In some laboratories, it is still common practice to cool a loop by touching the surface of the agar in an area where no growth is evident. We now know the unfortunate result of aerosolizing microscopic colonies of bacteria such as Mycobacterium tuberculosis, which has been shown to

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be capable of growing on most routine microbiology media including blood and chocolate agar (M. Gilchrist, C. Shaw, and D. Welch, Abstr. 94th Gen. Meet. Am. Soc. Microbiol. 1994, abstr. C-123, p. 512, 1994). The practice of “hot looping” should be discouraged, especially on plated media that have been inoculated and incubated. Moreover, the traditional practice of sniffing or smelling an agar plate in order to detect characteristic odors of various microorganisms should be discontinued. Generally, these odors are subjective in nature and there is no meaningful quality control procedure. Although smelling the colony may simply produce a little more aerosolization than lifting the plate lid, the practice is not justified. The greatest challenge lies with the safe implementation of automated microbiological instrumentation. Equipment used for dispensing suspensions into devices that are used for identification and susceptibility testing may create aerosols. It is recommended that vortexing of such suspensions be performed in a BSC. At minimum, the vortexing should be done in test tubes that have vapor-tight, as well as droplet-containing, sealed lids. A recent National Committee for Clinical Laboratory Standards document provides guidance on safe practices to be employed with automated laboratory instrumentation (42). Each laboratory should audit its practices, by identifying all instances in which aerosols potentially are formed. Routine operations will be made much safer by identifying means to contain the aerosols or to abandon the procedure in favor of a safer one. A laboratory working at BSL-2 with an unknown agent should be prepared to shift to BSL-3 when such an agent is identified or suspected. Once an organism has been identified as F. tularensis or a Brucella sp., the organism should be treated as a BSL-3 agent and manipulated solely in the BSC. If the organism is to be archived in the freezer, it should be placed in a box prominently labeled “BSL-3 organisms” so that correct biosafety precautions will be employed when it is subsequently manipulated. This logic is based on the concept that the laboratory normally encounters thousands of less-biohazardous agents for every one that it encounters at the higher hazard level. However, once the identification of the organism has been confirmed, the laboratory technologist who processes it for additional testing must recognize the risk and perform all manipulations at the appropriate level of containment. When a specimen is submitted with a request to isolate a BSL-3 agent, the culture should be prominently labeled as “BSL-3” and evaluated only in the BSC. Table 1 indicates that increasing volumes of cultures or potential for aerosolization call for increasing containment of microorganisms. Level B bioterrorism laboratories are expected to have greater exposure,

CUMITECH 33

because they will receive many more cultures of biological agents. They would also expect to receive most of the agents, as in most cases their submission to the B level laboratory will be based upon referral from a level A laboratory or on suspicion or announcement of an attack. Therefore, routine containment for the B level laboratories requires additional precautions. Whereas the level A laboratories are to implement strict BSL-2 practices in a BSL-2 facility, level B laboratories will employ BSL-3 practices in a BSL-2 or higher BSL facility. Because most of the cultures referred to the level B laboratories will be known or suspected biothreat agents, it will be easy for the routine manipulation of these cultures to be conducted in the BSC. Although preferable, it is not necessary for a level B laboratory to meet the requirements of a BSL-3 facility. With F. tularensis and Brucella spp., laboratory accidents involving exposed or infected laboratory staff tend to affect those working most closely with the agents. Dissemination to adjacent spaces throughout the building at infective levels is known with agents such as M. tuberculosis, for which the use of the BSL-3 facility is now required. With mycobacteria, however, the characteristic hardiness of the organism and the low infective dose render them a greater risk for remote aerosol transmission. The use of a BSL-3 facility does little to add protection to the personnel performing the work in the clinical microbiology laboratory. Rather, it contains the aerosol within the immediate laboratory space and protects those outside of the space. Since remotely acquired infections with F. tularensis and Brucella spp., are not generally recognized, a requirement for a BSL-3 facility is not justified. However, if such agents are suspected or likely to be present, it is best to automatically refer them to a BSL-3 facility. As the risk increases, so should the level of containment. Thus, the requirement for containment is increased for the level C laboratories. These laboratories will work with greater numbers of cultures and they will perform more sophisticated or complex procedures such as typing of the organisms and evaluating new diagnostic tests. The level C laboratories will employ BSL-3 practices and will contain the practices in BSL-3 facilities. As the extent and number of organisms in an aerosol increase, so does the possibility that the aerosol will affect those outside the immediate work area. Thus, the requirement for the increasing level of containment is perfectly justified. Level D laboratories will also employ such reasoning when they work with biothreat agents. BSL-3 or -4 is strongly indicated for most of the level D laboratory activities at the development and archiving level when working with biological agents so classified. BSL-4 containment may be indicated when molecular chime-

CUMITECH 33

ras (recombinant organisms) or viral agents such as smallpox and viral hemorrhagic fever agents are suspected. The containment of viral bioterrorist agents requires a different approach because these agents are considered extremely hazardous. As amplified cultures, these viruses should be manipulated at BSL-4, full containment, at the CDC, National Institutes of Health, U.S. Army Medical Research Institute of Infectious Diseases, or another laboratory possessing this containment capacity. When a specimen is suspected to contain either smallpox virus or one of the agents of viral hemorrhagic fever, it should not be managed in any of the level A through level C laboratories. Rather, the CDC should be contacted immediately for instructions on how to manage such a specimen. Additionally, the state public health laboratory should be notified of the suspicious diagnosis. Of great concern is the circumstance in which a specimen is submitted to the hospital virology laboratory for viral studies but no bioterrorism agent is suspected or mentioned on the test order form. Although inoculation of the viral media in a BSC using standard precautions (e.g., gloving) would provide some protection from the hazard contained in the specimen, there are greater concerns with infected tissue cultures. For example, smallpox virus will grow and amplify its numbers in most of the routine cell lines that are employed for herpesvirus and varicellazoster virus cultures. Once amplified, the agent would present an even greater threat. A laboratory technologist may neither suspect nor recognize a hazard. Failing to obtain a positive fluorescent stain for one of the more common viruses, the technologist may pass or manipulate the culture multiple times before abandoning the effort. Prolonged manipulation and passage would increase the biosafety hazard. If the identity of the agent is never recognized, or recognition is delayed, the technologist would be at risk. Early recognition of the agent would provide the possibility of an effective intervention. Vaccine, if available, could be administered and is judged likely to be efficacious to prevent or moderate the course of smallpox when administered within 3 days after exposure. The potential for exposure to smallpox or hemorrhagic fever viruses, while of low probability, represents a great opportunity for the virology laboratory to assess its practices and improve those that do not conform to the highest standards of safety; also included is the need for laboratory personnel to obtain continuing education related to the basic characteristics and recognition of these viruses. This is important not just for the prospect of encountering an agent of bioterrorism. For example, a routine virology laboratory that performs viral cultures for herpes simplex virus may become complacent because of this virus

