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Appendix A Biosafety in Microbiological and Biomedical Laboratories C O N T E N TS Section I. Introduction 85 Section II. Principles of Biosafety 86 Laboratory practice and technique 86 Safety equipment (primary barriers). 86 Facility design (secondary barriers) 87 Biosafety levels Animal Biosafety levels Importation and interstate shipment of certain 87 88 biomedical materials 89 Section III. Laboratory Biosafety Level Criteria ................ Biosafety Level 1............... Biosafety Level 2....................... Biosafety Level 3............ Biosafety Level 4............. . . 89 ... . . ... 89 90 .92 94 - Section IV. Vertebrate Animal Biosafety Level Criteria 98 Animal Biosafety Level 1 98 Animal Biosafety Level 2 99 Animal Biosafety Level 3 . Animal Biosafety Level 4 . . .............. 100 ..102 Section V. Recommended Biosafety Levels for Infectious Agents and Infected Animab 104 Riskassessment 106 · .... · ... ·· . ·· ... ···· ~ Agent Summary Statements Parasitic Agents......... Nematode parasites of humans . Protozoal parasites of humans . . Tremamde parasites of humans . Cestode parasites of humans . . . Fungal Agents ........................ Blastomyces dermatitidis ...... Coccidioides immitis ......... ..107 ..107 ..107 ..107 ..107 ..108 .108 ..108 ..108 .109 .109 109 Cryptococcus neoformans Histoplasma capsulatum Sporothrix schenc~i Pathogenic members of the genera Epidernmphyton, Microsporum, and Trichophyton 109 .110 110 Bacmnal Agents ...................... Bacillus anthracis .... Brucella (B. abortus, B. cants, B. rnelitensis, B. suds) .............. Chlamydia psittaci, C. trachomatis ..... Clostridium botulinum ............... Clostridium tetani................... Corynebacterium diphtheriae.......... Francisella tularensis................ Leptospira interrogans-fill strops .... Legionella pneumophila; other Legionella-like agents ............. Mycobacterium leprae ............... 0 0 111 111 2 2 2 3 3 Repnnted from J.H. Richardson and W.E. Baricley, Biosafety in Microbiological and Biomedical Laboratories, 1st ea., U.S. Public Health Service, HHS Publication No. (CDC) 84-8395, U.S. Govemment Printing Office, Washington, D.C., March 1984. Figures, tables, appendixes, and page numbers in this repented version have been renumbered to avoid confusion. 83
84 Mycobacterium spp. other than M. tuberculosis, M. bovis, or M. Ieprae . . 113 Mycobacterium tuberculosis, M. bovis 114 Neisseria gonorrhoeae ................ Neisseria meningitidis ................. Pseudomonas pseudomallei ............ Salmonella cholera-suds, S. enteritidis" aDserotypes 115 Salmonella typhi ~ 115 Shigellaspp 116 Treponemapallidum 116 Vibrionic enteritis (Campylobacterfetus subsp. jejuni, Vibrio cholerae, V. parahaemolyticus) .114 .115 .115 . . .. .. . . .. . Yersinia pestis ........................ Rickettsial Agents . Coxiella burnetii 116 117 117 ...................... 117 Rickettsia akari, Rochalimaea quintana, end Rochalimaeavinsonii 117 Rickettsia prowaze~i, Rickettsia typhi (R. mooseri), Rickettsia tsutsugamushi, Rickettsia canada, and Spoued Fever Group agents of human disease other than Rickettsia rickettsii and Rickettsia akari..................... Rickettsm rickettsii .................... Viral Agents ......... Hepatitis A virus ...................... Hepatitis B9 hepatitis non-A non-B Herpesvirus sirruae (B-virus) .... Herpesviruses ................ Influenza virus............. Lymphocytic choriomeningitis ~CM) virus.................. Poliovirus e~ ee Poxviruses e-----~e~ APPENDIX A Rabies virus . . . . . . . . . . . . . . . . . eeeeee122 Transmissible spongiform encephalopathies (Creutzfeldt-Jakob and kuru agen~. . . . . . . . . . . . ee..ee122 Vesicular stomatitis virus (VSV) e e 123 Arbonruses ~ eee123 Arboviruses Assigned to Biosafe~r Level 2~ eeeeel23 Arboviruses and Arenaviruses Assigned to Bio~ety Level 3e~ A1bov~u~e,AUenavOu~es, or Filovinlses Assigned to Biosafety Level 4 . Appendix A.1. Biological Safety .......... 126 128 Cabinets ~ eeeeeee129 Appendix A.2. Immunoprophylaxis eee~131 AppendLx A.3. Surveillance of Personnel for Laboratory-Associated RickettsialInfections 131 . 118 Appendix A.4. Importation andInterstate . 118 Shipment of Human Pathogens and 119 .119 119 119 .. 120 120 Related Materials 132 Appendix A.5. Restricted Animal Pathogens 132 Appendix A.6. Resources for Information 133 . 121 121 Literature Cited 121
