SECTION I. INTRODUCTION

Microbiology laboratories are special, often unique, work environments that may pose special infectious disease risks to persons in or near them. Personnel have contracted infections 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(¹²³ ). In 1941, Meyer and Eddie(⁷⁵ ) 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 Pike(¹¹³ ) 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 Pike(¹¹⁴ ) 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 out-numbered all other reported laboratory-acquired infections and together with tuberculosis, tularemia, typhoid, and streptococcal infection accounted for 72% of all bacteria 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(⁹³ ), adding 641 new or previously unreported cases, and again in 1976(⁹⁰ ), 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.(⁵³ ) 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, Skinhoj(¹⁰⁴ ) 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 Shannon(⁵⁵ ) indicated that medical laboratory workers in England had "a five times increased risk of acquiring tuberculosis compared with the general population." Hepatitis and shigellosis were also shown to be continuing occupational risks and together with tuberculosis were the three most commonly reported occupation-associated infections in Britain.

Although these reports suggest that laboratory personnel are 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 Shannon(⁵⁵ ) and of Skinhoj(¹⁰⁴ ) indicate that laboratory personnel have higher rates of tuberculosis, shigellosis, and hepatitis than 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 Center for Disease Control in 1947-1973(⁹⁷ ), no secondary cases were reported in family members or community contacts. The National Animal Disease Center has reported a similar experience(¹¹⁵ ), with no secondary cases occurring in laboratory and nonlaboratory contacts of 18 laboratory-associated cases occurring in 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 his dismissal from the hospital(⁷⁰ ). Three secondary cases of smallpox were reported in two laboratory-associated outbreaks in England in 1973(⁹⁶ ) and 1978(¹³⁰ ). There were earlier reports of six cases of Q fever in employees of a commercial laundry which handled linens and uniforms from a laboratory where work with the agent was conducted(⁸⁴ ), and two cases of Q fever in household contacts of a rickettsiologist(⁵ ). These cases are representative of the sporadic nature and infrequent association of community infections with laboratories working with infectious agents.

In his 1979 review(⁹² ), Pike concluded, "the knowledge, the techniques, and the equipment to prevent most laboratory infections are available." No single code of practice, standards, guidelines, or other publication, however, provides detailed descriptions of techniques, equipment, and other considerations or recommendations for the broad scope of laboratory activities conducted in the United States with a variety of indigenous and exotic infectious agents. The booklet Classification of Etiologic Agents on the Basis of Hazard(¹⁵ ) has, since 1969, served as a general reference for some laboratory activities utilizing infectious agents. That booklet and the concept of categorizing infectious agents and laboratory activities into four classes or levels served as a basic format for Biosafety in Microbiological and Biomedical Laboratories . This publication will provide specific descriptions of combinations of microbiological practices, laboratory facilities, and safety equipment and recommendations for use in four categories or biosafety levels of laboratory operation with selected infectious agents of man.

The descriptions of biosafety levels 1-4 parallel those of P1-4 in the NIH Guidelines for Research Involving Recombinant DNA Molecules (⁴³ ) and are consistent with the general criteria used in assigning agents to Classes 1-4 in Classification of Etiologic Agents on the Basis of Hazard(¹⁵ ). 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.

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. Primary containment, the protection of personnel and the immediate laboratory environment from exposure to infectious agents, is provided by 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. The purpose of containment is to reduce exposure of laboratory workers and other persons to, and to prevent escape into the outside environment of, potentially hazardous agents. The three elements of containment include laboratory practice and technique, safety equipment, and facility design.

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 infected materials must be aware of potential hazards and must be trained and proficient in the practices and techniques required for safely handling such material. The director or person in charge of the laboratory is responsible for providing or arranging for appropriate training of personnel.

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.

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.

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 and a variety of enclosed containers. The biological safety cabinet is the principal device used to provide containment of infectious 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.1. Open-fronted Class I and Class II biological safety cabinets are partial containment cabinets which offer significant levels of protection to laboratory personnel and to the environment when used with good microbiological techniques. The gastight Class III biological safety cabinet provides the highest attainable level of protection to personnel and the environment.

An example of an enclosed container is the safety centrifuge cup, which is designed to prevent aerosols from being released during centrifugation.

Safety equipment also includes items for personal protection such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, and safety glasses. These personal protective devices are 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 devices may form the primary barrier between personnel and the infectious materials. Examples of such activities include certain animal studies, animal necropsy, 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 in the facility but outside the laboratory and those 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. Three facility designs are described below, in ascending order by level of containment.

1.

The basic laboratory. This laboratory provides general space in which work is done with viable agents which are not associated with disease in healthy adults. Basic laboratories include those facilities described in the following pages as Biosafety Levels 1 and 2 facilities. This laboratory is also appropriate for work with infectious agents or potentially infectious materials when the hazard levels are low and laboratory personnel can be adequately protected by standard laboratory practice. While work is commonly conducted on the open bench, certain operations are confined to biological safety cabinets. Conventional laboratory designs are adequate. Areas known to be sources of general contamination, such as animal rooms and waste staging areas, should not be adjacent to patient care activities. Public areas and general offices to which nonlaboratory staff require frequent access should be separated from spaces which primarily support laboratory functions.

