Office of Environmental Health and Safety

Microbiology laboratories are special, often unique, work environments that may pose identifiable infectious disease risks to persons in or near them. Infections have been contracted in the laboratory throughout the history of microbiology. Published reports around the turn of the century described laboratory-associated cases of typhoid, cholera, glanders, brucellosis, and tetanus.¹⁹² In 1941 Meyer and Eddie¹²⁵ published a survey of 74 laboratory-associated brucellosis infections that had occurred in the United States, and concluded that the "handling of cultures or specimens or the inhalation of dust containing Brucella organisms is eminently dangerous to laboratory workers." A number of cases were attributed to carelessness or poor technique in the handling of infectious materials.

In 1949, Sulkin and Pike¹⁷⁹ published the first in a series of surveys of laboratory-associated infections summarizing 222 viral infections -- 21 of which were fatal. In at least a third of the cases the probable source of infection was considered to be associated with the handling of infected animals and tissues. Known accidents were recorded in 27 (12%) of the reported cases.

In 1951, Sulkin and Pike¹⁸⁰ published the second of a series of summaries of laboratory-associated infections based on a questionnaire sent to 5,000 laboratories. Only one-third of the 1,342 cases cited had been reported in the literature. Brucellosis outnumbered all other reported laboratory-acquired infections and, together with tuberculosis, tularemia, typhoid, and streptococcal infection, accounted for 72% of all bacterial infections and for 31% of infections caused by all agents. The overall case fatality rate was 3%. Only 16% of all infections reported were associated with a documented accident. The majority of these were related to mouth pipetting and the use of needle and syringe.

This survey was updated in 1965,¹⁵⁴ adding 641 new or previously unreported cases, and again in 1976,¹⁵¹ summarizing a cumulative total of 3,921 cases. Brucellosis, typhoid, tularemia, tuberculosis, hepatitis, and Venezuelan equine encephalitis were the most commonly reported. Fewer than 20% of all cases were associated with a known accident. Exposure to infectious aerosols was considered to be a plausible but unconfirmed source of infection for the more than 80% of the reported cases in which the infected person had "worked with the agent."

In 1967 Hanson et al.⁸⁶ reported 428 overt laboratory-associated infections with arboviruses. In some instances the ability of a given arbovirus to produce human disease was first confirmed as the result of unintentional infection of laboratory personnel. Exposure to infectious aerosols was considered the most common source of infection.

In 1974 Skinhoj¹⁷⁰ published the results of a survey which showed that personnel in Danish clinical chemistry laboratories had a reported incidence of hepatitis (2.3 cases per year per 1,000 employees), seven times higher than that of the general population. Similarly, a 1976 survey by Harrington and Shannon⁸⁸ indicated that medical laboratory workers in England had "a five times increased risk of acquiring tuberculosis compared with the general population". Hepatitis B and shigellosis were also shown to be continuing occupational risks and, along with tuberculosis, were the three most commonly reported occupation-associated infections in Britain.

Although these reports suggest that laboratory personnel were at increased risk of being infected by the agents they handle, actual rates of infection are typically not available. However, the studies of Harrington and Shannon⁸⁸ and of Skinhoj¹⁷⁰ indicate that laboratory personnel had higher rates of tuberculosis, shigellosis, and hepatitis B than does the general population.

In contrast to the documented occurrence of laboratory-acquired infections in laboratory personnel, laboratories working with infectious agents have not been shown to represent a threat to the community. For example, although 109 laboratory-associated infections were recorded at the Centers for Disease Control and Prevention from 1947-1973,¹⁵⁹ no secondary cases were reported in family members or community contacts. The National Animal Disease Center reported a similar experience,¹⁸¹ with no secondary cases occurring in laboratory and non-laboratory contacts of 18 laboratory-associated cases occurring from 1960-1975. A secondary case of Marburg disease in the wife of a primary case was presumed to have been transmitted sexually two months after dismissal from the hospital.¹¹⁷ Three secondary cases of smallpox were reported in two laboratory-associated outbreaks in England in 1973¹⁵⁷ and 1978.²⁰² There were earlier reports of six cases of Q fever in a commercial laundry cleaning linens and uniforms from a laboratory working with the agent,¹⁴⁰ one case of Q fever in a visitor to a laboratory,¹⁴⁰ and two cases of Q fever in household contacts of a rickettsiologist.¹⁰ One case of Monkey B virus transmission from an infected animal care giver to his wife has been reported, apparently due to contact of the virus with broken skin.⁹² These cases are representative of the sporadic nature and infrequency of community infections in laboratory personnel working with infectious agents.

In his 1979 review,¹⁵³ Pike concluded "the knowledge, the techniques, and the equipment to prevent most laboratory infections are available". In the United States, however, no single code of practice, standards, guidelines, or other publication provided detailed descriptions of techniques, equipment, and other considerations or recommendations for the broad scope of laboratory activities conducted with a variety of indigenous and exotic infectious agents. The booklet, Classification of Etiologic Agents on the Basis of Hazard,²³ served as a general reference for some laboratory activities utilizing infectious agents. This booklet, and the concept of categorizing infectious agents and laboratory activities into four classes or levels, served as a basic format for earlier editions of Biosafety in Microbiological and Biomedical Laboratories (BMBL). This third edition of the BMBL continues to specifically describe combinations of microbiological practices, laboratory facilities, and safety equipment, and recommend their use in four categories or biosafety levels of laboratory operation with selected agents infectious to humans.

The descriptions of Biosafety Levels 1-4 parallel those in the NIH Guidelines for Research Involving Recombinant DNA,^71,72,139 and are consistent with the general criteria originally used in assigning agents to Classes 1-4 in Classification of Etiologic Agents on the Basis of Hazards.²³ Four biosafety levels are also described for infectious disease activities utilizing small laboratory animals. Recommendations for biosafety levels for specific agents are made on the basis of the potential hazard of the agent and of the laboratory function or activity.

Since the early 1980's, laboratories have applied these fundamental guidelines in activities associated with manipulations involving the human immunodeficiency virus (HIV). Even before HIV was identified as the causative agent of acquired immunodeficiency syndrome (AIDS), the principles for manipulating a bloodborne pathogen were suitable for safe laboratory work. Guidelines were also promulgated for health care workers under the rubric of Universal Precautions.⁴³ Indeed, Universal Precautions and this publication have become the basis for safe handling of blood and body fluids, as described in the recent OSHA publication Bloodborne Pathogen Standard.¹⁸⁷

In the late 1980's, considerable public concern was expressed about medical wastes, which led to the promulgation of the Medical Waste Tracking Act of 1988.¹⁸⁶ The principles established in the earlier volumes of the BMBL for handling potentially infectious wastes as an occupational hazard were reinforced by the National Research Council's Biosafety in the Laboratory: Prudent Practices for the Handling and Disposal of Infectious Materials.¹²

As this edition goes to press, there is growing concern about safe practices, procedures and facilities integral to the issues associated with the re-emergence of tuberculosis and worker safety in laboratory and health care settings. The underlying principles of the BMBL are applicable in the control of this airborne pathogen, including multi-drug resistant strains of M. tuberculosis.^47,54

Experience has demonstrated the prudence of the Biosafety Level 1-4 practices, procedures and facilities described for manipulations of etiologic agents in laboratory settings and animal facilities. Although no national reporting system exists for reporting laboratory-associated infections, anecdotal information suggests that strict adherence to these guidelines does contribute to a healthier and safer work environment for laboratorians, their co-workers and the surrounding community. The guidelines presented here can be customized for each individual laboratory, and can be used in conjunction with other available scientific information on risk assessment, to further minimize the potential for laboratory-associated infections.

SECTION II

Principles of Biosafety

The term "containment" is used in describing safe methods for managing infectious agents in the laboratory environment where they are being handled or maintained. The purpose of containment is to reduce or eliminate exposure of laboratory workers, other persons, and the outside environment to potentially hazardous agents.

Primary containment, the protection of personnel and the immediate laboratory environment from exposure to infectious agents, is provided by both good microbiological technique and the use of appropriate safety equipment. The use of vaccines may provide an increased level of personal protection. Secondary containment, the protection of the environment external to the laboratory from exposure to infectious materials, is provided by a combination of facility design and operational practices. Therefore, the three elements of containment include laboratory practice and technique, safety equipment, and facility design. The risk assessment of the work to be done with a specific agent will determine the appropriate combination of these elements.

Laboratory Practice and Technique. The most important element of containment is strict adherence to standard microbiological practices and techniques. Persons working with infectious agents or potentially infected materials must be aware of potential hazards, and must be trained and proficient in the practices and techniques required for handling such material safely. The director or person in charge of the laboratory is responsible for providing or arranging for appropriate training of personnel.

Each laboratory should develop or adopt a biosafety or operations manual which identifies the hazards that will or may be encountered, and which specifies practices and procedures designed to minimize or eliminate risks. Personnel should be advised of special hazards and should be required to read and to follow the required practices and procedures. A scientist trained and knowledgeable in appropriate laboratory techniques, safety procedures, and hazards associated with handling infectious agents must direct laboratory activities.

When standard laboratory practices are not sufficient to control the hazard associated with a particular agent or laboratory procedure, additional measures may be needed. The laboratory director is responsible for selecting additional safety practices, which must be in keeping with the hazard associated with the agent or procedure.

Laboratory personnel, safety practices, and techniques must be supplemented by appropriate facility design and engineering features, safety equipment, and management practices.

Safety Equipment (Primary Barriers). Safety equipment includes biological safety cabinets (BSCs), enclosed containers, and other engineering controls designed to remove or minimize exposures to hazardous biological materials. The biological safety cabinet (BSC) is the principal device used to provide containment of infectious splashes or aerosols generated by many microbiological procedures. Three types of biological safety cabinets (Class I, II, III) used in microbiological laboratories are described and illustrated in Appendix A. Open-fronted Class I and Class II biological safety cabinets are primary barriers which offer significant levels of protection to laboratory personnel and to the environment when used with good microbiological techniques. The Class II biological safety cabinet also provides protection from external contamination of the materials (e.g., cell cultures, microbiological stocks) being manipulated inside the cabinet. The gas-tight Class III biological safety cabinet provides the highest attainable level of protection to personnel and the environment.

An example of another primary barrier is the safety centrifuge cup, an enclosed container designed to prevent aerosols from being released during centrifugation. To minimize this hazard, containment controls such as BSCs or centrifuge cups must be used for handling infectious agents that can be transmitted through the aerosol route of exposure.

Safety equipment also may include items for personal protection such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Personal protective equipment is often used in combination with biological safety cabinets and other devices which contain the agents, animals, or materials being worked with. In some situations in which it is impractical to work in biological safety cabinets, personal protective equipment may form the primary barrier between personnel and the infectious materials. Examples include certain animal studies, animal necropsy, agent production activities, and activities relating to maintenance, service, or support of the laboratory facility.

Facility Design (Secondary Barriers). The design of the facility is important in providing a barrier to protect persons working inside and outside of the laboratory within the facility, and to protect persons or animals in the community from infectious agents which may be accidentally released from the laboratory. Laboratory management is responsible for providing facilities commensurate with the laboratory's function and the recommended biosafety level for the agents being manipulated.

The recommended secondary barrier(s) will depend on the risk of transmission of specific agents. For example, the exposure risks for most laboratory work in Biosafety Level 1 and 2 facilities will be direct contact with the agents, or inadvertent contact exposures through contaminated work environments. Secondary barriers in these laboratories may include separation of the laboratory work area from public access, availability of a decontamination facility (e.g., autoclave), and handwashing facilities.

As the risk for aerosol transmission increases, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment. Such design features could include specialized ventilation systems to assure directional air flow, air treatment systems to decontaminate or remove agents from exhaust air, controlled access zones, airlocks as laboratory entrances, or separate buildings or modules for isolation of the laboratory. Design engineers for laboratories may refer to specific ventilation recommendations as found in the Applications Handbook for Heating, Ventilation, and Air-Conditioning (HVAC) published by the American Society of Heating, Refrigerating, and Air-Conditioning Engineers (ASHRAE).²

Biosafety Levels. Four biosafety levels (BSLs) are described which consist of combinations of laboratory practices and techniques, safety equipment, and laboratory facilities. Each combination is specifically appropriate for the operations performed, the documented or suspected routes of transmission of the infectious agents, and for the laboratory function or activity.

The recommended biosafety level(s) for the organisms in Section VII (Agent Summary Statements) represent those conditions under which the agent can ordinarily be safely handled. The laboratory director is specifically and primarily responsible for assessing risks and for appropriately applying the recommended biosafety levels. Generally, work with known agents should be conducted at the biosafety level recommended in Section VII. When specific information is available to suggest that virulence, pathogenicity, antibiotic resistance patterns, vaccine and treatment availability, or other factors are significantly altered, more (or less) stringent practices may be specified.

Biosafety Level 1 practices, safety equipment, and facilities are appropriate for undergraduate and secondary educational training and teaching laboratories, and for other facilities in which work is done with defined and characterized strains of viable microorganisms not known to cause disease in healthy adult humans. Bacillus subtilis, Naegleria gruberi, and infectious canine hepatitis virus are representative of those microorganisms meeting these criteria. Many agents not ordinarily associated with disease processes in humans are, however, opportunistic pathogens and may cause infection in the young, the aged, and immunodeficient or immunosuppressed individuals. Vaccine strains which have undergone multiple in vivo passages should not be considered avirulent simply because they are vaccine strains.

Biosafety Level 1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for handwashing.

Biosafety Level 2 practices, equipment, and facilities are applicable to clinical, diagnostic, teaching and other facilities in which work is done with the broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided the potential for producing splashes or aerosols is low. Hepatitis B virus, the salmonellae, and Toxoplasma spp. are representative of microorganisms assigned to this containment level. Biosafety Level 2 is appropriate when work is done with any human-derived blood, body fluids, or tissues where the presence of an infectious agent may be unknown. (Laboratory personnel working with human-derived materials should refer to the Bloodborne Pathogen Standard¹⁸⁷ for specific, required precautions).

Primary hazards to personnel working with these agents relate to accidental percutaneous or mucous membrane exposures, or ingestion of infectious materials. Extreme precaution with contaminated needles or sharp instruments must be emphasized. Even though organisms routinely manipulated at BSL2 are not known to be transmissible by the aerosol route, procedures with aerosol or high splash potential that may increase the risk of such personnel exposure must be conducted in primary containment equipment, or devices such as a BSC or safety centrifuge cups. Other primary barriers should be used as appropriate, such as splash shields, face protection, gowns, and gloves.

Secondary barriers such as handwashing and waste decontamination facilities must be available to reduce potential environmental contamination.

Biosafety Level 3 practices, safety equipment, and facilities are applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents with a potential for respiratory transmission, and which may cause serious and potentially lethal infection. Mycobacterium tuberculosis, St. Louis encephalitis virus, and Coxiella burnetii are representative of microorganisms assigned to this level. Primary hazards to personnel working with these agents relate to autoinoculation, ingestion, and exposure to infectious aerosols.

At Biosafety Level 3, more emphasis is placed on primary and secondary barriers to protect personnel in contiguous areas, the community, and the environment from exposure to potentially infectious aerosols. For example, all laboratory manipulations should be performed in a BSC or other enclosed equipment, such as a gas-tight aerosol generation chamber. Secondary barriers for this level include controlled access to the laboratory and a specialized ventilation system that minimizes the release of infectious aerosols from the laboratory.

Biosafety Level 4 practices, safety equipment, and facilities are applicable for work with dangerous and exotic agents which pose a high individual risk of life-threatening disease, which may be transmitted via the aerosol route, and for which there is no available vaccine or therapy. Additionally, agents with a close or identical antigenic relationship to Biosafety Level 4 agents should also be handled at this level. When sufficient data are obtained, work with these agents may continue at this level or at a lower level. Viruses such as Marburg or Congo-Crimean hemorrhagic fever are manipulated at Biosafety Level 4.

The primary hazards to personnel working with Biosafety Level 4 agents are respiratory exposure to infectious aerosols, mucous membrane exposure to infectious droplets, and autoinoculation. All manipulations of potentially infectious diagnostic materials, isolates, and naturally or experimentally infected animals pose a high risk of exposure and infection to laboratory personnel, the community, and the environment.

The laboratory worker's complete isolation of aerosolized infectious materials is accomplished primarily by working in a Class III BSC or a full-body, air-supplied positive-pressure per sonnel suit. The Biosafety Level 4 facility itself is generally a separate building or completely isolated zone with complex, specialized ventilation and waste management systems to prevent release of viable agents to the environment.

The laboratory director is specifically and primarily responsible for the safe operation of the laboratory. His/her knowledge and judgment are critical in assessing risks and appropriately applying these recommendations. The recommended biosafety level represents those conditions under which the agent can ordinarily be safely handled. Special characteristics of the agents used, the training and experience of personnel, and the nature or function of the laboratory may further influence the director in applying these recommendations.

Animal Facilities. Four biosafety levels are also described for activities involving infectious disease work with experimental mammals. These four combinations of practices, safety equipment, and facilities are designated Animal Biosafety Levels 1, 2, 3, and 4, and provide increasing levels of protection to personnel and the environment.

Clinical Laboratories. Clinical laboratories, especially those in health care facilities, receive clinical specimens with requests for a variety of diagnostic and clinical support services. Typically, the infectious nature of clinical material is unknown, and specimens are often submitted with a broad request for microbiological examination for multiple agents (e.g., sputa submitted for "routine," acid-fast, and fungal cultures). It is the responsibility of the laboratory director to establish standard procedures in the laboratory which realistically address the issue of the infective hazard of clinical specimens.

Except in extraordinary circumstances (e.g., suspected hemorrhagic fever), the initial processing of clinical specimens and identification of isolates can be done safely at Biosafety Level 2, the recommended level for work with bloodborne pathogens such as hepatitis B virus and HIV. The containment elements described in Biosafety Level 2 are consistent with the Occupational Exposure to Bloodborne Pathogens Standard¹⁸⁷ from the Occupational Safety and Health Administration (OSHA), that requires the use of specific precautions with all clinical specimens of blood or other potentially infectious material (Universal Precautions).⁴³ Additionally, other recommendations specific for clinical laboratories may be obtained from the National Committee for Clinical Laboratory Standards.¹³⁴

Biosafety Level 2 recommendations and OSHA requirements focus on the prevention of percutaneous and mucous membrane exposures to clinical material. Primary barriers such as biological safety cabinets (Class I or II) should be used when performing procedures that might cause splashing, spraying, or splattering of droplets. Biological safety cabinets should also be used for the initial processing of clinical specimens when the nature of the test requested or other information is suggestive that an agent readily transmissible by infectious aerosols is likely to be present (e.g., M. tuberculosis), or when the use of a biological safety cabinet (Class II) is indicated to protect the integrity of the specimen.

The segregation of clinical laboratory functions and limiting or restricting access to such areas is the responsibility of the laboratory director. It is also the director's responsibility to establish standard, written procedures that address the potential hazards and the required precautions to be implemented.

Importation and Interstate Shipment of Certain Biomedical Materials. The importation of etiologic agents and vectors of human diseases is subject to the requirements of the Public Health Service Foreign Quarantine regulations. Companion regulations of the Public Health Service and the Department of Transportation specify packaging, labeling, and shipping requirements for etiologic agents and diagnostic specimens shipped in interstate commerce (see Appendix D).

The U. S. Department of Agriculture regulates the importation and interstate shipment of animal pathogens and prohibits the importation, possession, or use of certain exotic animal disease agents which pose a serious disease threat to domestic livestock and poultry (see Appendix E).

TABLE 1 & 2
Summary of Recommended Biosafety Levels for Infectious Agents

Summary of Recommended Biosafety Levels for Activities in Which Experimentally or Naturally Infected Vertebrate Animals Are Used

SECTION III

Laboratory Biosafety Level Criteria

The essential elements of the four biosafety levels for activities involving infectious microorganisms and laboratory animals are summarized in Tables 1 and 2 (see pages 14 and 15). The levels are designated in ascending order, by degree of protection provided to personnel, the environment, and the community.

Biosafety Level 1

Biosafety Level 1 is suitable for work involving well-characterized agents not known to cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. The laboratory is not necessarily separated from the general traffic patterns in the building. Work is generally conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is not required nor generally used. Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology or a related science.

The following standard and special practices, safety equipment and facilities apply to agents assigned to Biosafety Level 1:

A. Standard Microbiological Practices

1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments or work with cultures and specimens are in progress.

2. Persons wash their hands after they handle viable materials and animals, after removing gloves, and before leaving the laboratory.

3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas where there is reasonable likelihood of exposure to potentially infectious materials. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.

4. Mouth pipetting is prohibited; mechanical pipetting devices are used.

5. All procedures are performed carefully to minimize the creation of splashes or aerosols.

6. Work surfaces are decontaminated at least once a day and after any spill of viable material.

7. All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. Materials to be decontaminated at off-site from the laboratory are packaged in accordance with applicable local, state, and federal regulations, before removal from the facility.

8. An insect and rodent control program is in effect.

B. Special Practices: None

C. Safety Equipment (Primary Barriers)

1. Special containment devices or equipment such as a biological safety cabinet are generally not required for manipulations of agents assigned to Biosafety Level 1.

2. It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.

3. Gloves should be worn if the skin on the hands is broken or if a rash exists.

4. Protective eyewear should be worn for anticipated

splashes of microorganisms or other hazardous materials to the face.

D. Laboratory Facilities (Secondary Barriers)

1. Each laboratory contains a sink for handwashing.

2. The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not appropriate, and should not be used because proper decontamination following a spill extremely difficult to achieve.

3. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.

4. Laboratory furniture is sturdy. Spaces between benches, cabinets, and equipment are accessible for cleaning.

5. If the laboratory has windows that open, they are fitted with fly screens.

Biosafety Level 2

Biosafety Level 2 is similar to Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs in that (1) laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists, (2) access to the laboratory is limited when work is being conducted, (3) extreme precautions are taken with contaminated sharp items, and (4) certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment.

The following standard and special practices, safety equipment, and facilities apply to agents assigned to Biosafety Level 2:

A. Standard Microbiological Practices

1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments are in progress.

2. Persons wash their hands after they handle viable materials and animals, after removing gloves, and before leaving the laboratory.

3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.

4. Mouth pipetting is prohibited; mechanical pipetting devices are used.

5. All procedures are performed carefully to minimize the creation of splashes or aerosols.

6. Work surfaces are decontaminated at least once a day and after any spill of viable material.

8. An insect and rodent control program is in effect.

B. Special Practices

1. Access to the laboratory is limited or restricted by the laboratory director when work with infectious agents is in progress. In general, persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. For example, persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. The laboratory director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.

2. The laboratory director establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet specific entry requirements (e.g., immunization) enter the laboratory or animal rooms.

3. When the infectious agent(s) in use in the laboratory require special provisions for entry (e.g., immunization), a hazard warning sign incorporating the universal biohazard symbol is posted on the access door to the laboratory work area. The hazard warning sign identifies the infectious agent, lists the name and telephone number of the laboratory director or other responsible person(s), and indicates the special requirement(s) for entering the laboratory.

4. Laboratory personnel receive appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing).

5. When appropriate, considering the agent(s) handled, baseline serum samples for laboratory and other at-risk personnel are collected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the facility.

6. A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures.

7. Laboratory personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural or policy changes.

8. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.

a. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.

b. Syringes which re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.

c. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass are decontaminated before disposal, according to any local, state, or federal regulations.

9. Cultures, tissues, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping.

10. Laboratory equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination by infectious materials. Contaminated equipment must be decontaminated according to any local, state, or federal regulations before it is sent for repair or maintenance or packaged for transport in accordance with applicable local, state, or federal regulations, before removal from the facility.

11. Spills and accidents which result in overt exposures to infectious materials are immediately reported to the laboratory director. Medical evaluation, surveillance, and treatment are provided as appropriate and written records are maintained.

12. Animals not involved in the work being performed are not permitted in the lab.

C. Safety Equipment (Primary Barriers)

1. Properly maintained biological safety cabinets, preferably Class II, or other appropriate personal protective equipment or physical containment devices are used whenever:

a. Procedures with a potential for creating infectious aerosols or splashes are conducted. These may include centrifuging, grinding, blending, vigorous shaking or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating animals intranasally, and harvesting infected tissues from animals or eggs.

b. High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory if sealed rotor heads or centrifuge safety cups are used, and if these rotors or safety cups are opened only in a biological safety cabinet.

