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BMBL Section II
Principles of Biosafety
The term "containment" is used in describing safe
methods for managing infectious materials in the laboratory environment where they are
being handled or maintained. The purpose of containment is to reduce or eliminate exposure
of laboratory workers, other persons, and the outside environment to potentially hazardous
agents.
Primary containment, the protection of personnel and the
immediate laboratory environment from exposure to infectious agents, is provided by both
good microbiological technique and the use of appropriate safety equipment. The use of
vaccines may provide an increased level of personal protection. Secondary containment, the
protection of the environment external to the laboratory from exposure to infectious
materials, is provided by a combination of facility design and operational practices.
Therefore, the three elements of containment include laboratory practice and technique,
safety equipment, and facility design. The risk assessment of the work to be done with a
specific agent will determine the appropriate combination of these elements.
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The most important element of containment is strict
adherence to standard microbiological practices and techniques. Persons working with
infectious agents or potentially infected materials must be aware of potential hazards,
and must be trained and proficient in the practices and techniques required to handle such
material safely. The director or person in charge of the laboratory is responsible for
providing or arranging the appropriate training of personnel. Each
laboratory should develop or adopt a biosafety or operations manual that identifies the
hazards that will or may be encountered, and that specifies practices and procedures
designed to minimize or eliminate exposures to these hazards. Personnel should be advised
of special hazards and should be required to read and 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 be
responsible for the conduct of work with any infectious agents or material. This
individual should consult with biosafety or other health and safety professionals with
regard to risk assessment.
When standard laboratory practices are not sufficient to control the
hazards 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 hazards 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 |
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Safety equipment includes biological safety cabinets
(BSCs), enclosed containers, and other engineering controls designed to remove or minimize
exposures to hazardous biological materials. The biological safety cabinet (BSC) is the
principal device used to provide containment of infectious splashes or aerosols generated
by many microbiological procedures. Three types of biological safety cabinets (Class I,
II, III) used in microbiological laboratories are described and illustrated in Appendix A.
Open-fronted Class I and Class II biological safety cabinets are primary barriers which
offer significant levels of protection to laboratory personnel and to the environment when
used with good microbiological techniques. The Class II biological safety cabinet also
provides protection from external contamination of the materials (e.g., cell cultures,
microbiological stocks) being manipulated inside the cabinet. The gas-tight Class III
biological safety cabinet provides the highest attainable level of protection to personnel
and the environment. An example of another primary barrier is
the safety centrifuge cup, an enclosed container designed to prevent aerosols from being
released during centrifugation. To minimize this hazard, containment controls such as BSCs
or centrifuge cups must be used when handling infectious agents that can be transmitted
through the aerosol route of exposure.
Safety equipment also may include items for personal protection,
such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety
glasses, or goggles. Personal protective equipment is often used in combination with
biological safety cabinets and other devices that contain the agents, animals, or
materials being handled. In some situations in which it is impractical to work in
biological safety cabinets, personal protective equipment may form the primary barrier
between personnel and the infectious materials. Examples include certain animal studies,
animal necropsy, agent production activities, and activities relating to maintenance,
service, or support of the laboratory facility. |
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The design and construction of the facility contributes
to the laboratory workers' protection, provides a barrier to protect persons outside the
laboratory, and protects persons or animals in the community from infectious agents which
may be accidentally released from the laboratory. Laboratory management is responsible for
providing facilities commensurate with the laboratory's function and the recommended
biosafety level for the agents being manipulated. The
recommended secondary barrier(s) will depend on the risk of transmission of specific
agents. For example, the exposure risks for most laboratory work in Biosafety Level 1 and
2 facilities will be direct contact with the agents, or inadvertent contact exposures
through contaminated work environments. Secondary barriers in these laboratories may
include separation of the laboratory work area from public access, availability of a
decontamination facility (e.g., autoclave), and handwashing facilities.
