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Appendix B 1988 Agent Summary Statement for HIVs, Including HTEV-IU, LAV, HIV-l, and HIV-2 CONTENTS Introduction HIV Agent Summary Statement: Agent: HIVs Including HTLV-III, LAV, H]V-1, and H]V-2 142 Laboratory Hazards 143 Recommended Precautions .. 143 ........... 142 Addendum 1 . e ee e e e e e e e e e e e e e e e e ee e e e e e 145 Laboratory Biosafety Level Criteria . . . 145 Biosafety Level 2 . . Biosafety Level 3 . . Vertebrate Animal Biosafety LevelCriterm Animal Biosafen,~ Level 2 Animal Biosafen,r Level 3 Addendum 2 ..... References ... 145 ... 147 .. 149 . 149 .. 150 ............. 152 ... 152 Reprinted from Morbidity and Mortality Weeldy Report, 1988; 37(no. S4):1-17. The information and r=endations contained in this appendix were developed and compiled by (1) the Division of Safety, National Institute of Allergy and Infectious Diseases, the National Cancer Institute, and the Clinical Center of the National Institutes of Health; (2) the Food and Drug Administration; and (3) the following units of the Centers for Disease Control: AIDS Program, Hospital Infections Program, and Office of the Director, Center for Infectious Diseases; the Training and Laboratory Program Office; and the Office of Biosafety, Office of the Centers Director. Representatives of the following organizations also collaborated in the effort: American Academy of Microbiology, American Biological Safety Association, American Society for Microbiology, American Society for Clinical Pathology, Association of State and Territorial Public Health Laboratory Directors, College of American Pathologists, Pharmaceutical Manufacturers Association, and Walter Reed Army Institute for Research. 141

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142 1988 AGENT SUMMARY STATEMENT FOR HIVs, INCLUDING HTLV-m, LAY, HIV-1, AND HIV-2 INTRODUCTION In l9X4, the Centers for Disease Control (CDC) and the National Institutes of Health ~H), in con- sultation with experts from academic institutions, industry, and government, published the book Bio- safety in Microbiological and Biomedical Laborato- ries ("Guidelines"~* (1~. These Guidelines are based on combinations of standard and special practices, equipment, and facili- ties recommended for use in working with infectious agents in various laboratory settings. The recommen- dations are advisory; they provide a general code for operating microbiologic and biomedical laboratories. One section of the Guidelines is devoted to stan- dard and special microbiologic practices, safety equip- ment, and facilities for biosafety levels (BSL) 1 through 4. Another section contains specific agent summary statements, each consisting of a brief de- scription of laboratory-associated infections, the na- ture of laboratory hazards, and recommended pre- cautions for working with the causative agent. The authors realized that the discovery of the availability of information about these agents would necessitate updating the agent summary. Such a statement for human immunodeficiency virus ~V) (called HTLV- III~AV when the Guidelines were published) was published in Morbidity and Mortality Weekly Report (MMW R) in 1986 (2~. The HIV agent summary state- ment printed in this Supplement updates the 1986 statement. Attached to He updated HIV agent summary state- ment are the essential elements for BSL 2 and 3 laboratories, reproduced from the Guidelines (1) (see Addendum 1, p. 145~. BSL 2 and 3 laboratory de- scriptions are included because they are recommended *Available from: Superintendent of Documents U.S. Govenunent Printing Office Washington, DC 20402 Stock ft01702300167-1 APPENDIX B for laboratory personnel working with HIV, depend- ing on the concentration or quantity of virus or the type of laboratory procedures used. Ibe HIV agent summary statement does not specifically address safety measures for collecting and handling clinical specimens. Nonetheless, it has been recommended Hat blood and body-fluid pre- cautions consistently be used for ALL specimens from ALL patients. This approach, referred to as "universal blood and body-fluid precautions" or "universal precautions," eliminates the need to iden- tify all patients infected with HIV (or other blood- borne pathogens) (3~. This subject is also covered in other publications (3-~. Laboratory directors, supervisors, and others are asked to attach a copy of this revised "1988 Agent Summary Statement for HIVs" to each copy of the Guidelines and to all copies of their laboratory bio- safety manual; they should review the recommended precautions with laboratory personnel, provide ap- propriate training in practices and operation of facili- ties, and ensure that all personnel demonstrate profi- ciency BEFORE being allowed to work with HIV. The laboratory director (or the designated laboratory supervisor) is responsible for biosafety in the labora- tory and must establish and implement practices, facilities, equipment, training, and work assignments as appropriate (9~. HIV AGENT SUMMARY STATEMENT AGENT: HIVS INCLUDING HTLV-m, LAY, HIV-1, AND HIV-2 In the period 1984-1986, several health-care workers (HCWs) who had no recognized risk behav- ior for acquired immunodeficiency syndrome (AIDS) were reported to have HIV infection (10-15~. Only one of these HCWs was identified as a laboratory worker. These and other reports assessed the risk of work-related HIV infection for all HCWs as being very low (3,6,10-12,14-18~. National Technical Infonnation SeIvice U.S. Department of Commerce 5282 Port Royal Road Springfield, VA 22161 Stock #PB84-206879