2.

The containment laboratory. This laboratory has special engineering features which make it possible for laboratory workers to handle hazardous materials without endangering themselves, the community, or the environment. The containment laboratory is described in the following pages as a Biosafety Level 3 facility. The unique features which distinguish this laboratory from the basic laboratory are the provisions for access control and a specialized ventilation system. The containment laboratory may be an entire building or a single module or complex of modules within a building. In all cases, the laboratory is separated by a controlled access zone from areas open to the public.

3.

The maximum containment laboratory. This laboratory has special engineering and containment features that allow activities involving infectious agents that are extremely hazardous to the laboratory worker or that may cause serious epidemic disease to be conducted safely. The maximum containment laboratory is described on the following pages as a Biosafety Level 4 facility. Although the maximum containment laboratory is generally a separate building, it can be constructed as an isolated area within a building. The laboratory's distinguishing characteristic is that it has secondary barriers to prevent hazardous materials from escaping into the environment. Such barriers include sealed openings into the laboratory, airlocks or liquid disinfectant barriers, a clothing-change and shower room contiguous to the laboratory ventilation system, and a treatment system to decontaminate exhaust air.

Biosafety levels. Four biosafety levels are described which consist of combinations of laboratory practices and techniques, safety equipment, and laboratory facilities appropriate for the operations performed and the hazard posed by the infectious agents and for the laboratory function or activity.

Biosafety Level 1. 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 2. 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 aerosols is low. Hepatitis B virus, the salmonellae, and Toxoplasma spp. are representative of microorganisms assigned to this containment level. Primary hazards to personnel working with these agents may include accidental autoinoculation, ingestion, and skin or mucous membrane exposure to infectious materials. Procedures with high aerosol potential that may increase the risk of exposure of personnel must be conducted in primary containment equipment or devices.

Biosafety Level 3. 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 where the potential for infection by aerosols is real and the disease may have serious or lethal consequences. Autoinoculation and ingestion also represent primary hazards to personnel working with these agents. Examples of such agents for which Biosafety Level 3 safeguards are generally recommended include Mycobacterium tuberculosis , St. Louis encephalitis virus, and Coxiella burnetii.

Biosafety Level 4. Biosafety Level 4 practices, safety equipment, and facilities are applicable to work with dangerous and exotic agents which pose a high individual risk of life-threatening disease. 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. Lassa fever virus is representative of the microorganisms assigned to Level 4.

Animal biosafety levels. Four biosafety levels are also described for activities involving infectious disease activities 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.

The laboratory director is directly 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.

Work with known agents should be conducted at the biosafety level recommended in Section V unless specific information is available to suggest that virulence, pathogenicity, antibiotic resistance patterns, and other factors are significantly altered to require more stringent or allow less stringent practices to be used.

Clinical laboratories, and especially those in health care facilities, receive clinical specimens with requests for a variety of diagnostic and clinical support services. Typically, clinical laboratories receive specimens without pertinent information such as patient history or clinical findings which may be suggestive of an infectious etiology. Furthermore, such specimens are often submitted with a broad request for microbiological examination for multiple agents (e.g., sputum samples 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 and are safely conducted using a combination of practices, facilities, and safety equipment described as Biosafety Level 2. Biological safety cabinets (Class I or II) should 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. Class II biological safety cabinets are also used to protect the integrity of the specimens or cultures by preventing contamination from the laboratory environment.

Segregating clinical laboratory functions and limiting or restricting access to laboratory areas are the responsibility of the laboratory director.

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 A.4).

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 A.5).

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 A.1 and A.2. The levels are designated in ascending order by degree of protection provided to personnel, the environment, and the community.

TABLE A.1

Summary of Recommended Biosafety Levels for Infectious Agents.

TABLE A.2

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

Biosafety Level 1. Biosafety Level 1 is suitable for work involving agents of no known or minimal potential hazard to laboratory personnel and the environment. The laboratory is not separated from the general traffic patterns in the building. Work is generally conducted on open bench tops. Special containment equipment is not required or 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 are in progress.

2.

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

3.

All contaminated liquid or solid wastes are decontaminated before disposal.

4.

Mechanical pipetting devices are used; mouth pipetting is prohibited.

5.

Eating, drinking, smoking, and applying cosmetics are not permitted in the work area. Food may be stored in cabinets or refrigerators designated and used for this purpose only. Food storage cabinets or refrigerators should be located outside of the work area.

6.

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

7.

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

8.

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

B. Special practices

1.

Contaminated materials that are to be decontaminated at a site away from the laboratory are placed in a durable leakproof container which is closed before being removed from the laboratory.

2.

An insect and rodent control program is in effect.

C. Containment equipment

Special containment equipment is generally not required for manipulations of agents assigned to Biosafety Level 1.

D. Laboratory facilities

1.

The laboratory is designed so that it can be easily cleaned.