When the risk of infection by exposure to an infectious aerosol is
present, higher levels of primary containment and multiple secondary barriers may become
necessary to prevent infectious agents from escaping into the environment. Such design
features include specialized ventilation systems to ensure directional air flow, air
treatment systems to decontaminate or remove agents from exhaust air, controlled access
zones, airlocks as laboratory entrances, or separate buildings or modules to isolate the
laboratory. Design engineers for laboratories may refer to specific ventilation
recommendations as found in the Applications Handbook for Heating, Ventilation, and
Air-Conditioning (HVAC) published by the American Society of Heating, Refrigerating,
and Air-Conditioning Engineers (ASHRAE).(1) |
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Four biosafety levels (BSLs) are described in Section
III, 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 the laboratory function or activity. The
recommended biosafety level(s) for the organisms in Section VII (Agent Summary Statements)
represent those conditions under which the agent ordinarily can be safely handled. The
laboratory director is specifically and primarily responsible for assessing the risks and
appropriately applying the recommended biosafety levels. Generally, work with known agents
should be conducted at the biosafety level recommended in Section VII. When specific
information is available to suggest that virulence, pathogenicity, antibiotic resistance
patterns, vaccine and treatment availability, or other factors are significantly altered,
more (or less) stringent practices may be specified.
Biosafety Level 1
practices, safety equipment, and facility design and construction are appropriate for
undergraduate and secondary educational training and teaching laboratories, and for other
laboratories in which work is done with defined and characterized strains of viable
microorganisms not known to consistently cause disease in healthy adult humans. Bacillus
subtilis, Naegleria gruberi, infectious canine hepatitis virus, and exempt organisms
under the NIH Recombinant DNA Guidelines are representative of 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 that have undergone
multiple in vivo passages should not be considered avirulent simply because they
are vaccine strains.
Biosafety Level 1 represents a basic level of containment that
relies on standard microbiological practices with no special primary or secondary barriers
recommended, other than a sink for handwashing.
Biosafety Level 2 practices,
equipment, and facility design and construction are applicable to clinical, diagnostic,
teaching, and other laboratories in which work is done with the broad spectrum of
indigenous moderate-risk agents that are present in the community and associated with
human disease of varying severity. With good microbiological techniques, these agents can
be used safely in activities conducted on the open bench, provided the potential for
producing splashes or aerosols is low. Hepatitis B virus, HIV, the salmonellae, and Toxoplasma
spp. are representative of microorganisms assigned to this containment level. Biosafety
Level 2 is appropriate when work is done with any human-derived blood, body fluids,
tissues, or primary human cell lines where the presence of an infectious agent may be
unknown. (Laboratory personnel working with human-derived materials should refer to the
OSHA Bloodborne Pathogen Standard(2)for
specific required precautions.)
Primary hazards to personnel working with these agents relate to
accidental percutaneous or mucous membrane exposures, or ingestion of infectious
materials. Extreme caution should be taken with contaminated needles or sharp instruments.
Even though organisms routinely manipulated at Biosafety Level 2 are not known to be
transmissible by the aerosol route, procedures with aerosol or high splash potential that
may increase the risk of such personnel exposure must be conducted in primary containment
equipment, or in devices such as a BSC or safety centrifuge cups. Other primary barriers
should be used as appropriate, such as splash shields, face protection, gowns, and gloves.
Secondary barriers such as handwashing sinks and waste
decontamination facilities must be available to reduce potential environmental
contamination.
Biosafety Level 3 practices,
safety equipment, and facility design and construction are applicable to clinical,
diagnostic, teaching, research, or production facilities in which work is done with
indigenous or exotic agents with a potential for respiratory transmission, and which may
cause serious and potentially lethal infection. Mycobacterium tuberculosis, St.
Louis encephalitis virus, and Coxiella burnetii are representative of the
microorganisms assigned to this level. Primary hazards to personnel working with these
agents relate to autoinoculation, ingestion, and exposure to infectious aerosols.
At Biosafety Level 3, more emphasis is placed on primary and
secondary barriers to protect personnel in contiguous areas, the community, and the
environment from exposure to potentially infectious aerosols. For example, all laboratory
manipulations should be performed in a BSC or other enclosed equipment, such as a
gas-tight aerosol generation chamber. Secondary barriers for this level include controlled
access to the laboratory and ventilation requirements that minimize the release of
infectious aerosols from the laboratory.
Biosafety Level 4 practices,
safety equipment, and facility design and construction are 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. Agents with a close or identical antigenic relationship to Biosafety Level 4
agents also should be handled at this level. When sufficient data are obtained, work with
these agents may continue at this level or at a lower level. Viruses such as Marburg or
Congo-Crimean hemorrhagic fever are manipulated at Biosafety Level 4.