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APPENDIX B In 1985, anecdotal reports were received indi- cating that workers in two different HIV-reagent- production laboratories had been exposed to droplets and splashed liquid from a vessel containing concen- trated virus. One of several workers had been cut by glass from a broken carboy that contained HIV-in- fected cells and medium. None of the persons ex- posed in these episodes had developed antibody to HIV or had clinical signs of infection 18 and 20 months, respectively, after the reported exposure. In 1987, CDC received reports that three HCWs had HIV infection; none of the infections were asso- ciated with needlesticks or cuts. Two of these HCWs were clinical laboratory workers (11~. One was a phlebotomist whose face and mouth were splattered with a patient's blood when the rubber stopper was suddenly expelled from a blood-collection tube. The second was a medical technologist who inadvertently spilled blood on her arms and forearms while using an apheresis apparatus to process blood from an HIV - seroposltlve patient. In September 1987, a production-laboratory worker was reported to have HIV infection (18~. This person worked with large concentrations of HIV in a BSL 3 facility. HIV was isolated from the worker's blood; the isolate was genetically indistin guishable from the strain of virus being cultivated in the laboratory. No risk factors were identified, and the worker recalled no specific incident that might have led to infection. However, there were instances of leakage of virus-positive culture fluid from equip ment and contamination of the work area and centri fuge rotors. The report concluded that the most plau sible source of exposure was contact of the worker's gloved hand with virus-culture supernatant, followed by inapparent exposure to skin. In October 1987, a second person who worked in another HIV production facility was reported to have HIV infection (18~. This laboratory was a well equipped BSL 3 facility, and BSL 3 practices were being followed. This worker reported having sus tained a puncture wound to a finger while cleaning equipment used to concentrate HIV. LABORATORY HAZARDS . 143 mentally infected nonhuman primates. In the labora- tory, virus should be presumed to be present in all HIV cultures, in all materials derived from HIV cul- tures, and in/on all equipment and devices coming into direct contact with any of these materials. In the laboratory, the skin (especially when scratches, cuts, abrasions, dermatitis, or other lesions are present) and mucous membranes of the eye, nose, mouth, and possibly the respiratory tract should be considered as potential pathways for entry of virus. Needles, sharp instruments, broken glass, and other sharp objects must be carefully handled and properly discarded. Care must be taken to avoid spilling and splashing infected cell-culture liquid and other virus- containing materials. RECOMMENDED PRECAUTIONS 1. BSL 2 standards and special practices, con- tainment equipment, and facilities, as described in the CDC-NIH publication Bioso~fety in Microbiologi- cal and Biomedical Laboratories (`'Guidelines"), are recommended for activities involving all clinical _ specimens, body fluids, and tissues from humans or from infected or inoculated laboratory animals. These are the same standards and practices recommended for handling all clinical specimens. For example, and for emphasis: a. Use of syringes, needles, and other sharp instruments should be avoided if possible. Used needles and disposable cutting instruments should be discarded into a puncture-resistant container with a lid. Needles should not be re-sheathed, bent, broken, removed from disposable syringes, or otherwise manipulated by hand. b. Protective gloves should be worn by all personnel engaged in activities that may involve direct contact of skin with potentially infectious specimens, cultures, or tissues. Gloves should be carefully removed and changed when they are visibly contaminated. Personnel who have dermatitis or other lesions on the hands and who may have indirect contact with potentially infec- tious material should also wear protective gloves. Hand washing with soap and water immediately after infectious materials are handled and after work is completed-EVEN WHEN GLOVES HAVE BEEN WORN as described above- should be a routine practice. Human immunodeficiency virus has been iso- lated from blood, semen, saliva, tears, urine, cerebro- spinal fluid, amniotic fluid, breast milk, cervical secretions, and tissue of infected persons and experi

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144 c. Generation of aerosols, droplets, splashes, and spills should be avoided. A bio logical safety cabinet should be used for all pro cedures that might generate aerosols or droplets and for all infected cell-culture manipulations. The Guidelines (pp. 11-13) contain additional precautions for operating at BSL 2. 2. Activities such as producing research-labo- ratory-scale amounts of HIV, manipulating concen- trated virus preparations, and conducting procedures that may produce aerosols or droplets should be per- formed in a BSL 2 facility with the additional prac- tices and containment equipment recommended for BSL 3 (19) (Guidelines, pp. 14-17~. 3. Activities involving industrial-scale, large- volume production or high concentration and ma- nipulation of concentrated HIV should be conducted in a BSL 3 facility using BSL 3 practices and equip- ment (19~. 