Bioterrorism Agents

5

being classified as a BSL-2 agent. We know, however, that some humans can be infected with simian herpes viruses and that these viruses can be extremely hazardous to humans. Should the laboratory encounter a simian herpes virus, the risk would be substantial, for this agent is classified at BSL-3 for simple culture and at BSL-4 for larger volumes or in cases of greater potential for aerosolization of the culture. Thus, good BSL-2 practices are essential to provide safety from unlikely exposures to unexpected pathogens from all types of circumstances. The virology laboratory should understand risks and assess potential problems today. Does the technical staff understand the difference between the laminarflow clean-bench hood that is used for transferring media in an aseptic fashion and the laminar-flow (class II) BSC? The former provides no worker protection whatsoever and may be more hazardous than working on a laboratory bench. Does the technologist understand that uninoculated cell lines may be safely “fed” with fresh medium using the former apparatus but that inoculated cell lines must be manipulated only in the BSC? Is the BSC vented to the outside or is some of the air recirculated into the room after HEPA filtration? Information on BSCs is available through the CDC web site (www.cdc.gov/od/ohs/biosfty/bsc/ bsc.htm). Are the technologists familiar with the cell lines that may promote growth of smallpox or other hazardous viruses, and are they familiar with types of the cytopathic effect that may be present? Now is the time to perform a risk assessment and determine if changes are needed in the containment of aerosols in the BSC and in the containment of fluids from exposure to skin or mucous membranes. The discovery of HIV in the mid-1980s catalyzed an improved safety protocol in most laboratories. The kinds of safety practices that were imposed greatly augmented the protection of laboratory personnel against a hazard that was greater than HIV. The hepatitis B virus (HBV), with its higher titer in human clinical specimens, has long been recognized as a source of illness and death in laboratory personnel. At long last, safety practices were adopted for the lesser potential hazard (HIV) that provided protection from the greater potential hazard (HBV). The protection that was required in the case of HIV and HBV focused more on contact and puncture of skin because transmission was primarily via these means. Little attention was paid to transmission via aerosol, which is characteristic of many of the agents likely to be used by bioterrorists. If, like HIV, the specter of bioterrorism inspires greater general laboratory protection from the aerosol-borne agents, it will have accomplished a great improvement in biosafety. The threat of acquisition of a bioterrorist agent should not impede the process of diagnostic microbiology but

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Table 2.

Microbiology biosafety BSL for:

Agent

Recommended precautions for level A laboratories

Specimen handling

Culture handling

Specimen exposure risk

B. anthracis

2

2

Brucella spp.a

2

3

C. botulinum b

2

2

Blood, skin lesion exudates, CSF, pleural fluid, sputum, and rarely urine and feces Blood, bone marrow, CSF, tissue, semen, and occasionally urine Toxin may be present in food specimens, clinical material (serum, gastric, and feces), and environmental samples (soil, surface water). Toxin is extremely poisonous!

F. tularensisc

2

3

Y. pestis d

2

2

Skin lesion exudates, respiratory secretions, CSF, blood, and urine; tissues from infected animals and fluids from infected arthropods Bubo fluid, blood, sputum, CSF, feces, and urine

BSL-2

BSL-3

Activities involving clinical material collection and diagnostic quantities of infectious cultures Activities limited to collection, transport, and plating of clinical material Activities with materials known to contain or potentially containing toxin must be handled in a BSC (class II) with a laboratory coat, disposable surgical gloves, and a face shield (as needed). Activities limited to collection, transport, and plating of clinical material

Activities with high potential for aerosol or droplet production

Activities involving clinical material collection and diagnostic quantities of infectious cultures

All activities involving manipulations of cultures Activities with high potential for aerosol or droplet production

All activities involving manipulations of cultures

Activities with high potential for aerosol or droplet production

a

Laboratory-acquired brucellosis has occurred by sniffing cultures; aerosols generated by centrifugation; mouth pipetting; accidental parenteral inoculations; sprays into eyes, nose, and mouth; and finally by direct contact with clinical specimens. b 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. c Laboratory-acquired tularemia has been more commonly associated with cultures than with clinical materials or animals. Direct skin or mucous membrane contact with cultures, parenteral inoculation, ingestion, and aerosol exposure have resulted in infection. d Special care should be taken to avoid the generation of aerosols.

rather bring about full recognition and remediation of hazardous practices that currently exist. Cases of endemic tularemia and brucellosis, while rare in the United States, occasionally result in transmission to laboratory personnel. Where this has happened, investigation often reveals that the practices being employed in the laboratory do not achieve fully the intent of BSL-2, much less BSL-3. Careful attention to containment of aerosols in the laboratory should be instituted and regularly monitored. This new recognition of the possibility of bioterrorism should be taken as an opportunity to achieve the correct level of biosafety for handling the more common agents that are encountered regularly in the clinical microbiology laboratory.

SPECIFIC GUIDELINES FOR SPECIMEN SAFETY Although many microbial agents can be considered potential biological threats, for various reasons a lim-

ited number of these agents are more likely to be intentionally used for a biothreat or biocrime. The agents discussed in this document are a part of the limited list. Laboratory personnel are aware that many more microbes can be used intentionally to constitute a biothreat or biocrime. In fact, some of the agents described in this document may be occasionally isolated in laboratories within the United States in the absence of any biothreat. Table 2 describes a limited number of organisms but lists those agents of primary concern and their BSLs. Virtually all clinical microbiology laboratories in the United States should be, at a minimum, BSL-2 facilities, and these laboratories should fully comply with safety standards described for the BSL-2 laboratory (Table 3) as summarized in Table 4 of BMBL (51). When any agent is worked with in a clinical microbiology laboratory, for any purpose, it is essential that laboratory personnel comply completely and fully with the safety standards for their BSL, using aseptic technique for all procedures in the laboratory. When

CUMITECH 33

Table 3.

Bioterrorism Agents

7

Summary of BSL 1 and 2 for infectious agents Containmenta

BSLe

Agents and comments

b

Primary containment Microbiology practices and techniquesd

Safety equipment (primary barriers)

c

Secondary containment facilities (secondary barriers)

1

Well-characterized agents not known to consistently cause disease in healthy adults, and of minimal potential hazard to laboratory personnel and environment. Appropriate for undergraduate and secondary educational training and teaching laboratories (example: B. subtilis).

Laboratory personnel have specific training in those procedures conducted in the laboratory Supervised by a scientist with general training in microbiology Limited access to laboratory when experiments are in progress Hand washing after handling cultures and before exiting laboratory Eating, drinking, applying contact lenses or cosmetics, and the storage of food prohibited Mouth pipetting prohibited “Sharps” policy instituted All procedures minimize the creation of aerosols Work surfaces decontaminated after spills and end of day Waste disposal policy instituted Biohazard sign posted at entrance when infectious agents are present, with name of agent(s) and name and phone number of supervisor Insect and rodent control program in effect

None required. Recommendations: Work performed on open bench top Laboratory coats, gowns, or uniforms to be worn to protect street clothes Gloves should be worn if skin on hands is broken or a rash is present. Alternatives to powdered latex gloves should be available. Protective eyewear should be worn for procedures in which splashes are anticipated. Persons wearing contact lenses should also wear goggles or a face shield.