2.

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

3.

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

4.

Each laboratory contains a sink for handwashing.

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, and (3) certain procedures in which infectious aerosols are 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 by the laboratory director when work with infectious agents is in progress.

2.

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

3.

All infectious liquid or solid wastes are decontaminated before disposal.

4.

Mechanical pipetting devices are used; mouth pipetting is prohibited.

5.

Eating, drinking, smoking, and applying cosmetics are not permitted in the work area. Food may be stored in cabinets or refrigerators designated and used for this purpose only. Food storage cabinets or refrigerators should be located outside of the work area.

6.

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

7.

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

B. Special practices

1.

Contaminated materials that are to be decontaminated at a site away from the laboratory are placed in a durable leakproof container which is closed before being removed from the laboratory.

2.

The laboratory director limits access to the laboratory. 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. 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 hazard and meet any specific entry requirements (e.g., immunization) enter the laboratory or animal rooms.

4.

When the infectious agent(s) in use in the laboratory require special provisions for entry (e.g., vaccination), 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.

5.

An insect and rodent control program is in effect.

6.

Laboratory coats, gowns, smocks, or uniforms are worn while in the laboratory. Before leaving the laboratory for nonlaboratory areas (e.g., cafeteria, library, administrative offices), this protective clothing is removed and left in the laboratory or covered with a clean coat not used in the laboratory.

7.

Animals not involved in the work being performed are not permitted in the laboratory.

8.

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

9.

All wastes from laboratories and animal rooms are appropriately decontaminated before disposal.

10.

Hypodermic needles and syringes are used only for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for the injection or aspiration of infectious fluids. Extreme caution should be used when handling needles and syringes to avoid autoinoculation and the generation of aerosols during use and disposal. Needles should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before discard or reuse.

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.

When appropriate, considering the agent(s) handled, base-line serum samples for laboratory and other at-risk personnel are collected periodically, depending on the agents handled or the function of the facility.

13.

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

C. Containment equipment

Biological safety cabinets (Class I or II) (see Appendix A.1) or other appropriate personal protective or physical containment devices are used whenever:

1.

Procedures with a high potential for creating infectious aerosols are conducted(⁸² ). 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.

2.

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

D. Laboratory facilities

1.

The laboratory is designed so that it can be easily cleaned.

2.

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

3.

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

4.

Each laboratory contains a sink for handwashing.

5.

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

6.

An autoclave for decontaminating infectious laboratory wastes is 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 material are conducted within biological safety cabinets or other physical containment devices or by personnel wearing appropriate personal protective clothing and devices. 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, 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 laboratories where facility features satisfy Biosafety Level 2 recommendations, provided the recommended "Standard Microbiological Practices," "Special Practices," and "Containment Equipment" for Biosafety Level 3 are 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.

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

2.

All infectious liquid or solid wastes are decontaminated before disposal.

3.

Mechanical pipetting devices are used; mouth pipetting is prohibited.

4.

Eating, drinking, smoking, storing food, and applying cosmetics are not permitted in the work area.

5.

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

6.

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

B. Special practices

1.

Laboratory doors are kept closed when experiments are in progress.

2.

Contaminated materials that are to be decontaminated at a site away from the laboratory are placed in a durable leakproof container which is closed before being removed from the laboratory.

3.

The laboratory director controls access to the laboratory and restricts access to persons whose presence is required for program or support purposes. 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.

4.

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.

5.

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.

6.

All activities 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.

7.

The work surfaces of biological safety cabinets and other containment equipment are decontaminated when work with infectious materials is finished. Plastic-backed paper toweling used on nonperforated work surfaces within biological safety cabinets facilitates cleanup.

8.

An insect and rodent control program is in effect.

9.

Laboratory clothing that protects street clothing (e.g., solid front or wrap-around gowns, scrub suits, coveralls) is worn in the laboratory. Laboratory clothing is not worn outside the laboratory, and it is decontaminated before being laundered.

10.

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

11.

Molded surgical masks or respirators are worn in rooms containing infected animals.

12.

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

13.

All wastes from laboratories and animal rooms are appropriately decontaminated before disposal.

14.

Vacuum lines are protected with high efficiency particulate air (HEPA) filters and liquid disinfectant traps.

15.

Hypodermic needles and syringes are used only for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for the injection or aspiration of infectious fluids. Extreme caution should be used when handling needles and syringes to avoid autoinoculation and the generation of aerosols during use and disposal. Needles should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before discard or reuse.

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.

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

18.

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

C. Containment equipment

Biological safety cabinets (Class I, II, or III) (see Appendix A.1) or other appropriate combinations of personal protective or physical containment devices (e.g., special protective clothing, masks, gloves, respirators, centrifuge safety cups, sealed centrifuge rotors, and containment caging for animals) are used for all activities with infectious materials which pose a threat of aerosol exposure. These include: manipulation 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.

D. Laboratory facilities

1.

The laboratory is separated from areas which are open to unrestricted traffic flow within the building. Passage through two sets of doors is the basic requirement for entry into the laboratory from access corridors or other contiguous areas. Physical separation of the high containment laboratory from access corridors or other laboratories or 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 laboratory.