2. Face protection (goggles, mask, faceshield or other splatter guards) is used for anticipated splashes or sprays of infectious or other hazardous materials to the face, when the microorganisms must be manipulated outside the BSC.

3. Protective laboratory coats, gowns, smocks, or uniforms designated for lab use are worn while in the laboratory. This protective clothing is removed and left in the laboratory before leaving for non-laboratory areas (e.g., cafeteria, library, administrative offices). All protective clothing is either disposed of in the laboratory or laundered by the institution; it should never be taken home by personnel.

4. Gloves are worn when handling infected animals and when hands may contact infectious materials, contaminated surfaces or equipment. Wearing two pairs of gloves may be appropriate; if a spill or splatter occurs, the hand will be protected after the contaminated glove is removed. Gloves are disposed of when contaminated, removed when work with infectious materials is completed, and are not worn outside the laboratory. Disposable gloves are not washed or reused.

D. Laboratory Facilities (Secondary Barriers)

1. Each laboratory contains a sink for handwashing.

2. The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not appropriate, and should not be used because proper decontamination following a spill is extremely difficult to achieve.

3. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.

4. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning.

5. If the laboratory has windows that open, they are fitted with fly screens.

6. A method for decontamination of infectious or regulated laboratory wastes is available (e.g., autoclave, chemical disinfection, incinerator, or other approved decontamination system).

7. An eyewash facility is readily available.

Biosafety Level 3

Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents which may cause serious or potentially lethal disease as a result of exposure by the inhalation route. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents, and are supervised by competent scientists who are experienced in working with these agents.

All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features.

It is recognized, however, that many existing facilities may not have all the facility safeguards recommended for Biosafety Level 3 (e.g. access zone, sealed penetrations, and directional airflow, etc.). In these circumstances, acceptable safety may be achieved for routine or repetitive operations (e.g. diagnostic procedures involving the propagation of an agent for identification, typing, and susceptibility testing) in Biosafety Level 2 facilities. However, the recommended Standard Microbiological Practices, Special Practices, and Safety Equipment for Biosafety Level 3 must be rigorously followed. The decision to implement this modification of Biosafety Level 3 recommendations should be made only by the laboratory director.

The following standard and special safety practices, equipment and facilities apply to agents assigned to Biosafety Level 3:

A. Standard Microbiological Practices

1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments are in progress.

2. Persons wash their hands after handling infectious materials and animals, after removing gloves, and when they leave the laboratory.

3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the laboratory. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.

4. Mouth pipetting is prohibited; mechanical pipetting devices are used.

5. All procedures are performed carefully to minimize the creation of aerosols.

6. Work surfaces are decontaminated at least once a day and after any spill of viable material.

8. An insect and rodent control program is in effect.

B. Special Practices

1. Laboratory doors are kept closed when experiments are in progress.

2. The laboratory director controls access to the laboratory and restricts access to persons whose presence is required for program or support purposes. For example, persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. Persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. The director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.

3. The laboratory director establishes policies and procedures whereby only persons who have been advised of the potential biohazard, who meet any specific entry requirements (e.g., immunization), and who comply with all entry and exit procedures, enter the laboratory or animal rooms.

4. When infectious materials or infected animals are present in the laboratory or containment module, a hazard warning sign, incorporating the universal biohazard symbol, is posted on all laboratory and animal room access doors. The hazard warning sign identifies the agent, lists the name and telephone number of the laboratory director or other responsible person(s), and indicates any special requirements for entering the laboratory, such as the need for immunizations, respirators, or other personal protective measures.

5. Laboratory personnel receive the appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing).

6. Baseline serum samples are collected and stored for all laboratory and other at-risk personnel. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the laboratory.

7. A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures.

8. Laboratory personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural changes.

9. The laboratory director is responsible for insuring that, before working with organisms at Biosafety Level 3, all personnel demonstrate proficiency in standard microbiological practices and techniques, and in the practices and operations specific to the laboratory facility. This might include prior experience in handling human pathogens or cell cultures, or a specific training program provided by the laboratory director or other competent scientist proficient in safe microbiological practices and techniques.

10. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.

b. Syringes which re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.

c. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass should be decontaminated before disposal, according to any local, state, or federal regulations.

11. All manipulations involving infectious materials are conducted in biological safety cabinets or other physical containment devices within the containment module. No work in open vessels is conducted on the open bench.

12. Laboratory equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination with infectious materials. Contaminated equipment should also be decontaminated before it is sent for repair or maintenance or package for transport in accordance with applicable local, state, or federal regulations, before removal from the facility. Plastic-backed paper toweling used on non-perforated work surfaces within biological safety cabinets facilitates clean-up.

13. Cultures, tissues, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping.

14. All potentially contaminated waste materials (e.g., gloves, lab coats, etc.) from laboratories or animal rooms are decontaminated before disposal or reuse.

15. Spills of infectious materials are decontaminated, contained and cleaned up by appropriate professional staff, or others properly trained and equipped to work with concentrated infectious material.

16. Spills and accidents which result in overt or potential exposures to infectious materials are immediately reported to the laboratory director. Appropriate medical evaluation, surveillance, and treatment are provided and written records are maintained.

17. Animals and plants not related to the work being conducted are not permitted in the laboratory.

C. Safety Equipment (Primary Barriers)

1. Properly maintained biological safety cabinets are used (Class II or III - see Appendix A) for all manipulation of infectious materials.

2. Outside of a BSC, appropriate combinations of personal protective equipment are used (e.g., special protective clothing, masks, gloves, face protection, or respirators), in combination with physical containment devices (e.g., centrifuge safety cups, sealed centrifuge rotors, or containment caging for animals).

3. This equipment must be used for manipulations of cultures and of those clinical or environmental materials which may be a source of infectious aerosols; the aerosol challenge of experimental animals; harvesting of tissues or fluids from infected animals and embryonated eggs, and necropsy of infected animals.

4. Face protection (goggles and mask, or faceshield) is worn for manipulations of infectious materials outside of a biological safety cabinet.

5. Respiratory protection is worn when aerosols cannot be safely contained (i.e, outside of a biological safety cabinet), and in rooms containing infected animals.

6. Protective laboratory clothing such as solid-front or wrap-around gowns, scrub suits, or coveralls must be worn in, and not worn outside, the laboratory. Reusable laboratory clothing is to be decontaminated before being laundered.

7. Gloves must be worn when handling infected animals and when hands may contact infectious materials and contaminated surfaces or equipment. Disposable gloves should be discarded when contaminated, and never washed for reuse.

D. Laboratory Facilities (Secondary Barriers)

1. The laboratory is separated from areas which are open to unrestricted traffic flow within the building. Passage through two sets of self-closing doors is the basic requirement for entry into the laboratory from access corridors or other contiguous areas. A clothes change room (shower optional) may be included in the passage way.

2. Each laboratory contains a sink for handwashing. The sink is foot, elbow, or automatically operated and is located near the laboratory exit door.

3. The interior surfaces of walls, floors, and ceilings are water resistant so that they can be easily cleaned. Penetrations in these surfaces are sealed or capable of being sealed to facilitate decontamination.

4. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.

5. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning.

6. Windows in the laboratory are closed and sealed.

7. A method for decontaminating all laboratory wastes is available, preferably within the laboratory (i.e, autoclave, chemical disinfection, incineration, or other approved decontamination method).

8. A ducted exhaust air ventilation system is provided. This system creates directional airflow that draws air from "clean" areas into the laboratory toward "contaminated" areas. The exhaust air is not recirculated to any other area of the building, and is discharged to the outside with filtration and other treatment optional. The outside exhaust must be dispersed away from occupied areas and air intakes. Laboratory personnel must verify that the direction of the airflow (into the laboratory) is proper.

9. The High Efficiency Particulate Air (HEPA)-filtered exhaust air from Class II or Class III biological safety cabinets is discharged directly to the outside or through the building exhaust system. If the HEPA-filtered exhaust air from Class II or III biological safety cabinets is to be discharged to the outside through the building exhaust air system, it is connected to this system in a manner (e.g., thimble unit connection)¹³⁶ that avoids any interference with the air balance of the cabinets or building exhaust system. Exhaust air from Class II biological safety cabinets may be recirculated within the laboratory if the cabinet is tested and certified at least every twelve months.

10. Continuous flow centrifuges or other equipment that may produce aerosols are contained in devices that exhaust air through HEPA filters before discharge into the laboratory.

11. Vacuum lines are protected with liquid disinfectant traps and HEPA filters, or their equivalent, which are routinely maintained and replaced as needed.

12. An eyewash facility is readily available.

Biosafety Level 4

Biosafety Level 4 is required for work with dangerous and exotic agents which pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease. Agents with a close or identical antigenic relationship to Biosafety Level 4 agents are handled at this level until sufficient data are obtained either to confirm continued work at this level, or to work with them at a lower level. Members of the laboratory staff have specific and thorough training in handling extremely hazardous infectious agents; and they understand the primary and secondary containment functions of the standard and special practices, the containment equipment, and the laboratory design characteristics. They are supervised by competent scientists who are trained and experienced in working with these agents. Access to the laboratory is strictly controlled by the laboratory director. The facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building. A specific facility operations manual is prepared or adopted.

Within work areas of the facility, all activities are confined to Class III biological safety cabinets, or Class II biological safety cabinets used with one-piece positive pressure personnel suits ventilated by a life support system. The Biosafety Level 4 laboratory has special engineering and design features to prevent microorganisms from being disseminated into the environment.

The following standard and special safety practices equipment, and facilities apply to agents assigned to Biosafety Level 4:

A. Standard Microbiological Practices

1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments are in progress.

2. Persons wash their hands after handling infectious materials and animals; they take a decontaminating shower when they leave the laboratory.

4. Mouth pipetting is prohibited; only mechanical pipetting devices are used.

5. All procedures are performed carefully to minimize the creation of aerosols.

6. Work surfaces are decontaminated at least once a day and after any spill of viable material.

7. An insect and rodent control program is in effect.

B. Special Practices

1. Only persons whose presence in the facility or individual laboratory rooms is required for program or support purposes are authorized to enter. Persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. Therefore, persons who may be at increased risk of acquiring infection or for whom infection may be unusually hazardous, such as children or pregnant women, are not allowed in the laboratory or animal rooms.

The supervisor has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory. Access to the facility is limited by means of secure, locked doors; accessibility is managed by the laboratory director, biohazards control officer, or other person responsible for the physical security of the facility. Before entering, persons are advised of the potential biohazards and instructed as to appropriate safeguards for insuring their safety. Authorized persons comply with the instructions and all other applicable entry and exit procedures. A logbook, signed by all personnel, indicates the date and time of each entry and exit. Practical and effective protocols for emergency situations are established.

2. When infectious materials or infected animals are present in the laboratory or animal rooms, hazard warning signs, incorporating the universal biohazard symbol, are posted on all access doors. The sign identifies the agent, lists the name of the laboratory director or other responsible person(s), and indicates any special requirements for entering the area (e.g., the need for immunizations or respirators).

3. The laboratory director is responsible for insuring that, before working with organisms at Biosafety Level 4, all personnel demonstrate a high proficiency in standard microbiological practices and techniques, and in the special practices and operations specific to the laboratory facility. This might include prior experience in handling human pathogens or cell cultures, or a specific training program provided by the laboratory director or other competent scientist proficient in these unique safe microbiological practices and techniques.

4. Laboratory personnel receive available immunizations for the agents handled or potentially present in the laboratory.

5. Baseline serum samples for all laboratory and other atrisk personnel are collected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the laboratory. The decision to establish a serologic surveillance program takes into account the availability of methods for the assessment of antibody to the agent(s) of concern. The program provides for the testing of serum samples at each collection interval and the communication of results to the participants.

6. A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures.

8. Personnel enter and leave the facility only through the clothing change and shower rooms, and shower each time they leave the facility. Personnel use the airlocks to enter or leave the laboratory only in an emergency.

9. Personal clothing is removed in the outer clothing change room and kept there. Complete laboratory clothing, including under garments, pants and shirts or jumpsuits, shoes, and gloves, is provided and used by all personnel entering the facility. When leaving the laboratory and before proceeding into the shower area, personnel remove their laboratory clothing in the inner change room. Soiled clothing is autoclaved before laundering.

10. Supplies and materials needed in the facility are brought in by way of the double-doored autoclave, fumigation chamber, or airlock, which is appropriately decontaminated between each use. After securing the outer doors, personnel within the facility retrieve the materials by opening the interior doors of the autoclave, fumigation chamber, or airlock. These doors are secured after materials are brought into the facility.

11. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments are restricted in the laboratory for use only when there is no alternative, such as for parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.

b. Syringes which re-sheath the needle, needle-less systems, and other safe devices should be used when appropriate.

c. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass should be decontaminated before disposal, according to any local, state, or federal regulations.

12. Biological materials to be removed from the Class III cabinet or from the Biosafety Level 4 laboratory in a viable or intact state are transferred to a nonbreakable, sealed primary container and then enclosed in a nonbreakable, sealed secondary container. This is removed from the facility through a disinfectant dunk tank, fumigation chamber, or an airlock designed for this purpose.

13. No materials, except for biological materials that are to remain in a viable or intact state, are removed from the Biosafety Level 4 laboratory unless they have been autoclaved or decontaminated before they leave the facility. Equipment or material which might be damaged by high temperatures or steam may be decontaminated by gaseous or vapor methods in an airlock or chamber designed for this purpose.

14. Laboratory equipment is decontaminated routinely after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination with infectious materials. Contaminated equipment is also decontaminated before it is sent for repair or maintenance.

15. Spills of infectious materials are contained and cleaned up by appropriate professional staff or others properly trained and equipped to work with concentrated infectious material.

16. A system is set up for reporting laboratory accidents and exposures and employee absenteeism, and for the medical surveillance of potential laboratory-associated illnesses. Written records are prepared and maintained. An essential adjunct to such a reporting-surveillance system is the availability of a facility for the quarantine, isolation, and medical care of personnel with potential or known laboratory-associated illnesses.

17. Materials (e.g., plants, animals, and clothing) not related to the experiment being conducted are not permitted in the facility.

C. Safety Equipment (Primary Barriers)

1. All procedures within the facility with agents assigned to Biosafety Level 4 are conducted in the Class III biological safety cabinet or in Class II biological safety cabinets used in conjunction with one-piece positive pressure personnel suits ventilated by a life support system.

Activities with viral agents that require Biosafety Level 4 secondary containment capabilities can be conducted within Class II biological safety cabinets within the facility, without the one-piece positive pressure personnel suit being used if (a) the facility has been decontaminated, (b) no work is being conducted in the facility with other agents assigned to Biosafety Level 4, (c) all personnel are immunized against the specific agent being manipulated and demonstrate protective antibody levels, and (d) all other standard and special practices are followed.

2. All personnel entering the facility will don complete laboratory clothing, including undergarments, pants, and shirts or jumpsuits, shoes, and gloves. All such personal protective equipment is removed in the change room before showering and leaving the laboratory.

D. Laboratory Facility (Secondary Barriers)

1. The Biosafety Level 4 facility consists of either a separate building or a clearly demarcated and isolated zone within a building. Outer and inner change rooms separated by a shower are provided for personnel entering and leaving the facility. A double-doored autoclave, fumigation chamber, or ventilated airlock is provided for passage of those materials, supplies, or equipment which are not brought into the facility through the change room.

2. Walls, floors, and ceilings of the facility are constructed to form a sealed internal shell which facilitates fumigation and is animal and insect proof. The internal surfaces of this shell are resistant to liquids and chemicals, thus facilitating cleaning and decontamination of the area. All penetrations in these structures and surfaces are sealed. Any drains in the floors contain traps filled with a chemical disinfectant of demonstrated efficacy against the target agent, and they are connected directly to the liquid waste decontamination system. Sewer vents and other ventilation lines contain HEPA filters.

3. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize the horizontal surface area on which dust can settle.

4. Bench tops have seamless surfaces which are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.

5. Laboratory furniture is of simple and sturdy construction, and spaces between benches, cabinets, and equipment are accessible for cleaning.

6. A foot, elbow, or automatically operated handwashing sink is provided near the door of each laboratory room in the facility.

7. If there is a central vacuum system, it does not serve areas outside the facility. In-line HEPA filters are placed as near as practicable to each use point or service cock. Filters are installed to permit in-place decontamination and replacement. Other liquid and gas services to the facility are protected by devices that prevent backflow.

8. If water fountains are provided, they are foot operated and are located in the facility corridors outside the laboratory. The water service to the fountain is not connected to the backflow-protected distribution system supplying water to the laboratory areas.

9. Access doors to the laboratory are self-closing and lockable.

10. Any windows are breakage resistant.

11. A double-doored autoclave is provided for decontaminating materials passing out of the facility. The autoclave door which opens to the area external to the facility is sealed to the outer wall, and automatically controlled so that the outside door can only be opened after the autoclave "sterilization" cycle has been completed.

12. A pass-through dunk tank, fumigation chamber, or an equivalent decontamination method is provided so that materials and equipment that cannot be decontaminated in the autoclave can be safely removed from the facility.

13. Liquid effluents from laboratory sinks, biological safety cabinets, floor drains (if used), and autoclave chambers are decontaminated by heat treatment before being discharged to the sanitary sewer. Effluents from showers and toilets may be discharged to the sanitary sewer without treatment. The process used for decontamination of liquid wastes must be validated physically and biologically by use of a constant recording temperature sensor in conjunction with an indicator microorganism having a defined heat susceptibility profile.

14. A dedicated non-recirculating ventilation system is provided. The supply and exhaust components of the system are balanced to assure directional airflow from the area of least hazard to the area(s) of greatest potential hazard. The differential pressure/directional airflow between adjacent areas is monitored and alarmed to indicate malfunction of the system. The airflow in the supply and exhaust components is monitored and the components interlocked to assure inward (or zero) airflow is maintained.

15. The general room exhaust air from a facility in which the work is conducted in a Class III cabinet system is treated by a passage through a HEPA filter(s) prior to discharge to the outside. The air is discharged away from occupied spaces and air intakes. The HEPA filter(s) are located as near as practicable to the source in order to minimize the length of potentially contaminated ductwork. The HEPA filter housings are designed to allow for in situ decontamination of the filter prior to removal, or removal of the filter in a sealed gas-tight primary container for subsequent decontamination and/or destruction by incineration. The design of the HEPA filter housing should facilitate validation of the filter installation. The use of pre-certified HEPA filters can be an advantage. The service-life of the exhaust HEPA filters can be extended through adequate filtration of the supply air.

16. A specially designed suit area may be provided in the facility to provide personnel protection equivalent to that provided by Class III cabinets. Personnel who enter this area wear a one-piece positive pressure suit that is ventilated by a life support system. The life support system includes alarms and emergency backup breathing air tanks. Entry to this area is through an airlock fitted with airtight doors. A chemical shower is provided to decontaminate the surface of the suit before the worker leaves the area. The exhaust air from the suit area is filtered by two sets of HEPA filters installed in series. A duplicate filtration unit, exhaust fan, and an automatically starting emergency power source are provided. The air pressure within the suit area is lower than that of any adjacent area. Emergency lighting and communication systems are provided. All penetrations into the internal shell of the suit area are sealed. A double-doored autoclave is provided for decontaminating waste materials to be removed from the suit area.

17. The treated exhaust air from Class II biological safety cabinets, located in a facility in which workers wear a positive pressure suit, may be discharged into the animal room environment or to the outside through the facility air exhaust system. The biological safety cabinets are tested and certified at 12-month intervals. The air exhausted from Class III biological safety cabinets is passaged through two HEPA filter systems (in series) prior to discharge to the outside. If the treated exhaust is discharged to the outside through the facility exhaust system, it is connected to this system in a manner that avoids any interference with the air balance of the cabinets or the facility exhaust system.

TABLE 3
Comparison of Biological Safety Cabinets

SECTION IV

Vertebrate Animal Biosafety Level Criteria

If experimental animals are used, institutional management must provide facilities and staff and establish practices which reasonably assure appropriate levels of environmental quality, safety, and care. Laboratory animal facilities in many ways are extensions of the laboratory. As a general principle, the biosafety level (facilities, practices, and operational requirements) recommended for working with infectious agents in vivo and in vitro are comparable. It is well to remember, however, that the animal room is not the laboratory, and can present some unique problems. In the laboratory, hazardous conditions are caused by personnel or the equipment that is being used. In the animal room the activities of the animals themselves can introduce new hazards. Animals may produce aerosols, and they may also infect and traumatize animal handlers by biting and scratching.

These recommendations presuppose that laboratory animal facilities, operational practices, and quality of animal care meet applicable standards and regulations and that appropriate species have been selected for animal experiments (e.g., Guide for the Care and Use of Laboratory Animals, HEW Publication No. (NIH) 86-23, Rev. 1985, and Laboratory Animal Welfare Regulations - 9 CFR, Subchapter A, Parts 1, 2 and 3).

Ideally, facilities for laboratory animals used for studies of infectious or noninfectious disease should be physically separate from other activities such as animal production and quarantine, clinical laboratories, and especially from facilities that provide patient care. Animal facilities should be designed and constructed to facilitate cleaning and housekeeping. Traffic flow that will minimize the risk of cross contamination should be considered in the plans. A "clean/dirty hall" layout is useful in achieving this. Floor drains should be installed in animal facilities only on the basis of clearly defined needs. If floor drains are installed, the drain trap should always contain water or a suitable disinfectant.

These recommendations describe four combinations of practices, safety equipment, and facilities for experiments on animals infected with agents which produce, or may produce, human infection. These four combinations provide increasing levels of protection to personnel and to the environment, and are recommended as minimal standards for activities involving infected laboratory animals. These four combinations, designated Animal Biosafety Levels (ABSL) 1-4, describe animal facilities and practices applicable to work on animals infected with agents assigned to corresponding Biosafety Levels 1-4.

Facility standards and practices for invertebrate vectors and hosts are not specifically addressed in standards written for commonly used laboratory animals. "Laboratory Safety for Arboviruses and Certain other Viruses of Vertebrates,"¹⁷⁸ prepared by the Subcommittee on Arbovirus Laboratory Safety of the American Committee on Arthropod-Borne Viruses, serves as a useful reference in the design and operation of facilities using arthropods.

Animal Biosafety Level 1

A. Standard Practices

1. Access to the animal facility is limited or restricted at the discretion of the laboratory or animal facility director.

2. Personnel wash their hands after handling cultures and animals, after removing gloves, and before leaving the animal facility.

3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in animal rooms. Persons who wear contact lenses in animal rooms should also wear goggles or a face shield.

4. All procedures are carefully performed to minimize the creation of aerosols.

5. Work surfaces are decontaminated after use or after any spill of viable materials.

6. Doors to animal rooms open inward, are self-closing and are kept closed when experimental animals are present.

7. All wastes from the animal room are appropriately decontaminated, preferably by autoclaving, before disposal. Infected animal carcasses are incinerated after being transported from the animal room in leakproof, covered containers.

8. An insect and rodent control program is in effect.

B. Special Practices

1. The laboratory or animal facility director limits access to the animal room to personnel who have been advised of the potential hazard and who need to enter the room for program or service purposes when work is in progress. In general, persons who may be at increased risk of acquiring infection, or for whom infection might be unusually hazardous, are not allowed in the animal room.

2. The laboratory or animal facility director establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet any specific requirements (e.g., immunization) may enter the animal room.

3. Bedding materials from animal cages are removed in such a manner as to minimize the creation of aerosols, and are disposed of in compliance with applicable institutional or local requirements.

4. Cages are washed manually or in a cage washer. Temperature of final rinse water in a mechanical washer should be 180^oF.

5. The wearing of laboratory coats, gowns, or uniforms in the animal facility is recommended. It is further recommended that laboratory coats worn in the animal facility not be worn in other areas.

6. A biosafety manual is prepared or adopted. Personnel are advised of special hazards, are required to read and to follow instructions on practices and procedures.