Sink for hand washing required. Recommendations: The laboratory is not necessarily separated from the general traffic patterns in the building. Laboratory should have donors for access control. 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 able to support anticipated loading and uses, with spacing between cabinets, benches, and equipment accessible for cleaning Windows fitted with screens

2

Associated with human disease (examples: B. anthracis, Shigella spp., Y. pestis). BSL-2 recommendations and OSHAf requirements focus on the prevention of percutaneous, ingestion, and mucous membrane exposure(s) to clinical materials.

BSL-1 practice plus: Laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists Policy and procedures whereby only persons meeting specific entry and training requirements may enter laboratory Individuals at increased risk of acquiring infection are limited or restricted from the laboratory area Biohazard sign as for BSL-1, plus biosafety level, required immunization, required personal protective equipment, and any procedures required for exiting laboratory Immunizations or tests provided for agents in laboratory (hepatitis B vaccines, tuberculosis skin testing) Personnel receive appropriate training in safety precautions, exposure prevention, “sharps” precautions, and annual updates for procedure and/or policy changes

Properly maintained class I or II BSC (preferably class II) for all manipulations involving splashes or aerosols of infectious materials Personal protective equipment: Protective laboratory clothing. This clothing is removed and left in the laboratory area before leaving for nonlaboratory areas. 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

BLS-1 facilities plus: Autoclave available Lockable doors for facilities that house restricted agents Laboratory is separated from general traffic patterns and away from public areas. Recommended that sinks for hand washing be equipped with foot, knee, or automatic faucet operation BSC located for optimal operation to maintain parameters for containment Eyewash station readily available

(Table continues)

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Table 3. Continued Containmenta b

Agents and comments

BSLe

Primary containment Microbiology practices and techniquesd

Safety equipment (primary barriers)

Biosafety manual defining infectious waste handling and decontamination and medical surveillance policies Decontamination policy for work surfaces, spills, and contaminated equipment An accident policy involving an accidental or overt exposure to infectious materials that requires immediate reporting to laboratory director for documentation, medical evaluation, surveillance, and necessary treatment

for manipulations outside the BSC.

c

Secondary containment facilities (secondary barriers)

a

A term used to describe 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. b The protection of personnel and the immediate laboratory environment from exposure to infectious agents. c The protection fo the environment external to the laboratory from the exposure to infectious materials, provided by the facility design and operational practices. d The most important element of containment, i.e., strict adherence to standard microbiological practices and techniques. e Risk 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 BSL. f OSHA, Occupational Safety and Health Administration.

these recommendations are followed, laboratory personnel will minimize any harmful exposure unless an unforseen accident occurs. When an agent is recognized or even suspected as being an organism requiring handling at the next higher level of safety, the culture and its preliminary test plates and tubes should be transferred immediately to a class II BSC and the supervisor should be notified of the potential biosafety hazard. For many small to mid-size laboratories, shipment of the specimen to a reference laboratory would then be appropriate. Table 4. BSL

The bacterial agents in Table 2 are BSL-2 agents when they are contained in environmental or clinical specimens that arrive in the laboratory. Strict adherence to BSL-2 practices in the laboratory should protect laboratory personnel from accidental exposure during early routine isolation procedures. Once isolated in pure culture, however, some agents, such as Brucella spp. and F. tularensis, are considered BSL-3 agents because their virulence and extremely low infectious dose pose a threat to laboratory personnel who might inadvertently create an aerosol by working

Summary of BSL-3 and -4 Agents

Practices

Safety equipment (primary barriers)

Facilities (secondary barriers)

3

Indigenous or exotic agents with potential for aerosol transmission; disease may have serious consequences

BSL-2 practice plus: Controlled access Decontamination of all waste Decontamination of laboratory clothing before laundering Baseline serum

Primary barriers: class I or II BSCs or other physical containment devices used for all open manipulations of agents; personal protective equipment: protective laboratory clothing and gloves; respiratory protection as needed

BSL-2 facilities plus: Physical separation from access corridors Self-closing, double-door access Exhausted air not recirculated Negative airflow into laboratory

4

Dangerous or exotic agents which pose high risk of lifethreatening disease and aerosol-transmitted laboratory infections, or related agents with unknown risk of transmission

BSL-3 practice plus: Clothing change before entering Shower on exit All material decontaminated on exit from facility

Primary barriers: all procedures conducted in class III BSCs or class I or II BSCs in combination with full-body, air-supplied, positive-pressure personnel suit

BSL-3 facilities plus: Separate building or isolated zone Dedicated supply and exhaust, vacuum, and decontamination systems Other requirements outlined in BMBL (51)

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with the agents on the open bench before they have been correctly identified (Table 4). These agents should only be manipulated in laboratories where BSL-3 capacity is readily available and personnel are fully prepared to work with them. Most community hospital laboratories are unprepared to work with these agents and should ship them to a reference laboratory. However, for practical purposes, smaller numbers of unknowns that turn out to be BSL-3 organisms can be safely handled with good BSL-2 practices. Therefore, BSL-3 safety practices should be used when a BSL-3 pathogen has been isolated. Once the identity of these BSL-3 agents is confirmed, particularly for Brucella spp. and F. tularensis, any laboratory personnel who were involved in their isolation and subsequent manipulation in the open laboratory should be made aware of its identity. This laboratory exposure should be reported to infection control personnel who will assure compliance with local institutional policy concerning exposure or whether a period of observation or antimicrobial prophylaxis of laboratory personnel is necessary. The laboratory supervisor should be alerted to any subsequent illness, fever, or suspicious symptoms of any laboratory employee and report this information to infection control personnel and the facility’s employee health officials. An institutional exposure control plan, similar to that for tuberculosis and HIV, should be available for dealing with biological agents. Laboratory personnel must know how to package and transport these critical agents to another site for further analysis. Most bacterial agents would be sent to the state public health laboratory. In the case of a true bioterrorist release of an agent, the Federal Bureau of Investigation (FBI) will require that a chain-ofcustody protocol be initiated. The FBI personnel may elect to personally transfer the clinical specimen or subsequent isolates to the receiving laboratory, or they may authorize signature-traced shipment by commercial carrier. In either case, the chain-of-custody protocol should be carefully followed. Specimens suspected of containing BSL-4 agents such as the smallpox and viral hemorrhagic fever viruses should be shipped directly to the CDC only after consultation with CDC authorities (Table 4). Packaging and shipping criteria are the same for all biologic agents, including the agents listed in Table 2, and can be found in the section on transportation and transfer of biological agents in the BMBL manual (51). One of the major changes in the shipping regulations for both clinical specimens submitted for infectious disease analysis and pure cultures of infectious disease isolates is that both are now considered “infectious substances,” not clinical specimens, and must be labeled as such. Shipping containers and labels should be purchased and kept on hand at the

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laboratory. The concept of safety in shipping is reinforced in the BMBL manual by the current requirements for primary, secondary, and outer packaging containing padding and absorbent material (51). In addition, every container must be labeled properly as illustrated in Fig. 2 and must exhibit the “infectious substance” label (Fig. 3). Following this packaging protocol offers optimum protection against breakage. In addition, the use of plastic rather than glass for organism storage and shipping is recommended for BSL-2 and BSL-3 agents and is required for shipping BSL-4 agents.