2.

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 decontaminating the area.

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.

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

6.

Windows in the laboratory are closed and sealed.

7.

Access doors to the laboratory or containment module are self-closing.

8.

An autoclave for decontaminating laboratory wastes is available, preferably within the laboratory.

9.

A ducted exhaust air ventilation system is provided. This system creates directional airflow that draws air into the laboratory through the entry area. The exhaust air is not recirculated to any other area of the building, is discharged to the outside, and is dispersed away from occupied areas and air intakes. Personnel must verify that the direction of the airflow (into the laboratory) is proper. The exhaust air from the laboratory room can be discharged to the outside without being filtered or otherwise treated.

10.

The HEPA-filtered exhaust air from Class I or Class II biological safety cabinets is discharged directly to the outside or through the building exhaust system. Exhaust air from Class I or II biological safety cabinets may be recirculated within the laboratory if the cabinet is tested and certified at least every 12 months. If the HEPA-filtered exhaust air from Class I or II 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[⁸⁰ ]) that avoids any interference with the air balance of the cabinets or building exhaust system.

Biosafety Level 4. Biosafety Level 4 is required for work with dangerous and exotic agents which pose a high individual risk of life-threatening disease. 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 I or Class II biological safety cabinets used along with one-piece positive-pressure personnel suits ventilated by a life support system. The maximum containment 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.

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

2.

Only mechanical pipetting devices are used.

3.

Eating, drinking, smoking, storing food, and applying cosmetics are not permitted in the laboratory.

4.

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

B. Special practices

1.

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

2.

No materials, except for biological materials that are to remain in a viable or intact state, are removed from the maximum containment 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 is decontaminated by gaseous or vapor methods in an airlock or chamber designed for this purpose.

3.

Only persons whose presence in the facility or individual laboratory rooms 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 may be unusually hazardous 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 ensuring 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.

4.

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

5.

Street 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, is provided and used by all personnel entering the facility. Head covers are provided for personnel who do not wash their hair during the exit shower. When leaving the laboratory and before proceeding into the shower area, personnel remove their laboratory clothing and store it in a locker or hamper in the inner change room.

6.

When infectious materials or infected animals are present in the laboratory or animal rooms, a hazard warning sign, incorporating the universal biohazard symbol, is posted on all access doors. The 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 area (e.g., the need for immunizations or respirators).

7.

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.

8.

An insect and rodent control program is in effect.

9.

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

10.

Hypodermic needles and syringes are used only for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral part of unit) are used for the injection or aspiration of infectious fluids. Needles should not be bent, sheared, replaced in the needle guard, or removed from the syringe following use. The needle and syringe should be placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before discard or reuse. Whenever possible, cannulas are used instead of sharp needles (e.g., for gavage).

11.

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 an 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.

C. Containment equipment

All procedures within the facility with agents assigned to Biosafety Level 4 are conducted in a Class III biological safety cabinet or in Class I or II biological safety cabinets used in conjunction with one-piece positive-pressure personnel suits ventilated by a life support system. Activities with viral agents (e.g., Rift Valley fever virus) that require Biosafety Level 4 secondary containment capabilities and for which highly effective vaccines are available and used can be conducted within Class I or Class II biological safety cabinets within the facility without the one-piece positive-pressure personnel suit being used, if (1) the facility has been decontaminated; (2) no work is being conducted in the facility with other agents assigned to Biosafety Level 4; and (3) all other standards and special practices are followed.

D. Laboratory facilities

1.

The maximum containment 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 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, floors, and autoclave chambers are decontaminated by heat treatment before being released from the maximum containment facility. Liquid wastes from shower rooms and toilets may be decontaminated with chemical disinfectants or by heat in the liquid waste decontamination system. The procedure used for heat decontamination of liquid wastes is evaluated mechanically and biologically by using a recording thermometer and an indicator microorganism with a defined heat susceptibility pattern. If liquid wastes from the shower rooms are decontaminated with chemical disinfectants, the chemical used is of demonstrated efficacy against the target or indicator microorganisms.

14.

An individual supply and exhaust air ventilation system is provided. The system maintains pressure differentials and directional airflow as required to assure flows inward from areas outside of the facility toward areas of highest potential risk within the facility. Manometers are used to sense pressure differentials between adjacent areas maintained at different pressure levels. If a system malfunctions, the manometers sound an alarm. The supply and exhaust airflow is interlocked to assure inward (or zero) airflow at all times.

15.

The exhaust air from the facility is filtered through HEPA filters and discharged to the outside so that it is dispersed away from occupied buildings and air intakes. Within the facility, the filters are located as near the laboratories as practicable in order to reduce the length of potentially contaminated air ducts. The filter chambers are designed to allow in situ decontamination before filters are removed and to facilitate certification testing after they are replaced. Coarse filters and HEPA filters are provided to treat air supplied to the facility in order to increase the lifetime of the exhaust HEPA filters and to protect the supply air system should air pressures become unbalanced in the laboratory.