C. Safety Equipment (Primary Barriers)

Special containment equipment is not required for animals infected with agents assigned to Biosafety Level 1.

D. Animal Facilities (Secondary Barriers)

1. The animal facility is designed and constructed to facilitate cleaning and housekeeping.

2. A handwashing sink is available in the animal facility.

3. If the animal facility has windows that open, they are fitted with fly screens.

4. Exhaust air is discharged to the outside without being recirculated to other rooms, and it is recommended, but not required, that the direction of airflow in the animal facility is inward.

Animal Biosafety Level 2

A. Standard Practices

1. Access to the animal facility is limited or restricted at the discretion of the laboratory or animal facility director.

2. Personnel wash their hands after handling cultures and animals, after removing gloves, and before leaving the animal facility.

4. All procedures are carefully performed to minimize the creation of aerosols.

5. Work surfaces are decontaminated after use or after any spill of viable materials.

6. Doors to animal rooms open inward, are self-closing and are kept closed when experimental animals are present.

8. An insect and rodent control program is in effect.

B. Special Practices

3. When the infectious agent(s) in use in the animal room requires special entry provisions (e.g., the need for immunizations and respirators) a hazard warning sign, incorporating the universal biohazard symbol, is posted on the access door to the animal room. The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the animal facility supervisor or other responsible person(s), and indicates the special requirement(s) for entering the animal room.

4. Laboratory personnel receive appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing).

5. When appropriate, considering the agents handled, baseline serum samples from animal care and other at-risk personnel are collected and stored. Additional serum samples may be collected periodically depending on the agents handled or the function of the facility. The decision to establish a serologic surveillance program must take into account the availability of methods for the assessment of antibody to the agent(s) of concern. The program should provide for the testing of serum samples at each collection interval and the communication of results to the participants.

6. A biosafety manual is prepared or adopted. Personnel are advised of special hazards, and are required to read and to follow instructions on practices and procedures.

8. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments are restricted in the animal facility for use only when there is no alternative, such as for parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.

b. Syringes which re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.

c. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass should be decontaminated before disposal, according to any local, state, or federal regulations.

9. Cultures, tissues, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping.

10. Cages are appropriately decontaminated, preferably by autoclaving, before they are cleaned and washed.

Equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination by infectious materials. Contaminated equipment must be decontaminated according to any local, state, or federal regulations before it is sent for repair or maintenance or packaged for transport in accordance with applicable local, state, or federal regulations, before removal from the facility.

12. Animals not involved in the work being performed are not permitted in the lab.

C. Safety Equipment (Primary Barriers)

1. Biological safety cabinets, other physical containment devices, and/or personal protective equipment (e.g., respirators, face shields) are used whenever procedures with a high potential for creating aerosols are conducted.¹³⁹ These include necropsy of infected animals, harvesting of tissues or fluids from infected animals or eggs, intranasal inoculation of animals, and manipulations of high concentrations or large volumes of infectious materials.

2. Appropriate face/eye and respiratory protection is worn by all personnel entering animal rooms housing nonhuman primates.

3. Laboratory coats, gowns, or uniforms are worn while in the animal room. This protective clothing is removed before leaving the animal facility.

4. Special care is taken to avoid skin contamination with infectious materials; gloves are worn when handling infected animals and when skin contact with infectious materials is unavoidable.

D. Animal Facilities (Secondary Barriers)

1. The animal facility is designed and constructed to facilitate cleaning and housekeeping.

2. A handwashing sink is available in the room where infected animals are housed.

3. If the animal facility has windows that open, they are fitted with fly screens.

4. If floor drains are provided, the drain traps are always filled with water or a suitable disinfectant.

5. Exhaust air is discharged to the outside without being recirculated to other rooms, and it is recommended, but not required, that the direction of airflow in the animal facility is inward.

6. An autoclave which can be used for decontaminating infectious laboratory waste is available in the building with the animal facility.

Animal Biosafety Level 3

A. Standard Practices

1. Access to the animal facility is limited or restricted at the discretion of the laboratory or animal facility director.

2. Personnel wash their hands after handling cultures and animals, after removing gloves, and before leaving the animal facility.

4. All procedures are carefully performed to minimize the creation of aerosols.

5. Work surfaces are decontaminated after use or after any spill of viable materials.

6. Doors to animal rooms open inward, are self-closing and are kept closed when experimental animals are present.

8. An insect and rodent control program is in effect.

B. Special Practices

1. The laboratory director or other responsible person restricts access to the animal room to personnel who have been advised of the potential hazard and who need to enter the room for program or service purposes when infected animals are present. Persons who are at increased risk of acquiring infection, or for whom infection might be unusually hazardous, are not allowed in the animal room. Persons at increased risk may include children, pregnant women, and persons who are immunodeficient or immunosuppressed. The supervisor has the final responsibility for assessing each circumstance and determining who may enter or work in the facility.

2. The laboratory director or other responsible person establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet any specific requirements (e.g., for immunization) may enter the animal room.

4. Laboratory personnel receive appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing).

5. Baseline serum samples from all personnel working in the facility and other at-risk personnel should be collected and stored. Additional serum samples may be collected periodically and stored. The serum surveillance program must take into account the availability of methods for the assessment of antibody to the agent(s) of concern. The program should provide for the testing of serum samples at each collection interval and the communication of results to the participants.

6. A biosafety manual is prepared or adopted. Personnel are advised of special hazards, and are required to read and to follow instructions on practices and procedures.

8. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments are restricted in the laboratory for use only when there is no alternative, such as for parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.

a. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container, preferably containing a suitable disinfectant, for transport to a processing area for decontamination, preferably by autoclaving.

b. Syringes which re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.

c. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass should be decontaminated before disposal, according to any local, state, or federal regulations.

9. Cultures, tissues, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping.

10. Cages are autoclaved or thoroughly decontaminated before bedding is removed or before they are cleaned and washed. Equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination by infectious materials. Contaminated equipment must be decontaminated according to any local, state, or federal regulations before it is sent for repair or maintenance or packaged for transport in accordance with applicable local, state, or federal regulations, before removal from the facility.

12. All wastes from the animal room are autoclaved before disposal. All animal carcasses are incinerated. Dead animals are transported from the animal room to the incinerator in leakproof covered containers.

13. Animals not involved in the work being performed are not permitted in the lab.

C. Safety Equipment (Primary Barriers)

1. Personal protective equipment is used for all activities involving manipulations of infectious materials or infected animals.

a. Wrap-around or solid-front gowns or uniforms are worn by personnel entering the animal room. Front-button laboratory coats are unsuitable. Protective gowns should be appropriately contained until decontamination or disposal.

b. Personnel wear gloves when handling infected animals. Gloves are removed aseptically and autoclaved with other animal room wastes before disposal.

c. Appropriate face/eye and respiratory protection is worn by all personnel entering animal rooms housing nonhuman primates.

d. Boots, shoe covers, or other protective footwear, and disinfectant footbaths are available and used when indicated.

2. Physical containment devices and equipment appropriate for the animal species are used for all procedures and manipulations of infectious materials or infected animals.

3. The risk of infectious aerosols from infected animals or their bedding also can be reduced if animals are housed in partial containment caging systems, such as open cages placed in ventilated enclosures (e.g., laminar flow cabinets), solid wall and bottom cages covered with filter bonnets, or other equivalent primary containment systems.

D. Animal Facilities (Secondary Barriers)

1. The animal facility is designed and constructed to facilitate cleaning and housekeeping, and is separated from areas which are open to unrestricted personnel traffic within the building. Passage through two sets of doors is the basic requirement for entry into the animal room from access corridors or other contiguous areas. Physical separation of the animal room from access corridors or other activities may also be provided by a double-doored clothes change room (showers may be included), airlock, or other access facility which requires passage through two sets of doors before entering the animal room.

2. The interior surfaces of walls, floors, and ceilings are water resistant so that they may be easily cleaned. Penetrations in these surfaces are sealed or capable of being sealed to facilitate fumigation or space decontamination.

3. A foot, elbow, or automatically operated handwashing sink is provided in each animal room near the exit door.

4. If vacuum service (i.e., central or local) is provided, each service connection should be fitted with liquid disinfectant traps and a HEPA filter.

5. If floor drains are provided, they are protected with liquid traps that are always filled with water or disinfectant.

6. Windows in the animal room are non-operating and sealed.

7. Animal room doors are self-closing and are kept closed when infected animals are present.

8. An autoclave for decontaminating wastes is available, preferably within the animal facility. Materials are transferred to the autoclave in a covered leakproof container whose outer surface has been decontaminated.

9. A non-recirculating ventilation system is provided. The supply and exhaust components of the system are balanced to provide for directional flow of air into the animal room. The exhaust air is discharged directly to the outside and clear of occupied areas and air intakes. Exhaust air from the room can be discharged without filtration or other treatment. Personnel must periodically validate that proper directional airflow is maintained.

10. The HEPA filtered exhaust air from Class I or Class II biological safety cabinets or other primary containment devices is discharged directly to the outside or through the building exhaust system. Exhaust air from these primary containment devices may be recirculated within the animal room if the device is tested and certified at least every 12 months. If the HEPA filtered exhaust air from Class I or Class II biological safety cabinets is discharged to the outside through the building exhaust system, it is connected to this system in a manner (e.g., thimble unit connection)¹³⁴ that avoids any interference with the performance of either the cabinet or building exhaust system.

Animal Biosafety Level 4

A. Standard Practices

1. Access to the animal facility is limited or restricted at the discretion of the laboratory or animal facility director.

2. Personnel wash their hands after handling cultures and animals, after removing gloves, and before leaving the animal facility.

4. All procedures are carefully performed to minimize the creation of aerosols.

5. Work surfaces are decontaminated after use or after any spill of viable materials.

6. Doors to animal rooms open inward, are self-closing and are kept closed when experimental animals are present.

8. Cages are autoclaved before bedding is removed and before they are cleaned and washed. When feasible, disposable cages that do not require cleaning are recommended; however, these cages also autoclaved before disposal. Equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination by infectious materials. Contaminated equipment must be decontaminated according to any local, state, or federal regulations before it is sent for repair or maintenance or packaged for transport in accordance with applicable local, state, or federal regulations, before removal from the facility.

9. An insect and rodent control program is in effect.

B. Special Practices

1. Only persons whose entry into the facility or individual animal room is required for program or support purposes are authorized to enter. Persons who may be at increased risk of acquiring infection or for whom infection might be unusually hazardous are not allowed in the animal facility. Persons at increased risk may include children, pregnant women, and persons who are immunodeficient or immunosuppressed. The supervisor has the final responsibility for assessing each circumstance and determining who may enter or work in the facility. Access to the facility is limited by secure, locked doors; accessibility is controlled by the animal facility supervisor, biohazards control officer, or other person responsible for the physical security of the facility. Before entering, persons are advised of the potential biohazards and instructed as to appropriate safeguards. Personnel comply with the instructions and all other applicable entry and exit procedures. Practical and effective protocols for emergency situations are established.

2. Laboratory personnel receive appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing).

3. Baseline serum samples are collected and stored for all laboratory and other at-risk personnel. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the laboratory. The decision to establish a serologic surveillance program takes into account the availability of methods for the assessment of antibody to the agent(s) of concern. The program provides for the testing of serum samples at each collection interval and the communication of results to the participants.

4. A biosafety manual is prepared or adopted. Personnel are advised of special hazards, and are required to read and to follow instructions on practices and procedures.

5. When the infectious agent(s) in use in the animal room requires special entry provisions (e.g., the need for immunizations and respirators) a hazard warning sign, incorporating the universal biohazard symbol, is posted on the access door to the animal room. The hazard warning sign identifies the infectious agent(s) in use, lists the name and telephone number of the animal facility supervisor or other responsible person(s), and indicates the special requirement(s) for entering the animal room.

6. Laboratory personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural or policy changes.

7. Hypodermic needles and syringes are used only for gavage, for parenteral injection, and aspiration of fluids from diaphragm bottles or well-restrained laboratory animals.

a. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments are restricted in the laboratory for use only when there is no alternative, such as for parenteral injection, blood collection, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware is substituted for glassware whenever possible.

b. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles are not bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they are carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps are placed in a hard-walled container, preferably containing a suitable disinfectant, for transport to a processing area for decontamination, preferably by autoclaving.

c. Syringes which re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.

d. Broken glassware is not handled directly by hand, but is removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass are decontaminated before disposal, according to any local, state, or federal regulations.

8. Cultures, tissues, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping.

9. Spills and accidents which result in overt exposures to infectious materials are immediately reported to the laboratory director. Medical evaluation, surveillance, and treatment are provided as appropriate, and written records are maintained.

10. Personnel enter and leave the facility only through the clothing change and shower rooms. Personnel shower each time they leave the facility. Head covers are provided to personnel who do not wash their hair during the exit shower. Except in an emergency, personnel do not enter or leave the facility through the airlocks.

11. Personal clothing is removed in the outer clothing change room and kept there. Complete laboratory clothing, including undergarments, pants and shirts or jumpsuits, shoes, and gloves, are provided and used by all personnel entering the facility. When exiting, personnel remove laboratory clothing in the inner change room before entering the shower area. Soiled clothing is autoclaved before laundering.

12. Supplies and materials are brought into the facility by way of a double-door autoclave, fumigation chamber, or airlock. After securing the outer doors, personnel inside the facility retrieve the materials by opening the interior door of the autoclave, fumigation chamber, or airlock. This inner door is secured after materials are brought into the facility. The autoclave fumigation chamber or airlock is decontaminated before the outer door is opened.

13. A system is established for the reporting of animal facility accidents and exposures, employee absenteeism, and for the medical surveillance of potential laboratory-associated illnesses. An essential adjunct to such a reporting-surveillance system is the availability of a facility for the quarantine, isolation, and medical care of persons with potential or known laboratory-associated illnesses.

14. Materials (e.g., plants, animals, clothing) not related to the experiment are not permitted in the facility.

C. Safety Equipment (Primary Barriers)

Laboratory animals, infected with agents assigned to Biosafety Level 4, are housed a Class III biological safety cabinet or in a partial containment caging system (such as open cages placed in ventilated enclosures, solid wall and bottom cages covered with filter bonnets, or other equivalent primary containment systems), in specially designed areas in which all personnel are required to wear one-piece positive pressure suits ventilated with a life support system.

Animal work with viral agents that require Biosafety Level 4 secondary containment, and for which highly effective vaccines are available and used, may be conducted with partial containment cages and without the one-piece positive pressure personnel suit if: the facility has been decontaminated, no concurrent experiments are being done in the facility which require Biosafety Level 4 primary and secondary containment, and all other standard and special practices are followed.

D. Animal Facility (Secondary Barriers)

1. The animal rooms are located in a separate building or in a clearly demarcated and isolated zone within a building. Outer and inner change rooms separated by a shower are provided for personnel entering and leaving the facility. A double-doored autoclave, fumigation chamber, or ventilated airlock is provided for passage of materials, supplies, or equipment which are not brought into the facility through the change room.

2. Walls, floors, and ceilings of the facility are constructed to form a sealed internal shell which facilitates decontamination and is animal and insect proof. The internal surfaces of this shell are resistant to liquids and chemicals, thus facilitating cleaning and decontamination of the area. All penetrations in these structures and surfaces are sealed.

3. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize horizontal surface areas on which dust can settle.

4. A foot, elbow, or automatically operated handwashing sink is provided in each animal room near the exit door.

5. If there is a central vacuum system, it does not serve areas outside of the facility. The vacuum system has in-line HEPA filters placed as near as practicable to each use point or service cock. Filters are installed to permit in-place decontamination and replacement. Other liquid and gas services for the facility are protected by devices that prevent backflow.

6. External animal facility doors are self-closing and selflocking.

7. Any windows must be resistant to breakage and sealed.

8. A double-doored autoclave is provided for decontaminating materials that leave the facility. The autoclave door which opens to the area external to the facility is automatically controlled so that it can only be opened after the autoclave "sterilization" cycle is completed.

9. A pass-through dunk tank, fumigation chamber, or an equivalent decontamination method is provided so that materials and equipment that cannot be decontaminated in the autoclave can be safely removed from the facility.

10. Liquid effluents from laboratory sinks, biological safety cabinets, floor drains (if used), and autoclave chambers are decontaminated by heat treatment before being discharged to the sanitary sewer. Effluents from showers and toilets may be discharged to the sanitary sewer without treatment. The process used for decontamination of liquid wastes must be validated physically and biologically by use of a constant recording temperature sensor in conjunction with an indicator microorganism having a defined heat susceptibility profile.

11. A dedicated non-recirculating ventilation system is provided. The supply and exhaust components of the system are balanced to assure directional airflow from the area of least hazard to the area(s) of greatest potential hazard. The differential pressure/directional airflow between adjacent areas is monitored and alarmed to indicate malfunction of the system. The airflow in the supply and exhaust components is monitored and the components interlocked to assure inward (or zero) airflow is maintained.

12. The general room exhaust air from a facility in which the work is conducted in a Class III cabinet system is treated by a passage through a HEPA filter(s) prior to discharge to the outside. The air is discharged away from occupied spaces and air intakes. The HEPA filter(s) are located as near as practicable to the source in order to minimize the length of potentially contaminated ductwork. The HEPA filter housings are designed to allow for in situ decontamination of the filter prior to removal, or removal of the filter in a sealed gas-tight primary container for subsequent decontamination and/or destruction by incineration. The design of the HEPA filter housing should facilitate validation of the filter installation. The use of pre-certified HEPA filters can be an advantage. The service-life of the exhaust HEPA filters can be extended through adequate filtration of the supply air.

13. The treated exhaust air from Class II biological safety cabinets located in a facility in which workers wear a positive pressure suit may be discharged into the animal room environment or to the outside through the facility air exhaust system. The biological safety cabinets are tested and certified at 9-month intervals. The air exhausted from Class III biological safety cabinets is passaged through two HEPA filter systems (in series) prior to discharge to the outside. If the treated exhaust is discharged to the outside through the facility exhaust system, it is connected to this system in a manner that avoids any interference with the air balance of the cabinets or the facility exhaust system.

14. A specially designed suit area may be provided in the facility. Personnel who enter this area wear a one-piece positive pressure suit that is ventilated by a life support system. The life support system is provided with alarms and emergency backup breathing air tanks. Entry to this area is through an airlock fitted with airtight doors. A chemical shower is provided to decontaminate the surface of the suit before the worker leaves the area. The exhaust air from the area in which the suit is used is filtered by two sets of HEPA filters installed in series. Duplicate filtration units and exhaust fans are provided. An automatically starting emergency power source is provided. The air pressure within the suit area is lower than that of any adjacent area. Emergency lighting and communication systems are provided. All penetrations into the inner shell of the suit area are sealed. A doubledoored autoclave is provided for decontaminating waste materials to be removed from the suit area.

SECTION V

Recommended Biosafety Levels For Infectious Agents and Infected Animals

Selection of an appropriate biosafety level for work with a particular agent or animal study depends upon a number of factors. Some of the most important are: the virulence, pathogenicity, biological stability, route of spread, and communicability of the agent; the nature or function of the laboratory; the procedures and manipulations involving the agent; the endemicity of the agent; and the availability of effective vaccines or therapeutic measures.

Agent summary statements in this section provide guidance for the selection of appropriate biosafety levels. Specific information on laboratory hazards associated with a particular agent, and recommendations regarding practical safeguards that can significantly reduce the risk of laboratory-associated diseases, are included. Agent summary statements are presented for agents which meet one or more of the following criteria: the agent is a proven hazard to laboratory personnel working with infectious materials (e.g., hepatitis B virus, M. tuberculosis); the potential for laboratory associated infections is high, even in the absence of previously documented laboratory-associated infections (e.g., exotic arboviruses); or, the consequences of infection are grave.

Recommendations for the use of vaccines and toxoids are included in agent summary statements when such products are available, either as licensed or Investigational New Drug (IND) products. When applicable, recommendations for the use of these products are based on current recommendations of the Public Health Service Advisory Committee on Immunization Practice, and are specifically targeted to at-risk laboratory personnel and others who must work in or enter laboratory areas. These specific recommendations should in no way preclude the routine use of such products as diphtheria-tetanus toxoids, poliovirus vaccine, influenza vaccine and others, because of the potential risk of community exposures irrespective of any laboratory risks. Appropriate precautions should be taken in the administration of live attenuated virus vaccines in individuals with altered immunocompetence, or other medical condition (e.g., pregnancy), in which a viral infection could result in adverse consequences.

Risk assessments and biosafety levels recommended in the agent summary statements presuppose a population of immunocompetent individuals. Persons with altered immunocompetence may be at a increased risk when exposed to infectious agents. Immunodeficiency may be hereditary, congenital, or induced by a number of neoplastic or infectious diseases, by therapy, or by radiation. The risk of becoming infected or the consequence of infection may also be influenced by such factors as age, sex, race, pregnancy, surgery (e.g., splenectomy, gastrectomy), predisposing diseases (e.g., diabetes, lupus erythematosus) or altered physiological function. These and other variables must be considered in applying the generic risk assessments of the agent summary statements to specific activities of selected individuals.

The biosafety level assigned to an agent is based on the activities typically associated with the growth and manipulation of the quantities and concentrations of infectious agents required to accomplish identification or typing. If activities with clinical materials pose a lesser risk to personnel than those activities associated with manipulation of cultures, a lower biosafety level is recommended. On the other hand, if the activities involve large volumes and/or concentrated preparations ("production quantities"), or manipulations which are likely to produce aerosols or which are otherwise intrinsically hazardous, additional personnel precautions and increased levels of primary and secondary containment may be indicated.

"Production quantities" refers to large volumes or concentrations of infectious agents considerably in excess of those typically used for identification and typing activities. Propagation and concentration of infectious agents as occurs in large-scale fermentations, antigen and vaccine production, and a variety of other commercial and research activities, clearly deal with significant masses of infectious agents that are reasonably considered "production quantities". However, in terms of potentially increased risk as a function of the mass of infectious agents, it is not possible to define "production quantities" in finite volumes or concentrations for any given agent. Therefore, the laboratory director must make an assessment of the activities conducted and select practices, containment equipment, and facilities appropriate to the risk, irrespective of the volume or concentration of agent involved.

Occasions will arise when the laboratory director should select a biosafety level higher than that recommended. For example, a higher biosafety level may be indicated by the unique nature of the proposed activity (e.g., the need for special containment for experimentally generated aerosols for inhalation studies) or by the proximity of the laboratory to areas of special concern (e.g., a diagnostic laboratory located near patient care areas). Similarly, a recommended biosafety level may be adapted to compensate for the absence of certain recommended safeguards. For example, in those situations where Biosafety Level 3 is recommended, acceptable safety may be achieved for routine or repetitive operations (e.g., diagnostic procedures involving the propagation of an agent for identification, typing and susceptibility testing) in laboratories where facility features satisfy Biosafety Level 2 recommendations, provided the recommended Standard Microbiological Practices, Special Practices, and Safety Equipment for Biosafety Level 3 are rigorously followed.

One example involves work with the Human Immunodeficiency Viruses (HIVs). Routine diagnostic work with clinical specimens can be done safely at Biosafety Level 2, using Biosafety Level 2 practices and procedures. Research work (including co-cultivation, virus replication studies, or manipulations involving concentrated virus) can be done in a BSL-2 facility, using BSL-3 practices and procedures. Virus production activities, including virus concentrations, require a BSL-3 facility and use of BSL-3 practices and procedures (see Agent Summary Statement).

The decision to adapt Biosafety Level 3 recommendations in this manner should be made only by the laboratory director. This adaptation, however, is not suggested for agent production operations or activities where procedures are frequently changing. The laboratory director should also give special consideration to selecting appropriate safeguards for materials that may contain a suspected agent. For example, sera of human origin may contain hepatitis B virus, and therefore, all blood or blood-derived fluids should be handled under conditions which reasonably preclude cutaneous, mucous membrane or parenteral exposure of personnel. Sputa submitted to the laboratory for tubercle bacilli assay should be handled under conditions which reasonably preclude the generation of aerosols during the manipulation of clinical materials or cultures.