CLINICAL FEATURES AND LABORATORY DIAGNOSIS OF BIOLOGICAL AGENTS TARGETED FOR USE IN ACTS OF BIOTERRORISM Whether a bioterrorism incident is announced (overt) or unannounced (covert), the clinical laboratory may be requested to analyze human or environmental specimens or to forward them to the appropriate reference laboratory. In either case, it is crucial to coordinate activities with local and state health departments and the FBI. A chain-of-custody document should accompany any specimen(s) from the moment of collection to maintain documentation for any criminal investigation. Alternatively, the hospital laboratory may receive a call regarding the collection and transport of specimens that a clinician suspects may contain a potential agent of bioterrorism. As a first responder, the laboratory may be the first to encounter a biological agent. In these cases, the laboratory personnel should know what steps to take to assist in the diagnosis of each disease. Anthrax Clinical Features of Infection The causative agent of anthrax is B. anthracis. Infection of humans can occur in one of three forms depending on the route of acquisition. (i) Cutaneous anthrax (sometimes referred to as malignant pustule) occurs following exposure of skin, usually hands and forearms, to spores carried by infected animals, chiefly cattle, sheep, or goats. This form is rarely fatal and has not been reported in the United States since 1992 (12). (ii) Gastrointestinal anthrax occurs by eating meat from infected animals (now rarely encountered). (iii) Inhalation anthrax occurs by the deposition of spores in the respiratory tract. Naturally occurring inhalation infection, associated with exposure to hides, wool, or other animal products during processing, is known as woolsorter’s disease and has been essentially eliminated among workers in the developed countries by use of the anthrax vaccine. Following inhalational exposure, the spores enter pulmo-

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FIGURE 2.

Packing and labeling of infectious substances. (Courtesy of Air Sea Atlanta, Atlanta, Ga.)

nary macrophages and the mediastinal lymph nodes. After germination, the vegetative phase begins with necrosis of the nodes, followed by bacterial entry into the general circulation and ensuing fatal sepsis with generalized hemorrhage and necrosis. It is anticipated that a bioterrorist attack would attempt human infection by this route. Following an incubation period of 1 to 6 days after respiratory exposure, a nonspecific prodrome of fever, malaise, myalgia, fatigue, cough, and chest pain develops (7). An interval of improvement over the next 2 to 3 days usually ensues before the final phase is initiated by high fever, dyspnea, and cyanosis, followed by septic shock, hematogenous dissemination of infection, and death within 36 h (26). The physical findings in human anthrax are nonspecific. The chest X ray may show mediastinal widening with pleural effusions and manifestations of a necrotizing hemorrhagic mediastinitis (1, 6, 17, 26). There are usually no pulmonary infiltrates. Clinical conditions which produce similar radiographic findings—such as blunt chest trauma or deceleration inFIGURE 3.

Infectious substance label.

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jury, especially following motor vehicle accidents, and the postthoracic surgery state—should be easily distinguishable (14). Death is certain to occur without treatment, and only 5% of those infected by the respiratory route will survive if therapy is delayed more than 48 h following the onset of symptoms (14). Anthrax has not shown significant resistance to erythromycin, chloramphenicol, ciprofloxacin, or gentamicin, and historically, intravenous (i.v.) treatment with 2 million U of penicillin every 2 h (24 million U per day) has been the standard therapy. However, the currently preferred antibiotic treatment is i.v. administration of ciprofloxacin (400 mg) every 8 to 12 h (24, 25). If individuals can be reached soon after exposure, the use of ciprofloxacin (500 mg) orally twice a day (BID) or doxycycline (100 mg) orally BID should be given together with initiation of anthrax immunization (24). Antibiotic prophylaxis should continue for at least 4 weeks and until three doses of vaccine have been given (24). Bacillus anthracis Safety Considerations

The laboratory diagnosis of anthrax is permissible in a BSL-2 facility except in cases where a dry powder is to be examined (22). Dry powder was a consideration, for example, in the case of the letters received by several abortion clinics with notes that said that the envelopes contained anthrax spores. Powdery samples should only be examined in BSL-3 facilities or BSL-2 facilities if the technologist is wearing full-face protection in addition to standard personal protective equipment. In addition, contaminated items such as pipettes, needles, plastic loops, and microscopic slides should be soaked in 10% bleach (0.5% hypochlorite solution) or 10 to 30% formalin for 24 h before autoclaving. Extreme caution should be used during specimen handling to avoid creation of aerosols. Collection of Human Specimens

The specimen of choice depends on the disease presentation. Inhalation anthrax is the most likely syndrome in the case of a bioterrorist event (38). If patients have a productive cough, the specimen of choice early in the course of disease is sputum, which should be collected and transported to the laboratory on ice. Two to eight days postexposure, blood cultures are the specimens of choice. These should be collected into the routine blood culture bottles being used in the laboratory. The specimen of choice for cutaneous anthrax depends on the stage of the disease. In the early, vesicular stage, the vesicle should be unroofed and two sterile, dry swabs should be soaked in the vesicular fluid. In the later, eschar stage, two sterile dry swabs should be rotated beneath the edge of the eschar

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without removing it. For the gastrointestinal form of anthrax, stool should be collected early in the disease, followed by blood culture in the later stages. Some patients with anthrax may present with symptoms of meningitis. Cerebrospinal fluid (CSF) should be collected for culture in these instances. Processing of Human Specimens

B. anthracis grows well on routine laboratory media, including 5% sheep blood and chocolate agars (32). It does not grow well on MacConkey agar. Trypticase soy broth is a good enrichment broth. Plates and broth should be incubated at 35 to 37oC in ambient air. Plates should be examined within 18 to 24 h. However, growth may be observed as early as 6 to 8 h after inoculation. If two swabs have been collected, one should be used for the culture and one for the preparation of a Gram stain. In cases of suspected sepsis, organisms may actually be visualized in direct blood smears. In fact, the presence of large, encapsulated (clear zone around bacilli) gram-positive rods in blood is strong presumptive evidence for infection with B. anthracis. Of interest, no spores will be detected in primary blood smears, as CO2 levels within the body inhibit sporulation (32). Stool specimens should be plated onto phenyl ethyl alcohol agar or the cefoperazone-vancomycin-amphotericin B agar used to isolate Campylobacter spp. Collection of Environmental Specimens