16.

The treated exhaust air from Class I and II biological safety cabinets can be discharged into the laboratory room environment or to the outside through the facility air exhaust system. If exhaust air from Class I or II biological safety cabinets is discharged into the laboratory, the cabinets are tested and certified at 6-month intervals. The treated exhaust air from Class III biological safety cabinets is discharged, without recirculation through two sets of HEPA filters in series, via the facility exhaust air system. If the treated exhaust air from any of these cabinets is discharged to the outside through the facility exhaust air system, it is connected to this system in a manner (e.g., thimble unit connection[⁸⁰ ]) that avoids any interference with the air balance of the cabinets or the facility exhaust air system.

17.

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 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, an 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 communications 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.

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 are extensions of the laboratory and in some situations are integral to and inseparable from 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.

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] 78-23, Rev. 1978, 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. A "clean hall/dirty hall" layout is very useful in reducing cross contamination. 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.

These recommendations describe four combinations of practices, safety equipment, and facilities for experiments on animals infected with agents which are known or believed to produce infections in humans. 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 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"(¹¹² ), 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.

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

2.

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

3.

Eating, drinking, smoking, and storing food for human use are not permitted in animal rooms.

4.

Personnel wash their hands after handling cultures and animals and before leaving the animal room.

5.

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

6.

An insect and rodent control program is in effect.

B. Special practices

1.

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

2.

Cages are washed manually or in a cagewasher. Temperature of final rinse water in a mechanical washer should be 180°F.

3.

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

C. Containment equipment

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

D. Animal facilities

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.

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

Animal Biosafety Level 2

A. Standard practices

1.

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

2.

Work surfaces are decontaminated after use or spills of viable materials.

3.

Eating, drinking, smoking, and storing of food for human use are not permitted in animal rooms.

4.

Personnel wash their hands after handling cultures and animals and before leaving the animal room.

5.

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

6.

An insect and rodent control program is in effect.

B. Special practices

1.

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

2.

Surgical-type masks are 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.

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.

5.

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., for immunization) may enter the animal room.

6.

When the infectious agent(s) in use in the animal room requires special entry provisions (e.g., vaccination), 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, 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.

7.

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

8.

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.

9.

Hypodermic needles and syringes are used only for the parenteral injection or aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable needle-syringe units (i.e., the needle is integral to the syringe) are used for the injection or aspiration of infectious fluids. Needles should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before discard or reuse.

10.

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

11.

When appropriate, considering the agents handled, base-line 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.

C. Containment equipment

Biological safety cabinets, other physical containment devices, and/or personal protective devices (e.g., respirators, face shields) are used whenever procedures with a high potential for creating aerosols are conducted(⁸² ). These include necropsy of infected animals, harvesting of infected tissues or fluids from animals or eggs, intranasal inoculation of animals, and manipulations of high concentrations or large volumes of infectious materials.

D. Animal facilities

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.

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

5.

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.

Doors to animals rooms open inward, are self-closing, and are kept closed when work with infected animals is in progress.

2.

Work surfaces are decontaminated after use or spills of viable materials.

3.

Eating, drinking, smoking, and storing of food for human use are not permitted in the animal room.

4.

Personnel wash their hands after handling cultures and animals and before leaving the laboratory.

5.

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

6.

An insect and rodent control program is in effect.

B. Special practices

1.

Cages are autoclaved before bedding is removed and before they are cleaned and washed.

2.

Surgical-type masks or other respiratory protection devices (e.g., respirators) are worn by personnel entering rooms housing animals infected with agents assigned to Biosafety Level 3.

3.

Wrap-around or solid-front gowns or uniforms are worn by personnel entering the animal room. Front-button laboratory coats are unsuitable. Protective gowns must remain in the animal room and must be decontaminated before being laundered.

4.

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. 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.

5.

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.

6.

Hazard warning signs, incorporating the universal biohazard warning symbol, are posted on access doors to animal rooms containing animals infected with agents assigned to Biosafety Level 3. The hazard warning sign should identify the agent(s) in use, list the name and telephone number of the animal room supervisor or other responsible person(s), and indicate any special conditions of entry into the animal room (e.g., the need for immunizations or respirators).

7.

Personnel wear gloves when handling infected animals. Gloves are removed aseptically and autoclaved with other animal room wastes before being disposed of or reused.

8.

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.

9.

Hypodermic needles and syringes are used only for gavage or for parenteral injection or aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable needle-syringe units (e.g., the needle is integral to the syringe) are used. Needles should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before discard or reuse. Whenever possible, cannulas should be used instead of sharp needles (e.g., for gavage).

10.

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

11.

If vacuum lines are provided, they are protected with HEPA filters and liquid disinfectant traps.

12.

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

C. Containment equipment

1.

Personal protective clothing and equipment and/or other physical containment devices are used for all procedures and manipulations of infectious materials or infected animals.

2.

The risk of infectious aerosols from infected animals or their bedding 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 by filter bonnets, or other equivalent primary containment systems.