The infectious agents which meet the previously stated criteria are listed by category of agent in Section VII. To use these summaries, first locate the agent in the listing under the appropriate category of agent. Second, utilize the practices, safety equipment, and type of facilities recommended in the agent summary statement as described in Section VII for working with clinical materials, cultures or infectious agents, or infected animals.

The laboratory director is also responsible for appropriate risk assessment and for utilization of appropriate practices, containment equipment, and facilities for agents not included in the agent summary statements.

SECTION VI

Risk Assessment

The assessment of risks associated with laboratory activities involving the use of infectious microorganisms is ultimately a subjective process. The risks associated with the agent, as well as with the activity to be conducted, must be considered in the assessment. The characteristics of infectious agents and the primary laboratory hazards of working with the agents are described generically for agents in Biosafety Levels 1-4 and specifically for individual agents or groups of agents in Section VII of this publication.

Hepatitis B (HBv) is an appropriate model for illustrating the risk assessment process. HBv is among the most ubiquitous of human pathogens and most prevalent of laboratory-associated infections. The agent has been demonstrated in a variety of body secretions and excretions. Blood, saliva, and semen have been shown to contain the virus. Natural transmission is associated with parenteral inoculation or with contamination of broken skin or mucous membranes with infectious body fluids. There is no evidence of airborne or interpersonal spread through casual contact. Prophylactic measures include the use of a licensed vaccine in high-risk groups and the use of hepatitis B immune globulin following overt exposures.

The primary risks of HBv infection for laboratory personnel are accidental parenteral inoculation, exposure of broken skin or the mucous membranes of eyes, nose, or mouth. These risks are typical of those described for Biosafety Level 2 agents, and are addressed by using the recommended standard and special microbiological practices to minimize or eliminate these overt exposures.

The routes of infection with hepatitis C virus (non A-non B) and human immunodeficiency virus are similar for laboratory personnel. The prudent practices recommended for HBv are applicable to these two pathogens, as well as to the routine laboratory manipulation of clinical materials of domestic origin.

The described risk assessment process is also applicable to laboratory operations other than those involving the use of primary agents of human disease. Microbiological studies of animal host-specific pathogens, soil, water, food, feeds, and other natural or manufactured materials, by comparison, pose substantially lower risks for the laboratory worker. Microbiologists and other scientists working with such materials may, nevertheless, find the practices, containment equipment, and facility recommendations described in this publication of value in developing operational standards to meet their own assessed needs.

SECTION VII

Agent Summary Statements

Parasitic Agents

Agent: Nematode Parasites of Humans

Laboratory-associated infections with Ascaris spp.; Strongyloides spp.; hookworms; and Enterobius spp. have been reported.^91,110,151 Allergic reactions to various antigenic components of nematodes (e.g., aerosolized Ascaris antigens) may represent an individual risk to sensitized persons. Laboratory animal-associated infections (including arthropods) have not been reported, but infective larvae in the feces of nonhuman primates infected with Strongyloides spp. are a potential infection hazard for laboratory and animal care personnel.

Laboratory Hazards: Eggs and larvae in freshly passed feces of infected hosts are usually not infective; development to the infective stages may take periods of one day to several weeks. Trichinella is of concern since fresh or digested tissue may contain larvae and would be infective if ingested. Ingestion of the infective eggs or skin penetration of infective larvae are the primary hazards to laboratory and animal care personnel. Arthropods infected with filarial parasites pose a potential hazard to laboratory personnel. In laboratory personnel with frequent exposure to aerosolized antigens of Ascaris spp. development of hypersensitivity is common.

Recommended Precautions: Biosafety Level 2 practices and facilities are recommended for activities with infective stages listed. Exposure to aerosolized sensitizing antigens of Ascaris spp. should be avoided. Primary containment (e.g., biological safety cabinet) may be required for work with these materials by hypersensitive individuals. Appropriate treatment for most nematode infections exists, and information on dosage, source of drugs, etc. is available.⁵

Agent: Protozoal Parasites of Humans

Laboratory-associated infections with Toxoplasma spp.; Plasmodium spp. (including P. cynomologi); Trypanosoma spp.; Entamoeba spp.; Coccidia spp.; Giardia spp.; Leishmania spp.; Sarcocystis spp.; and Cryptosporidia spp. have been reported.^{29,68,91,110,151,162} In addition, no laboratory infections with Babesia spp. or Microsporidia spp. have been reported but could result from accidental needlestick or ingestion of cysts, oocysts, or spores in feces.

Although laboratory animal-associated infections have not been reported, a direct source of infection for laboratory personnel may be contact with lesion material from rodents with cutaneous leishmaniasis and with feces or blood of experimentally or naturally infected animals.

Laboratory-related infections with Cryptosporidia have occurred with regularity in almost every laboratory working with this agent, especially those in which calves are utilized as the source of oocysts. Other experimentally-infected animals pose potential risks as well. There is circumstantial evidence that airborne transmission of oocysts of this small organism may occur. Rigid adherence to protocol should reduce the occurrence in laboratory and animal care personnel.

Laboratory Hazards: Infective stages may be present in blood, feces, lesion exudates, and infected arthropods. Depending on the parasite, ingestion, skin penetration through wounds or microabrasions, accidental parenteral inoculation, and transmission by arthropod vectors are the primary laboratory hazards. Aerosol or droplet exposure of the mucous membranes of the eyes, nose, or mouth with trophozoites are potential hazards when working with cultures of Naegleria fowleri, Leishmania spp., T. cruzi, or with tissue homogenates or blood containing hemoflagellates. Immunocompromised individuals should avoid working with live organisms. Because of the grave consequences of toxoplasmosis in the developing fetus, serologically negative women of childbearing age who might become pregnant should not work with Toxoplasma in the same laboratory room where these materials are handled.

Recommended Precautions: Biosafety Level 2 practices and facilities are recommended for activities with infective stages of the parasites listed. Infected arthropods should be maintained in facilities which reasonably preclude the exposure of personnel or their escape to the outside. Primary containment (e.g., biological safety cabinet) or personal protection (e.g., face shield) may be indicated when working with cultures of Naegleria fowleri, Leishmania spp., T. cruzi or with tissue homogenates or blood containing hemoflagellates.⁸¹ Gloves are recommended for activities where there is the likelihood of direct skin contact with infective stages of the parasites listed. Appropriate treatment for most protozoal infections exists, and information on dosage, source of drugs, etc., is available.⁵

Agent: Trematode Parasites of Humans (Schistosoma spp. and Fasciola spp.)

Laboratory-associated infections with Schistosoma spp. and Fasciola spp. have been reported, none associated directly with laboratory animals.^91,110,151

Laboratory Hazards: Infective stages of Schistosoma spp. (cercariae) and Fasciola spp. (metacercaria) may be found, respectively, in the water or encysted on aquatic plants in laboratory aquaria used to maintain snail intermediate hosts. Skin penetration by schistosome cercariae and ingestion of fluke metacercaria are the primary laboratory hazards. Dissection or crushing of schistosome-infected snails may also result in exposure of skin or mucous membrane to cercariae-containing droplets. Additionally, metacercaria may be inadvertently transferred from hand to mouth by fingers or gloves following contact with contaminated aquatic vegetation or surfaces of aquaria. Most laboratory exposures to Schistosoma spp. would predictably result in low worm burdens with minimal disease potential. Safe and effective drugs are available for the treatment of schistosomiasis.

Recommended Precautions: Biosafety Level 2 practices and facilities are recommended for activities with infective stages of the parasites listed. Gloves should be worn when there may be direct contact with a water containing cercariae, or vegetation containing metacercaria from naturally or experimentally infected snail intermediate hosts. Long-sleeved laboratory coats or other protective garb should be worn when working arouquaria or other water sources that may contain schistosome cercariae. Snails and cercariae in the water of laboratory aquaria should be killed by chemicals (e.g., hypochlorites, iodine) or heat before discharge to sewers. Appropriate treatment for most trematode infections exists, and information on source of drugs, dosage, etc. is available.⁵

Agent: Cestode Parasites of Humans - Echinococcus granulosus, Taenia solium (cysticercus cellulosae) and Hymenolepsis nana.

Although no laboratory-associated infections with either E. granulosus or T. solium have been reported, the consequences of such infections following the ingestion of infective eggs of E. granulosus or T. solium are potentially grave. H. nana is a very cosmopolitan parasite, does not require an intermediate host, and is directly transmissible by ingestion of feces of infected humans or rodents.

Laboratory Hazards: Infective eggs may be present in the feces of dogs or other canids (the definitive hosts of E. granulosus), or in the feces of humans (the definitive host of T. solium). Ingestion of infective eggs from these sources are the primary laboratory hazard. Cysts and cyst fluids of E. granulosus are not infectious for humans. Ingestion of cysts containing the larval stage of T. solium (Cysticercus cellulosae) readily produces human infection with the adult tapeworm. With either parasite, the ingestion of a single infective egg from the feces of the definitive host could potentially result in serious disease. Ingestion of the eggs of H. nana in the feces of the definitive host could result in intestinal infection.

Recommended Precautions: Biosafety Level 2 practices and facilities are recommended for work with infective stages of these parasites. Special attention should be given to personal hygiene practices (e.g., handwashing) and avoidance of ingestion of infective eggs. Gloves are recommended when there may be direct contact with feces or surfaces contaminated with fresh feces of dogs infected with E. granulosus, humans infected with T. solium adults, or humans or rodents infected with H. nana. Appropriate treatment for many cestode infections exists, and information concerning source of drugs, dosage, etc., is available.⁵

Fungal Agents

Agent: Blastomyces dermatitidis

Laboratory-associated local infections following accidental parenteral inoculation with infected tissues or cultures containing yeast forms of B. dermatitidis^{67,87,107,108,168,199} have been reported. Pulmonary infections have occurred following the presumed inhalation of conidia; two developed pneumonia and one had an osteolytic lesion from which B. dermatitidis was cultured.^9,58 Presumably, pulmonary infections are associated only with sporulating mold forms (conidia).

Laboratory Hazards: Yeast forms may be present in the tissues of infected animals and in clinical specimens. Parenteral (subcutaneous) inoculation of these materials may cause local granulomas. Mold form cultures of B. dermatitidis containing infectious conidia may pose a hazard of aerosol exposure.

Recommended Precautions: Biosafety Level 2 and Animal Biosafety Level 2 practices and facilities are recommended for activities with clinical materials, animal tissues, cultures, and infected animals.

Agent: Coccidioides immitis

Laboratory-associated coccidioidomycosis is a documented hazard.^{18,56,59,60,61,105,113,133,171,172,173} Smith reported that 28 of 31 (90%) laboratory-associated infections in his institution resulted in clinical disease, whereas more than half of infections acquired in nature were asymptomatic.²⁰⁰

Laboratory Hazards: Because of the size (2-5 millimicrons), the arthroconidia are conducive to ready dispersal in air and retention in the deep pulmonary spaces. The much larger size of the spherule (30-60 millimicrons) considerably reduces the effectiveness of this form of the fungus as an airborne pathogen.

Spherules of the fungus may be present in clinical specimens and animal tissues, and infectious arthroconidia in mold cultures and soil samples. Inhalation of arthroconidia from soil samples, mold cultures, or following transformation from the spherule form in clinical materials, is the primary laboratory hazard. Accidental percutaneous inoculation of the spherule form may result in local granuloma formation.¹⁸⁴ Disseminated disease occurs at a much greater frequency in blacks and Filipinos than whites.

Recommended Precautions: Biosafety Level 2 practices and facilities are recommended for handling and processing clinical specimens, identifying isolates, and processing animal tissues. Animal Biosafety Level 2 practices and facilities are recommended for experimental animal studies when the route of challenge is parenteral.

Biosafety Level 3 practices and facilities are recommended for propagating and manipulating sporulating cultures already identified as C. immitis and for processing soil or other environmental materials known or likely to contain infectious arthroconidia.

Agent: Cryptococcus neoformans

A single account is reported of a laboratory exposure to Cryptococcus neoformans as a result of a laceration by a scalpel blade heavily contaminated with encapsulated cells.⁸³ This vigorous exposure, which did not result in local or systemic evidence of infection, suggests that the level of pathogenicity for normal immunocompetent adults is low. Respiratory infections as a consequence of laboratory exposure have not been recorded.

Laboratory Hazards: Accidental parenteral inoculation of cultures or other infectious materials represents a potential hazard to laboratory personnel -- particularly to those that may be immunocompromised. Bites by experimentally infected mice and manipulations of infectious environmental materials (e.g., pigeon droppings) may also represent a potential hazard to laboratory personnel.

Recommended Precautions: Biosafety Level 2 and Animal Biosafety Level 2 practices and facilities are recommended, respectively, for activities with known or potentially infectious clinical, environmental, or culture materials and with experimentally infected animals.

The processing of soil or other environmental materials known or likely to contain infectious yeast cells should be conducted in a Class I or Class II biological safety cabinet. This precaution is also indicated for culture of the perfect or sexual state of the agent.

Agent: Histoplasma capsulatum

Laboratory-associated histoplasmosis is a documented hazard in facilities conducting diagnostic or investigative work.^151,152 Pulmonary infections have resulted from handling mold form cultures.¹³² Local infection has resulted from skin puncture during autopsy of an infected human¹⁸⁵ and from accidental needle inoculation of a viable culture.¹⁸² Collecting and processing soil samples from endemic areas has caused pulmonary infections in laboratory workers. Encapsulated spores are resistant to drying and may remain viable for long periods of time. The small size of the infective conidia (less than 5 microns) is conducive to airborne

dispersal and intrapulmonary retention. Furcolow reported that 10 spores were almost as effective as a lethal inoculum in mice as 10,000 to 100,000 spores.⁷⁵

Laboratory Hazards: The infective stage of this dimorphic fungus (conidia) is present in sporulating mold form cultures and in soil from endemic areas. The yeast form in tissues or fluids from infected animals may produce local infection following parenteral inoculation.

Recommended Precautions: Biosafety Level 2 and Animal Biosafety Level 2 practices and facilities are recommended for handling and processing clinical specimens, identifying isolates, animal tissues and mold cultures, identifying cultures in routine diagnostic laboratories, and for experimental animal studies when the route of challenge is parenteral.

Biosafety Level 3 practices and facilities are recommended for propagating and manipulating cultures already identified as H. capsulatum, as well as processing soil or other environmental materials known or likely to contain infectious conidia.

Agent: Sporothrix schenckii

S. schenckii has caused a substantial number of local skin or eye infections in laboratory personnel. Most cases have been associated with accidents and have involved splashing culture material into the eye,^69,197 scratching²¹ or injecting¹⁸³ infected material into the skin or being bitten by an experimentally infected animal.^100,101 Skin infections have resulted also from handling cultures^57,124,137 or necropsy of animals⁷³ without a known break in technique. No pulmonary infections have been reported to result from laboratory exposure, although naturally occurring lung disease is thought to result from inhalation.

Recommended Precautions: Biosafety Level 2 and Animal Biosafety Level 2 practices and facilities are recommended for all laboratory and experimental animal activities with S. schenckii. Gloves should be worn when handling experimentally infected animals, and during operations with broth cultures that might result in hand contamination.

Agents: Pathogenic Members of the Genera Epidermophyton, Microsporum and Trichophyton

Although skin, hair and nail infections by these dermatophytid molds are among the most prevalent of human infections, the processing of clinical material has not been associated with laboratory infections. Infections have been acquired through contacts with naturally or experimentally infected laboratory animals (mice, rabbits, guinea pigs, etc.) and, rarely, with handling cultures.^84,119,151

Laboratory Hazards: Agents are present in the skin, hair and nails of human and animal hosts. Contact with infected laboratory animals with inapparent or apparent infections is the primary hazard to laboratory personnel. Cultures and clinical materials are not an important source of human infection.

Recommended Precautions: Biosafety Level 2 and Animal Biosafety Level 2 practices and facilities are recommended for all laboratory and experimental animal activities with dermatophytes. Experimentally infected animals should be handled with disposable gloves.

Agent: Miscellaneous Molds

Several molds have caused serious infection in immunocompetent hosts following presumed inhalation or accidental subcutaneous inoculation from environmental sources. These agents are Cladosporium (Xylohypha) trichoides, Cladosporium bantianum, Penicillium marnefii, Exophiala (Wangiella) dermatitidis, Fonsecaea pedrosoi and Dactylaria gallopava (Ochroconis gallopavum). Even though no laboratory acquired infections appear to have been reported with most of these agents, the gravity of naturally acquired illness is sufficient to merit special precautions in the laboratory. Penicillium marnefii has caused a local inoculation infection in a laboratory worker.¹⁶⁹

Laboratory Hazards: Inhalation of conidia from sporulating mold cultures or accidental injection into the skin during infection or experimental animals is a theoretical risk to laboratory personnel.

Recommended Precautions: Biosafety Level 2 practices and facilities are recommended for propagating and manipulating cultures known to contain these agents.

Bacterial Agents

Agent: Bacillus anthracis

Forty (40) cases of laboratory-associated anthrax, occurring primarily at facilities conducting anthrax research, have been reported.^66,151 No laboratory-associated cases of anthrax have been reported in the United States since the late 1950's when human anthrax vaccine was introduced.

Naturally and experimentally infected animals pose a potential risk to laboratory and animal care personnel.

Laboratory Hazards: The agent may be present in blood, skin lesion exudates, cerebrospinal fluid, pleural fluid, sputum, and rarely, in urine and feces. Direct and indirect contact of the intact and broken skin with cultures and contaminated laboratory surfaces, accidental parenteral inoculation, and rarely, exposure to infectious aerosols are the primary hazards to laboratory personnel.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for activities using clinical materials and diagnostic quantities of infectious cultures. Animal Biosafety Level 2 practices, containment equipment and facilities are recommended for studies utilizing experimentally infected laboratory rodents. A licensed vaccine is available through the Centers for Disease Control and Prevention; however, immunization of laboratory personnel is not recommended unless frequent work with clinical specimens or diagnostic cultures is anticipated (e.g., animal disease diagnostic laboratory). Biosafety Level 3 practices, containment equipment and facilities are recommended for work involving production volumes or concentrations of cultures, and for activities which have a high potential for aerosol production. In these facilities immunization is recommended for all persons working with the agent, all persons working in the same laboratory room where the cultures are handled, and persons working with infected animals.

Agent: Bordetella pertussis

Bordetella pertussis, a human respiratory pathogen of worldwide distribution, is the causative agent of whooping cough. The disease is typically a childhood illness; however, the agent has been associated, with increased frequency, in adult illness.^106,112,130 Several outbreaks in health-care workers have been reported in the literature.^106,112 Adolescents and adults with atypical or undiagnosed disease can serve as reservoirs of infection and transmit the organism to infants and children.¹³⁵ Eight cases of infection with B. pertussis in adults have been documented at a large research institution. The individuals involved did not work directly with the organism, but had access to common laboratory spaces where the organism was manipulated. One case of secondary transmission to a family member was documented.¹²² A similar incident occurred at a large midwestern university resulting in two documented cases of laboratory-acquired infection and one documented case of secondary transmission.¹⁴⁶ Other laboratory-acquired infections with B. pertussis have been reported, as well as adult-to-adult transmission in the workplace.^19,35 Laboratory-acquired infections resulting from the manipulation of clinical specimens or isolates have not been reported. The attack rate of this airborne infection is influenced by intimacy and frequency of exposure of susceptible individuals.

Laboratory Hazards: The agent may be present in respiratory secretions, but is not found in blood or tissues. Since the natural mode of transmission is by the respiratory route, the greatest potential hazard is aerosol generation during the manipulation of cultures or concentrated suspensions of the organism.

Recommended Precautions: Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities involving the use or manipulation of known or potentially infectious clinical materials or cultures. Animal Biosafety Level 2 should be used for the housing of infected animals. Primary containment devices and equipment (e.g., biological safety cabinets, centrifuge safety cups, or specially designed safety centrifuges) should be used for activities likely to generate potentially infectious aerosols. Biosafety Level 3 practices, procedures, and facilities are appropriate when engaged in large scale production operations. The current pertussis vaccine may not provide complete and permanent immunity; however, a booster dose of pertussis vaccine is not recommended for use in persons who have passed their seventh birthday.⁵⁰

Agent: Brucella (B. abortus, B. canis, B. melitensis, B. suis)

B. abortus, B. canis, B. melitensis, and B. suis have all caused illness in laboratory personnel.^129,151,176 Brucellosis is the most commonly reported laboratory-associated bacterial infection.^127,143,151 Hypersensitivity to Brucella antigens is also a hazard to laboratory personnel. Occasional cases have been attributed to exposure to experimentally and naturally infected animals or their tissues.

Laboratory Hazards: The agent may be present in blood, cerebrospinal fluid, semen, and occasionally urine. Most laboratory-associated cases have occurred in research facilities and have involved exposure to Brucella organisms being grown in large quantities. Cases have also occurred in a clinical laboratory setting: direct skin contact with cultures or with infectious clinical specimens from animals (e.g., blood, uterine discharges) are commonly implicated in these cases. Aerosols generated during laboratory procedures have caused large outbreaks.⁹⁵ Mouth pipetting, accidental parenteral inoculations, and sprays into eyes, nose and mouth have also resulted in infection.

Recommended Precautions: Biosafety Level 2 practices are recommended for activities with clinical specimens of human or animal origin containing or potentially containing pathogenic Brucella spp. Biosafety Level 3 and Animal Biosafety Level 3 practices, containment equipment and facilities are recommended, respectively, for all manipulations of cultures of the pathogenic Brucella spp. listed in this summary, and for experimental animal studies. Vaccines are not available for use in humans.

Agent: Campylobacter (C. jejuni/C. coli, C. fetus subsp. fetus)

C. jejuni/C. coli gastroenteritis is rarely a cause of laboratory associated illness. Three laboratory-acquired cases have been documented.^138,149,155 Numerous domestic and wild animals, including poultry, pets, farm animals, laboratory animals, and wild birds are known reservoirs and are a potential source of infection for laboratory and animal care personnel. Experimentally infected animals are also a potential source of infection.¹⁵⁵

Laboratory Hazards: Pathogenic campylobacters may occur in fecal specimens in large numbers. C. fetus subsp. fetus may also be present in blood, exudates from abscesses, tissues, and sputa. Ingestion or parenteral inoculation of C. jejuni constitute the primary laboratory hazards. The oral ingestion of 500 organisms caused infection in one individual.¹⁶³ The importance of aerosol exposure is not known.

Recommended Precautions: Biosafety level 2 practices, containment equipment and facilities are recommended for activities with cultures or potentially infectious clinical materials. Animal Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with naturally or experimentally infected animals. Vaccines are not available for use in humans.

Agent: Chlamydia psittaci, C. pneumoniae, C. trachomatis

Infections with psittacosis, lymphogranuloma venereum (LGV), and trachoma are documented hazards and among the most commonly reported laboratory-associated bacterial infection. In one report,¹⁵¹ the majority of cases were psittacosis, occurred before 1955, and had the highest case fatality rate of all groups of infectious agents. Additional cases of laboratory-acquired psittacosis have been documented more recently.¹²⁷ Contact with and exposure to infectious aerosols in the handling, care, or necropsy of naturally or experimentally infected birds are the major sources of laboratory-associated psittacosis. Infected mice and eggs are less important sources of C. psittaci. Laboratory animals are not a reported source of human infection with C. trachomatis.

Laboratory Hazards: C. psittaci may be present in the tissues, feces, nasal secretions and blood, of infected birds and in blood, sputum, and tissues of infected humans. C. trachomatis may be present in genital, bubo, and conjunctival fluids of infected humans. Exposure to infectious aerosols and droplets, created during the handling of infected birds and tissues, are the primary hazards to laboratory personnel working with psittacosis. The primary laboratory hazards of C. trachomatis are accidental parenteral inoculation and direct and indirect exposure of mucous membranes of the eyes, nose, and mouth to genital, bubo, or conjunctival fluids, cell culture materials, and fluids from infected eggs. Infectious aerosols may also pose a potential source of infection.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for activities involving the necropsy of infected birds and the diagnostic examination of tissues or cultures known or potentially infected with C. psittaci or C. trachomatis. Wetting the feathers of infected birds with a detergent-disinfectant prior to necropsy can appreciably reduce the risk of aerosols of infected feces and nasal secretions on the feathers and external surfaces of the bird. Animal Biosafety Level 2 practices, containment equipment and facilities and respiratory protection are recommended for personnel working with naturally or experimentally infected caged birds. Gloves are recommended for the necropsy of birds and mice, the opening of inoculated eggs, and when there is the likelihood of direct skin contact with infected tissues, bubo fluids, and other clinical materials. Additional primary containment and personnel precautions, such as those recommended for Biosafety Level 3, may be indicated for activities with high potential for droplet or aerosol production and for activities involving production quantities or concentrations of infectious materials. Vaccines are not available for use in humans.