Soil, bone, hair, swabs, water, or letters are specimens that may be examined for B. anthracis. These are usually collected during the course of a criminal investigation. While the hospital laboratory would usually not process these specimens, in cases where the FBI needs rapid turnaround, clinical laboratories may be asked to examine these samples for the purpose of ruling out the presence of this organism. Processing of Environmental Specimens

Soil, bone, hair, or powdery substances should first be weighed. A portion (2 g) should then be placed into a 15-ml centrifuge tube to which is added 2 ml of 0.3% Tween 20 in phosphate-buffered saline. The sample should be vortexed for 1 min and allowed to settle for 1 min. One milliliter of supernatant should then be cultured on 5% sheep blood agar, and the remainder should be placed in a temperature-safe container and stored at ⫺70oC. Swabs should be placed in 3 ml of 0.3% Tween 20 –phosphate-buffered saline and vortexed for 1 min. One-half of the specimen should then be heat shocked by placing it in a 65oC water bath for 10 min. Finally, the unheated and heated specimens are each inoculated to 5% sheep blood agar. B. anthracis spores will survive heat treatment and grow before and after heat treatment. Water should be filtered through a 0.45-␮m-pore-

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size filter. The filter should then be placed in a 50-ml centrifuge tube containing 4 ml of sterile distilled water. After vortexing for 1 min, one-half of the specimen should be heat shocked; the heated and unheated portions should then be plated onto 5% sheep blood agar (as above). Identification

Colonial morphology. At 16 to 18 h, colonies of B. anthracis are 2 to 5 ␮m in diameter and flat or slightly convex, with a ground-glass appearance. There are often comma-shaped projections from the edge of the colony, producing a colony that resembles the Medusa head of Greek mythology. The organism is nonhemolytic and has a tenacious consistency on blood agar. This means that when the colony is moved with a loop, the growth will stand up like a beaten egg white. Gram stain morphology. B. anthracis is a large gram-positive rod (1 to 1.5 ␮m by 3 to 5 ␮m) which forms oval, central to subterminal spores which do not swell the vegetative cell. Stains from colonies grown on blood agar show long chains of bacilli. Presumptive identification. A direct wet mount (gloves must be worn during preparation) and penicillin susceptibility test should be performed on any suspicious isolate. B. anthracis is nonmotile, nonhemolytic, and inhibited by a 10-U penicillin disk (zone of inhibition, 15 to 20 mm). Isolates suspected of being B. anthracis should be reported to the patient’s physician, and the isolate should be forwarded to the state health department for confirmatory testing. Confirmation at level B and above includes testing for the susceptibility to gamma phage, the production of a capsule in bicarbonate medium (nutrient agar with 0.8% sodium bicarbonate), and direct fluorescent antibody testing of the isolate using a reagent available from the CDC. Plague Clinical Features of Infection The causative agent of plague is Y. pestis. Three forms of plague infection are recognized in the human host: (i) bubonic, transmitted by the bite of infected fleas, leading to regional lymph node infection with pain, tenderness, swelling, and, rarely, meningitis; (ii) septicemia, beginning commonly with gastrointestinal symptoms and then proceeding to systemic symptoms with disseminated intravascular coagulation, adult respiratory distress syndrome, and circulatory collapse; and (iii) pneumonic, the form following respiratory exposure and the most likely to be encountered in a bioterrorist attack (9, 37). Following an incubation period of 2 to 3 days, primary respiratory infection with plague bacilli causes the rapid onset of fever, chills, malaise, myal-

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gia, headache, and an acute pneumonia syndrome, accompanied by chest pain, dyspnea, and cough (24). Sputum production is usually watery and bloodtinged, but may be frankly bloody (10). There follows a rapid progression to respiratory failure and shock. The chest X ray of pneumonic plague victims shows patchy or consolidated airspace disease with involvement of a single lobe or multiple lobes on both sides of the chest; cavitation of the lungs may be evident early on in the course of the disease (10). Pneumonic plague is fatal if not treated within the first 24 h. Appropriate antibiotic therapy includes streptomycin, 30 mg/kg of body weight intramuscularly (i.m.) per day, in two divided doses, or gentamicin i.v. for 10 days. Chloramphenicol may be given i.v. for plague meningitis or in sepsis syndrome. Doxycycline, 100 mg i.v. every 12 h, for 10 to 14 days is also effective and may be administered after an initial loading dose of 200 mg (24). Unfortunately, there is no proven benefit of the plague vaccine against pneumonic infection (11). Exposed individuals should be treated prophylactically with 100 mg of doxycycline orally every 12 h for 7 days (24). Y. pestis Collection of Specimens

The specimen(s) of choice depends on the disease presentation. Material from an infected bubo (lymph node) or a series of blood cultures collected within 24 h are the specimens of choice in bubonic plague (3). Tracheal or lung aspirates are the specimens of choice in pneumonic plague. Suitable autopsy specimens include lymphoid tissue, lung tissue, and bone marrow. Processing of Specimens

Y. pestis grows well on 5% sheep blood agar or chocolate agar; it is not a fastidious organism, although it grows more slowly than other Enterobacteriaceae at 35 to 37°C (45). Therefore, it is important to inoculate two blood agar plates and incubate one at 35 to 37°C and the other at 28oC (at which temperature Y. pestis grows faster than most enteric bacteria). Identification

Colonial morphology. Y. pestis produces pinpoint gray-white translucent colonies at 24 h at 35oC; by 48 h, the colony resembles that of a typical enteric gramnegative rod, i.e., 1 to 2 mm long, gray-white, and opaque. Y. pestis produces structures resembling stalactites in broth at 24 h; these resemble crumbly clumps at the side and bottom of the tube while the remainder of the broth is clear. The stalactites disappear after 48 h of incubation (3). Staining characteristics. Y. pestis is a small (1- by 0.5-␮m) gram-negative bipolar-staining rod. Plague should be expected if the direct stain shows small