D. Animal facilities

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 near each animal room exit door.

4.

Windows in the animal room are closed and sealed.

5.

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

6.

An autoclave for decontaminating wastes is available, preferably within the animal room. Materials to be autoclaved outside the animal room are transported in a covered leakproof container.

7.

An exhaust air ventilation system is provided. This system creates directional airflow that draws air into the animal room through the entry area. The building exhaust can be used for this purpose if the exhaust air is not recirculated to any other area of the building, is discharged to the outside, and is dispersed away from occupied areas and air intakes. Personnel must verify that the direction of the airflow (into the animal room) is proper. The exhaust air from the animal room that does not pass through biological safety cabinets or other primary containment equipment can be discharged to the outside without being filtered or otherwise treated.

8.

The HEPA-filtered exhaust air from Class I or Class III 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 cabinet 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[⁸⁰ ]) that avoids any interference with the air balance of the cabinets or building exhaust system.

Animal Biosafety Level 4

A. Standard practices

1.

Doors to animal rooms open inward and are self-closing.

2.

Work surfaces are decontaminated after use or spills of viable materials.

3.

Eating, drinking, smoking, and storing of food for human use are not permitted in the animal room.

4.

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

5.

An insect and rodent control program is in effect.

6.

Cages are autoclaved before bedding is removed and before they are cleaned and washed.

B. Special practices

1.

Only persons whose entry into the facility or individual animal rooms 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 laboratory. 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.

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.

3.

Street 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, is provided and used by all personnel entering the facility. When exiting, personnel remove laboratory clothing and store it in a locker or hamper in the inner change room before entering the shower area.

4.

When infectious materials or infected animals are present in the animal rooms, a hazard warning sign, incorporating the universal biohazard symbol, is posted on all access doors. The sign identifies the agent, lists the name and telephone number of the animal facility supervisor or other responsible person(s), and indicates any special conditions of entry into the area (e.g., the need for immunizations and respirators).

5.

Supplies and materials to be taken into the facility enter by way of the double-door autoclave, fumigation chamber, or airlock, which is appropriately decontaminated between each use. After securing the outer doors, personnel inside the facility retrieve the materials by opening the interior doors of the autoclave, fumigation chamber, or airlock. This inner door is secured after materials are brought into the facility.

6.

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

7.

Hypodermic needles and syringes are used only for gavage or for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral part of unit) are used. Needles should not be bent, sheared, replaced in the guard or sheath, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before discard or reuse. Whenever possible, cannulas should be used instead of sharp needles (e.g., for gavage).

8.

A system is developed and is operational 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.

9.

Base-line 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.

C. Containment equipment

Laboratory animals infected with agents assigned to Biosafety Level 4 are housed in a Class III biological safety cabinet or in partial-containment caging systems (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, if no concurrent experiments are being done in the facility which require Biosafety Level 4 primary and secondary containment, and if all other standard and special practices are followed.

D. Animal facility

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 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.

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.

A foot-, elbow-, or automatically operated handwashing sink is provided near the door of each animal room within the facility.

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 self-locking.

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, cabinets, floors, and autoclave chambers are decontaminated by heat treatment before being discharged. Liquid wastes from shower rooms and toilets may be decontaminated with chemical disinfectants or by heat in the liquid waste decontamination system. The procedure used for heat decontamination of liquid wastes must be evaluated mechanically and biologically by using a recording thermometer and an indicator microorganism with a defined heat susceptibility pattern. If liquid wastes from the shower rooms are decontaminated with chemical disinfectants, the chemicals used must have documented efficacy against the target or indicator microorganisms.

11.

An individual supply and exhaust air ventilation system is provided. The system maintains pressure differentials, and directional airflow is required to assure inflow from areas outside the facility toward areas of highest potential risk within the facility. Manometers are provided to sense pressure differentials between adjacent areas and are maintained at different pressure levels. The manometers sound an alarm when a system malfunctions. The supply and exhaust airflow is interlocked to assure inward (or zero) airflow at all times.

12.

Air can be recirculated within an animal room if it is filtered through a HEPA filter.

13.

The exhaust air from the facility is filtered by HEPA filters and discharged to the outside so that it is dispersed away from occupied buildings and air intakes. Within the facility, the filters are located as near to the laboratories as practicable in order to reduce the length of potentially contaminated air ducts. The filter chambers are designed to allow in situ decontamination before filters are removed and to facilitate certification testing after they are replaced. Coarse filters are provided for treatment of air supplied to the facility in order to increase the lifetime of the HEPA filters.

14.

The treated exhaust air from Class I or Class II biological safety cabinets can be discharged into the animal room environment or to the outside through the facility air exhaust system. If exhaust air from Class I or II biological safety cabinets is discharged into the animal room, the cabinets are tested and certified at 6-month intervals. The treated exhaust air from Class III biological safety cabinets is discharged without recirculation via the facility exhaust air system. If the treated exhaust air from any of these cabinets is discharged to the outside through the facility exhaust air 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 air system.

15.