Agent: Clostridium botulinum

While there is only one report¹⁷⁷ of botulism associated with the handling of the agent or toxin in the laboratory or working with naturally or experimentally infected animals, the consequences of such intoxications must still be considered quite grave.

Laboratory Hazards: C. botulinum or its toxin may be present in a variety of food products, clinical materials (serum, feces) and environmental samples (soil, surface water). Exposure to the toxin of C. botulinum is the primary laboratory hazard. The toxin may be absorbed after ingestion or following contact with the skin, eyes, or mucous membranes, including the respiratory tract.⁹³ Accidental parenteral inoculation may also represent a significant exposure to toxin. Broth cultures grown under conditions of optimal toxin production may contain 2 X 10⁶ mouse LD₅₀ per mL.¹⁷⁷

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities with materials known or potentially containing the toxin. A pentavalent (ABCDE) botulism toxoid is available through the Centers for Disease Control and Prevention, as an investigational new drug (IND). This toxoid is recommended for personnel working with cultures of C. botulinum or its toxins. Solutions of sodium hypochlorite (0.1%) or sodium hydroxide (0.1N) readily inactivate the toxin and are recommended for decontaminating work surfaces and spills of cultures or toxin. Additional primary containment and personnel precautions, such as those recommended for Biosafety Level 3, are indicated for activities with a high potential for aerosol or droplet production, and those involving production quantities of toxin. Animal Biosafety Level 2 practices, containment equipment and facilities are recommended for diagnostic studies and titration of toxin.

Agent: Clostridium tetani

Although the risk of infection to laboratory personnel is negligible, Pike¹⁵¹ has recorded 5 incidents related to exposure of personnel during manipulation of the toxin.

Laboratory Hazards: Accidental parenteral inoculation and ingestion of the toxin are the primary hazards to laboratory personnel. It is uncertain if tetanus toxin can be absorbed through mucous membranes; consequently, the hazards associated with aerosols and droplets remain unclear.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for activities involving the manipulation of cultures or toxin. While the risk of laboratory-associated tetanus is low, the administration of an adult diphtheria-tetanus toxoid at 10-year intervals further reduces the risk to laboratory and animal care personnel of toxin exposures and wound contamination, and is therefore highly recommended.³²

Agent: Corynebacterium diphtheria

Laboratory-associated infections with C. diphtheria are documented. Pike¹⁵¹ lists 33 cases reported in the world literature. Laboratory animal-associated infections have not been reported.

Laboratory Hazards: The agent may be present in exudates or secretions of the nose, throat (tonsil), pharynx, larynx, wounds, in blood, and on the skin. Inhalation, accidental parenteral inoculation, and ingestion are the primary laboratory hazards.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities utilizing known or potentially infected clinical materials or cultures. Animal Biosafety Level 2 facilities are recommended for studies utilizing infected laboratory animals. While the risk of laboratory-associated diphtheria is low, the administration of an adult diphtheria-tetanus toxoid at 10-year intervals may further reduce the risk to laboratory and animal care personnel of toxin exposures and work with infectious materials.³²

Agent: Francisella tularensis

Tularemia is the third most commonly reported laboratoryassociated bacterial infection.¹⁵¹ Almost all cases occurred at facilities involved in tularemia research. Occasional cases have been related to work with naturally or experimentally infected animals or their ectoparasites. Although not reported, cases have occurred in clinical laboratories.

Laboratory Hazards: The agent may be present in lesion exudate, respiratory secretions, cerebrospinal fluid, blood, urine, tissues from infected animals, and fluids from infected arthropods. Direct contact of skin or mucous membranes with infectious materials, accidental parenteral inoculation, ingestion, and exposure to aerosols and infectious droplets have resulted in infection. Cultures have been more commonly associated with infection than clinical materials and infected animals. The human 25% to 50% infectious dose is approximately 10 organisms by the respiratory route.¹⁶

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with clinical materials of human or animal origin containing or potentially containing Francisella tularensis. Biosafety Level 3 and Animal Biosafety Level 3 practices, containment equipment and facilities are recommended, respectively, for all manipulations of cultures and for experimental animal studies. An investigational live attenuated vaccine¹⁶ is available. It is recommended for persons working with the agent or infected animals, and for persons working in or entering the laboratory or animal room where cultures or infected animals are maintained.

Agent: Leptospira interrogans - all serovars

Leptospirosis is a well-documented laboratory hazard. Pike¹⁵¹ reported 67 laboratory-associated infections and 10 deaths, and three additional cases have been reported elsewhere.¹²⁷

An experimentally infected rabbit was identified as the source of an infection with L. interrogans serovar icterohemorrhagiae.¹⁵⁹ Direct and indirect contact with fluids and tissues of experimentally or naturally infected mammals during handling, care, or necropsy is a potential source of infection. In animals with chronic kidney infections, the agent is shed in the urine in enormous numbers for long periods of time.

Laboratory Hazards: The agent may be present in urine, blood, and tissues of infected animals and humans. Ingestion, accidental parenteral inoculation, and direct and indirect contact of skin or mucous membranes with cultures or infected tissues or body fluids -- especially urine -- are the primary laboratory hazards. The importance of aerosol exposure is not known.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities involving the use or manipulation of known or potentially infectious tissues, body fluids, and cultures, and for the housing of infected animals. Gloves are recommended for the handling and necropsy of infected animals, and when there is the likelihood of direct skin contact with infectious materials. Vaccines are not available for use in humans.

Agent: Legionella pneumophila; other Legionella-like agents

A single documented nonfatal laboratory-associated case of legionellosis due to presumed aerosol or droplet exposure during animal challenge studies with Pontiac Fever agent (L. pneumophila) has been recorded.²⁴ Human-to-human spread has not been documented.

Experimental infections are readily produced in guinea pigs and embryonated chicken eggs.¹²¹ Challenged rabbits develop antibodies but not clinical disease. Mice are refractory to parenteral exposure. Unpublished studies by Kaufmann, Feeley and others at the Centers for Disease Control and Prevention have shown that animal-to-animal transmission did not occur in a variety of experimentally infected mammalian and avian species.

Laboratory Hazards: The agent may be present in pleural fluid, tissue, sputum, and environmental sources (e.g., cooling tower water). Because the natural mode of transmission appears to be airborne, the greatest potential hazard is the generation of aerosols during the manipulation of cultures or of other materials containing high concentrations of infectious microorganisms (e.g., infected yolk sacs and tissues).

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities involving the use or manipulation of known or potentially infectious clinical materials or cultures, and for the housing of infected animals. Biosafety Level 3 practices with primary containment devices and equipment (e.g., biological safety cabinets, centrifuge safety cups) are used for activities likely to generate potentially infectious aerosols and for activities involving production quantities of microorganisms. Vaccines are not available for use in humans.

Agent: Mycobacterium leprae

Inadvertent parenteral human to human transmission of leprosy following an accidental needle stick in a surgeon¹¹⁵ and the use of a presumably contaminated tattoo needle¹⁴⁷ have been reported. There are no cases reported as a result of working in a laboratory with biopsy or other clinical materials of human or animal origin. While naturally occurring leprosy or leprosy-like diseases have been reported in armadillos¹⁸⁹ and in nonhuman primates^63,126 humans are the only known important reservoir of this disease.

Laboratory Hazards: The infectious agent may be present in tissues and exudates from lesions of infected humans and experimentally or naturally infected animals. Direct contact of the skin and mucous membranes with infectious materials, and accidental parenteral inoculation, are the primary laboratory hazards associated with handling infectious clinical materials.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities with known or potentially infectious clinical materials from infected humans and animals. Extraordinary care should be taken to avoid accidental parenteral inoculation with contaminated sharp instruments. Animal Biosafety Level 2 practices, containment equipment and facilities are recommended for animal studies utilizing rodents, armadillos, and nonhuman primates.

Agent: Mycobacterium spp. other than M. tuberculosis, M. bovis or M. leprae

Pike reported 40 cases of nonpulmonary "tuberculosis" thought to be related to accidents or incidents in the laboratory or autopsy room.¹⁵¹ Presumably these infections were due to mycobacteria other than M. tuberculosis or M. bovis. A number of mycobacteria which are ubiquitous in nature are associated with diseases, other than tuberculosis or leprosy, in humans, domestic animals, and wildlife. Characteristically, these organisms are infectious but not contagious. Clinically, the diseases associated with infections by these "atypical" mycobacteria can be divided into three general categories:

1. Pulmonary diseases resembling tuberculosis which may be associated with infection by M. kansasii, M. avium complex, and rarely, by M. xenopi, M. malmoense, M. asiaticum, M. simiae and M. szulgai.

2. Lymphadenitis which may be associated with infection by M. scrofulaceum, M. avium complex, and rarely, by M. fortuitum and M. kansasii.

3. Skin ulcers and soft tissue wound infections which may be associated with infection by M. ulcerans, M. marinum, M. fortuitum, and M. chelonei.

Laboratory Hazards: The agents may be present in sputa, exudates from lesions, tissues, and in environmental samples (e.g., soil and water). Direct contact of skin or mucous membranes with infectious materials, ingestion, and accidental parenteral inoculation are the primary laboratory hazards associated with clinical materials and cultures. Infectious aerosols, created during the manipulation of broth cultures or tissue homogenates of these organisms associated with pulmonary disease, also pose a potential infection hazard to laboratory personnel.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with clinical materials and cultures of Mycobacterium spp. other than M. tuberculosis or M. bovis. Animal Biosafety Level 2 practices, containment equipment and facilities are recommended for animal studies with mycobacteria other than M. tuberculosis, M. bovis, or M. leprae.

Agent: Mycobacterium tuberculosis, M. bovis

Mycobacterium tuberculosis and M. bovis infections are a proven hazard to laboratory personnel as well as others who may be exposed to infectious aerosols in the laboratory.^{79,127,131,151,154} The incidence of tuberculosis in laboratory personnel working with M. tuberculosis has been reported to be three times higher than those not working with the agent.¹⁵⁶ Naturally or experimentally infected nonhuman primates are a proven source of human infection (e.g., the annual tuberculin conversion rate in personnel working with infected nonhuman primates is about 70/10,000 compared with less than 3/10,000 in the general population).¹⁰² Experimentally infected guinea pigs or mice do not pose the same problem since droplet nuclei are not produced by coughing in these species; however, litter from infected animals may become contaminated and serve as a source of infectious aerosols.

Laboratory Hazards: Tubercle bacilli may be present in sputum, gastric lavage fluids, cerebrospinal fluid, urine, and in lesions from a variety of tissues.⁶ Exposure to laboratory-generated aerosols is the most important hazard encountered. Tubercle bacilli may survive in heat-fixed smears¹, and may be aerosolized in the preparation of frozen sections and during manipulation of liquid cultures. Because of the low infective dose of M. tuberculosis for humans (i.e., ID₅₀ <10 bacilli)^160,161 and in some laboratories a high rate of isolation of acid-fast organisms from clinical specimens (>10%),⁷⁷ sputa and other clinical specimens from suspected or known cases of tuberculosis must be considered potentially infectious and handled with appropriate precautions.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are required for activities at American Thoracic Society (ATS) laboratory level I,^3,4 preparation of acid-fast smears, and culturing of sputa or other clinical specimens, provided that aerosol generating manipulations of such specimens are conducted in a Class I or II biological safety cabinet. Liquification and concentration of sputa for acid-fast staining may also be conducted safely on the open bench by first treating the specimen (in a Class I or II safety cabinet) with an equal volume of 5% sodium hypochlorite solution (undiluted household bleach) and waiting 15 minutes before centrifugation.^142,174

Biosafety Level 3 practices, containment equipment and facilities are required for laboratory activities of ATS levels II and III)^3,4 in the propagation and manipulation of cultures of M. tuberculosis or M. bovis, and for animal studies utilizing nonhuman primates experimentally or naturally infected with M. tuberculosis or M. bovis. Animal studies utilizing guinea pigs or mice can be conducted at Animal Biosafety Level 3. Skin testing with purified protein devivatie (PPD) of previously skin-tested-negative laboratory personnel can be used as a surveillance procedure. A licensed attenuated live vaccine (BCG) is available but is not routinely used in the United States for laboratory personnel.

Agent: Neisseria gonorrhoeae

Four cases of laboratory-associated gonorrhoea have been reported in the United States.^62,151

Laboratory Hazards: The agent may be present in conjunctival, urethral and cervical exudates, synovial fluid, urine, feces, and cerebrospinal fluid. Accidental parenteral inoculation and direct or indirect contact of mucous membranes with infectious clinical materials are the known primary laboratory hazards. The importance of aerosols is not determined.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities involving the use or manipulation of clinical materials or cultures. Gloves should be worn when handling infected laboratory animals and when there is the likelihood of direct skin contact with infectious materials. Additional primary containment and personnel precautions, such as those described for Biosafety Level 3, may be indicated for aerosol or droplet production, and for activities involving production quantities or concentrations of infectious materials. Vaccines are not available for use in humans.

Agent: Neisseria meningitidis

Meningococcal meningitis is a demonstrated but rare hazard to laboratory workers.^8,49,153

Laboratory Hazards: The agent may be present in pharyngeal exudates, cerebrospinal fluid, blood, and saliva. Parenteral inoculation, droplet exposure of mucous membranes, infectious aerosol and ingestion are the primary hazards to laboratory personnel.

Agent: Pseudomonas pseudomallei

Two laboratory-associated cases of melioidosis are reported: one associated with a massive aerosol and skin exposure;⁷⁸ the second resulting from an aerosol created during the open-flask sonication of a culture presumed to be Ps. cepacia.^166a

Laboratory Hazards: The agent may be present in sputum, blood, wound exudates and various tissues depending on the infection's site of localization. Direct contact with cultures and infectious materials from humans, animals, or the environment, ingestion, autoinoculation, and exposure to infectious aerosols and droplets are the primary laboratory hazards. The agent has been demonstrated in blood, sputum, and abscess materials and may be present in soil and water samples from endemic areas.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities utilizing known or potentially infectious body fluids, tissues, and cultures. Gloves should be worn when handling infected animals, during their necropsy, and when there is the likelihood of direct skin contact with infectious materials. Additional primary containment and personnel precautions, such as those described for Biosafety Level 3, may be indicated for activities with a high potential for aerosol or droplet production, and for activities involving production quantities or concentrations of infectious materials.

Agent: Salmonella - all serotypes except typhi

Salmonellosis is a documented hazard to laboratory personnel.^80,127,151 Primary reservoir hosts include a broad spectrum of domestic and wild animals, including birds, mammals, and reptiles, all of which may serve as a source of infection to laboratory personnel.

Laboratory Hazards: The agent may be present in feces, blood, urine, and in food, feed, and environmental materials. Ingestion or parenteral inoculation are the primary laboratory hazards. The importance of aerosol exposure is not known. Naturally or experimentally infected animals are a potential source of infection for laboratory and animal care personnel, and for other animals.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with clinical materials and cultures known or potentially containing the agents. Animal Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with experimentally or naturally infected animals.

Agent: Salmonella typhi

Typhoid fever is a demonstrated hazard to laboratory personnel.^13,80,153

Laboratory Hazards: The agent may be present in feces, blood, gallbladder (bile) and urine. Humans are the only known reservoir of infection. Ingestion or parenteral inoculation of the organism represent the primary laboratory hazards. The importance of aerosol exposure is not known.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities utilizing known or potentially infectious clinical materials and cultures. Biosafety Level 3 practices and procedures are recommended for activities likely to generate aerosols or for activities involving production quantities of organisms.

Licensed vaccines, which have been shown to protect 70-90% of recipients, may be a valuable adjunct to good safety practices in personnel regularly working with cultures or clinical materials which may contain S. typhi.¹³

Agent: Shigella spp.

Shigellosis is a demonstrated hazard to laboratory personnel with dozens of cases reported in the United States and Great Britain alone.^79,80,97,151 While outbreaks have occurred in captive nonhuman primates, humans are the only significant reservoir of infection. However, experimentally infected guinea pigs, other rodents, and nonhuman primates are also proven sources of infection.

Laboratory Hazards: The agent may be present in feces, and rarely, in blood of infected humans or animals. Ingestion or parenteral inoculation of the agent are the primary laboratory hazards. The oral 25%-50% infectious dose of S. flexneri for humans is approximately 200 organisms.¹⁹¹ The importance of aerosol exposure is not known.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities utilizing known or potentially infectious clinical materials or cultures. Animal Biosafety Level 2 facilities and practices are recommended for activities with experimentally or naturally infected animals. Vaccines are not available for use in humans.

Agent: Treponema pallidum

Syphilis is a documented hazard to laboratory personnel who handle or collect clinical material from cutaneous lesions. Pike lists 20 cases of laboratory-associated infection.¹⁵¹ Humans are the only known natural reservoir of the agent. Syphilis has been transmitted to laboratory personnel working with a concentrated suspension of T. pallidum obtained from an experimental rabbit orchitis.⁷⁴ Hematogenous transfer of syphilis has occurred from the transfusion of a unit of fresh blood obtained from a patient with secondary syphilis. T. pallidum is present in the circulation during primary and secondary syphilis. The minimum number (LD₅₀) of T. pallidum organisms needed to infect by subcutaneous injection is 23.¹¹⁴ The concentration of T. pallidum in patients' blood during early syphilis, however, has not been determined.

No cases of laboratory animal-associated infections are reported; however, rabbit-adapted strains of T. pallidum (Nichols and possibly others) retain their virulence for humans.

Laboratory Hazards: The agent may be present in materials collected from primary and secondary cutaneous and mucosal lesions and in blood. Accidental parenteral inoculation, contact of mucous membranes or broken skin with infectious clinical materials, and possibly infectious aerosols, are the primary hazards to laboratory personnel.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities involving the use or manipulation of blood or lesion materials from humans or infected rabbits. Gloves should be worn when there is a likelihood of direct skin contact with lesion materials. Periodic serological monitoring should be considered in personnel regularly working with infectious materials. Vaccines are not available for use in humans.

Agent: Vibrionic enteritis (Vibrio cholerae, V. para-haemolyticus)

Vibrionic enteritis due to Vibrio cholerae or Vibrio parahaemolyticus is a documented but rare cause of laboratory-associated illness.¹⁵³ Naturally and experimentally infected animals are a potential source of infection.

Laboratory Hazards: All pathogenic vibrios may occur in feces. Ingestion of V. cholerae, and ingestion or parenteral inoculation of other vibrios constitute the primary laboratory hazard. The human oral infecting dose of V. cholerae in healthy non-achlorhydric individuals is approximately 10⁶ organisms.¹¹¹ The importance of aerosol exposure is not known. The risk of infection following oral exposure may be increased in achlorhydric individuals.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with cultures or potentially infectious clinical materials. Animal Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with naturally or experimentally infected animals. Although vaccines have been shown to provide partial protection of short duration (3-6 months) to nonimmune individuals in highly endemic areas,¹³ the routine use of cholera vaccine in laboratory staff is not recommended.

Agent: Yersinia pestis

Plague is a proven but rare laboratory hazard. Four cases have been reported in the United States.^17,151

Laboratory Hazards: The agent may be present in bubo fluid, blood, sputum, cerebrospinal fluid (CSF), feces, and urine from humans, depending on the clinical form and stage of the disease. Primary hazards to laboratory personnel include: direct contact with cultures and infectious materials from humans or rodents; infectious aerosols or droplets generated during the manipulation of cultures, infected tissues, and in the necropsy of rodents; accidental autoinoculation; ingestion; and bites by infected fleas collected from rodents.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities involving the handling of potentially infectious clinical materials and cultures. Special care should be taken to avoid the generation of aerosols from infectious materials, and during the necropsy of naturally or experimentally infected rodents. Gloves should be worn when handling field-collected or infected laboratory rodents, and when there is the likelihood of direct skin contact with infectious materials. Necropsy of rodents is ideally conducted in a biological safety cabinet. Although field trials have not been conducted to determine the efficacy of a licensed inactivated vaccine, experience with this product has been favorable.²² Immunization is recommended for personnel working regularly with cultures of Y. pestis or infected rodents.³⁴

Additional primary containment and personnel precautions, such as those described for Biosafety Level 3, are recommended for activities with high potential for droplet or aerosol production, for work with antibiotic-resistant strains and for activities involving production quantities or concentrations of infectious materials.

Rickettsial Agents

Agent: Coxiella burnetii

Of the rickettsial agents, Coxiella burnetii probably presents the greatest risk of laboratory infection. The organism is highly infectious and remarkably resistant to drying and environmental conditions.¹⁹⁰ The infectious dose of virulent, Phase I organisms in laboratory animals has been calculated to be as small as a single organism. The estimated human ID_25-50 (inhalation) for Q fever is 10 organisms.¹⁹¹ Pike's summary indicates that Q fever is the second most commonly reported laboratory-associated infection, with outbreaks involving 15 or more persons recorded in several institutions.^141,151 A broad range of domestic and wild mammals are natural hosts for Q fever, and may serve as potential sources of infection for laboratory and animal care personnel. Exposure to naturally infected, often asymptomatic sheep, and to their birth products, is a documented hazard to personnel.^28,175 Although rare, C. burnetii is known to cause chronic infections such as endocarditis or granulomatous hepatitis. Genetic analyses¹⁶⁵ as well as structural analysis of the lipopolysaccharides⁸² of endocarditis-associated isolates of C. burnetii suggest that specific strains may be associated with endocarditis.

Laboratory Hazards: The necessity of using embryonated eggs or cell culture techniques for the propagation of C. burnetii leads to extensive purification procedures. Exposure to infectious aerosols or parenteral inoculation are the most likely sources of infection to laboratory and animal care personnel.¹⁴¹ The agent may be present in infected arthropods, and in the blood, urine, feces, milk, and tissues of infected animal or human hosts. The placenta of infected sheep may contain as many as 10⁹ organisms per gram of tissue¹⁹⁵ and milk may contain 10⁵ organisms per gram.

Recommended Precautions: Biosafety Level 2 practices and facilities are recommended for nonpropagative laboratory procedures including serological examinations and staining of impression smears. Biosafety Level 3 practices and facilities are recommended for activities involving the inoculation, incubation, and harvesting of embryonated eggs or cell cultures, the necropsy of infected animals and the manipulation of infected tissues. Since infected guinea pigs and other rodents may shed the organisms in urine or feces,¹⁵¹ experimentally infected rodents should be maintained under Animal Biosafety Level 3.

Recommended precautions for facilities using sheep as experimental animals are described by Spinelli¹⁷⁵ and by Bernard.¹¹ An investigational new Phase I Q fever vaccine (IND) is available from the Special Immunizations Program, U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID), Fort Detrick, Maryland. The use of this vaccine should be limited to those at high risk of exposure and who have no demonstrated sensitivity to Q fever antigen. Individuals with valvular heart disease should not work with C. burnetii.

Agent:Rickettsia prowazekii, Rickettsia typhi (R. mooseri), Rickettsia tsutsugamushi, Rickettsia canada, and Spotted Fever Group agents of human disease; Rickettsia rickettsii, Rickettsia conorii, Rickettsia akari, Rickettsia australis, Rickettsia siberica.

Pike reported 57 cases of laboratory-associated typhus (type not specified), 56 cases of epidemic typhus with 3 deaths, and cases of murine typhus.¹⁵¹ More recently 3 cases of murine typhus were reported from a research facility.²⁶ Two of these 3 cases were associated with handling of infectious materials on the open bench; the third case resulted from an accidental parenteral inoculation. These 3 cases represented an attack rate of 20% in personnel working with infectious materials.