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gram-negative bipolar staining rods from bubo, blood, or tracheal or lung aspirates from a patient with compatible clinical symptoms. The Wayson stain (a combination of basic fuchsin and methylene blue) or Wright-Giemsa stain can be used to demonstrate the classic closed safety pin appearance of Y. pestis. Presumptive identification. Since Y. pestis grows more slowly than other enteric bacteria at 35 to 37oC, the inoculum for rapid or automated identification systems must be heavier than usual in order to detect biochemical activity within 24 h. The organism is included in the database of the most commonly used enteric identification systems including automated devices. However, an identification of Y. pestis should be considered presumptive only and should not be ruled out until the isolate has been referred to a reputable reference laboratory for confirmation by specific bacteriophage analysis and direct fluorescent antibody stain using a conjugate available from the CDC. Alternatively, a single serum specimen exhibiting a titer of ⱖ1:128 from a patient with no known exposure to plague and no vaccination history is also evidence of current infection. Unfortunately, it is not possible to make a rapid serologic diagnosis when the titer is ⬍1:128, since convalescent-phase sera are usually not drawn for 1 to 4 months after antibiotic therapy is completed. PCR for Y. pestis DNA directly from clinical specimens is available from some reference laboratories but has not been validated for widespread use (45). In the United States, suspicious isolates should be sent for confirmation to the Plague Section, Bacterial Zoonosis Branch, Centers for Disease Control and Prevention, P.O. Box 2087, Fort Collins, CO 80522; phone, (970) 221-6450. Botulism Clinical Features of Infection Botulinum toxin causes illness in humans by binding to presynaptic nerve endings and blocking acetylcholine release, thus interrupting neurotransmission and causing weakness of the muscles supplied by the affected nerves (13, 50). While natural intoxication most often follows ingestion, the inhalation route is most likely to be used by bioterrorists (24). Symptoms usually begin within 24 to 36 h but may require several days after respiratory exposure depending on the magnitude of the dose. Early symptoms involve abnormalities of cranial nerve function (23, 39) that manifest as ocular symptoms, with blurred and double vision as well as light-induced pain; disordered speech, as evidenced by articulation problems, nasalized speech, and difficulty in swallowing; and a descending, symmetrical, skeletal muscle paralysis (24). The clinical examination of persons with botulism

Bioterrorism Agents

13

reveals them to be alert, awake, and oriented, with no fever or sensory findings. Involvement of the autonomic nervous system may be shown by orthostatic hypotension (reduction of blood pressure when patient is in a standing position). Eyes may show disconjugate gaze, pupillary dilatation, and eyelid drooping. The gag reflex is absent. Constipation and urinary retention are common (2). Cyanosis with advancing respiratory compromise signals severe impairment of phrenic nerve function and the need for ventilatory support. If adequate treatment of respiratory failure is instituted promptly, less than 5% of cases are fatal (24). A variety of clinical conditions may be confused with botulism. Guillain-Barre´ syndrome typically causes an ascending paralysis with notable sensory findings and elevated protein in the cerebrospinal fluid. Myasthenia gravis is usually, but not always, distinguished by a positive Tensilon (edrophonium chloride) test, used as a presumptive test in the differentiation of myasthenia gravis from other neurological disorders (for example, botulism) and has a typical electromyographic profile and induces antibodies to acetylcholine receptors. Additionally, the LambertEaton syndrome (progressive proximal muscle weakness in patients with carcinoma), acute poliomyelitis, tick paralysis, diphtheria, hypermagnesemia, and mushroom or chemical intoxications may be confused with botulism (2). Treatment of active botulism involves the use of antitoxin if the disease is in a phase of progression. Antitoxin is most effective if given immediately postexposure, before the appearance of symptoms (23). A trivalent antitoxin is available from the CDC through a 24-h emergency response system; a heptavalent product developed by the U.S. Army with as yet undetermined efficacy in humans is available as an investigational new drug (23, 49). All antitoxin products are of equine origin, so skin testing is required to avoid the occurrence of anaphylaxis or serum sickness. C. botulinum Botulism is a public health emergency even in the situation where criminal activity is not suspected. State health departments and the CDC offer 24-h diagnostic consultation, epidemiological assistance, and diagnostic laboratory services (14). The Foodborne and Diarrheal Diseases Branch of the CDC can be contacted by phone 24 h/day, 365 days/year, at (404) 639-2206 (Monday through Friday, 8:30 a.m. to 4:30 p.m., eastern standard time) or (404) 6392888 (after hours or on weekends). Collection of Specimens

In the event of a bioterrorist attack, C. botulinum is most likely to be dispersed via aerosolizaton or inges-

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tion. In the case of aerosolization, serum, for toxigenicity studies, feces or the return from a sterile water or saline enema, and environmental swabs should be collected for laboratory analysis (4). In the case of food-borne illness, serum, gastric contents, vomitus, stool, or the return from a sterile water or saline enema, and the suspected food source are considered optimal specimens for collection. If possible, food should be left in the original container. If the original container is not available, the food should be placed in a sterile, unbreakable, leakproof container. Each container should be placed in an individual plastic bag to prevent cross-contamination during shipment. It would be highly unusual for commercially prepared food to contain botulinum toxin. If a commercial product is suspected, an unopened container of food should be forwarded immediately to the Food and Drug Administration; the Food and Drug Administration should be contacted and notified prior to shipment. Environmental swabs should be collected in plastic containers without transport medium. Human specimens should be collected as soon as possible in the course of the infection and before the administration of antitoxin. A walnut-sized (10 to 50 g) sample of feces should be collected. If the patient is constipated, no more than 20 ml of sterile bacteriostatic water or saline should be infused as an enema and the return should be collected. The smallest volume of liquid should be used in the enema to prevent dilution of the toxin. Approximately 20 ml of gastric washings or vomitus may also be collected. Usually 20 ml of whole blood must be collected (red stopper tube) in order to provide at least 10 ml of serum for mouse toxicity studies. Transport and Shipment of Specimens

With the exception of environmental swabs, all specimens should be packaged in an insulated container with refrigerant; environmental swabs should be shipped at ambient temperature. All specimens for culture should be stored and shipped under anaerobic conditions; this is not necessary for toxin studies. If there is an unavoidable delay in transport, serum or feces may be frozen and shipped on dry ice. However, freezing may compromise the isolation and recovery of the organism but will not affect the detection of botulinum toxin. Processing of Specimens

C. botulinum grows on commercially available anaerobic media such as CDC anaerobic blood agar, brucella agar with 5% sheep blood, and phenyl ethyl alcohol blood agar at 35 to 37oC in an anaerobic atmosphere.