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. A duplicate filtration unit and exhaust fan 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 double-doored 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 quantity and concentration of 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, tubercle bacilli); the potential for laboratory-associated infection is high even in the absence of previously documented laboratory-associated infections (e.g., exotic arboviruses); or the consequences of infection are grave (e.g., Creutzfeldt-Jakob disease, botulism).

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.

Risk assessments and Biosafety Levels recommended in the agent summary statements presuppose a population of immunocompetent individuals. Those with altered immunocompetence may be at increased risk when exposed to infectious agents. Immunodeficiency may be hereditary, congenital, or induced by a number of neoplastic diseases, by therapy, or by radiation. The risk of becoming infected or the consequences 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 individualizing the generic risk assessments of the agent summary statements for specific activities.

The basic biosafety level assigned to an agent is based on the activities typically associated with the growth and manipulation of quantities and concentrations of infectious agents required to accomplish identification of typing. If activities with clinical materials pose a lower 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 or highly 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 in 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 a risk 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 "Containment Equipment" for Biosafety Level 3 are rigorously followed. 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 should be handled under conditions which reasonably preclude cutaneous, mucous membrane, or parenteral exposure of personnel, and sputa submitted to the laboratory for assay for tubercle bacilli should be handled under conditions which reasonably preclude the generation of aerosols or which contain any aerosols that may be generated during the manipulation of clinical materials or cultures.

The infectious agents which meet the previously stated criteria are listed by category of agent on the following pages. 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 for working with clinical materials, cultures of infectious agents, or infected animals recommended in the agent summary statement and described in Section V.

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

Risk Assessment. The risk assessment of laboratory activities involving the use of infectious microorganisms is ultimately a subjective process. Those 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 agent are described generically for agents in Biosafety Levels 1-4 and specifically for individual agents or groups of agents on pages 87-88 and in Section V, respectively, of this Appendix.

Hepatitis B virus (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 be infectious. Natural transmission is associated with parenteral inoculation or with contamination of the broken skin or of 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 exposure.

The primary risk of HBv infection in laboratory personnel is associated with accidental parenteral inoculation, exposure of the broken skin or mucous membranes of the eyes, nose, or mouth, or ingestion of infectious body fluids. 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.

Hepatitis non-A non-B and AIDS—acquired immune deficiency syndrome—pose similar infection risks to laboratory personnel. The prudent practices recommended for HBv are applicable to these two disease entities, 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 of laboratory infection. Microbiologists and other scientists working with such materials may nevertheless find the practices, containment equipment, and facilities recommendations described in this publication of value in developing operational standards to meet their own assessed needs.

AGENT SUMMARY STATEMENTS

Rickettsial Agents

Viral Agents

ARBOVIRUSES

APPENDIX A.1. BIOLOGICAL SAFETY CABINETS

Biological safety cabinets are among the most effective, as well as the most commonly used, primary containment devices in laboratories working with infectious agents. Each of the three types—Class I, II, and III—has performance characteristics which are described below. In addition to the design, construction, and performance standards for vertical laminar flow biological safety cabinets (Class III), the National Sanitation Foundation has also developed a list of such products which meet the reference standard. Utilization of this standard(⁸⁰ ) and list should be the first step in selection and procurement of a biological safety cabinet.

Class I and II biological safety cabinets, when used in conjunction with good microbiological techniques, provide an effective partial containment system for safe manipulation of moderate and high-risk microorganisms (i.e., Biosafety Level 2 and 3 agents). Both Class I and II biological safety cabinets have comparable inward face velocities (75 linear ft/min) and provide comparable levels of containment in protecting the laboratory worker and the immediate laboratory environment from infectious aerosols generated within the cabinet.

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 cabinet 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. The slide-sound training film developed by NIH (Effective Use of the Laminar Flow Biological Safety Cabinet) provides a thorough training and orientation guide. 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 briskly walking past the biological safety cabinet while it is in use are demonstrated causes of the escape of aerosolized particles from within the cabinet. Strict adherence to recommended practices for the use of biological safety cabinets is as important in attaining the maximum containment capability of the equipment as is the mechanical performance of the equipment itself.

Horizontal laminar flow ''clean benches" are present in a number of clinical, pharmacy, and laboratory facilities. These "clean benches" provide a high-quality environment within the work chamber for manipulation of nonhazardous materials. Caution: Since the operator sits in the immediate downstream exhaust from the "clean bench," this equipment must never be used for the handling of toxic, infectious, or sensitizing materials.

Class I. The Class I biological safety cabinet (Figure A.1) is an open-fronted, negative-pressure, ventilated cabinet with a minimum inward face velocity at the work opening of at least 75 ft/min. The exhaust air from the cabinet may be used in three operational modes: with a full-width open front, with an installed front closure panel not equipped with gloves, and with an installed front closure panel equipped with arm-length rubber gloves.

