III. Principles of Biosafety
A. Risk Groups
Infectious agents may be classified into risk groups based on their relative hazard. The table below, which was excerpted from the NIH Recombinant DNA Guidelines, presents the "Basis for the Classification of Biohazardous Agents by Risk Group."
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Risk Group 1 (RG1) |
Agents that are not associated with disease in healthy adult humans |
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Risk Group 2 (RG2) |
Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available |
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Risk Group 3 (RG3) |
Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk) |
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Risk Group 4 (RG4) |
Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk) Work with Risk Group 4 agents IS PROHIBITED at the University of Pennsylvania. |
Laboratories and animal facilities are classified according to their design features, construction and containment capabilities. Combinations of these design characteristics represent levels of containment appropriate for work with agents in various risk groups. Containment appropriate for given work is the biosafety level or animal biosafety level respectively that provides appropriate containment for the various risk group agents. Biosafety Levels and Animal Biosafety Levels are discussed in greater detail below.
B. Containment
The term "containment" is used in describing safe methods for managing infectious agents in the laboratory environment where they are being handled or maintained. The purpose of containment is to reduce or eliminate exposure of laboratory workers, other people and the outside environment to potentially hazardous agents. The three elements of containment include laboratory practice and technique, safety equipment, and facility design.
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 risk assessment of the work to be done with a specific agent will determine the appropriate combination of work practices, safety equipment and facility design to provide adequate containment.
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 handling such material safely. The PI or laboratory supervisor is responsible for providing or arranging for appropriate training of personnel.
Each laboratory should develop an operational manual which identifies specific 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 the handling of infectious agents must direct laboratory activities.
When standard laboratory practices are not sufficient to control the hazard associated with a particular agent or laboratory procedure, additional measures may be needed. The PI is responsible for selecting additional safety practices, which must be in keeping with the hazard associated with the agent or procedure.
Laboratory personnel, safety practices and techniques must be supplemented by appropriate facility design and engineering features, safety equipment and management practices.
Safety Equipment (Primary Barriers) -- Safety equipment includes biological safety cabinets, enclosed containers (i.e., safety centrifuge cups) and other engineering controls designed to remove or minimize exposures to hazardous biological materials. The biological safety cabinet (BSC) is the principal device used to provide containment of infectious splashes or aerosols generated by many microbiological procedures. More information on BSC’s may be found at the EHRS website and CDC website.
Safety equipment also may include items for personal protection such as personal protective clothing, respirators, face shields, safety glasses or goggles. Personal protective equipment is often used in combination with other safety equipment when working with biohazardous materials. In some situations, personal protective clothing may form the primary barrier between personnel and the infectious materials.
Facility Design (Secondary Barriers) -- The design of a facility is important in providing a barrier to protect people working inside and outside the laboratory, and to protect people or animals in the community from infectious agents which may be accidentally released from the laboratory. Facilities must be commensurate with the laboratory's function and the recommended biosafety level for the agent being manipulated.
The recommended secondary barrier(s) will depend on the risk of transmission of specific agents. For example, the exposure risks for most laboratory work in Biosafety Level 1 and 2 facilities will be direct contact with the agents, or inadvertent contact exposures through contaminated work environments. Secondary barriers in these laboratories may include separation of the laboratory work area from public access, availability of a decontamination facility (e.g., autoclave) and hand washing facilities.
As the risk for aerosol transmission increases, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment. Such design features could include specialized ventilation systems to assure directional airflow, air treatment systems to decontaminate or remove agents from exhaust air, controlled access zones, airlocks at laboratory entrances, or separate buildings or modules for isolation of the laboratory.
C. Biosafety Levels
CDC describes four biosafety levels (BSLs) which consist of combinations of laboratory practices and techniques, safety equipment, and laboratory facilities. Each combination is specifically appropriate for the operations performed, the documented or suspected routes of transmission of the infectious agents, and for the laboratory function or activity. The recommended biosafety level for an organism represents the conditions under which the agent can be ordinarily handled safely. When specific information is available to suggest that virulence, pathogenicity, antibiotic resistance patterns, vaccine and treatment availability, or other factors are significantly altered, more (or less) stringent practices may be specified.
Biological Safety Level 1 (BSL-1) -- is appropriate for work done with defined and characterized strains of viable microorganisms not known to cause disease in healthy adult humans. It represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for hand washing.
Biological Safety Level 2 (BSL-2) -- is applicable to work done with a broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. Agents can be used safely on the open bench, provided the potential for producing splashes or aerosols is low. Primary hazards to personnel working with these agents relate to accidental percutaneous or mucous membrane exposures or ingestion of infectious materials. Procedures with high aerosol or splash potential must be conducted in primary containment equipment such as biosafety cabinets. Primary barriers such as splash shields, face protection, gowns and gloves should be used as appropriate. Secondary barriers such as hand washing and waste decontamination facilities must be available.
Biological Safety Level 3 (BSL-3) -- is applicable to work done with indigenous or exotic agents with a potential for respiratory transmission and which may cause serious and potentially lethal infection. Primary hazards to personnel working with these agents (i.e., Mycobacterium tuberculosis, St. Louis encephalitis virus and Coxiella burnetii) include auto-inoculation, ingestion and exposure to infectious aerosols. Greater emphasis is placed on primary and secondary barriers to protect personnel in adjoining areas, the community and the environment from exposure to infectious aerosols. For example, all laboratory manipulations should be performed in a biological safety cabinet or other enclosed equipment. Secondary barriers include controlled access to the laboratory and a specialized ventilation system that minimizes the release of infectious aerosols from the laboratory.