4. BSL 2 practices, containment equipment, and facilities for animals are recommended for activities involving nonhuman primates and any animals ex- perimentally infected or inoculated with HIV. Be- cause laboratory animals may bite, throw feces or urine, or expectorate at humans, animal-care person- nel, investigators, technical staff, and other persons who enter the animal rooms should wear coats, pro- tective gloves, coveralls or uniforms, and-as appro- priate-face shields or surgical masks and eye shields to protect the skin and mucous membranes of the eyes, nose, and mouth. 5. All laboratory glassware, disposable mate- rial, and waste material suspected or known to con- tain HIV should be decontaminated, preferably in an autoclave, before it is washed, discarded, etc. An alternate method of disposing of solid wastes is in- cineration. 6. Laboratory workers should wear laboratory coats, gowns, or uniforms when working with HIV or with material known or suspected to contain HIV. There is no evidence that laboratory clothing poses a risk for HIV transmission; however, clothing that becomes contaminated with HIV preparations should be decontaminated before being laundered or dis- carded. Laboratory personnel must remove labora- tory clothing before going to nonlaboratory areas. 7. Work surfaces should be decontaminated with an appropriate chemical germicide after proce- dures are completed, when surfaces are overdy con APPENDIX B laminated, and at the end of each work day. Many commercially available chemical disinfectants (5,20- 23) can be used for decontaminating laboratory work surfaces, for some laboratory instruments, for spot cleaning of contaminated laboratory clothing, and for spills of infectious materials. Prompt decontami- nation of spills should be standard practice. 8. Universal precautions are recommended for handling all human blood specimens for hematol- ogic, microbiologic, chemical, and serologic testing; these are the same precautions for preventing trans- mission of all bloodborne infections, including hepa- titis B (17,21,24,25~. It is not certain how effective 56C-60C heat is in destroying HIV in serum (22,23,26), but heating small volumes of serum for 30 minutes at 56C before serologic testing reduces residual infectivity to below detectable levels. Such treatment causes some false-positi~re results in HIV enzyme immunoassays (27-30) and may also affect some biochemical assays performed on serum (27,31,32~. 9. Human serum from any source that is used as a control or reagent in a test procedure should be handled at BSL 2 (Guidelines, pp. 11-13~. Adden- dum 2 (p. 152) to this report is a statement issued by CDC on the use of all human control and reagent serum specimens shipped to other laboratories. The Food and Drug Administration requires that manu- facturers of human serum reagents use a similarly worded statement. 10. Medical surveillance programs should be in place in all laboratories that test specimens, do re- search, or produce reagents involving HIV. The na- ture and scope of a surveillance program will vary according to institutional policy and applicable local, state, and Federal regulations (9~. 11. If a laboratory worker has a parenteral or mucous-membrane exposure to blood, body fluid, or viral-culture material, the source material should be identified and, if possible, tested for the presence of virus. If the source material is positive for HIV anti- body, virus, or antigen, or is not available for exami- nation, the worker should be counseled regarding the risk of infection and should be evaluated clinically and serologically for evidence of HIV infection. The worker should be advised to report on and to seek medical evaluation of any acute febrile illness that occurs within 12 weeks after the exposure (3~. Such an illness-particularly one characterized by fever, rash, or lymphadenopathy~nay indicate recent HIV

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APPENDIX B infection. If seronegative, the worker should be re- tested 6 weeks after the exposure and periodically thereafter (e.g., at 12 weeks and 6 months after expo- sure). During this follow-up periokespecially dur- ing the fast 6-12 weeks after exposure, when most infected persons are expected to show serologic evi- dence of infection-exposed workers should be coun- seled to follow Public Health Service recommenda- tions for preventing transmission of HIV (3,14,25,33~. It is recommended that all institutions establish writ- ten policies regarding the management of laboratory exposure to HIV; such policies should deal with con- O~dentiality, counseling, and other related issues. 12. Other primary and opportunistic pathogenic agents may be present in the body fluids and tissues of persons infected with HIV. Laboratory workers should follow accepted biosafety practices to ensure maximum protection against inadvertent laboratory exposure to agents that may also be present in clini- cal specimens (34-36~. 13. Unless otherwise dictated by institutional policy, the laboratory director (or designated labora- tory supervisor) is responsible for carrying out the biosafety program in the laboratory. In this regard, the laboratory director or designated supervisor should establish the biosafety level for each component of the work to be done and should ensure that facilities and equipment are adequate and in good working order, that appropriate initial and periodic training is provided to the laboratory staff, and that recom- mended practices and procedures are strictly followed (9~. 