APPENDIX A SECTIONI. INTRODUCTION Microbiology laboratories are special, often unique, work environments that may pose special infectious disease risks to persons in or near them. Personnel have contracted infections in the labora- tory throughout the history of microbiology. Pub- lished reports around the turn of the century described laboratory-associated cases of typhoid, cholera, "landers, brucellosis, and tetanus (123~. In 1941, Meyer and Eddie (75) published a survey of 74 laboratory-associated brucellosis infections that had occurred in the United States and concluded that the "handling of cultures or specimens or the inhalation of dust containing Brucella organisms is eminently dangerous to laboratory workers." A number of cases were attributed to carelessness or poor technique in the handling of infectious materials. In 1949, Sulkin and Pike (113) published the first in a series of surveys of laboratory-associated infections summarizing 222 viral infections 21 of which were fatal. In at least a third of the cases the probable source of infection was considered to be associated with the handling of infected animals and tissues. Known accidents were recorded in 27 (12%) of the reported cases. In 1951, Sulkin and Pike (114) published the second of a series of summaries of laboratory-associ- ated infections based on a questionnaire sent to 5,000 laboratories. Only one-third of the 1,342 cases cited had been reported in the literature. Brucellosis out- numbered all other reported laboratory-acquired in- fections and together with tuberculosis, tularemia, typhoid, and streptococcal infection accounted for 72% of all bacteria infections and for 31% of infec- tions caused by all agents. The overall case fatality rate was 3%. Only 16% of all infections reported were associated with a documented accident. The majority of these were related to mouth pipetting and the use of needle and syringe. This survey was updated in 1965 (93), adding 641 new or previously unreported cases, and again in 1976 (903, summarizing a cumulative total of 3,921 cases. Brucellosis, typhoid, tularemia, tuberculosis, hepatitis, and Venezuelan equine encephalitis were the most commonly reported. Fewer than 20% of all cases were associated with a known accident. Expo- sure to infectious aerosols was considered to be a plausible but unconfirmed source of infection for the 85 more than 80% of the reported cases in which the infected person had "worked with the agent." In 1967, Hanson et al. (53) reported 428 overt laboratory-associated infections with arboviruses. In some instances the ability of a given arbovirus to produce human disease was first confirmed as the result of unintentional infection of laboratory per- sonnel. Exposure to infectious aerosols was consid- ered the most common source of infection. In 1974, Skinhoj (104) published the results of a survey which showed that personnel in Danish clini- cal chemistry laboratories had a reported incidence of hepatitis (2.3 cases per year per 1,000 employees) seven times higher than that of the general popula- tion. Similarly, a 1976 survey by Hamngton and Shannon (55) indicated that medical laboratory work- ers in England had "a five times increased risk of acquiring tuberculosis compared with the general population." Hepatitis and shigellosis were also shown to be continuing occupational risks and together with tuberculosis were the three most commonly reported occupation-associated infections in Britain. Although these reports suggest that laboratory personnel are at increased risk of being infected by the agents they handle, actual rates of infection are typically not available. However, the studies of Har- rington and Shannon (55) and of Skinhoj (104) indi- cate that laboratory personnel have higher rates of tu- berculosis, shigellosis, and hepatitis than the general population. In contrast to the documented occurrence of labo- ratory-acquired infections in laboratory personnel, laboratories working with infectious agents have not been shown to represent a threat to the community. For example, although 109 laboratory-associated in- fections were recorded at the Center for Disease Control in 1947-1973 (97), no secondary cases were reported in family members or community contacts. The National Animal Disease Center has reported a similar experience (115), with no secondary cases occurring in laboratory and nonlaboratory contacts of 18 laboratory-associated cases occurring in 1960- 1975. A secondary case of Marburg disease in the wife of a primary case was presumed to have been transmitted sexually two months after his dismissal from the hospital (70~. Three secondary cases of smallpox were reported in two laboratoqy-associated outbreaks in England in 1973 (96) and 1978 (1303. There were earlier reports of six cases of Q fever in