Rocky Mountain spotted fever is a documented hazard to laboratory personnel. Pike¹⁵¹ reported 63 laboratory-associated cases, 11 of which were fatal. Oster¹⁴⁴ reported 9 cases occurring over a 6-year period in one laboratory, which were believed to have been acquired as a result of exposure to infectious aerosols.

Laboratory Hazards: Accidental parenteral inoculation and exposure to infectious aerosols are the most likely sources of laboratory-associated infection.⁸⁹ Successful aerosol transmission of R. rickettsii has been experimentally documented in nonhuman primates.¹⁶⁶ Five cases of rickettsial pox recorded by Pike¹⁵¹ were associated with exposure to bites of infected mites.

Naturally and experimentally infected mammals, their ectoparasites, and their infected tissues are potential sources of human infection. The organisms are relatively unstable under ambient environmental conditions.

Recommended Precautions: Biosafety Level 2 practices and facilities are recommended for nonpropagative laboratory procedures, including serological and fluorescent antibody procedures, and for the staining of impression smears. Biosafety Level 3 practices and facilities are recommended for all other manipulations of known or potentially infectious materials including necropsy of experimentally infected animals and trituration of their tissues, and inoculation, incubation, and harvesting of embryonated eggs or tissue cultures. Animal Biosafety Level 2 practices and facilities are recommended for the holding of experimentally infected mammals other than arthropods.

Level 3 practices and facilities are recommended for animal studies with arthropods naturally or experimentally infected with rickettsial agents of human disease.

Because of the proven value of antibiotic therapy in the early stages of infection, it is essential that laboratories working with rickettsiae have an effective system for reporting febrile illnesses in laboratory personnel, medical evaluation of potential cases and, when indicated, institution of appropriate antibiotic therapy. Vaccines are not currently available for use in humans (see Appendix C).

Viral Agents (other than arboviruses)

Agent: Hepatitis A Virus, Hepatitis E Virus

Laboratory-associated infections with hepatitis A or E viruses do not appear to be an important occupational risk among laboratory personnel. However, the disease is a documented hazard in animal handlers and others working with chimpanzees and other nonhuman primates which are naturally or experimentally infected.¹⁵³ Hepatitis E virus appears to be less of a risk to personnel than hepatitis A virus, except during pregnancy, when infection can result in severe or fatal disease. Workers handling other recently captured, susceptible primates (owl monkeys, marmosets) may also be at risk.

Laboratory Hazards: The agents may be present in feces, saliva, and blood of infected humans and nonhuman primates. Ingestion of feces, stool suspensions, and other contaminated materials is the primary hazard to laboratory personnel. The importance of aerosol exposure has not been demonstrated. Attenuated or avirulent strains of hepatitis A viruses have been described resulting from serial passage in cell culture.

Recommended Precautions: Biosafety Level 2 practices, safety equipment, and facilities are recommended for activities with known or potentially infected feces from humans or nonhuman primates. Animal Biosafety Level 2 practices and facilities are recommended for activities using naturally or experimentally infected nonhuman primates. Animal care personnel should wear gloves and take other appropriate precautions to avoid possible fecal-oral exposure. A licensed inactivated vaccine against hepatitis A is available in Europe; it is available as an investigational vaccine in the U.S., and is recommended for laboratory personnel. Vaccines against hepatitis E are not available for use in humans.

Agent: Hepatitis B Virus, Hepatitis C Virus, (formerly known as nonA nonB Virus), Hepatitis D Virus

Hepatitis B has been one of the most frequently occurring laboratory-associated infections,¹⁵³ and laboratory workers are recognized as a high risk group for acquiring such infections.¹⁷⁰ Individuals who are infected with hepatitis B virus are at risk of infection with hepatitis D (delta) virus, which is defective and requires the presence of hepatitis B virus for replication.

Hepatitis C infection can occur in the laboratory situation. The prevalence of antibody to hepatitis C is slightly higher in medical care workers than in the general population. Epidemiologic evidence indicates that hepatitis C is spread predominantly by the parenteral route.^94,128

Laboratory Hazards: Hepatitis B virus may be present in blood and blood products of human origin, in urine, semen, cerebrospinal fluid, and saliva. Parenteral inoculation, droplet exposure of mucous membranes, and contact exposure of broken skin are the primary laboratory hazards. The virus may be stable in dried blood or blood components for several days. Attenuated or avirulent strains have not been identified.

Hepatitis C virus has been detected primarily in blood and serum, less frequently in saliva and rarely or not at all in urine or semen. It appears to be relatively unstable to storage at room temperature, repeated freezing and thawing, etc.

Recommended Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities utilizing known or potentially infectious body fluids and tissues. Additional primary containment and personnel precautions, such as those described for Biosafety Level 3, may be indicated for activities with potential for droplet or aerosol production and for activities involving production quantities or concentrations of infectious materials. Animal Biosafety Level 2 practices, containment equipment and facilities are recommended for activities utilizing naturally or experimentally infected chimpanzees or other nonhuman primates. Gloves should be worn when working with infected animals and when there is the likelihood of skin contact with infectious materials. Licensed recombinant vaccines against hepatitis B are available and are highly recommended for laboratory personnel.⁴⁶ Vaccines against hepatitis C and D are not yet available for use in humans.

Agent: Herpesvirus simiae (B-virus)

B-virus is a naturally occurring alphaherpesvirus infecting free-living or captive Macaca mulatta, M. fasicularis, and other members of the genus. It is associated with acute vesicular oral lesions, as well as latent and often recrudescent infection.¹⁹³ Human infection has been documented in 25 instances, usually with a lethal outcome or serious sequelae from encephalitis.^7,92,145

Although B-virus presents a potential hazard to laboratory personnel working with the agent, laboratory-associated human infections with B-virus have, with rare exceptions, been limited to those having direct contact with macaques. Primary macaque cell cultures, including commercially-prepared rhesus monkey kidney cells, occasionally are inapparently infected with B-virus and have been implicated in one human case.⁹²

Sixteen fatal cases of human infections with B-virus have been reported.¹⁹³

Laboratory Hazards: The highest risk of acquiring B-virus from macaques is through the bite of an infected monkey with active lesions. Contamination of broken skin or mucous membranes with oral, ocular, or genital secretions from animals with lesions, or experiencing clinically silent virus shedding, is also dangerous. Stability of viral infectivity on cages and other surfaces is not known, but the potential hazard must be recognized. The importance of aerosol exposure is thought to be minimal. Attenuated or avirulent strains have not been identified.

The agent also may be present in thoracic and abdominal viscera and nervous tissues of naturally infected macaques. These tissues, and the cultures prepared from them, are potential hazards.¹⁹⁶

Recommended Precautions: Biosafety Level 2 practices and facilities are recommended for all activities involving the use or manipulation of tissues, body fluids, and primary tissue culture materials from macaques. Additional practices and personnel precautions, such as those detailed for Biosafety Level 3, are recommended for activities involving the use or manipulation of any material known to contain Herpesvirus simiae. In vitro propagation of the virus for diagnosis may be conducted under the same guidelines, but it would be prudent to confine manipulations of positive cultures which would contain high-titered virus to a Class 3 biosafety cabinet or BSL-4 facility, depending on the judgement of the laboratory director.

Biosafety Level 4 practices and facilities are recommended for activities involving the propagation and manipulation of production quantities or concentrates of H. simiae.

The wearing of gloves, masks, and laboratory coats is re-

commended for all personnel working with non-human primates -- especially macaques and other Old World species -- and for all persons entering animal rooms where non-human primates are housed. Any macaque colony not known to be free of B-virus infection should be presumed to be naturally infected. Guidelines are available for safely working with macaques and should be consulted.^39,150 Animals with oral lesions suggestive of active B-virus infection should be isolated and handled with extreme caution. Studies with animals experimentally infected with H. simiae should be conducted at ABSL-3.

Vaccines are not available for use in humans. Human to human transmission has only occurred in one case, suggesting that precautions should be taken with vesicle fluids, oral secretions, and conjunctival secretions of infected persons.⁹²

Antiviral drugs have shown promise in the therapy of rabbits infected with H. simiae, and anecdotal observations suggest this may extend to man.^7,92 Because of the seriousness of infection with this virus, experienced medical personnel should be available for consultation to manage incidents involving exposure to the agent or suspected infections.

Agent: Human Herpesviruses

The herpesviruses are ubiquitous human pathogens and are commonly present in a variety of clinical materials submitted for virus isolation. While few of these viruses are demonstrated causes of clinical laboratory-associated infections, they are primary as well as opportunistic pathogens, especially in immunocompromised hosts. Herpes simplex viruses 1 and 2 and varicella virus pose some risk via direct contact and/or aerosols; cytomegalovirus and Epstein-Barr virus pose relatively low infection risks to laboratory personnel. The risk of laboratory infection from herpesviruses 6 and 7 is not known. Although this diverse group of indigenous viral agents does not meet the criteria for inclusion in agent-specific summary statements (i.e., demonstrated or high potential hazard for laboratory-associated infection; grave consequences should infection occur), the frequency of their presence in clinical materials and their common use in research warrants their inclusion in this publication.

Laboratory Hazards: Clinical materials and isolates of herpesviruses may pose a risk of infection following ingestion, accidental parenteral inoculation, droplet exposure of the mucous membranes of the eyes, nose, or mouth, or inhalation of concentrated aerosolized materials. Clinical specimens containing the more virulent Herpesvirus simiae (B-virus) may be inadvertently submitted for diagnosis of suspected herpes simplex infection. This virus has also been found in cultures of primary rhesus monkey kidney cells. Cytomegalovirus may pose a special risk during pregnancy because of potential infection of the fetus.

Recommended Precautions: Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities utilizing known or potentially infectious clinical materials or cultures of indigenous viral agents which are associated or identified as a primary pathogen of human disease. Although there is little evidence that infectious aerosols are a significant source of laboratory-associated infections, it is prudent to avoid the generation of aerosols during the handling of clinical materials or isolates, or during the necropsy of animals. Primary containment devices (e.g., biological safety cabinets) constitute the basic barrier protecting personnel from exposure to infectious aerosols.

Agent: Influenza

Laboratory-associated infections with influenza are not normally documented in the literature, but are known to occur by informal accounts and published reports, particularly when new strains showing antigenic drift or shift are introduced into a laboratory for diagnostic/research purposes.⁶⁴

Laboratory animal-associated infections are not reported; however, there is a high possibility of human infection from infected ferrets and vice-versa.

Laboratory Hazards: The agent may be present in respiratory tissues or secretions of humans or most infected animals, and in the cloaca of many infected avian species. The virus may be disseminated in multiple organs in some infected animal species.

The primary laboratory hazard is inhalation of virus from aerosols generated by infected animals, or by aspirating, dispensing, or mixing virus-infected samples. Genetic manipulation has the potential for altering the host range, pathogenicity, and antigenic composition of influenza viruses. There is unknown potential for introducing into man transmissible viruses with novel antigenic composition.

Recommended Precautions: Biosafety Level 2 practices and facilities are recommended when receiving and inoculating routine laboratory diagnostic specimens. Autopsy material should be handled in a biological safety cabinet using Biosafety Level 2 procedures.

Activities Utilizing Noncontemporary Virus Strains: Biosafety considerations should take into account the available information about infectiousness and virulence of the strains being used, and the potential for harm to the individual or society in the event that laboratory-acquired infection and subsequent transmission occurs. Research or production activities utilizing contemporary strains may be safely performed using Biosafety Level 2 containment practices. Susceptibility to infection with older noncontemporary human strains, with recombinants, or with animal isolates warrant the use of Biosafety Level 2 containment procedures. However, there is no evidence for laboratory-acquired infection with reference strains A/PR/8/34 and A/WS/33, or its commonly used neurotropic variants.

Agent: Lymphocytic Choriomeningitis Virus

Laboratory-associated infections with LCM virus are well- documented in facilities where infections occur in laboratory rodents -- especially mice, hamsters and guinea pigs.^14,98,151 Nude and SCID mice may pose a special risk of harboring silent chronic infections. Cell cultures that inadvertently have become infected represent a potential source of infection and dissemination of the agent. Natural infections are found in nonhuman primates, including macaques and marmosets (Callitrichid hepatitis virus is a lymphocytic choriomeningitis virus) and may be fatal to marmoset monkeys. Swine and dogs are less important vectors.

Laboratory Hazards: The agent may be present in blood, cerebrospinal fluid, urine, secretions of the nasopharynx, feces and tissues of infected animal hosts and possibly man. Parenteral inoculation, inhalation, contamination of mucous membranes or broken skin with infectious tissues or fluids from infected animals, are common hazards. Aerosol transmission is well documented.¹⁴ The virus may pose a special risk during pregnancy because of potential infection of the fetus.

Recommended Precautions: Biosafety Level 2 practices and facilities are suitable for activities utilizing known or potentially infectious body fluids, and for tissue culture passage of laboratory-adapted, mouse brain-passaged strains. Animal Biosafety Level 2 practices and facilities are suitable for studies in adult mice with mouse brain-passaged strains. However, additional primary containment and personnel precautions, such as those described for Biosafety Level 3, are indicated for activities with high potential for aerosol production, or involving production quantities or concentrations of infectious materials; and for manipulation of infected transplantable tumors, field isolates and clinical materials from human cases. Animal Biosafety Level 3 practices and facilities are recommended for work with infected hamsters. Vaccines are not available for use in humans.⁹⁸

Agent: Poliovirus

Laboratory-associated infections with polioviruses are uncommon and have been limited to unvaccinated laboratory personnel working directly with the agent.¹⁵¹ Laboratory animal-associated infections have not been reported;²³ however, naturally or experimentally infected nonhuman primates could provide a source of infection to exposed unvaccinated persons. Transgenic mice expressing the human receptor for polioviruses can be infected with virulent polioviruses and are a potential source of human infection.

Laboratory Hazards: The agent is present in the feces and in throat secretions of infected persons. Ingestion or parenteral inoculation of infectious tissues or fluids by non-immunized personnel are the primary hazards in the laboratory. The importance of aerosol exposure is not known; it has not been reported as a hazard. Laboratory exposures pose negligible risk to appropriately immunized persons.

Recommended Precautions: Biosafety Level 2 practices and facilities are recommended for all activities utilizing known or potentially infectious culture fluids and clinical materials involving known or suspected wild-type strains. All laboratory personnel working directly with the agent must have documented polio vaccination or demonstrated serologic evidence of immunity to all three poliovirus types.³³ Animal Biosafety Level 2 practices and facilities are recommended for studies of virulent viruses in animals. Unless there are strong scientific reasons for working with virulent polioviruses (which have been eradicated from the United States), laboratories should use the attenuated Sabin oral poliovirus vaccine strains. These pose no significant risk to immunized laboratory personnel.

Agent: Poxviruses

Sporadic cases of laboratory-associated infections with pox viruses (smallpox, vaccinia, yaba, tanapox) have been reported.¹⁵¹ Epidemiological evidence suggests that transmission to humans of monkeypox virus from nonhuman primates or rodents to humans may have occurred in nature, but not in the laboratory setting. Naturally or experimentally infected laboratory animals are a potential source of infection to exposed unvaccinated laboratory personnel. Genetically engineered recombinant vaccinia viruses pose an additional potential risk to laboratory personnel, through direct contact or contact with clinical materials from infected volunteers or animals.

Laboratory Hazards: The agents may be present in lesion fluids or crusts, respiratory secretions, or tissues of infected hosts. Ingestion, parenteral inoculation, and droplet or aerosol exposure of mucous membranes or broken skin with infectious fluids or tissues, are the primary hazards to laboratory and animal care personnel. Some poxviruses are stable at ambient temperature when dried and may be transmitted by fomites.

Recommended Precautions: The possession and use of variola viruses is restricted to the World Health Organization Collaborating Center for Smallpox and Other Poxvirus Infections, located at the Centers for Disease Control and Prevention, Atlanta, Georgia. Biosafety Level 2 practices and facilities are recommended for all activities involving the use or manipulation of poxviruses, other than variola, that pose an infection hazard to humans. All persons working in or entering laboratory or animal care areas where activities with vaccinia, monkey pox, or cow pox viruses are being conducted should have documented evidence of satisfactory vaccination within the preceding ten years.^31,52 Activities with vaccinia, cow pox, or monkey pox viruses, in quantities or concentrations greater than those present in diagnostic cultures, may also be conducted at Biosafety Level 2 by immunized personnel, provided that all manipulations of viable materials are conducted in Class I or II biological safety cabinets or other primary containment equipment. Immunosuppressed individuals are at greater risk of severe disease if infected with a poxvirus.⁵²

Agent: Rabies Virus

Laboratory-associated infections are extremely rare. Two have been documented. Both resulted from presumed exposure to high titered infectious aerosols generated in a vaccine production facility²⁰¹ and a research facility,²⁵ respectively. Naturally or experimentally infected animals, their tissues, and their excretions are a potential source of exposure for laboratory and animal care personnel.

Laboratory Hazards: The agent may be present in all tissues of infected animals. Highest titers are present in CNS tissue, salivary glands, and saliva. Accidental parenteral inoculation, cuts, or sticks with contaminated laboratory equipment, bites by infected animals, and exposure of mucous membranes or broken skin to infectious tissue or fluids, are the most likely sources for exposure of laboratory and animal care personnel. Infectious aerosols have not been a demonstrated hazard to personnel working with clinical materials and conducting diagnostic examinations. Fixed and attenuated strains of virus are presumed to be less hazardous, but the only two recorded cases of laboratory associated rabies resulted from exposure to a fixed Challenge Virus Standard (CVS) and an attenuated strain derived from SAD (Street Alabama Dufferin) strain, respectively.^25,201

Recommended Precautions: Biosafety Level 2 practices and facilities are recommended for all activities utilizing known or potentially infectious materials. Immunization is recommended for all individuals prior to working with rabies virus or infected animals, or engaging in diagnostic, production, or research activities with rabies virus. Immunization is also recommended for all individuals entering or working in the same room where rabies virus or infected animals are used. While it is not always feasible to open the skull or remove the brain of an infected animal within a biological safety cabinet, it is pertinent to wear heavy protective gloves to avoid cuts or sticks from cutting instruments or bone fragments, and to wear a face shield to protect the mucous membranes of the eyes, nose, and mouth from exposure to infectious droplets or tissue fragments. If a Stryker saw is used to open the skull, avoid contacting the brain with the blade of the saw. Additional primary containment and personnel precautions, such as those described for Biosafety Level 3, may be indicated for activities with a high potential for droplet or aerosol production, and for activities involving production quantities or concentrations of infectious materials.

Agent: Retroviruses, including Human and Simian Immunodeficiency Viruses (HIV and SIV)

Data on occupational HIV transmission in laboratory workers are collected through two CDC-supported national surveillance systems: surveillance for 1) AIDS and 2) HIV-infected persons who may have acquired their infection through occupational exposures. For surveillance purposes, laboratory workers are defined as those persons, including students and trainees, who have worked in a clinical or HIV laboratory setting at anytime since 1978. Persons reported from these two systems are classified as cases of either documented or possible occupational transmission. Those classified as documented occupational transmission had evidence of HIV seroconversion (a negative HIV-antibody test at the time of the exposure which converted to positive) following a discrete percutaneous or mucocutaneous occupational exposure to blood, body fluids, or other clinical or laboratory specimens. Those persons classified as possible occupational transmission do not have behavioral or transfusion risks for HIV infection which could be identified during follow-up investigation; each reported past percutaneous or mucocutaneous occupational exposures to blood, body fluids, or laboratory specimens, but seroconversion to HIV was not documented.

As of September 30, 1992, CDC had reports of 12 (11 clinical and 1 nonclinical) laboratory workers in the United States with documented occupational transmission and 13 (12 clinical and 1 nonclinical) classified as possible occupational transmission.⁵⁵

Among those with documented occupational transmission, 8 had percutaneous exposure, 3 had mucocutaneous exposure, and 1 had both percutaneous and mucocutaneous exposures. Eleven were exposed to HIV-infected blood and one to concentrated live HIV. The procedure most often associated with transmission was phlebotomy. Three of these workers have developed AIDS.

Of the 13 laboratory workers classified as possible occupational transmission, 9 have developed AIDS.

In 1992, two workers were reported to have developed antibodies to simian immunodeficiency virus (SIV) following exposures in different laboratories. One was associated with a needle stick which occurred while the worker was manipulating a blood-contaminated needle after bleeding an SIV-infected macaque monkey.¹⁰³ The other involved a laboratory worker who handled macaque SIV-infected blood specimens without gloves. Though no specific incident was recalled, this worker had dermatitis on the forearms and hands while working with the infected blood specimens.⁵³ As of October 1992 neither of the two workers had developed any illness.

Laboratory Hazards: HIV has been isolated from blood, semen, saliva, tears, urine, cerebrospinal fluid, amniotic fluid, breast milk, cervical secretion, and tissue of infected persons and experimentally infected nonhuman primates.¹⁶⁷ CDC has recommended that blood and body fluid precautions be used consistently when handling any blood-contaminated specimens.^43,45 This approach, referred to as "universal precautions", precludes the need to identify clinical specimens obtained from HIV⁺ patients or to speculate as to the HIV status of a specimen.

Although the risk of occupationally acquired HIV is primarily through exposure to infected blood, it is also prudent to wear gloves when manipulating other body fluids such as feces, saliva, urine, tears, sweat, vomitus and human breast milk. This reduces the potential for exposure to other microorganisms that may cause other types of infections.

In the laboratory, virus should be presumed to be present in all blood or clinical specimens contaminated with blood, in any unfixed tissue or organ (other than intact skin) from a human (living or dead), in HIV cultures, in all materials derived from HIV cultures, and in/on all equipment and devices coming into direct contact with any of these materials.

SIV has been isolated from blood, cerebrospinal fluid, and a variety of tissues of infected nonhuman primates. Limited data exist on the concentration of virus in semen, saliva, cervical secretions, urine, breast milk, and amniotic fluid. In the laboratory, virus should be presumed to be present in all SIV cultures, in animals experimentally infected or inoculated with SIV, in all materials derived from HIV or SIV cultures, and in/on all equipment and devices coming into direct contact with any of these materials.⁴⁴

In the laboratory, the skin (especially when scratches, cuts, abrasions, dermatitis, or other lesions are present) and mucous membranes of the eye, nose, and mouth should be considered as potential pathways for entry of these retroviruses. Whether infection can occur via the respiratory tract is unknown. Needles, sharp instruments, broken glass, and other sharp objects must be carefully handled and properly discarded. Care must be taken to avoid spilling and splashing infected cell-culture liquid and other virus-containing or potentially-infected materials.

Recommended Precautions:

In addition to these recommended precautions, persons working with HIV, SIV, or other bloodborne pathogens should consult the OSHA Bloodborne Pathogen Standard.¹⁸⁷ Questions related to interpretation of this Standard should be directed to Federal, regional or state OSHA offices.

1. BSL-2 standard and special practices, containment equipment and facilities are recommended for activities involving all blood-contaminated clinical specimens, body fluids and tissues from all humans or from HIV- or SIV-infected or inoculated laboratory animals.

2. Activities such as producing research-laboratory-scale quantities of HIV or SIV, manipulating concentrated virus preparations, and conducting procedures that may produce droplets or aerosols, are performed in a BSL-2 facility, but using the additional practices and containment equipment recommended for BSL-3.

3. Activities involving industrial-scale volumes or preparation of concentrated HIV or SIV are conducted in a BSL-3 facility, using BSL-3 practices and containment equipment.

4. Nonhuman primates or other animals infected with HIV or SIV are housed in ABSL-2 facilities using ABSL-2 special practices and containment equipment.

Additional Comments:

1. There is no evidence that laboratory clothing poses a risk for retrovirus transmission; however, clothing that becomes contaminated with HIV or SIV preparations should be decontaminated before being laundered or discarded. Laboratory personnel must remove laboratory clothing before going to non-laboratory areas.

2. Work surfaces are decontaminated with an appropriate chemical germicide after procedures are completed, when surfaces are overtly contaminated, and at the end of each work day. Many commercially available chemical disinfectants^{70,116,158,164,188} can be used for decontaminating laboratory work surfaces and some laboratory instruments, for spot cleaning of contaminated laboratory clothing, and for spills of infectious materials. Prompt decontamination of spills should be standard practice.