14. Attention is directed to a "Joint Advisory Notice" of the Departments of Labor and Health and Human Services (9) that describes the responsibility of employers to provide "safe and healthful working conditions" to protect employees against occupational infection with HIV. The notice defines three expo- sure categories of generic job-related tasks and describes the protective measures required for em- ployees involved in each exposure category. These measures are: administrative measures, training and education programs for employees, engineering controls, work practices, medical and health-care practices, and record-keeping. The recommendations in this report are consistent with the "Joint Advisory Notice"; managers/directors of all biomedical labora- tories are urged to read this notice. }45 ADDENDUM 1 LABORATORY BIOSAFETY LEVEL CRITERIA Biosafeb Level 2 Biosafety Level 2 is similar to Level 1 and is suitable for work involving agents that represent a moderate hazard for personnel and the environment. It differs in that a. laboratory personnel have specific training in handling pathogenic agents and are di- rected by competent scientists, b. access to the laboratory is limited when work is being conducted, and c. certain procedures in which infectious aero- sols 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 as- signed to Biosafety Level 2: A. Standard microbiologicalpractices 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 waste is decon- taminated before being disposed of. 4. Mechanical pipetting devices are used; mouth pipetting is prohibited. 5. Eating, drinking, smoking, and applying cos- metics are not permitted in the work area. Food must be stored in cabinets or refrigerators designed and used for this purpose only. Food storage cabinets or refrigerators should be located outside the work area. 6. Persons are to wash their hands when they leave the laboratory after handling infectious mate- rial or animals. 7. All procedures are performed carefully to minimize the creation of aerosols.

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146 B. Specialpractices 1. Contaminated materials that are to be decon- taminated away from the laboratory are placed in a durable, leakproof container that 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 labo- ratory. 3. The laboratory director establishes policies or procedures whereby only persons who have been advised of the potential hazard and who meet any specific entry requirements (e.g., vaccination) enter the laboratory or animal rooms. 4. When an infectious agent teeing worked with in the laboratory requires special provisions for entry (e.g., vaccination), a hazard warning sign that incor- porates 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 labora- tory director or other responsible personas), and indi- cates the special requirements for entering the labo- ratory. 5. An insect and rodent control program is in effect. 6. Laboratory coats, gowns, smocks, or uni- forms are worn while in the laboratory. Before leav- ing the laboratory for nonlaboratory areas (e.g., cafe- teria, 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 per- formed are not permitted in the laboratory. S. Special care is taken to avoid having skin be contaminated with infectious material; gloves should be worn when handling infected animals and when skin contact with infectious material is unavoidable. 9. All waste from laboratories and animal rooms is 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 APPENDIX B needle-locking syringes or disposable syringe-needle units (i.e., the needle is integral to the syringe) are used for the injection or aspiration of infectious fluid. Extreme caution should be used when handling needles and syringes to avoid autoinoculation and the generation of aerosols during use and disposal. A needle should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe follow- ing use. The needle and syringe should be promptly placed in a puncture-resistant container and decon- taminated, preferably by autoclaving, before discard or reuse. 11. Spills and accidents that result in overt expo- sures to infectious material are immediately reported to the laboratory director. Medical evaluation, sur- veillance, and treatment are provided as appropriate, and written records are maintained. 12. When appropriate, considering the agents handled, baseline serum samples for laboratory and other at-risk personnel are collected and stored. Ad- ditional serum specimens may be collected periodi- cally, depending on the agents handled or on the function of the facility. 13. A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are re- quired to read instructions on practices and proce- dures and to follow them. C. Containment equipment Biological safety cabinets (Class I or II) or other appropriate personal-protection or physical-contain- ment devices are used when: 1. Procedures with a high potential for creating infectious aerosols are conducted. These may in- clude centrifuging, grinding, blending, vigorous shak- ing or mixing, sonic disruption, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating ani- mals intranasally, and harvesting infected tissues from animals or eggs. 2. High concentrations or large volumes of in- fectious agents are used. Some types of materials may be centrifuged in the open laboratory if sealed heads or centrifuge safety cups are used and if the containers are opened only in a biological safety cabinet.