86 employees of a commercial laundry which handled linens and uniforms from a laboratory where work with the agent was conducted (84), and two cases of Q fever in household contacts of a rickettsiologist (5~. These cases are representative of the sporadic nature and infrequent association of community in- fections with laboratories working with infectious agents. In his 1979 review (92), Pike concluded, "the knowledge, the techniques, and the equipment to prevent most laboratory infections are available." No single code of practice, standards, guidelines, or other publication, however, provides detailed descriptions of techniques, equipment, and other considerations or recommendations for the broad scope of labora- tory activities conducted in the United States with a variety of indigenous and exotic infectious agents. The booklet Classification of Etiologic Agents on the Basis of Hazard (15) has, since 1969, served as a general reference for some laboratory activities util- izing infectious agents. That booklet and the concept of categorizing infectious agents and laboratory ac- tivities into four classes or levels served as a basic format for Biosafety in Microbiological and Biomedi- cal Laboratories. This publication will provide spe- cific descriptions of combinations of microbiological practices, laboratory facilities, and safety equipment and recommendations for use in four categories or biosafety levels of laboratory operation with selected infectious agents of man. The descriptions of biosafety levels 14 parallel those of P14 in the NIH Guidelines for Research Involving Recombinant DNA Molecules (43) and are consistent with the general criteria used in assigning agents to Classes 14 in Classification of Etiologic Agents on the Basis of Hazard (15~. Four biosafety levels are also described for infectious disease activi- ties utilizing small laboratory animals. Recommen- dations for biosafety levels for specific agents are made on the basis of the potential hazard of the agent and of the laboratory function or activity. SECTION II. PRINCIPLES OF BIOSAFETY The term 4'containment" is used in describing safe methods for managing infectious agents in the laboratory environment where they are being handled or maintained. Primary containment, the protection of personnel and the immediate laboratory environ- ment from exposure to infectious agents, is provided APPENDIX A by good microbiological technique and the use of appropriate safety equipment. The use of vaccines may provide an increased level of personal protec- tion. Secondary containment, the protection of the environment external to the laboratory from expo- sure to infectious materials, is provided by a combi- nation of facility design and operational practices. The purpose of containment is to reduce exposure of laboratory workers and other persons to, and to pre- vent escape into the outside environment of, poten- tially hazardous agents. The three elements of con- tainment include laboratory practice and technique, safety equipment, and facility design. Laboratory practice and technique. The most important element of containment is strict adherence to standard microbiological practices and techniques. Persons working with infectious agents or infected materials must be aware of potential hazards and must be trained and proficient in the practices and techniques required for safely handling such mate- rial. The director or person in charge of the labora- tory is responsible for providing or arranging for appropriate training of personnel. When standard laboratory practices are not suf- f~cient to control the hazard associated with a par- ticular agent or laboratory procedure, additional measures may be needed. The laboratory director is responsible for selecting additional safety practices, which must be in keeping with the hazard associated with the agent or procedure. Each laboratory should develop or adopt a bio- safety or operations manual which identifies the haz- ards that will or may be encountered and which speci- f~es practices and procedures designed to minimize or eliminate risks. Personnel should be advised of special hazards and should be required to read and to follow the required practices and procedures. A sci- entist trained and knowledgeable in appropriate labm ratory techniques, safety procedures, and hazards associated with handling infectious agents must di- rect laboratory activities. Laboratory personnel, safety practices, and tech- niques must be supplemented by appropriate facility design and engineering features, safety equipment, and management practices. Safety equipment (primary barriers). Safety equipment includes biological safety cabinets and a variety of enclosed containers. The biological safety