Biological Safety Level 4 (BSL-4) -- is applicable for work with dangerous and exotic agents that pose a high individual risk of life-threatening disease, which may be transmitted via the aerosol route and for which there is no available vaccine or therapy. All BSL-4 work IS PROHIBITED at the University of Pennsylvania. Agents with close or identical antigenic relationship to Biosafety Level 4 agents should also be handled at this level. Primary hazards to workers include respiratory exposure to infectious aerosols, mucous membrane exposure to infectious droplets and auto-inoculation. All manipulations of potentially infected materials and isolates pose a high risk of exposure and infection to personnel, the community and the environment. Isolation of aerosolized infectious materials is accomplished primarily by working in a Class III biological safety cabinet or a full-body, air-supplied positive pressure personnel suit. The facility is generally a separate building or a completely isolated zone within a complex with specialized ventilation and waste management systems to prevent release of viable agents to the environment.
D. Vertebrate Animal Biosafety Levels
There are four animal biosafety levels (ABSLs), designated Animal Biosafety Level 1 through 4, for work with infectious agents in mammals. The levels are combinations of practices, safety equipment and facilities for experiments on animals infected with agents that produce or may produce human infection. In general, the biosafety level recommended for working with an infectious agent in vivo and in vitro is comparable.
Animal Biological Safety Level 1 (ABSL-1) -- is suitable for work involving well characterized agents that are not known to cause disease in healthy adult humans, and that are of minimal potential hazard to laboratory personnel and the environment.
Animal Biological Safety Level 2 (ABSL-2) -- is suitable for work with those agents associated with human disease. It addresses hazards from ingestion as well as from percutaneous and mucous membrane exposure.
Animal Biological Safety Level 3 (ABSL-3) -- is suitable for work with animals infected with indigenous or exotic agents that present the potential of aerosol transmission and of causing serious or potentially lethal disease.
Animal Biological Safety Level 4 (ABSL-4) -- is suitable for addressing dangerous and exotic agents that pose high risk of like threatening disease, aerosol transmission, or related agents with unknown risk of transmission. All ABSL-4 work IS PROHIBITED at the University of Pennsylvania.
Animal biosafety levels have been adapted for work in agricultural animals by EHRS and ULAR. The use of any barn for research with infectious agents in farm animals must be approved by EHRS and ULAR prior to experimentation.
Complete descriptions of all Biosafety Levels and Animal Biosafety Levels are outlined in the 4th edition of Biosafety in Microbiological and Biomedical Laboratories published by the U. S. Department of Health and Human Services (CDC/NIH).
E. Risk Assessment
It is the responsibility of the principal investigator or laboratory director to conduct a risk assessment to determine the proper work practices and containment requirements for work with biohazardous material. The risk assessment process should identify features of microorganisms as well as host and environmental factors that influence the potential for workers to have a biohazard exposure. This responsibility cannot be shifted to inexperienced or untrained personnel.
The principal investigator or laboratory director should consult with a Biosafety Officer to ensure that the laboratory is in compliance with established guidelines and regulations. When performing a risk assessment, it is advisable to take a conservative approach if there is incomplete information available. Factors to consider when evaluating risk include the following:
Pathogenicity: The more severe the potentially acquired disease, the higher the risk. Salmonella, a Risk Group 2 agent, can cause diarrhea, septicemia if ingested. Treatment is available. Viruses such as Ebola, Marburg, and Lassa fever cause diseases with high mortality rates. There are no vaccines or treatment available. These agents belong to Risk Group 4.
Route of transmission: Agents that can be transmitted by the aerosol route have been known to cause the most laboratory-acquired infections. The greater the aerosol potential, the higher the risk of infection. Work with Mycobacterium tuberculosis is performed at Biosafety Level 3 because disease is acquired via the aerosol route.
Agent stability: The greater the potential for an agent to survive in the environment, the higher the risk. Consider factors such as desiccation, exposure to sunlight or ultraviolet light, or exposure to chemical disinfections when looking at the stability of an agent.
Infectious dose: Consider the amount of an infectious agent needed to cause infection in a normal person. An infectious dose can vary from one to hundreds of thousands of organisms or infectious units. An individual’s immune status can also influence the infectious dose.
Concentration: Consider whether the organisms are in solid tissue, viscous blood, sputum, etc., the volume of the material and the laboratory work planned (amplification of the material, sonication, centrifugation, etc.). In most instances, the risk increases as the concentration of microorganisms increases.
Origin: This may refer to the geographic location (domestic or foreign), host (infected or uninfected human or animal), or nature of the source (potential zoonotic or associated with a disease outbreak).
Availability of data from animal studies: If human data is not available, information on the pathogenicity, infectivity, and route of exposure from animal studies may be valuable. Use caution when translating infectivity data from one species to another.
Availability of an effective prophylaxis or therapeutic intervention: Effective vaccines, if available, should be offered to laboratory personnel in advance of their handling of infectious material. However, immunization does not replace engineering controls, proper practices and procedures and the use of personal protective equipment (PPE). The availability of post-exposure prophylaxis should also be considered.
Medical surveillance: Medical surveillance programs may include monitoring employee health status, participating in post-exposure management, employee counseling prior to offering vaccination, and annual physicals.
Experience and skill level of at-risk personnel: Laboratory workers must become proficient in specific tasks prior to working with microorganisms. Laboratory workers may have to work with non-infectious materials to ensure they have the appropriate skill level prior to working with biohazardous materials. Laboratory workers may have to go through additional training (e.g., HIV training, BSL-3 training, etc.) before they are allowed to work with materials or in a designated facility.
Refer to the following resources to assist in your risk assessment:
NIH Recombinant DNA Guidelines
Biosafety in Microbiological & Biomedical Laboratories, 5th ed (CDC/NIH)