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Identification

Colonial morphology. Colonies of C. botulinum are gray-white with a circular or irregular edge and are usually beta hemolytic. Gram stain. C. botulinum is a gram-positive, straight rod (0.3 to 2.0 ␮m by 1.5 to 20.0 ␮m) with oval, subterminal spores that resemble a tennis racquet. Presumptive identification. C. botulinum can be presumptively identified following aerotolerance testing, Gram stain, spore stain, and growth on egg yolk agar (lipase-positive; variable for lecithinase activity). Definitive identification involves demonstrating toxigenicity. Therefore, the isolate must be transported to a reference laboratory under anaerobic conditions and examined using the toxin neutralization test. Tularemia Clinical Features of Infection The clinical forms of F. tularensis infection recognized in humans include glandular (or ulceroglandular or oculoglandular), pulmonary, gastrointestinal, and typhoidal. These result from direct penetration of the skin or exposure of mucous membranes with blood or tissue of infected animals or indirectly from bites of deerflies, ticks, or mosquitos; inhalation; or ingestion of contaminated food or water. The typhoidal and pulmonary forms result primarily from aerosol exposures (30). Following an incubation period of 2 to 10 days, patients present with fever, headache, myalgia, weight loss, and fatigue but no lymphadenopathy (20, 36, 40). Chest pain, nonproductive cough, and pneumonia with pleural effusions may occur. Radiographically, such infection may result in patchy, lobar, or cavitary infiltrates; mortality in untreated cases is approximately 35% (19, 20). Despite evident pulmonary involvement, secondary human-to-human transmission is unusual. High fever, meningitis, hepatitis, endocarditis, osteomyelitis, and septic shock may occur in the final stages of typhoidal tularemia. The differential diagnosis of pulmonary tularemia should include all of the atypical pneumonias, including psittacosis, legionellosis, Q fever, mycoplasma, and Chlamydia pneumoniae infections (30). Beta-lactam antibiotics are ineffective for the treatment of tularemia. Streptomycin (30 mg/kg/day i.m. in two divided doses for 10 to 14 days) is the drug of choice (44). Gentamicin (3 to 5 mg/kg/day i.m. in two divided doses for 10 to 14 days) is also effective (40, 47). Prophylaxis with doxycycline (100 mg BID for 14 days) is recommended immediately following a recognized exposure (24). For long-term prophylaxis, a live attenuated vaccine is available in the United States under an investigational new drug protocol (8).

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Francisella tularensis Collection of Specimens

In a bioterrorism event, F. tularensis would most likely be dispersed by aerosolization with subsequent onset of systemic or pneumonic tularemia. In such cases, the specimens of choice are bronchial or tracheal washings, sputum, and blood (52). Processing of Specimens

F. tularensis is a fastidious organism that usually requires cysteine supplementation for optimal growth. In this regard, cysteine heart agar with 9% chocolatized sheep red blood cells is the medium of choice (52). Most hospital (level A) laboratories would not normally maintain this medium in their inventory. Therefore, it is important that laboratory personnel be capable of recognizing this organism on commonly available media. F. tularensis grows on chocolate agar containing IsoVitaleX, Thayer-Martin agar, and buffered charcoal yeast extract agar (commonly used for the recovery of Legionella pneumophila). The organism may initially be recovered on 5% sheep blood agar, but it will not survive following subculture. Cultures suspected of containing F. tularensis should be incubated at 35 to 37oC for 72 h under ambient (aerobic) conditions; the growth of this organism is not enhanced by CO2. If tularemia is suspected, plates should be sealed and opened only in a BSC at a level B or higher laboratory equipped to handle this organism. The Septi-Chek blood culture system has been used successfully to isolate F. tularensis from blood as has lysis centrifugation with subsequent subculture to chocolate agar (16). It is recommended that a terminal subculture of other commercially available blood culture broths be performed prior to discarding the bottles. Identification

Colonial morphology. Colonies of F. tularensis are usually too small to be visualized at 24 h. Thereafter, colonial appearance depends on the growth medium. Colonies on cysteine heart agar are 2 to 4 mm in diameter, greenish-white, and dense, with a butyrous consistency; an opalescent sheen may be observed on the surface of the colony under oblique light if the plate is incubated for 48 to 72 h. After 48 h, colonies on 5% sheep blood agar will be 1 to 2 mm in diameter, grayish-white, and gamma-hemolytic. After the same period of time, colonies on chocolate or Thayer-Martin agars will be gray and flat with a smooth, entire edge and a shiny surface. Gram stain morphology. F. tularensis is a tiny, pleomorphic, poorly staining gram-negative coccobacillus (0.2 to 0.7 ␮m by ␮m). Presumptive identification. The hospital (level A) laboratory should be capable of isolating F. tularensis

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and recognize it by Gram stain and colonial characteristics. The organism is oxidase-negative, weakly catalase-positive, and nonmotile. F. tularensis grows slowly at 35 to 37oC and poorly at 28oC; these are useful features in differentiating it from Y. pestis, whose clinical presentation is very similar (52). Suspicious isolates should be sent to a reference laboratory for both presumptive and confirmatory identification. Testing must be performed in a BSL-3 facility. Presumptive identification is performed using one of three tests: (i) positive direct immunofluorescence test using a reagent available only from the CDC, (ii) agglutination by hyperimmune antiserum, or (iii) cellular fatty acid analysis. Confirmatory testing involves additional performance of standard biochemical tests. Serological testing may also be used for the diagnosis of tularemia. A single microagglutination titer of ⱖ1:128 or a tube agglutination titer of ⱖ1:160 is presumptive evidence of infection. A fourfold rise in titer between acute and convalescent serum specimens is confirmatory evidence of disease. Molecular diagnostic tests, especially PCR, have been used experimentally to detect F. tularenis directly from patient specimens but are not routinely available (27, 33). Brucellosis Clinical Features of Infection Human brucellosis may occur following enteric, percutaneous, or respiratory exposure, and subsequent patterns of illness are similar in all forms of infection. This fact reflects the distribution of these intracellular organisms to macrophages in bone, joints, brain, liver, spleen, and lung and accounts for the wide range of involved organ systems and the protean manifestations of illness. After an incubation period ranging from 5 to 60 days, fever, chills, diaphoresis, headache, myalgia, fatigue, anorexia, joint and lower back pain, weight loss, constipation, sore throat, or dry cough are common (35). Although respiratory symptoms consisting of cough and pleuritic chest pain occur in 20% of patients, overt pneumonia is uncommon (24). Skeletal involvement is evidenced by osteomyelitis of the vertebrae as well as by major joint infections, including the knees, hips, shoulders, and sacroiliac joints (5, 28, 41). While bone marrow involvement is reduced in all hematopoietic cell lines and hepatitis, and genitourinary tract infections may also occur, most fatalities result from central nervous system infection and endocarditis, which occur in only 5% of untreated patients (43). Symptoms may persist for weeks to months, but most patients will eventually recover within a year, regardless of treatment; however, subsequent relapses are frequent (18). The treatment of choice for severe brucellosis (bone, joint, heart, and central nervous system infec-

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tions) is a combination of doxycycline (100 mg BID) plus an aminoglycoside for 4 weeks, followed by the same dose of doxycycline plus rifampin, 600 to 900 mg/day for 6 weeks (31, 34). The latter regimen may be used as prophylaxis for 3 weeks following recognized exposure. At present, no vaccine is licensed in the United States for prevention of brucellosis among high-risk individuals (24). Brucella spp. Collection of Specimens

Brucellae are likely to be dispersed via aerosol during a bioterrorism incident; aerosols containing these organisms are considered to be highly infectious, with as few as 10 to 100 bacteria being capable of causing disease. Blood and bone marrow are the diagnostic specimens of choice. Collection of serum for serological studies is also recommended. An acutephase specimen should be collected as soon as possible following the onset of illness, followed by a convalescent-phase specimen collected 3 weeks later (48). Processing of Specimens