APPENDIX A cabinet is the principal device used to provide con- tainment of infectious aerosols generated by many microbiological procedures. Three types of biologi- cal safety cabinets (Class I, II, III) used in microbio- logical laboratories are described and illustrated in Appendix A.1. Open-fronted Class I and Class II biological safety cabinets are partial containment cabinets which offer significant levels of protection to laboratory personnel and to the environment when used with good microbiological techniques. The gas- tight Class III biological safety cabinet provides the highest attainable level of protection to personnel and the environment. An example of an enclosed container is the safety centrifuge cup, which is designed to prevent aerosols from being released during centrifugation. Safety equipment also includes items for per- sonal protection such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, and safety glasses. These personal protective devices are often used in combination with biological safety cabinets and other devices which contain the agents, animals, or materials being worked with. In some situations in which it is impractical to work in biological safety cabinets, personal protective devices may form the primary barrier between personnel and the infectious materials. Examples of such activities include certain animal studies, animal necropsy, production activi- ties, and activities relating to maintenance, service, or support of the laboratory facility. Facility design (secondary barriers). The de- sign of the facility is important in providing a barrier to protect persons working in the facility but outside the laboratory and those in the community from in- fectious agents which may be accidentally released from the laboratory. Laboratory management is re- sponsible for providing facilities commensurate with the laboratory's function. Three facility designs are described below, in ascending order by level of con- tainment. 1. The basic laboratory. This laboratory pro- vides general space in which work is done with vi- able agents which are not associated with disease in healthy adults. Basic laboratories include those fa- cilities described in the following pages as Biosafety Levels 1 and 2 facilities. This laboratory is also appropriate for work with 87 infectious agents or potentially infectious materials when the hazard levels are low and laboratory per- sonnel can be adequately protected by standard labm ratory practice. While work is commonly conducted on the open bench, certain operations are confined to biological safety cabinets. Conventional laboratory designs are adequate. Areas known to be sources of general contamination, such as animal rooms and waste staging areas, should not be adjacent to patient care activities. Public areas and general offices to which nonlaboratory staff require frequent access should be sepal from spaces which primarily support laboratory functions. 2. The containment laboratory. This laboratory has special engineering features which make it pos- sible for laboratory workers to handle hazardous materials without endangering themselves, the com- munity, or the environment. The containment labora- tory is described in the following pages as a Bio- safety Level 3 facility. The unique features which distinguish this laboratory from the basic laboratory are the provisions for access control and a special- ized ventilation system. The containment laboratory may be an entire building or a single module or complex of modules within a building. In all cases, the laboratory is separated by a controlled access zone from areas open to the public. 3. The maximum containment laboratory. This laboratory has special engineering and containment features that allow activities involving infectious agents that are extremely hazardous to the laboratory worker or that may cause serious epidemic disease to be conducted safely. The maximum containment labs ratory is described on the following pages as a Bio- safety Level 4 facility. Although the maximum con- tainment laboratory is generally a separate building, it can be constructed as an isolated area within a building. The laboratory's distinguishing character- istic is that it has secondary barriers to prevent haz- ardous materials from escaping into the environment. Such barriers include sealed openings into the labo- ratory, airlocks or liquid disinfectant barriers, a cloth- ing-change and shower room contiguous to the labo- ratory ventilation system, and a treatment system to decontaminate exhaust air. Biosafety levels. Four Biosafety levels are de- scribed which consist of combinations of laboratory practices and techniques, safety equipment, and labm ratory facilities appropriate for the operations per
