Laboratory Biosafety Level 4 (BSL 4)

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

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

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

A. Standard Microbiological Practices

1. Access to the laboratory is limited by the laboratory director when experiments are in progress.

2. Policies for safe handling of sharps are instituted.

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

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

5. All waste is decontaminated before disposal by an approved method such as autoclaving.

6. An insect and rodent control program is in effect (see Appendix G).

B. Special Practices

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

The supervisor has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory. Access to the facility is limited by means of secure, locked doors; accessibility is managed by the laboratory director, biohazard 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.

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

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

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

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

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

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

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

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

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

11. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels.

a. Needles and syringes or other sharp instruments are restricted in the laboratory for use only when there is no alternative, such as for parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.

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

c. Syringes that re-sheath the needle, needleless systems, and other safety devices are used when appropriate.

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

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

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

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

15. Spills of infectious materials are contained and cleaned up by appropriate professional staff or others properly trained and equipped to work with concentrated infectious material. A spill procedure is developed and posted within the laboratory.

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

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

C. Safety Equipment (Primary Barriers) All procedures within the facility are conducted in the Class III biological safety cabinet or in Class II biological safety cabinets used in conjunction with one-piece positive pressure personnel suits ventilated by a life support system.

D. Laboratory Facility (Secondary Barriers) There are two models for Biosafety Level 4 laboratories: (A) the Cabinet Laboratory where all handling of the agent is performed in a Class III Biological Safety Cabinet, and (B) the Suit Laboratory where personnel wear a protective suit. Biosafety Level-4 laboratories may be based on either model or a combination of both models in the same facility. If a combination is used, each type must meet all the requirements identified for that type.

(A) Cabinet Laboratory (See Appendix A)

1. The Biosafety Level 4 facility consists of either a separate building or a clearly demarcated and isolated zone within a building. The rooms in the facility are arranged to ensure passage through a minimum of two doors prior to entering the room(s) containing the Class III biological safety cabinet (cabinet room). Outer and inner change rooms separated by a shower are provided for personnel entering and leaving the cabinet room. A double-door autoclave, dunk tank, fumigation chamber, or ventilated anteroom for decontamination is provided at the containment barrier for passage of those materials, supplies, or equipment that are not brought into the cabinet room through the change room.

2. Daily inspections of all containment parameters (e.g., directional airflow) and life support systems are completed before laboratory work is initiated to ensure that the laboratory is operating according to its operating parameters.

3. Walls, floors, and ceilings of the cabinet room and inner change room are constructed to form a sealed internal shell which facilitates fumigation and is resistant to entry and exit of animals and insects. Floors are integrally sealed and coved. The internal surfaces of this shell are resistant to liquids and chemicals to facilitate cleaning and decontamination of the area. All penetrations in these structures and surfaces are sealed. Openings around doors into the cabinet room and inner change room are minimized and are capable of being sealed to facilitate decontamination. Any drains in the cabinet room floor are connected directly to the liquid waste decontamination system. Sewer vents and other service lines contain HEPA filters and protection against vermin.

4. Bench tops have seamless or sealed surfaces which are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surfaces and equipment.

5. Laboratory furniture is of simple open construction, capable of supporting anticipated loading and uses. Spaces between benches, cabinets, and equipment are accessible for cleaning and decontamination. Chairs and other furniture used in laboratory work should be covered with a non-fabric material that can be easily decontaminated.

6. A hands-free or automatically operated handwashing sink is provided near the door of the cabinet room(s) and the outer and inner change rooms.

7. If there is a central vacuum system, it does not serve areas outside the cabinet room. 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 cabinet room are protected by devices that prevent backflow.

8. If water fountains are provided, they are automatically or foot-operated and are located in the facility corridors outside the laboratory. The water service to the fountain is isolated from the distribution system supplying water to the laboratory areas and is equipped with a backflow preventer.