FIGURE A.1

Class I biological safety cabinet

Class II. The Class II vertical laminar-flow biological cabinet (Figure A.2) is an open-fronted, ventilated cabinet with an average inward face velocity at the work opening of at least 75 ft/min. This cabinet provides a HEPA-filtered, recirculated mass airflow within the work space. The exhaust air from the cabinet is also filtered by HEPA filters. Design, construction, and performance standards for Class II cabinets have been developed by and are available from the National Sanitation Foundation, Ann Arbor, Michigan(⁸⁰ ).

FIGURE A.2

Class II biological safety cabinets

Class III. The Class III cabinet (Figure A.3) is a totally enclosed ventilated cabinet of gas-tight construction. Operations within the Class III cabinet are conducted through attached rubber gloves. When in use, the Class III cabinet is maintained under negative air pressure of at least 0.5 in. water gauge. Supply air is drawn into the cabinet through HEPA filters. The cabinet exhaust air is filtered by two HEPA filters, installed in series, before discharge outside of the facility. The exhaust fan for the Class III cabinet is generally separate from the exhaust fans of the facility's ventilation system.

FIGURE A.3

Class III biological safety cabinet

Use of cabinets. Personnel protection provided by Class I and Class II cabinets is dependent on the inward airflow. Since the face velocities are similar, they generally provide an equivalent level of personnel protection. The use of these cabinets alone, however, is not appropriate for containment of highest-risk infectious agents because aerosols may accidentally escape through the open front.

The use of a Class II cabinet in the microbiological laboratory offers the additional capability and advantage of protecting materials contained within it from extraneous airborne contaminants. This capability is provided by the HEPA-filtered, recirculated mass airflow within the work space.

The Class III cabinet provides the highest level of personnel and product protection. This protection is provided by the physical isolation of the space in which the infectious agent is maintained. When these cabinets are required, all procedures involving infectious agents are contained within them. Several Class III cabinets are therefore typically set up as an interconnected system. All equipment required by the laboratory activity, such as incubators, refrigerators, and centrifuges, must be an integral part of the cabinet system. Double-doored autoclaves and chemical dunk tanks are also attached to the cabinet system to allow supplies and equipment to be safely introduced and removed.

Personnel protection equivalent to that provided by Class III cabinets can also be obtained with a personnel suit area and Class I or Class II cabinets. This area is one in which the laboratory worker is protected from a potentially contaminated environment by a one-piece positive-pressure suit ventilated by a life-support system. This area is entered through an airlock fitted with airtight doors. A chemical shower is provided 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 units installed in series.

APPENDIX A.2. IMMUNOPROPHYLAXIS

An additional level of protection for at-risk personnel may be achieved with appropriate prophylactic vaccinations. 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 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 the Public Health Service Advisory Committee on Immunization Practices, are included in the agent summary statements in Section V.

APPENDIX A.3. 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.

Recent 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 nonpunitive 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 A.4. 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 71.56). 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.

The interstate shipment of indigenous etiologic agents, diagnostic specimens, and biological 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 A.4.

Additional information on the importation and interstate shipment of etiologic agents of human disease and other related materials may be obtained by writing to:

Centers for Disease Control

Attention: Office of Biosafety

1600 Clifton Road, N.E.

Atlanta, GA 30333

Telephone: (404) 329-3883

FTS: 236-3883

APPENDIX A.5. 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 virus

African swine fever virus

Besnoitia besnoiti

Borna disease virus

Bovine ephemeral fever

Bovine infectious petechial fever agent

Camelpox virus

Foot and mouth disease virus

Fowl plague virus

Histoplasma (Zymonema) farciminosum

Hog cholera virus

Louping ill virus

Lumpy skin disease virus

Mycoplasma agalactiae

Mycoplasma mycoides

Nairobi sheep disease virus (Ganjam virus)

Newcastle disease virus (velogenic strains)

Pseudomonas mallei

Rickettsia ruminantium

Rift Valley fever virus

Rinderpest virus

Swine vesicular disease virus

Teschen disease virus

Theileria annulata

Theileria bovis

Theileria hirci

Theileria lawrencei

Trypanosoma evansi

Trypanosoma vivax

Vesicular exanthema virus

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:

Chief Staff Veterinarian

Organisms and Vectors

Veterinary Services

Animal and Plant Health Inspection Service

U.S. Department of Agriculture

Hyattsville, MD 20782

Telephone: (301) 436-8017

FTS 436-8017

APPENDIX A.6. 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

Attention: Office of Biosafety

Atlanta, GA 30333

Telephone: (404) 329-3883

FTS 236-3883

National Institutes of Health

Attention: Division of Safety

Bethesda, MD 20892

Telephone: (301) 496-1357

FTS 496-1357

National Animal Disease Center

U.S. Department of Agriculture

Ames, IA 50010

Telephone: (515) 862-8258

FTS 862-8258

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Footnotes

Reprinted from J.H. Richardson and W.E. Barkley, Biosafety in Microbiological and Biomedical Laboratories, 1st ed., U.S. Public Health Service, HHS Publication No. (CDC) 84-8395, U.S. Government Printing Office, Washington, D.C., March 1984. Figures, tables, appendixes, and page numbers in this reprinted version have been renumbered to avoid confusion.