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APPENDIX B D. Laboratoryfaciliiies 1. The laboratory is designed so that it can be easily cleaned. 2. Bench tops are impervious to water and resis- tant to acids, alkalis, organic solvents, and moderate heat. 3. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are acces- sible for cleaning. 4. Each laboratory contains a sink for hand- washing. 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, diag- nostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents that may cause serious or potentially lethal disease as a result of exposure by inhalation. Laboratory per- sonnel have specific training in handling pathogenic and/or potentially lethal agents and are supervised by competent scientists who are experienced in working with these agents. All procedures involving the ma- nipulation of infectious material are conducted within biological safety cabinets or other physical contain- ment devices or by personnel wearing appropriate personal-protection clothing and devices. The labo- ratory 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 pene- trations, and directional airflow). In these circum- stances, acceptable safety may be achieved for routine or repetitive operations (e.g., diagnostic pro- cedures involving the propagation of an agent for identification, typing, and susceptibility testing) in laboratories in which facility features satisfy Bio- safety Level 2 recommendations if the recommended "Standard Microbiological Practices," "Special Prac- tices," 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 labo- ratory director. |47 The following standard and special practices, safety equipment, and facilities apply to agents as- signed to Biosafety Level 3: A. Stand`~rd~rucrobiologicalpractices 1. Work surfaces are decontaminated at least once a day and after any spill of viable material. 2. All infectious liquid or solid waste is decon- taminated before being disposed of. 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 in- fectious materials and animals and every time they leave the laboratory. 6. All procedures are performed carefully to minimize the creation of aerosols. B. Specialpractices 1. Laboratory doors are kept closed when ex- periments are in progress. 2. Contaminated materials that are to be decon- taminated at a site away from the laboratory are placed in a durable, leakproof container that is closed before being removed from the laboratory. 3. The laboratory director controls access to the laboratory and limits access only to persons whose presence is required for program or support pur- poses. 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., vaccination), and who comply with all entry and exit procedures enter the laboratory or animal rooms. 5. When infectious materials or infected ani- mals are present in the laboratory or containment module, a hazard warning sign (incorporating the universal biohazard symbol) is posted on all labora- tory and animal-room access doors. The hazard warn

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148 ing sign identifies the agent, lists the name and tele- phone number of the laboratory director or other re- sponsible personas), and indicates any special re- quirements for entering the laboratory, such as the need for vaccinations, respirators, or other personal- protection measures. 6. All activities involving infectious materials are conducted in biological safety cabinets or other physical-containment devices within the containment module. No work is conducted in open vessels on the open bench. 7. The work surfaces of biological safety cabi- nets and other containment equipment are decon- taminated when work with infectious materials is finished. Plastic-backed paper toweling used on non- perforated work surfaces within biological safety cabinets facilitates clean-up. 8. An insect and rodent control program is in effect. 9. Laboratory clothing that protects street cloth- ing (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 contami- nation with infectious materials; gloves are worn when handling infected animals and when skin con- tact 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 waste from laboratories and animal rooms is appropriately decontaminated before being dis- posed of. 14. Vacuum lines are protected with high-effi- ciency particulate air (HEPA) filters and liquid disin- fectant 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., the needle is integral to the syringe) are used for the injection or aspiration of infectious fluids. Extreme caution is used when handling needles and syringes to avoid autoinoculation and the generation of aerosols during use and disposal. A needle 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 APPENDIX B puncture-resistant container and decontaminated, preferably by autoclaving, before being discarded or reused. 16. Spills and accidents that result in overt or potential exposures to infectious material are imme- diately reported to the laboratory director. Appropri- ate medical evaluation, surveillance, and treatment are provided, and written records are maintained. 17. Baseline serum samples for all laboratory and other at-risk personnel are collected and stored. Additional serum specimens may be collected peri- odically, 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 re- quired to read instructions on practices and proce- dures and to follow them. C. Containment equipment Biological safety cabinets (Class I, II, or III) or other appropriate combinations of personal-protec- tion or physical-containment devices (e.g., special protective clothing, masks, gloves, respirators, cen- trifuge safety cups, sealed centrifuge rotors, and con- tainment caging for animals) are used for all activi- ties with infectious materials that pose a threat of aerosol exposure. These include: manipulation of cultures and of clinical or environmental material that may be a source of infectious aerosols; the aero- sol challenge of experimental animals; harvesting of tissues or fluids from infected animals and embryon- ated eggs; and necropsy of infected animals. D. Laboratoryfacilities 1. The laboratory is separated from areas that are open to unrestricted traffic flow within the build- ing. Passage through two sets of doors is the basic re- quirement for entry into the laboratory from access corridors or other contiguous areas. Physical separa- tion 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 that requires passing 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