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

10. Any windows are breakage-resistant and sealed.

11. Double-door autoclaves are provided for decontaminating materials passing out of both the Class III biological safety cabinet(s) and the cabinet room(s). Autoclaves that open outside of the containment barrier must be sealed to the wall of the containment barrier. The autoclave doors are automatically controlled so that the outside door can only be opened after the autoclave "sterilization" cycle has been completed.

12. Pass-through dunk tanks, fumigation chambers, or equivalent decontamination methods are provided so that materials and equipment that cannot be decontaminated in the autoclave can be safely removed from both the Class III biological safety cabinet(s) and the cabinet room(s).

13. Liquid effluents from the dirty-side inner change room (including toilets) and cabinet room sinks, floor drains (if used), autoclave chambers, and other sources within the cabinet room are decontaminated by a proven method, preferably heat treatment, before being discharged to the sanitary sewer. Effluents from showers and clean-side toilets may be discharged to the sanitary sewer without treatment. The process used for decontamination of liquid wastes must be validated physically and biologically.

14. A dedicated non-recirculating ventilation system is provided. The supply and exhaust components of the system are balanced to ensure directional airflow from the area of least hazard to the area(s) of greatest potential hazard. The differential pressure/directional airflow between adjacent areas is monitored and alarmed to indicate any system malfunction. An appropriate visual pressure monitoring device that indicates and confirms the pressure differential of the cabinet room is provided and located at the entry to the clean change room. The airflow in the supply and exhaust components is monitored and the HVAC control system is designed to prevent sustained positive pressurization of the laboratory. The Class III cabinet should be directly connected to the exhaust system. If the Class III cabinet is connected to the supply system, it is done in a manner that prevents positive pressurization of the cabinet.

15. The supply air to and exhaust air from the cabinet room, inner change room, and anteroom pass through HEPA filter(s). The air is discharged away from occupied spaces and air intakes. The HEPA filter(s) are located as near as practicable to the source in order to minimize the length of potentially contaminated ductwork. All HEPA filters need to be tested and certified annually. The HEPA filter housings are designed to allow for in situ decontamination of the filter prior to removal, or removal of the filter in a sealed, gas-tight primary container for subsequent decontamination and/or destruction by incineration. The design of the HEPA filter housing should facilitate validation of the filter installation. The use of pre-certified HEPA filters can be an advantage. The service life of the exhaust HEPA filters can be extended through adequate prefiltration of the supply air.

16. The Biosafety Level 4 facility design and operational procedures must be documented. The facility must be tested for verification that the design and operational parameters have been met prior to operation. Facilities should be re-verified annually against these procedures as modified by operational experience.

17. Appropriate communication systems are provided between the laboratory and the outside (e.g., voice, fax, computer).

(B) Suit Laboratory

1. The Biosafety Level 4 facility consists of either a separate building or a clearly demarcated and isolated zone within a building. The rooms in the facility are arranged to ensure passage through the changing and decontamination areas prior to entering the room(s) where work is done with BSL-4 agents (suit area). Outer and inner change rooms separated by a shower are provided for personnel entering and leaving the suit area. A specially designed suit area is maintained in the facility to provide personnel protection equivalent to that provided by Class III biological safety cabinets. Personnel who enter this area wear a one-piece positive pressure suit that is ventilated by a life-support system protected by HEPA filtration. The life support system includes redundant breathing air compressors, 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. An automatically starting emergency power source is provided at a minimum for the exhaust system, life support systems, alarms, lighting, entry and exit controls, and BSCs.. The air pressure within the suit is positive to the surrounding laboratory. The air pressure within the suit area is lower than that of any adjacent area. Emergency lighting and communication systems are provided. All penetrations into the internal shell of the suit area, chemical shower, and airlocks, are sealed.

2. A daily inspection of all containment parameters (e.g., directional airflow, chemical showers) and life support systems is completed before laboratory work is initiated to ensure that the laboratory is operating according to its operating parameters.