3. Human serum from any source that is used as a control or reagent in a test procedure should be handled at BSL-2.

4. It is recommended that all institutions establish written policies regarding the management of laboratory exposure to HIV and SIV in conjunction with applicable federal, state and local laws. Such policies should consider confidentiality, consent for testing, administration of appropriate prophylactic drug therapy,⁴⁸ counseling, and other related issues. If a laboratory worker has a parenteral or mucous-membrane exposure to blood, body fluid, or viral-culture material, the source material should be identified and, if possible, tested for the presence of virus. If the source material is positive for HIV antibody, virus, or antigen, or is not available for examination, the worker should be counseled regarding the risk of infection and should be evaluated clinically and serologically for evidence of HIV infection. The worker should be advised to report and seek medical evaluation of any acute febrile illness that occurs within 12 weeks after the exposure.⁴⁰ Such an illness -- particularly one characterized by fever, rash, or lymphadenopathy -- may indicate recent HIV infection. If seronegative, the worker should be retested 6 weeks after the exposure and periodically thereafter (i.e., at 12 weeks and 6, 9 and 12 months after exposure). During this follow-up period exposed workers should be counseled to follow Public Health Service recommendations for preventing transmission of HIV.^{37,40,41,48,120}

5. Other primary and opportunistic pathogenic agents may be present in the body fluids and tissues of persons infected with HIV. Laboratory workers should follow accepted biosafety practices to ensure maximum protection against inadvertent laboratory exposure to agents that may also be present in clinical specimens or in specimens obtained from non-human primates.^36,38,42

Research involving other human (i.e., human T-lymphotrophic virus types I and II) and simian retroviruses occurs in many laboratories. Transmission of such viruses has not been reported in the laboratory setting. The precautions outlined above are sufficient while working with these agents.

Between 1989 and 1992, 69 persons with CD₄⁺ T-lymphocyte depletion, but without evident HIV infection, were identified in the United States. This condition has been provisionally termed "Idiopathic CD₄⁺ T-lymphocytopenia (ICL). To date, investigations of persons with idiopathic CD₄⁺ T-cell depletion indicate that ICL is rare, that these findings may represent various disorders, and in some cases, normal or transient variations in CD₄⁺ T-lymphocyte counts. No epidemiologic or laboratory evidence of a transmissible agent of immunodeficiency has been found as of October 1992.^172a

Agent: Transmissible Spongiform Encephalopathies (Creutzfeldt-Jakob, kuru and related agents)

Laboratory-associated infections with the transmissible spongiform encephalopathies (prion diseases) have not been documented. However, there is evidence that Creutzfeldt-Jakob disease (CJD) has been transmitted iatrogenically to patients by corneal transplants, dura mater grafts and growth hormone extracted from human pituitary glands, and by exposure to contaminated electroencephalographic electrodes.⁹⁹ Infection is always fatal. There is no known nonhuman reservoir for CJD or kuru. Nonhuman primates and other laboratory animals have been infected by inoculation, but there is no evidence of secondary transmission. Scrapie of sheep and goats, bovine spongiform encephalopathy and mink encephalopathy are transmissible spongiform encephalopathies of animals that are similar to the human transmissible diseases. However, there is no evidence that the animal diseases can be transmitted to man.

Laboratory Hazards: High titers of a transmissible agent have been demonstrated in the brain and spinal cord of persons with kuru. In persons with Creutzfeldt-Jakob disease and its Gerstmann-Sträussler-Schenker Syndrome variants, a similar transmissible agent has been demonstrated in the brain, spleen, liver, lymph nodes, lungs, spinal cord, kidneys, cornea and lens, and in spinal fluid and blood. Accidental parenteral inoculation, especially of nerve tissues, including formalin-fixed specimens, is extremely hazardous. Although non-nerve tissues are less often infectious, all tissues of humans and animals infected with these agents should be considered potentially hazardous. The risk of infection from aerosols, droplets, and exposure to intact skin, gastric and mucous membranes is not known; however, there is no evidence of contact or aerosol transmission. These agents are characterized by extreme resistance to conventional inactivation procedures including irradiation, boiling, dry heat and chemicals (formalin, betapropiolactone, alcohols); however, they are inactivated by 1 N NaOH, sodium hypochlorite (2% free chlorine concentration) and steam autoclaving at 134^oC for 1 hour.

Recommended Precautions: Biosafety Level 2 practices and facilities are recommended for all activities utilizing known or potentially infectious tissues and fluids from naturally-infected humans and from experimentally infected animals. Extreme care must be taken to avoid accidental autoinoculation, or other traumatic parenteral inoculations of infectious tissues and fluids.⁷⁶ Although there is no evidence to suggest that aerosol transmission occurs in the natural disease, it is prudent to avoid the generation of aerosols or droplets during the manipulation of tissues or fluids, and during the necropsy of experimental animals. It is further strongly recommended that gloves be worn for activities that provide the opportunity for skin contact with infectious tissues and fluids. Formaldehyde-fixed and paraffin-embedded tissues, especially of the brain, remain infectious. It is recommended that formalin-fixed tissues from suspected cases of transmissible encephalopathy be immersed in 96% formic acid for 30 minutes before histopathologic processing.¹⁵ Vaccines are not available for use in humans.⁵¹

Agent: Vesicular Stomatitis Virus

A number of laboratory-associated infections with indigenous strains of VSV have been reported.¹⁷⁸ Laboratory activities with such strains present two different levels of risk to laboratory personnel and are related, at least in part, to the passage history of the strains utilized. Activities utilizing infected livestock, their infected tissues, and virulent isolates from these sources are a demonstrated hazard to laboratory and animal care personnel.^85,148 Rates of seroconversion and clinical illness in personnel working with these materials are high.¹⁴⁸ Similar risks may be associated with exotic strains such as Piry.¹⁷⁸

In contrast, anecdotal information indicates that activities with less virulent laboratory-adapted strains (e.g., Indiana, San Juan and Glascow) are rarely associated with seroconversion or illness. Such strains are commonly used by molecular biologists, often in large volumes and high concentrations, under conditions of minimal or no primary containment. Some strains of VSV are considered restricted organisms by USDA regulations (9CFR 122.2). Experimentally infected mice have not been a documented source of human infection.

Laboratory Hazards: The agent may be present in vesicular fluid, tissues, and blood of infected animals and in blood and throat secretions of infected humans. Exposure to infectious aerosols, infected droplets, direct skin and mucous membrane contact with infectious tissues and fluids, and accidental autoinoculation, are the primary laboratory hazards associated with virulent isolates. Accidental parenteral inoculation and exposure to infectious aerosols represent potential risks to personnel working with less virulent laboratory-adapted strains.

Recommended Precautions: Biosafety Level 3 practices and facilities are recommended for activities involving the use or manipulation of infected tissues and virulent isolates from naturally or experimentally infected livestock. Gloves and respiratory protection are recommended for the necropsy and handling of 157 infected animals. Biosafety Level 2 practices and facilities are recommended for activities utilizing laboratory-adapted strains of demonstrated low virulence. Vaccines are not available for use in humans.

Agent: Arboviruses

Arboviruses Assigned to Biosafety Level 2

The American Committee on Arthropod-Borne Viruses (ACAV) registered 535 arboviruses as of December 1991. In 1979, the ACAV's Subcommittee on Arbovirus Laboratory Safety (SALS) categorized each of the 424 viruses then registered in the Catalogue of Arboviruses and Certain other Viruses of Vertebrates⁹⁶ into 1 of 4 recommended practices, safety equipment, and facilities described in this publication as Biosafety Levels 1-4.¹⁷⁸ Since 1980, SALS has periodically updated the 1980 publication by providing a supplemental listing and recommended levels of practice and containment for arboviruses registered since 1979. SALS categorizations were based on risk assessments from information provided by a worldwide survey of 585 laboratories working with arboviruses. SALS recommended that work with the majority of these agents should be conducted at the equivalent of Biosafety Level 2, Table A. SALS also recognizes five commonly used vaccine strains, for which attenuation is firmly established, which may be handled safely at BSL 2, provided that personnel working with these vaccine strains are immunized, Table B. SALS has classified all registered viruses for which insufficient laboratory experience exists as BSL 3, Table C, and reevaluates the classification whenever additional experience is reported.

The viruses classified as BSL 2 are listed alphabetically in Table A on pages 126 through 129, and include the following agents which are reported to cause laboratory-associated infections.^86,151,178

Virus Cases

Vesicular stomatitis 46

Colorado tick fever 16

Dengue 11

Pinchinde 17

Western equine encephalomyelitis 7 (2 deaths)

Rio Bravo 7

Kunjin 6

Catu 5

Caraparu 5

Ross River 5

Bunyamwera 4

Eastern equine encephalomyelitis 4

Zika 4

Apeu 2

Marituba 2

Tacaribe 2

Muructucu 1

O'nyong nyong 1

Modoc 1

Oriboca 1

Ossa 1

Keystone 1

Bebaru 1

Bluetongue 1

The result of the SALS survey clearly indicate that the suspected source of the laboratory-associated infections listed above was other than exposure to infectious aerosols. Recommendations that work with the 341 arboviruses, listed in Table A, should be conducted at Biosafety Level 2 was based on the existence of adequate historical laboratory experience to assess risks for the virus which indicate that (a) no overt laboratory-associated infections are reported or (b) infections resulted from exposures other than to infectious aerosols or (c) if disease from aerosol exposure is documented, it is uncommon.

Laboratory Hazards: Agents listed in this group may be present in blood, CSF, central nervous system and other tissues, and infected arthropods, depending on the agent and the stage of infection. While the primary laboratory hazards are accidental parenteral inoculation, contact of the virus with broken skin or mucous membranes, and bites of infected laboratory rodents or arthropods, infectious aerosols may also be a potential source of infection.

Recommended Precautions: Biosafety Level 2 practices, safety equipment, and facilities are recommended for activities with potential infectious clinical materials and arthropods and for manipulations of infected tissue cultures, embryonated eggs, and rodents. Infection of newly hatched chickens with eastern and western equine encephalomyelitis viruses is especially hazardous and should be undertaken under Biosafety Level 3 conditions by immunized personnel. Investigational vaccines (IND) against eastern equine encephalomyelitis and western equine encephalomyelitis viruses are available through the Centers for Disease Control and Prevention and the U.S. Army Medical Research Institute for Infectious Diseases, (USAMRIID) Fort Detrick, Maryland. The use of these vaccines is recommended for personnel who work directly and regularly with these two agents in the laboratory. Western equine encephalomyelitis immune globulin (human) is also available from the Centers for Disease Control and Prevention. The efficacy of this product has not been established.

Prior to 1988, 12 laboratory-acquired dengue infections had been reported. However, from 1988 through 1991, four additional cases have been documented. In all four cases, proper protective gear (long sleeve lab gowns tying in back, gloves, masks, safety glasses) were not worn; and, in three instances, containment of potential aerosols in a laminar flow biosafety cabinet was ignored. These aerosols or infected fluids most likely produced contamination of broken, unprotected skin. An additional factor in these cases was work with highly concentrated amounts of virus. Safe manipulations of dengue viruses in the laboratory (particularly in concentrated preparations) requires strict adherence to Biosafety Level 2 recommendations.

Table A. Arboviruses and Arenaviruses Assigned to Biosafety Level 2 Click here to see this table.

^d. A vaccine is available and is recommended for all persons working with this agent.

Table B. Vaccine Strains of BSL 3/4 Viruses Which May be Handled at BSL 2

Virus	Vaccine Strain
Chikungunya	131/25
Junin	Candid #1
Rift Valley fever²⁰	MP-12
Venezuelan equine encephalomyelitis	TC-83
Yellow fever	17-D

Arboviruses and Arenaviruses Assigned to Biosafety Level 3

SALS has recommended that work with the 171 arboviruses included in the two alphabetical listings on pages 126 and 132 should be conducted at the equivalent of Biosafety Level 3 practices, safety equipment and facilities. These recommendations are based on the following criteria: for Table C (pages 132-133), SALS considered the laboratory experience inadequate to assess risk, regardless of the available information regarding disease severity. For Table D (page 134), SALS recorded overt laboratory-associated infections with these agents which occurred by the aerosol route if protective vaccines were not used or were unavailable, and that the natural disease in humans is potentially severe, life threatening, or causes residual damage. Arboviruses were also classified BSL 3 if they cause diseases in domestic animals in countries outside the USA. Laboratory or laboratory animal-associated infections have been reported with the following BSL agents:^86,151,178

Virus Cases (SALS)

Venezuelan equine encephalomyelitis 150 deaths

Rift Valley fever 47 (1 death)

Chikungunya 39

Yellow fever 38 (8 deaths)

Japanese encephalitis 22

Louping ill 22

West Nile 18

Lymphocytic choriomeningitis 15

Orungo 13

Pery 13

Wesselsbron 13

Mucambo 10

Oropouche 7

Germiston 6

Bhanja 6

Hantaan 6

Mayaro 5

Spondweni 4

Murray Valley encephalitis 3

Semliki Forest 3 (1 death)

Powassan 2

Dugbe 2

Issyk-kul 1

Koutango 1

Large quantities and high concentrations of Semliki Forest virus are commonly used or manipulated by molecular biologists under conditions of moderate or low containment. Although antibodies have been demonstrated in individuals working with this virus the first overt (and fatal) laboratory-associated infection with this virus was reported in 1979.¹⁹⁸ Because the outcome of this infection may have been influenced by a compromised host, an unusual route of exposure or high dosage, or a mutated strain of the virus, this case and its outcome are not typical. More recently, SFV was associated with an outbreak of febrile illness among European soldiers stationed in Bangui.¹¹⁸ The route of exposure was not determined in the fatal laboratory infection; for the natural infections, mosquitoes were the probable vector. SALS continues to classify SFV as a BL 3 virus, with the caveat that most activities with this virus can be safety conducted at Biosafety Level 2.

Some viruses (e.g., Akabane, Israel turkey meningoencepha-litis) are listed in Level 3, not because they pose a threat to human health, but because they are exotic diseases of domestic livestock or poultry.

Laboratory Hazards: The agents listed in this group may be present in blood, cerebrospinal fluid, urine and exudates depending on the specific agent and stage of disease. The primary laboratory hazards are exposure to aerosols of infectious solutions and animal bedding, accidental parenteral inoculation, and broken skin contact. Some of these agents (e.g., VEE) may be relatively stable in dried blood or exudates. Attenuated vaccine strains for a number of these agents are listed in Table B.

Recommended Precautions: Biosafety Level 3 practices, safety equipment, and facilities are recommended for activities using potentially infectious clinical materials and infected tissue cultures, animals, or arthropods.

A licensed attenuated live virus is available for immunization against yellow fever and is recommended for all personnel who work with this agent or with infected animals, and those who enter rooms where the agents or infected animals are present. An investigational vaccine (IND) available for immunization against Venezuelan equine encephalomyelitis is recommended for all personnel working with VEE (and the related Everglades, Mucambo, Tonate, and Cabassou viruses), infected animals, or entering rooms where these agents or infected animals are present. Likewise, investigational vaccines for Rift Valley fever and Junin viruses are available from USAMRIID. Work with Hantaan (Korean hemorrhagic fever) virus and related viruses (Puumala and Seoul) in rats, voles, and other laboratory rodents should be conducted with special caution, because of the extreme hazard of aerosol infection.

Table C. Arboviruses and Certain Other Viruses Assigned to Biosafety Level 3 (on the basis of insufficient experience)

Adelaide River
Agua Preta
Alenquer
Almeirim
Altamira
Andasibe
Antequera
Araguari
Aransas Bay
Arbia
Arboledas
Babanki
Batken
Belem
Berrimah
Bimbo
Bobaya
Bobia
Bozo
Buenaventura
Cabassue ^(c,d)
Cacipacore
Calchaqui
Cananeia
Caninde
Chim

Coastal Plains
Connecticut
Corfou
Dabakala
Douglas
Enseada
Estero Real
Fomede
Forecariah
Fort Sherman
Gabek Forest
Gadgets Gully
Garba
Gordil
Gray Lodge
Gurupi
Iaco
Ibaraki
Ife
Ingangapi
Inini
Issyk-Kul
Itaituba
Itimirim
Itupiranga
Jacareacanga

Jamanxi
Jari
Kedougou
Khasan
Kindia
Kyzylagach
Lake Clarendon
Llano Seco
Macaua
Mapuera
Mboke
Meaban
Mojui Dos Compos
Monte Dourado
Munguba
Naranjal
Nariva
Nasoule
Ndelle
New Minto
Ngari
Ngoupe
Nodamura
Northway
Odrenisrou

Omo
Oriximina
Ouango
Oubangui
Oubi
Ourem
Palestina
Para
Paramushir
Paroo River
Perinet
Petevo
Picola
Playas
Pueblo Viejo
Purus
Radi
Razdan
Resistencia
Rochambeau
Salanga
San Juan
Santa Rosa
Santarem
Saraca
Saumarez Reef

Sedlec
Sena Madureira
Sepik
Shokwe
Slovakia
Somone
Spipur
Tai
Tamdy
Telok Forest
Termeil
Thiafora
Tilligerry
Tinaroo
Tlacotalpan
Tonate ^(c,d)
Ttinga
Xiburema
Yacaaba
Yaounde
Yoka
Yug Bogkanovac

^c. SALS recommends that work with this agent should be conducted only in Biosafety Level 3 facilities which provide for HEPA filtration of all exhaust air prior to discharge from the laboratory.

^d. A vaccine is available and is recommended for all persons working with this agent.

Table D. Arboviruses and Certain Other Viruses Assigned to Biosafety Level 3

Aino
Akabane
Bhanja
Chikungunya ^(c,d)
Cocal
Dhori
Dugbe
Everglades ^(c,d)
Flexal
Germiston ^(c)
Getah
Hantaan
Israel Turkey mening.
Japanese enc.
Junin ^(c,d)
Kairi
Kimberley
Koutango
Louping Ill ^(a,c)

Mayaro
Middelburg
Mobala
Mopeia ^(e)
Mucambo ^(c,d)
Murray Valley enc.
Nairobi sheep disease^(a)
Ndumu
Negishi
Oropouche ^(c)
Orungo
Peaton
Piry
Powassan
Puumala
Rift Valley fever ^(a,b,c,d)
Sagiyama

Sal Vieja
San Perlita
Semliki Forest
Seoul
Spondweni
St. Louis enc.
Thogoto
Tocio ^(c)
Turuna
Venezuelan equine^(c,d)encephalitis
Vesicular Stomatitus (alagoas)
Wesselsbron
^(a,c)
West Nile
Yellow fever
^(c,d)
Zinga ^(b)

^a.The importation, possession, or use of this agent is restricted by USDA regulation or administrative policy. See Appendix E.

^b. Zinga virus is now recognized as being identical to Rift Valley Fever virus.

^c. SALS recommends that work with this agent should be conducted only in Biosafety Level 3 facilities which provide for HEPA filtration of all exhaust air prior to discharge from the laboratory.

^d. A vaccine is available and is recommended for all persons working with this agent.

^e. This virus is presently being registered in the Catalogue of Arboviruses.⁹⁶

Arboviruses, Arenaviruses, or Filoviruses Assigned to Biosafety Level 4

SALS has recommended that work with the 15 arboviruses, arenaviruses, or filoviruses¹⁰⁴ included in the alphabetical listing that follows should be conducted at the equivalent of Biosafety Level 4 practices, safety equipment, and facilities. These recommendations are based on documented cases of severe and frequently fatal naturally occurring human infections and aerosoltransmitted laboratory infections. SALS recommended that certain agents with a close antigenic relationship to the Biosafety Level 4 agents (e.g., Absettarov and Kumlinge viruses) also be handled at this level provisionally until sufficient laboratory experience was obtained to retain these agents at this level or to work with them at a lower level. Laboratory or laboratory animal-associated infections have been reported with the following agents:^{65,86,90,109,151,178,194}

Virus Cases (SALS)

Junin 21 (1 death)

Marburg 25 (5 deaths)

Russian Spring-Summer encephalitis 8

Congo-Crimean hemorrhagic fever 8 (1 death)

Omsk hemorrhagic fever 5

Lassa 2 (1 death)

Machupo 1 (1 death)

Ebola 1

Rodents are natural reservoirs of Lassa fever virus (Mastomys natalensis), Junin and Machupo viruses (Calomys spp.) and perhaps other members of this group. Nonhuman primates were associated with the initial outbreaks of Kyasanur Forest disease (Presbytis spp.) and Marburg disease (Cercopithecus spp.); more recently, filoviruses related to Ebola were associated with Macaca spp. Arthropods are the natural vectors of the tick-borne encephalitis complex agents. Work with or exposure to rodents, nonhuman primates, or vectors naturally or experimentally infected with these agents represents a potential source of human infection.

Laboratory Hazards: The infectious agents may be present in blood, urine, respiratory and throat secretions, semen and tissues from human or animal hosts, and in arthropods, rodents, and nonhuman primates. Respiratory exposure to infectious aerosols, mucous membrane exposure to infectious droplets, and accidental parenteral inoculation are the primary hazards to laboratory or animal care personnel.^109,194

Recommended Precautions: Biosafety Level 4 practices and facilities are recommended for all activities utilizing known or potentially infectious materials of human, animal, or arthropod origin. A new, live attenuated investigational (IND) Junin virus vaccine (Candid #1) is available from the U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID) and is recommended for all laboratory and animal care personnel working with the agent or infected animals and for all personnel entering laboratories or animal rooms when the agent is in use. SALS has lowered the biohazard classification of Junin virus to BSL 3, provided all at risk personnel are immunized and the laboratory is equipped with HEPA filtered exhaust. Clinical specimens from persons suspected of being infected with one of the agents listed in this summary should be submitted to a laboratory with a Biosafety Level 4 maximum containment facility.^23,141

Arboviruses, Arenaviruses and Filoviruses Assigned To Biosafety Level 4

Congo-Crimean hemorrhagic fever	Marburg
Tick-borne encephalitis virus complex	Ebola
(Absettarov, Hanzalova, Hypr,	Lassa
Kumlinge, Kyasanur Forest disease,	Junin
Omsk hemorrhagic fever, and	Machupo
Russian Spring-Summer encephalitis	Guanarito

APPENDIX A

Biological Safety Cabinets

Biological Safety Cabinets (BSCs) are among the most effective, as well as the most commonly used primary containment devices in laboratories working with infectious agents. The three general types available (Class I, II, III) have performance characteristics and applications which are described in this appendix.

Properly maintained Class I and II BSCs, when used in conjunction with good microbiological techniques, provide an effective containment system for safe manipulation of moderate and high-risk microorganisms (Biosafety Level 2 and 3 agents). Both Class I and II BSCs have inward face velocities (75-100 linear feet per minute) that provide comparable levels of containment for laboratory workers and the immediate environment from infectious aerosols generated within the cabinet. Class II BSCs have the additional advantage of providing protection to the research material by high-efficiency particulate air (HEPA)-filtration of the air flow down across the work surface (vertical laminar flow). Class III cabinets offer the maximum protection to laboratory personnel, the community, and the environment because all hazardous materials are contained in a totally enclosed, ventilated cabinet.

CLASS I

(Note: Class I BSCs are no longer being manufactured on a regular basis; many have been replaced by Class II BSCs.)

The Class I Biological Safety Cabinet (Fig. 1) is a negative-pressure, ventilated cabinet usually operated with an open front and a minimum face velocity at the work opening of at least 75 linear feet per minute (lfpm). All of the air from the cabinet is exhausted through a HEPA filter either into the laboratory, or to the outside. The Class I BSC is designed for general microbiological research with low and moderate risk agents, and is useful for containment of mixers, blenders, and other equipment. These cabinets are not appropriate for handling research materials that are vulnerable to airborne contamination, since the inward flow of unfiltered air from the laboratory can carry microbial contaminants into the cabinet.

The Class I BSC can also be used with an installed front closure panel without gloves that will increase the inward flow velocity to approximately 150 lfpm. If such equipped cabinets are ducted to the outside exhaust, they may be used for toxic or radiolabelled materials used as an adjunct to microbiological research. Additionally, arm-length rubber gloves may be attached to the front panel with an inlet air pressure release for further protection.