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APPENDIX B capable of being sealed to facilitate decontaminating the area. 3. Bench tops are impervious to water and re- sistant to acids, alkalis, organic solvents, and moder- ate heat. 4. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are acces- sible for cleaning. 5. Each laboratory contains a sink for washing hands. 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 contain- ment 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 dis- persed away from occupied areas and air intakes. Personnel must verify that the direction of the airflow is proper (i.e., into the laboratory). 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 di- rectly to the outside or through the building exhaust system. Exhaust air from Class I or II biological safety cabinets may be recirculated within the labora- tory if the cabinet is tested and certified at least every 12 months. If the HEPA-f~ltered exhaust air from Class I or II biological safety cabinets is to be dis- charged to the outside through the building exhaust system, it is connected to this system in a manner (e.g., thimble-unit connection) that avoids any inter- ference with the air balance of the cabinets or build- ing exhaust system. VERTEBRATE ANIMAL BIOSAFETY LEVEL CRITERIA 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. |49 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. Specialpractices 1. Cages are decontaminated, preferably by autoclaving, before being cleaned and washed. 2. Surgical-type masks are worn by all person- nel entering animal rooms housing nonhuman pri- mates. 3. Laboratory coats, gowns, or uniforms are worn while in the animal room. This protective cloth- ing is removed before leaving the animal facility. 4. The laboratory or animal-facility director limits access to the animal room only 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, per- sons who may be at increased risk of acquiring infec- tion or for whom infection might be unusually haz- ardous are not allowed in the animal room. 5. The laboratory or animal-facility director es- tablishes policies and procedures whereby only per- sons who have been advised of the potential hazard and who meet any specific requirements (e.g., vacci- nation) may enter the animal room. 6. When an infectious agent in use In the an~- mal room requires special-entry provisions (e.g., vac- cination), 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 personas), and indicates the spe- cial requirements for entering the animal room. 7. Special care is taken to avoid contaminating skin with infectious material; gloves should be worn when handling infected animals and when skin con- tact with infectious materials is unavoidable. 8. All waste from the animal room is appropri- ately decontaminated preferably by autoclaving

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lS0 before being disposed of. Infected animal carcasses are incinerated after being transported from the ani- mal 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 syringe-needle units (i.e., the needle is integral to the syringe) are used for the injection or aspiration of infectious fluids. A needle should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe follow- ing use. The needle and syringe should be promptly placed in a puncture-resistant container and decon- taminated, preferably by autoclaving, before being discarded or reused. 10. If floor drains are provided, the drain taps are always fUled with water or a suitable disinfec- tant. 11. When appropriate, considering the agents handled, baseline serum samples from animal-care and other at-risk personnel are collected and stored. Additional serum samples may be collected periodi- cally, depending on the agents handled or the func- tion of the facility. C. Containment equipment Biological safety cabinets, other physical-contain- ment devices, and/or personal-protection devices (e.g., respirators, face shields) are used when procedures with a high potential for creating aerosols are con- ducted. These include necropsy of infected animals, harvesting of infected tissues or fluids from animals or eggs, intranasal inoculation of animals, and ma- nipulation of high concentrations or large volumes of infectious materials. D. Animalfacilities 1. The animal facility is designed and con- structed to facilitate cleaning and housekeeping. 2. A sink for washing hands is available in the room that houses infected animals. 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 with- out being recirculated to other rooms. S. An autoclave that can be used for decon APPENDIX B laminating infectious laboratory waste is available in the same building that contains the animal facility. Animal Biosafety Level 3 A. Standard practices 1. Doors to animal 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 after 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 or animals and before leaving the labora- tory. 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 re- moved and before they are cleaned and washed. 2. Surgical-type masks or other respiratory pro- tection devices (e.g., respirators) are worn by person- nel entering rooms that house animals infected with agents assigned to Biosafety Level 3. 3. Wrap-around or solid-front gowns or uni- forms 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 limits access to the animal room only to per- sonnel 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., vac cination) may enter the animal room.