3. A double-doored autoclave is provided at the containment barrier for decontaminating waste materials to be removed from the suit area. The autoclave door, which opens to the area external to the suit area, is sealed to the outer wall of the suit area and is automatically controlled so that the outside door can be opened only after the autoclave "sterilization" cycle. A dunk tank, fumigation chamber, or ventilated airlock for decontamination is provided for passage of materials, supplies, or equipment that are not brought into the suit area through the change room. These devices can be also used for the safe removal of materials, supplies, or equipment from the laboratory that cannot be decontaminated in the autoclave.

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

5. Internal facility appurtenances in the suit area, such as light fixtures, air ducts, and utility pipes, are arranged to minimize the horizontal surface area.

6. Bench tops have seamless surfaces which are impervious to water and are resistant to moderate heat and the organic solvents, acids, alkalis, and chemicals used to decontaminate the work surfaces and equipment.

7. Laboratory furniture is of simple open construction capable of supporting anticipated loading and uses. Non-porous materials are preferable. Spaces between benches, cabinets, and equipment are accessible for cleaning and decontamination. Chairs and other furniture used in laboratory work should be covered with a non-fabric material that can be easily decontaminated.

8. A hands-free or automatically operated handwashing sink is provided in the suit area(s); handwashing sinks in the outer and inner change rooms should be considered based on the risk assessment.

9. If there is a central vacuum system, it does not serve areas outside the suit area. 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 suit area are protected by devices that prevent backflow.

10. Access doors to the laboratory are self-closing and lockable. Inner and outer doors to the chemical shower and inner and outer doors to airlocks are interlocked to prevent both doors from being opened simultaneously.

11. Any windows are breakage-resistant and are sealed.

12. Liquid effluents from sinks, floor drains (if used), autoclave chambers and other sources within the containment barrier are decontaminated by a proven method, preferably heat treatment, before being discharged to the sanitary sewer. Effluents from showers and toilets may be discharged to the sanitary sewer without treatment. The process used for decontamination of liquid wastes must be validated physically and biologically.

13. A dedicated non-recirculating ventilation system is provided. The supply and exhaust components of the system are balanced to ensure directional airflow from the area of least hazard to the area(s) of greatest potential hazard. Redundant supply fans are recommended. Redundant exhaust fans are required. The differential pressure/directional airflow between adjacent areas is monitored and alarmed to indicate malfunction of the system. An appropriate visual pressure monitoring device that indicates and confirms the pressure differential of the suit area must be provided and located at the entry to the clean change room. The airflow in the supply and exhaust components is monitored and an HVAC control system is installed to prevent positive pressurization of the laboratory.

14. The supply air to the suit area, decontamination shower, and decontamination airlock is protected by passage through a HEPA filter. The general room exhaust air from the suit area, decontamination shower and decontamination airlock is treated by a passage through two HEPA filters in series prior to discharge to the outside. The air is discharged away from occupied spaces and air intakes. The HEPA filters are located as near as practicable to the source in order to minimize the length of potentially contaminated ductwork. All HEPA filters need to be tested and certified annually. The HEPA filter housings are designed to allow for in situ decontamination of the filter prior to removal. Alternatively, the filter can be removed in a sealed, gas-tight primary container for subsequent decontamination and/or destruction by incineration. The design of the HEPA filter housing should facilitate validation of the filter installation. The use of pre-certified HEPA filters can be an advantage. The service life of the exhaust HEPA filters can be extended through adequate prefiltration of the supply air.

15. The positioning of the supply and exhaust points should be such that dead air space in the suit room is minimized.

16. The treated exhaust air from Class II biological safety cabinets, located in a facility where workers wear a positive pressure suit, may be discharged into the room environment or to the outside through the facility air exhaust system. If the treated exhaust is discharged to the outside through the facility exhaust system, it is connected to this system in a manner that avoids any interference with the air balance of the cabinets or the facility exhaust system.

17. The Biosafety Level 4 facility design and operational procedures must be documented. The facility must be tested for verification that the design and operational parameters have been met prior to operation. Facilities should be re-verified annually against these procedures as modified by operational experience.

18. Appropriate communication systems should be provided between the laboratory and the outside.