Brucella spp. grow on 5% sheep blood agar, chocolate agar, and supplemented media such as brucella agar or other infusion-supplemented media. Blood or bone marrow should be inoculated into a commercial blood culture system and incubated at 35 to 37oC for a minimum of 21 days. Blood culture broths rarely become turbid; thus, terminal subcultures should be performed before the broth is discarded and all subculture plates should be held a minimum of 7 days (48). Alternatively, whole blood may be processed by lysis centrifugation and plated onto the aforementioned agars. All specimens suspected of harboring brucellae should be handled in a BSC, and all plates should be sealed. Identification

Colonial morphology. Brucella spp. produce pinpoint, convex, smooth, translucent colonies after 48 h; they are nonhemolytic. Young colonies are slightly yellow and opalescent and may darken, becoming brownish, with age (48). Gram stain. Brucella spp. are tiny, faintly staining gram-negative coccobacilli (0.5 to 0.7 ␮m by 1.5 ␮m); their appearance has been described as resembling fine particles of sand (48). Presumptive identification. Most brucellae are oxidase positive and catalase positive, and all are nonmotile. Suspicious colonies should be heavily inoculated onto a Christensen’s urea agar slant; brucellae will usually hydrolyze urea within 2 h (48). These few simple tests are sufficient for differentiating Brucella spp. from Y. pestis (which grows quickly at 28oC), Haemophilus influenzae (which is urea negative),

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Acinetobacter spp. (which are oxidase negative), and Bordetella bronchiseptica (which is motile). The laboratory diagnosis of brucellosis can also be made serologically using a tube agglutination test. A single titer of ⱖ1:160 or a fourfold rise in titer is considered evidence of brucellosis (48). An enzymelinked immunosorbent assay has also been developed but is not commercially available. Molecular diagnostic tests, primarily PCR, have been used experimentally to detect brucellae in peripheral blood (46). Smallpox Clinical Features of Infection Transmission of smallpox to humans may occur via fomites but generally occurs when aerosolized particles enter the respiratory tract. After an incubation period of 12 to 14 days, symptoms begin with the abrupt onset of malaise, fever, chills, vomiting, headache, backache, and occasionally mental status changes or an erythematous macular rash (29). After 2 to 3 or more days, an enanthem and discrete papular rash of the face, hands and arms begin, later spreading to the legs and finally the trunk. The papules evolve into vesicles and finally pustules as fever and pain persist. Most lesions develop on the face and extremities and all are synchronous, that is, in the same phase of development. The pustules form scabs which are infectious until separation, and form deep, depigmented areas. Mortality from smallpox is 3% among vaccinated persons and 30% overall among nonvaccinated individuals (21). Among those with a secondary bacterial pneumonia, mortality rises to 50%. Death usually occurs in the second week of illness, although 5 to 10% of cases with a more fulminant course progress to death within 7 days. The most important infection to be distinguished from smallpox is primary varicella, or chickenpox. As opposed to smallpox, the lesions of chickenpox are more superficial and appear in “crops” or waves and thus are asynchronous in development, with groups of vesicles, pustules, and scabs appearing adjacent to one another. The lesions of chickenpox are denser over the trunk and do not appear on palms or soles. Also to be distinguished are monkeypox, which typically causes cervical and inguinal adenopathy, and skin eruptions from exposures to certain drugs or skin contact agents, such as erythema multiforme and allergic dermatitis. Any person exposed to smallpox should be immunized immediately with either the calf lymph-derived or cell culture vaccine developed by the Department of Defense. Vaccination against smallpox is effective in preventing death if given up to 5 days after exposure and in preventing illness if given within 72 h. Quarantine of those exposed should continue for 17 days,

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as they may transmit infection asymptomatically through oral and pharyngeal secretions. Treatment of active smallpox should be attempted with cidofovir, an agent with activity against many poxviruses though no proven efficacy against smallpox. Four other agents, adefovir dipivoxide, cyclic cidofovir, marboran, and ribavirin may also be candidates for use in this setting. The laboratory management of specimens suspected of harboring the agent of smallpox, variola major virus, is not discussed in this Cumitech since the handling of this virus requires a BSL-4 facility. In the event that this agent is suspected, the sample (specimen and/or tissue culture) should be shipped directly to the CDC and, in addition, the local health department should be notified. If packaged correctly, emergency medical specimens can usually be shipped on most major airlines. In the absence of appropriate flights, courier services such as World Courier [(800) 214-1350] which deliver 24 hours/day may be utilized. Finally, the Bioterrorism Emergency Number at the CDC (located in the Emergency Response Office) is (770) 488-7100.

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2. Abrutyn, E. 1998. Botulism, p. 904 –906. In A. S. Fauci, E. Braunwald, K. J. Isselbacher, J. D. Wilson, J. B. Martin, D. L. Kasper, S. L. Hauser, and D. L. Longo (ed.), Harrison’s Principles of Internal Medicine, 14th ed. McGraw Hill, New York, N.Y. 3. Aleksic, S. and J. Bockemuhl. 1999. Yersinia and other Enterobacteriaceae, p. 483– 496. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 7th ed. American Society for Microbiology, Washington, D.C. 4. Allen, S. D., C. L. Emery, and J. A. Siders. 1999. Clostridium, p. 654 – 671. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 7th ed. American Society for Microbiology, Washington, D.C. 5. Ariza, J., F. Gudiol, J. Valverde, R. Pallares, P. Fernandez-Viladrich, G. Rufi, L. Espadaler, and F. Fernandez-Nogues. 1985. Brucellar spondylitis: a detailed analysis based on current findings. Rev. Infect. Dis. 7:656 – 664. 6. Brachman, P. S. 1980. Inhalation anthrax. Ann. N. Y. Acad. Sci. 353:83–93.

ADDITIONAL INFORMATION

7. Brachman, P. S., and A. M. Friedlander. Anthrax, p. 729 –739. In S. A. Plotkin and E. A. Mortimer, Jr. (ed.), Vaccines. W. B. Saunders, Philadelphia, Pa.

For additional information not included in this Cumitech, consult the following websites:

8. Burke, D. S. 1997. Immunization against tularemia. J. Infect. Dis. 135:55– 60.

●

www.dot.gov (U.S. Department of Transportation shipping regulations)

9. Butler, T. 1983. Plague and Other Yersinia Infections. Plenum Press, New York, N.Y.

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www.cdc.gov/od/ohs/biosfty/biosfty.htm (biosafety information)

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www.cdc.gov/ncidod/hip/ISOLAT/Isolat.htm (Guideline for Isolation Precautions)

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www.hopkins-biodefense.org (Center for Civilian Biodefense Studies)

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www.cdc.gov/ncidod/dbmd/diseaseinfo/botulism .pdf

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www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm (BMBL)

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www.apic.org (Association for Professionals in Infection Control and Epidemiology)

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www.nbc-med.org (Nuclear, Biological, and Chemical Information Service)

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www.oep-ndms.dhhs.gov (Office of Emergency Preparedness: Counter Bioterrorism Program)

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