The primary hazards to personnel working with Biosafety Level 4
agents are respiratory exposure to infectious aerosols, mucous membrane or broken skin
exposure to infectious droplets, and autoinoculation. All manipulations of potentially
infectious diagnostic materials, isolates, and naturally or experimentally infected
animals, pose a high risk of exposure and infection to laboratory personnel, the
community, and the environment.
The laboratory worker's complete isolation from aerosolized
infectious materials is accomplished primarily by working in a Class III BSC or in a
full-body, air-supplied positive-pressure personnel suit. The Biosafety Level 4 facility
itself is generally a separate building or completely isolated zone with complex,
specialized ventilation requirements and waste management systems to prevent release of
viable agents to the environment.
The laboratory director is specifically and primarily responsible
for the safe operation of the laboratory. His/her knowledge and judgment are critical in
assessing risks and appropriately applying these recommendations. The recommended
biosafety level represents those conditions under which the agent can ordinarily be safely
handled. Special characteristics of the agents used, the training and experience of
personnel, and the nature or function of the laboratory may further influence the director
in applying these recommendations. |
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Four biosafety levels are also described
for activities involving infectious disease work with experimental animals. 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. |
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Clinical laboratories, especially those in health care
facilities, receive clinical specimens with requests for a variety of diagnostic and
clinical support services. Typically, the infectious nature of clinical material is
unknown, and specimens are often submitted with a broad request for microbiological
examination for multiple agents (e.g., sputa submitted for "routine," acid-fast,
and fungal cultures). It is the responsibility of the laboratory director to establish
standard procedures in the laboratory which realistically address the issue of the
infective hazard of clinical specimens. Except in
extraordinary circumstances (e.g., suspected hemorrhagic fever), the initial processing of
clinical specimens and serological identification of isolates can be done safely at
Biosafety Level 2, the recommended level for work with bloodborne pathogens such as
hepatitis B virus and HIV. The containment elements described in Biosafety Level 2 are
consistent with the OSHA standard, "Occupational Exposure to Bloodborne
Pathogens"(3)(4)
from the Occupational Safety and Health Administration. This requires the use of specific
precautions with all clinical specimens of blood or other potentially
infectious material (Universal or Standard Precautions).(5)
Additionally, other recommendations specific for clinical laboratories may be obtained
from the National Committee for Clinical Laboratory Standards.(6)
Biosafety Level 2 recommendations and OSHA requirements focus on the
prevention of percutaneous and mucous membrane exposures to clinical material. Primary
barriers such as biological safety cabinets (Class I or II) should be used when performing
procedures that might cause splashing, spraying, or splattering of droplets. Biological
safety cabinets also should be used for the initial processing of clinical specimens when
the nature of the test requested or other information suggests the likely presence of an
agent readily transmissible by infectious aerosols (e.g., M. tuberculosis), or
when the use of a biological safety cabinet (Class II) is indicated to protect the
integrity of the specimen.
The segregation of clinical laboratory functions and limited or
restricted access to such areas is the responsibility of the laboratory director. It is
also the director's responsibility to establish standard, written procedures that address
the potential hazards and the required precautions to be implemented. |
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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 C).
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 D). |
1. American Society of Heating,
Refrigerating, and Air-Conditioning Engineers, Inc. 1999. "Laboratories." In:
ASHRAE Handbook, Heating, Ventilation, and Air-Conditioning Applications, Chapter 13.
2. U.S. Department of Labor,
Occupational Safety and Health Administration. 1991. Occupational Exposure to Bloodborne
Pathogens, Final Rule. Fed. Register 56:64175-64182.
3. U.S. Department of Labor,
Occupational Safety and Health Administration. 1991. (2)
4. Richmond, J.Y. 1994. "HIV
Biosafety: Guidelines and Regulations." In (G. Schochetman, J. R. George, Eds.), AIDS
Testing, Edition 2 (pp. 346-360). Springer-Verlag New York, Inc.
5. Centers for Disease Control. 1988.
Update: Universal Precautions for Prevention of Transmission of Human Immunodeficiency
Virus, Hepatitis B Virus and Other Bloodborne Pathogens in Healthcare Settings. MMWR, 37:377-382,
387, 388.
6. National Committee for Clinical
Laboratory Standards (NCCLS). 1997. Protection of laboratory workers from instrument
biohazards and infectious disease transmitted by blood, body fluids, and tissue. Approved
guideline. Dec. 1977, NCCLS Doc. M29-A (ISBN1-56238-339-6. |