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APPENDIX B 6. Hazard warning signs (incorporating the uni- versal biohazard warning symbol) are posted on ac- cess doors to animal rooms containing animals in- fected with agents assigned to Biosafety Level 3 are present. The hazard warning sign should identify the agents in use, list the name and telephone number of the animal room supervisor or other responsible personas), and indicate any special conditions of en- tq into the animal room (e.g., the need for vaccina- tions or respirators). 7. Personnel wear gloves when handling in- fected animals. Gloves are removed aseptically and autoclaved with other animal room waste before being disposed of or reused. 8. All wastes from the animal room are auto- claved before being disposed of. All animal car- casses 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 Savage or parenteral injection or aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or disposable synnge-needle units (i.e., the needle is integral to the syringe) are used. A needle should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use. The needle and sy- ringe should be promptly placed in a puncture-resis- tant container and decontaminated, preferably by autoclaving, before being discarded or reused. When possible, cannulas should be used instead of sharp needles (e.g., for Savage). 10. If floor drains are provided, the drain traps are always filled with water or a suitable disinfec- tant. 11. If vacuum lines are provided, they are pro- tected with HEPA filters and liquid disinfectant traps. 12. Boots, shoe covers, or other protective foot- wear and disinfectant footbaths are available and used when indicated. C. Containment equipment 1. Personal-protection clothing and equipment andfor 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, IS! 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. Animalfacilities 1. The animal facility is designed and con- smucted to facilitate cleaning and housekeeping and is separated from areas that 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 from other activities may also be provided by a double-doored clothes change room (showers may be included), airlock, or other access facility that 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 can be cleaned easily. Penetrations in these surfaces are sealed or capable of being sealed to facilitate fumigation or space decontamination. 3. A foot-, elbow-, or automatically operated sink for handwashing is provided near each animal- room exit door. sealed. 4. Windows in the animal room are closed and 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. Mate- rials 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 dis- persed away from occupied areas and air intakes. Personnel must verify that the direction of the air- flow is proper (i.e., into the animal room). The ex- haust air from the animal room that does not pass through biological safety cabinets or other primary containment equipment can be discharged to the out- side without being filtered or otherwise treated. 8. The HEPA-filtered exhaust air from Class I or Class II biological safety cabinets or other primary

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152 containment devices is discharged directly to the outside or through the building's 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-f~tered 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) that avoids any interference with the air balance of the cabinets or building exhaust sys tem. ADDENDUM 2: CDC CAUTIONARY NOTICE CDC cautionary notice for all human-serum-derived reagents used as controls: WARNING: Because no test method can offer complete assurance that laboratory specimens do not contain HIV, hepatitis B virus, or other infectious agents, this speci- men should be handled at the BSL 2 as rec- ommended for any potentially infectious human serum or blood specimen in the CDC- NIH manual, Biosafety in Microbiological and Biomedical Laboratories, 1984, pages 11-13. If additional statements describing the results of any heat treatment or serologic procedures already performed on the human-serum reagent or control are used in conjunction with the above cautionary notice, these statements should be worded so as not to diminish the impact of the warning that empha- sizes the need for universal precautions. REFERENCES 1. Richardson JH, Barkley WE, eds. Biosafety in microbiological and biomedical laboratories, 1984. Washington, DC: Public Health Service, 1984; DHHS publication no. (CDC)84-8395. 2. CDC. Human T-lymphotropic virus type IIV lymphadenopathy-assoc iated vines agent sum APPENDIX B mary statement. MMWR 1986;35:540-2, 547- 9. CDC. Recommendations for prevention of HIV transmission in health-care settings. WAR 1987;36(suppl 2~:3S-18S. Isenberg HD, Washington JA, Balows A, Son- nenwirth AC. Collection, handling and proc- essing of specimens. In: Lennette EH, Balows A, Hausler WJ, Shadomy HI, eds. Manual of clinical microbiology, 4th ed. Washington, DC: American Society for Microbiology, 1985:73- 98. 5. CDC. Acquired immune deficiency syndrome (AIDS): precautions for clinical and laboratory workers. WAR 1982;32:577-80. 6. CDC. Recommendations for preventing trans- mission of infection with human T-lym- photropic virus type III/lymphadenopathy- associated Virus in the workplace. MMWR 1985;34:681. 7. CDC. Recommendations for preventing trans- mission of infection with human T-lym- photropic virus type III/lymphadenopathy- associated virus during invasive procedures. MMWR 1986;35:221-3. 8. CDC. Recommendations for preventing trans- mission of infection with human T-lym- photropic virus type IIVlymphadenopathy- associated virus in the workplace. MMWR 1985;34:682-6, 691-5. 9. U.S. Department of Health and Human Serv- ices, Public Health Service. Joint advisory no- tice: BY/HIV. Federal Register 1987;52 (October 30~: 41818-24. 10. CDC. Update: evaluation of human T-lym- photropic virus type III/lymphadenopathy- associated vims infection in health-care person- nel United States. MMW R 1985;34:575-8. 11. CDC. Human immunodeD~ciency virus infec- tions in health-care workers exposed to blood of infected patients. MMWR 1987;36:285-9.