CLASS II

The Class II Biological Safety Cabinet (Fig. 2) is designed with inward air flow at a velocity to protect personnel (75-100 lfpm), HEPA-filtered vertical laminar airflow for product protection, and HEPA-filtered exhaust air for environmental protection. Design, construction and performance standards for Class II BSCs, as well as a list of products that meet these standards, have been developed by and are available from the National Sanitation Foundation International,¹³⁶ Ann Arbor, Michigan. Utilization of this standard and list should be the first step in selection and procurement of a Class II BSC.

Class II BSCs are classified into two types (A and B) based on construction, air flow velocities and patterns, and exhaust systems. Basically, Type A cabinets are suitable for work with microbiological research in the absence of volatile or toxic chemicals and radionuclides, since air is recirculated within the work area. Type A cabinets may be exhausted through HEPA filters into the laboratory, or to the outside via a "thimble" connection to the exhaust ductwork.

Type B cabinets are further sub-typed into types B1, B2, and B3. A comparison of the design features and applications are found in Table 1 of this appendix. Type B cabinets are hard-ducted to the exhaust system, and contain negative pressure plena. These features, plus an increased face velocity of 100 lfpm, allow work to be done with toxic chemicals or radionuclides.

It is imperative that Class I and II biological safety cabinets are tested and certified in situ at the time of installation within the laboratory, at any time the BSC is moved, and at least annually thereafter. Certification at locations other than the final site may attest to the performance capability of the individual cabinet or model but does not supersede the critical certification prior to use in the laboratory.

As with any other piece of laboratory equipment, personnel must be trained in the proper use of the biological safety cabinets. Of particular note are those activities which may disrupt the inward directional airflow through the work opening of Class I and II cabinets. Repeated insertion and withdrawal of the workers' arms in and from the work chamber, opening and closing doors to the laboratory or isolation cubicle, improper placement or operation of materials or equipment within the work chamber, or brisk walking past the BSC while it is in use are demonstrated causes of the escape of aerosolized particles from within the cabinet. Class I and II cabinets should be located away from traffic patterns and doors. Fans, heating and air conditioning registers, and other air handling devices can also disrupt airflow patterns if located adjacent to the BSC. Strict adherence to recommended practices for the use of BSCs and proper placement in the laboratory are important in attaining the maximum containment capability of the equipment as is the mechanical performance of the equipment itself.

CLASS III

The Class III Biological Safety Cabinet (Fig. 3) is a totally enclosed, ventilated cabinet of gas-tight construction and offers the highest degree of personnel and environmental protection from infectious aerosols, as well as protection of research materials from microbiological contaminants. Class III cabinets are most suitable for work with hazardous agents that require Biosafety Level 3 or 4 containment.

All operations in the work area of the cabinet are performed through attached rubber gloves. The Class III cabinet is operated under negative pressure. Supply air is HEPA-filtered, and the cabinet exhaust air is filtered by two HEPA filters in series, or HEPA filtration followed by incineration, before discharge outside of the facility.

All equipment required by the laboratory activity, such as incubators, refrigerators, and centrifuges, must be an integral part of the cabinet system, The Class III cabinet must be connected to double-doored autoclaves and chemical dunk tanks to sterilize or disinfect all materials exiting the cabinet, and to allow supplies to enter the cabinet. Several Class III cabinets are therefore typically set up as an interconnected system.

POSITIVE-PRESSURE PERSONNEL SUIT

Personnel protection equivalent to that provided by Class III cabinets can also be obtained with the use of a one-piece, ventilated suit for the laboratory worker when working with Biosafety Level 3 or 4 agents in a "suit area" and using Class I or II BSCs. The personnel suit is maintained under positive pressure with a life support system to prevent leakage into the suit. In this containment system, the worker is isolated from the work materials.

The personnel suit area must be essentially equivalent to a large Class III cabinet. The area is entered through an air-lock fitted with airtight doors. A chemical shower is provided as a "dunk tank" to decontaminate the surfaces of the suit as the worker leaves the area. The exhaust air from the suit area is filtered by two HEPA filter units installed in series. The entire area must be under negative pressure.

As in the case with Class III BSCs, the gloves of the personnel suit are the most vulnerable component of the system, as they are subject to punctures by sharps or animal bites.

(Caution: Horizontal laminar flow "clean benches" are used in clinical, pharmaceutical, and laboratory facilities strictly for product protection. Since the worker sits in the immediate downstream exhaust from the "clean bench", this equipment must never be used for handling toxic, infectious, radioactive, or sensitizing materials.)

Figure 1. Class I Biological Safety Cabinet. A. front opening, B. work surface, C. window, D. exhaust plenum, E. HEPA

Figure 2(a). Class II, Type A BSC. A. blower, B. rear plenum, C. supply HEPA filter, D. exhaust, E. sash, F. work surface

Figure 2(b). Class II, Type B1 BSC. A. blowers, B. supply HEPA filters, C. sliding sash, D. positive pressure plenums, E. additional supply HEPA filter or back-pressure plate, F. exhaust HEPA filter, G. negative pressure exhaust plenum, H. work surface

Figure 2(c). Class II, Type B2 BSC. A. storage cabinet, B. work surface, C. sliding sash, D. lights, E. supply HEPA filter, F. exhaust HEPA filter, G. supply blower, H. control panel, I. filter screen, J. negative pressure plenum

Figure 2(d). Table-top model of a Class II, Type B3 BSC. A. front opening, B. sliding sash, C. light, D. supply HEPA filter, E. positive pressure plenum, F. exhaust HEPA filter, G. control panel, H. negative pressure plenum, I. work surface.

Figure 3. Class III BSC. A. stand, B. glove ports, C. O-ring for attaching arm-length gloves to cabinet, D. slopped glass viewing window, E. supply HEPA filter, F. exhaust HEPA filter (Note that the second exhaust HEPA filter required for Class III cabinets is not depicted in this diagram), G. double-ended autoclave

APPENDIX B

Immunoprophylaxis

An additional level of protection for at-risk personnel may be achieved with appropriate prophylactic immunizations. A written organizational policy which defines at-risk personnel, which specifies risks as well as benefits of specific vaccines, and which distinguishes between required and recommended vaccines is essential. In developing such an organizational policy, these recommendations and requirements should be specifically targeted at infectious diseases known or likely to be encountered in a particular facility.

Vaccines for which the benefits (levels of antibody considered to be protective) clearly exceed the risks (local or systemic reactions) should be required for all clearly identified at-risk personnel. Examples of such preparations include vaccines against hepatitis B, yellow fever, rabies, and poliomyelitis. Recommendations for giving less efficacious vaccines, those associated with high rates of local or systemic reactions, or those that produce increasingly severe reactions with repeated use should be carefully considered. Products with these characteristics (e.g., cholera, tularemia, and typhoid vaccines) may be recommended but should not ordinarily be required for employment. A complete record of vaccines received on the basis of occupational requirements or recommendations should be maintained in the employee's permanent medical file.

Recommendations for the use of vaccines, adapted from those of Public Health Service Advisory Committee on Immunization Practices, are included in the agent summary statements in Section VII. Particular attention must be given to individuals who are or may become immunocompromised, as recommendations for vaccine administration may be different than for immunologically competent adults.

APPENDIX C

Surveillance of Personnel for Laboratory-Associated Rickettsial Infections

Under natural circumstances, the severity of disease caused by rickettsial agents varies considerably. In the laboratory, very large inocula which might produce unusual and perhaps very serious responses are possible. Surveillance of personnel for laboratory-associated infections with rickettsial agents can dramatically reduce the risk of serious consequences of disease.

Experience indicates that infections treated adequately with specific anti-rickettsial chemotherapy on the first day of disease do not generally present serious problems. Delay in instituting appropriate chemotherapy, however, may result in debilitating or severe acute disease ranging from increased periods of convalescence in typhus and scrub typhus to death in R. rickettsii infections. The key to reducing the severity of disease from laboratory-associated infections is a reliable surveillance system which includes (1) round-the-clock availability of an experienced medical officer, (2) indoctrination of all personnel into the potential hazards of working with rickettsial agents and advantages of early therapy, (3) a reporting system for all recognized overt exposures and accidents, (4) the reporting of all febrile illnesses, especially those associated with headache, malaise, prostration, when no other certain cause exists and, (5) a non-punitive atmosphere that encourages reporting of any febrile illness.

Rickettsial agents can be handled in the laboratory with minimal real danger to life when an adequate surveillance system complements a staff who are knowledgeable about the hazards of rickettsial infections and who put to use the safeguards recommended in the agent summary statements.

APPENDIX D

Importation and Interstate Shipment of Human Pathogens and Related Materials

The importation or subsequent receipt of etiologic agents and vectors of human disease is subject to the Public Health Service Foreign Quarantine Regulations (42 CFR, Section 71.156). Permits authorizing the importation or receipt of regulated materials and specifying conditions under which the agent or vector is shipped, handled, and used are issued by the Centers for Disease Control and Prevention.

The interstate shipment of indigenous etiologic agents, diagnostic specimens, and biologic products is subject to applicable packaging, labeling, and shipping requirements of the Interstate Shipment of Etiologic Agents (42 CFR Part 72). Packaging and labeling requirements for interstate shipment of etiologic agents are summarized and illustrated in Figure 4.

Persons needing to report leaking damaged packages of etiologic agents may call 1-800-232-0124.

Additional information on the importation and interstate shipment of etiologic agents of human disease, diagnostic specimens, and other related materials may be obtained by contacting:

Centers for Disease Control and Prevention
Attention: Biosafety Branch
Office of Health and Safety, Mail Stop F-05
1600 Clifton Road N.E.
Atlanta, Georgia 30333
Telephone: (404) 639-3883
Fax: (404) 639-2294

Figure 4. Packing and Labeling of Etiologic Agents

Figures 4a and 4b diagram the packaging and labelling of etiologic agents in volumes of less than 50 ml. in accordance with the provisions of subparagraph 72.3(a) of the regulation on Interstate Shipment of Etiologic Agents (42 CFR, Part 72). A revision has been proposed that may result in additional package labeling requirements, but this has not been issued as of the publication of this third edition of BMBL.

For further information on any provision of this regulation contact:

Centers for Disease Control and Prevention
Mail Stop F-05
1600 Clifton Road N.E.
Atlanta, GA 30333
Telephone: (404) 639-3883
FAX: (404) 639-2294

Note that the shipper's name, address and telephone number must be on the outer and inner containers. The reader is also advised to refer to additional provisions of the Department of Transportation (49 CFR, Parts 171-180) Hazardous Materials Regulations.

APPENDIX E

Restricted Animal Pathogens

Nonindigenous pathogens of domestic livestock and poultry may require special laboratory design, operation, and containment features not generally addressed in this publication. The importation, possession, or use of the following agents is prohibited or restricted by law or by U.S. Department of Agriculture regulations or administrative policies:

African horse sickness
African Swine fever virus
Akabane virus
Besnoitia besnoiti
Borna disease virus
Bovine spongiform en- cephalopathy
Bovine infectious petechial fever agent
Brucellosis melitensis
Camelpox virus
Cochliomyia hominivorax (screwworn)
Ephermeral fever virus
Foot and mouth disease virus
Fowl plague virus (lethal avian influenza)
Hog cholera virus
Histoplasma (Zymonema) farciminosum
Louping ill virus
Lumpy skin disease virus
Mycoplasma agalactiae
Mycoplasma mycoides
Nairobi sheep disease virus (Ganjam virus)
Newcastle disease virus (velogenic strains)
Peste des petits ruminants (pest of small ruminants)
Pseudomonas ruminantium (heartwater)
Rift Valley fever virus
Riderspest virus
Sheep ant goat pox
Swine vesicular disease virus
Teschen disease virus
Theileria annulata
Theileria lawrencei
Theileria bovis
Theileria hirci
Trypanosoma evansi
Trypanosoma vivax
Vesicular exanthema virus
Viral hemorrhagic disease of rabbits
Wesselsbron disease virus

The importation, possession, use, or interstate shipment of animal pathogens other than those listed above may also be subject to regulations of the U.S. Department of Agriculture.

Additional information may be obtained by writing to:

U.S. Department of Agriculture
Animal and Plant Health Inspection Service
Veterinary Services, Import-Export Products Staff
Room 756, Federal Building
6505 Belcrest Road
Hyattsville, Maryland 20782
Telephone:

(301) 436-7830 or

(301) 436-8499

Fax: (301) 436-8226

APPENDIX F

Resources for Information

Resources for information, consultation, and advice on biohazard control, decontamination procedures, and other aspects of laboratory safety management include:

Centers for Disease Control and Prevention
Attention: Biosafety Branch
Atlanta, Georgia 30333
Telephone: (404) 639-3883

National Institutes of Health
Attention: Division of Safety
Bethesda, Maryland 20205
Telephone: (301) 496-1357
National Animal Disease Center
U.S. Department of Agriculture
Ames, Iowa 50010
Telephone: (515) 862-8258

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INDEX

African Horse sickness 150

African Swine Fever virus 150

Akabane virus 150

Allergic reactions 74

Animal Biosafety Levels (ABSL) 45

Animal Biosafety Level 1: 45

Animal Biosafety Level 2: 47, 78-82, 84, 85, 87-90, 93, 94, 96, 98, 100, 101, 105-107, 112, 113

Animal Biosafety Level 3: 52, 86, 91, 103, 113

Animal Biosafety Level 4: 59

Animal pathogens 13, 150

Anthrax 83, 158

Arboviruses and Arenaviruses 126, 130

Assigned to Biosafety Level 2: 18, 124, 126

Assigned to Biosafety Level 3: 25, 130, 132, 134

Assigned to Biosafety Level 4: 33, 38, 64, 135, 137

Armadillos 93, 169

Ascaris spp. 74

B virus (herpesvirus simiae) 161

Bacillus anthracis 83

Bacillus subtilis 9

Bacterial agents 83

Besnoitia besnoiti 150

Biosafety levels (BSL)

Biosafety Level 1: 4, 8, 9, 16, 17, 45, 47

Biosafety Level 2: 10, 12, 13, 18, 25, 47, 70, 72, 74, 76, 78, 79-103, 105-108, 110-115, 122-126, 131, 138

Biosafety Level 3: 10, 11, 24, 25, 27, 52, 70, 79, 81, 84, 85, 86, 88, 89, 91, 92, 96-99, 102, 103, 105, 107, 108, 112, 113, 116, 123, 126, 130, 132, 134, 135, 140, 141

Biosafety Level 4: 11, 32, 33, 35, 37-39, 59, 64, 109, 135, 136, 137

Blastomyces dermatitidis 78

Bloodborne pathogens 12, 118, 156, 169

BMBL 3, 4, 149

Bordetella pertussis 84, 154

Borna disease virus 150

Botulism 88, 89, 161

Bovine 121, 150

ephermeral fever virus 150

spongiform encephalopathy 121

Brucella 1, 85, 86, 161, 164, 165

abortus 85

canis 85, 164

melitensis 85, 150, 161, 165

suis 85

Brucella melitensis 3, 11, 135-137, 160, 162, 163

Brucellosis 1, 85, 150, 160, 161, 164, 168

BSC 7, 10, 11, 23, 30, 138-140, 143-145

C virus 72, 106, 107, 164

Calomys spp. 136

Camelpox virus 150

Campylobacter 86

coli 86

fetus 75, 86, 110, 112

jejuni 86, 166, 167

Cercopithecus spp. 136

Cestode parasites 77

hymenolepsis nana 77

taenia solium 77

Chimpanzees 105, 107

Chlamydia 87

pneumoniae 87

psittaci 87, 88

trachomatis 87, 88

Cholera 1, 101, 146, 150

Clostridium

botulinum 88, 89, 168

tetani 89

Coccidia spp. 75

Coccidioides immitis 79

Coccidioidomycosis 79, 154, 158, 162, 164, 167, 168, 170

Cochliomyia hominivorax 150

Congo-Crimean hemorrhagic fever 11, 135, 137

Conidia 78, 81, 83

Containment 6-10, 12, 16-18, 22, 24, 27, 29, 30, 33, 38, 47, 51, 57, 58, 64, 69-71, 73, 74, 76, 83-102, 107, 110, 112, 115, 116, 118, 119, 123, 124, 126, 131, 136, 138, 140, 141, 150

Corynebacterium diphtheria 90

Coxiella burnetii 11, 102, 160, 167, 170

Creutzfeldt-Jakob 121, 159, 161

Cryptococcus neoformans 80

Cysticercus cellulosae 77

Cytomegalovirus 110

Decontamination 8, 10, 17-19, 21, 24, 26, 28, 31, 37, 39-41, 50, 55, 56, 57, 62, 64-66, 119, 152

Dengue 124, 126, 127

Dermatophytes 82

Diagnostic specimens 13, 111, 148

Diphtheria 68, 89, 90, 155, 157

Ebola 135-137, 158, 162

Entamoeba spp. 75

Enteroviruses

non-polio 163

Ephermeral fever virus 150

Epidermophyton 82, 163

Equine encephalomyelitis 124-126, 130, 132

Fasciola spp. 76

Filoviruses 135-137

Foot and mouth disease 150

Fowl plague virus 150

Francisella tularensis 90, 91, 154

Fungal agents 78

Giardia spp. 75

Gloves 7, 10, 16, 17, 19, 23, 25, 29, 30, 36, 38, 45, 47, 51, 52, 56, 59, 63, 76-78, 82, 88, 92, 96, 98, 101, 102, 106, 107, 109, 116, 117, 122, 123, 126, 139-141, 145

Gonorrhoeae 96

Heartwater 150

HEPA filter 41, 42, 58, 66, 138, 141-145

Hepatitis iii, 1, 2, 9, 10, 12, 20, 27, 49, 54, 60, 68, 70, 72, 103, 105, 106, 107, 112, 146, 156, 157, 160, 161, 164

A virus 105, 106

B virus 10, 12, 68, 70, 106, 107, 156, 157

C virus 72, 106, 107, 164

D virus 106

E virus 105, 106

Herpesvirus simiae 108-110, 156, 161, 165, 170

Histoplasma

capsulatum 80

farciminosum 150

Hog cholera virus 150

Human Immunodeficiency Virus 4, 72, 156, 157

Hypr 137

Immunoprophylaxis 146

Importation and interstate shipment 13, 148

Infectious canine hepatitis virus 9

Influenza 68, 111, 150, 158

Investigational New Drug 68, 89

Junin virus 136, 169

Kuru 121

Kyasanur Forest disease 136, 137

Laboratory coats 17, 23, 46, 51, 56, 77, 109

Laboratory hazards 68, 72, 74-83, 85-104, 106-108, 110-115, 117, 121, 123, 125, 132, 136

Lassa fever virus 136

Legionella pneumophila 92, 163

Leishmania spp. 75, 76

Leprosy 93, 94, 158, 164, 165, 169

Leptospira interrogans 91

Leptospirosis 91

Louping ill virus 150

Lumpy skin disease virus 150

Lymphocytic choriomeningitis 112, 131, 154, 161

Lymphogranuloma venereum (LGV) 87

Macaque 108, 109, 117, 169

Machupo 135-137

Malaria 158

Marburg 3, 11, 135-137, 160, 162, 163

Mask 23, 30

Mastomys natalensis 136

Metacercaria 76, 77

Mice 80-82, 87, 88, 92, 95, 96, 112, 113, 123

Microsporum 82

Miscellaneous molds 82

Monkey pox 114

Mycobacterium 10, 93-95

asiaticum 94

avium complex 94

bovis 93-96, 150

chelonei 94

fortuitum 94

kansasii 94

leprae 93, 94

malmoense 94

marinum 94

scrofulaceum 94

simiae 94, 108-110, 156, 161, 165, 170

szulgai 94

tuberculosis 95

ulcerans 94

xenopi 94

Mycoplasma agalactiae 150

Mycoplasma mycoides 150

Mycoplasme mycoides 150

Naegleria

fowleri 75, 76

gruberi 9

Nairobi sheep disease 150

Needles and syringes 21, 28, 36, 49, 54, 61, 62

Neisseria

gonorrhoeae 96

meningitidis 97

Nematode parasites 74

Newcastle disease virus 150

Omsk hemorrhagic fever 135, 137

Parasitic agents 74

Pathogens iii, 9, 12, 13, 27, 35, 72, 73, 110, 118, 148, 150, 156, 169

Penicillium marnefii 82, 83, 167

Personal protective equipment 7, 8, 22, 30, 39, 51, 56

Personnel suits 33, 38

Peste des petits ruminants 150

Pipetting 1, 17, 19, 25, 33, 86

Plague 101, 150, 154, 155

Plasmodium spp. 75

Plastic-backed paper toweling 29

Poliovirus 68, 113, 114

Pontiac fever 92

Poxviruses 114

Presbytis spp. 136

Primates 51, 57, 74, 93, 95, 96, 99, 104-107, 109, 112-114, 117, 118, 119-121, 136, 161

Production quantities 69, 88, 89, 93, 96-99, 102, 107, 109, 113, 116

Protozoal parasites 75

Pseudomonas

pseudomallei 97

Psittacosis 87

Q fever 3, 103, 104, 153, 155, 165, 168, 170

Rabies virus 115

Retroviruses 116, 118, 120

Rickettsia 104, 167

akari 104

australis 104

canada 104

conorii 104

mooseri 104

prowazekii 104

rickettsii 104, 147, 167

siberica 104

tsutsugamushi 104

typhi 98, 99, 104, 153

Rickettsial agents 102, 105, 147

Rickettsial pox 104

Riderspest virus 150

Rift Valley Fever virus 134, 150, 154

Risk Assessment 5, 6, 71-73

Rocky Mountain spotted fever 104, 160, 165

Safety Equipment 3, 6, 7, 9-12, 16-18, 22, 25, 30, 38, 44, 47, 51, 56, 64, 70, 71, 106, 124, 126, 130, 132, 135

Salmonella 98, 99, 153

Salmonellosis 98

SALS 124, 125, 130, 131, 134-136

Schistosoma spp. 76

Serum 20, 27, 35, 49, 54, 61, 88, 107, 119

Sharp items 18, 21, 28, 36, 49, 54, 61

Sheep ant goat pox 150

Shigella spp. 99

Shigellosis 2, 99, 160

Shipment 13, 148-150

Simian Immunodeficiency Virus (SIV) 117

Smallpox 3, 114, 155, 157, 166, 170

Sporothrix schenckii 81

Spotted fever group 104

Strongyloides spp. 74

Surveillance 22, 29, 35, 38, 49, 51, 54, 56, 61-63, 96, 116, 147, 156, 157, 170

Swine vesicular disease 150

Syphilis 100, 163

T-lymphotrophic virus 120, 156

Taenia solium 77

Tanapox 114

Teschen disease virus 150

Tetanus 1, 68, 89, 90, 155, 157

Theileria

annulata 150

bovis 150

hirci 150

lawrencei 150

Thimble unit connection 32, 59

Toxoids 68

Toxoplasma spp. 10, 75

Trachoma 87

Transmissible spongiform encephalopathies 121

Trematode parasites 76

Fasciola 76

Schistosoma 76

Treponema pallidum 76, 100, 159

Trichophyton 82, 163

Trypanosoma 75, 150, 160

cruzi 75, 76, 150, 160

evansi 75, 150, 160

vivax 75, 150, 158, 160

Tuberculosis 1, 2, 4, 10, 13, 68, 93-96, 153, 157, 160, 161, 166

Tularemia 1, 90, 146, 154

Typhoid fever 99

Typhus 104, 147, 155

Universal precautions 4, 13, 117, 156

Vaccines 6, 64, 68, 86-88, 92, 93, 96, 99-101, 105-107, 109, 113, 122, 123, 126, 130, 132, 146, 155

Vaccinia 114, 157

Variola 114

Venezuelan equine encephalomyelitis 130

Vesicular exanthema virus 150

Vesicular stomatitis virus 122

Vibrio 101

cholerae 101

parahaemolyticus 101

Vibrionic enteritis 101

Viral agents 38, 64, 105, 110

Viral hemorrhagic disease 150

Warning sign 20, 27, 48, 49, 54, 61

Wesselsbron disease virus 150

Yaba 114

Yellow fever 130, 132, 134, 146

Yersinia pestis 101