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APPENDIX B 12. Anonymous. Needlestick transmission of HTLV-III from a patient infected in Africa. Lancet 1984;2:1376-7. 13. Stricof RL, Morse DL. HTLV-III/LAV sero- conversion following a deep intramuscular needlestick injury. New Engl J Med 1986; 314:1115. 14. McCray E, Cooperative Needlestick Study Group. Occupational risk of the acquired im- munodeficiency syndrome among health-care workers. New Engl J Med 1986;314:1127-32. 15. Weiss SH, Saxinger WC, Richtman D, et al. HTLV-III infection among health-care work- ers: association with needlestick injuries. JAMA 1985;254:2089-93. 16. Henderson DK, Saab AT, Zak BJ, et al. Risk of nosocomial infection with human T-cell lym- photropic virus type III/lymphadenopathy-as- sociated virus in a large cohort of intensively exposed health care workers. Ann Intern Med 1986; 104:644-7. 17. Gerberding JL, Bryant-Le Blanc CE, Nelson K, et al. Risk of transmitting the human immu- nodef~ciency virus, cytomegalovirus, and hepa- titis B virus to health-care workers exposed to patients with AIDS and AIDS-related condi- tions. J Infect Dis 1987;156:1-8. 18. Weiss SH, Goedert JJ, Gartner S. et al. Risk of human immunodeficiency virus (HIV-1) infec- tion among laboratory workers. Science 1988;239:68-71. 19. U.S. Department of Health and Human Serv- ices, Public Health Service. Biosafety guide- lines for use of HTLV-III and related viruses. Federal Register 1984 (October 16~;49:40556. U.S. Environmental Protection Agency. EPA guide for infectious waste management. Wash- ington, DC: U.S. Environmental Protection Agency, 1986; Publication no. EPA/530-SW- 86-014. 21. Favero MS. Sterilization, disinfection and anti- sepsis in the hospital. In: Lennette EH, Balows A, Hausler WJ, Shadomy HJ, eds. Manual of clinical microbiology, 4th ed. Washington, DC: American Society for Microbiology, 1985: 129- 37. 22. Martin LS, McDougal JS, Loskoski SL. Disin- fection and inactivation of the human T lymphotropic virus ~e III/lymphadenopathy- associated virus. J Infect Dis 1985;152:400-3. Resnick L, Veren K, Salahuddin, SZ, Tondreau S. Markham PD. Stability and inactivation of HTLV-IIVLAV under clinical and laboratory environments. JAMA 1986;255: 1887-91. 24. Favero MS, Petersen NJ, Bond WW. Trans- mission and control of laboratory-acquired hepatitis infection. In: Miller BM, Groschel DHM, Richardson JH, et al., eds. Laboratory safety: principl~s and practice. Washington, DC: American Society for Microbiology, 1986:49- 58. CDC. Public Health Service guidelines for counseling and antibody testing to prevent HIV infections and AIDS. MMWR 1987;36:509-15. 26. Ronalds CJ, Grint PCA, Kangro HD. Disinfec- tion and inactivation of HTLV-III/LAV (Let- ter). J Infect Dis 1986;153:996. 27. Evans RP, Shanson DC. Effect of heat on sero- logic tests for hepatitis B and syphilis and on aminoglycoside assays (Letter). Lancet 1985;1:1458. 28. Van den Akker R. Held~er AC, Osterhaus ADME. Heat inactivation of serum may inter- fere with HTLV-III/LAV serology (Letter). Lancet 1985;2:672. 29. Mortimer PP, Parry JV, Mortimer JY. Which anti-HTLV III/LAY assays for screening and conElrmatory testing? Lancet 1985;2:873-7.

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154 30. Jungkind DL, DiRenzo SA, Young SJ. Effect of using heat-inactivated serum with the Ab bott human T-cell lymphotropic vines type III antibody test. J Clin Microbiol 1986;23:381-2. 34. 31. Goldie DJ, McConnell AA, Cooke PR. Heat treatment of whole blood and serum before chemical analysis (Letter3. Lancet 1985; 1: 1 161 . 32. Lai L, Ball G. Stevens J. Shanson D. Effect of heat treatment of plasma and send on bio- chemical indices (Letter). Lancet 1985;1:1457- 8. 33. CDC. Additional recommendations to reduce sexual and drug abuse-related transmission of human T-lymphotropic virus type III/lymphad APPENDIX B enopathy-associated virus. MMW R 1986; 35:152-5. CDC. Revision of the case definition of ac- quired immunode0~ciency syndrome for national reporting-United States. MMWR 1985; 34:373-5. 35. CDC. Diagnosis and management of myco- bacterial infection and disease in persons with human T-lymphotropic virus type III/lymphad- enopathy-associated virus infection. MMWR 1986;35:448-52. 36. CDC. Revision of the CDC surveillance case definition for acquired immunodeficiency syn- drome. MMWR 1987;36(suppl D: lS-lSS.