88 formed and the hazard posed by the infectious agents and for the laboratory function or activity. Biosafety Level 1. Biosafety level 1 practices, safety equipment, and facilities are appropriate for undergraduate and secondary educational training and teaching laboratories and for other facilities in which work is done with defined and characterized strains of viable microorganisms not known to cause disease in healthy adult humans. Bacillus subtilis, Naegleria gruberi, and infectious canine hepatitis virus are rep- resentative of those microorganisms meeting these criteria. Many agents not ordinarily associated with disease processes in humans are, however, opportun- istic pathogens and may cause infection in the young, the aged, and immunodef~cient or immunosuppressed individuals. Vaccine strains which have undergone multiple in viva passages should not be considered av~rulent simply because they are vaccine strains. Biosafety Level 2. Biosafety Level 2 practices, equipment, and facilities are applicable to clinical, diagnostic, teaching, and other facilities in which work is done with the broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided the potential for producing aerosols is low. Hepatitis B virus, the salmonellae, and Toxoplasma spp. are representative of microor- ganisms assigned to this containment level. Primary hazards to personnel working with these agents may include accidental autoinoculation, ingestion, and skin or mucous membrane exposure to infectious materi- als. Procedures with high aerosol potential that may increase the risk of exposure of personnel must be conducted in primary containment equipment or de- vices. Biosafety Level 3. Biosafety Level 3 practices, safety equipment, and facilities are applicable to clini- cal, diagnostic, teaching, research, or production fa- cilities in which work is done with indigenous or exotic agents where the potential for infection by aerosols is real and the disease may have serious or lethal consequences. Autoinoculation and ingestion also represent primary hazards to personnel working with these agents. Examples of such agents for which Biosafety Level 3 safeguards are generally recom- mended include Mycobacterium tuberculosis, St. Louis encephalitis virus, and Coxiella burnetii. APPENDIX A Biosafety Level 4. Biosafety Level 4 practices, safety equipment, and facilities are applicable to work with dangerous and exotic agents which pose a high individual risk of life-threatening disease. All ma- nipulations of potentially infectious diagnostic materials, isolates, and naturally or experimentally infected animals pose a high risk of exposure and infection to laboratory personnel. Lassa fever virus is representative of the microorganisms assigned to Level 4. Animal Biosafety levels. Four Biosafety levels are also described for activities involving infectious disease activities with experimental mammals. These four combinations of practices, safety equipment, and facilities are designated Animal Biosafety Levels 1, 2, 3, and 4 and provide increasing levels of protec- tion to personnel and the environment. The laboratory director is directly and primarily responsible for the safe operation of the laboratory. Hisser knowledge and judgment are critical in as- sessing risks and appropriately applying these rec- ommendations. The recommended Biosafety level represents those conditions under which the agent can ordinarily be safely handled. Special characteris- tics of the agents used, the training and experience of personnel, and the nature or function of the labora- tory may further influence the director in applying these recommendations. Work with known agents should be conducted at the Biosafety level recommended in Section V unless specific information is available to suggest that viru- lence, pathogenicity, antibiotic resistance patterns, and other factors are significantly altered to require more stringent or allow less stringent practices to be used. Clinical laboratories, and especially those in health care facilities, receive clinical specimens with requests for a variety of diagnostic and clinical sup- port services. Typically, clinical laboratories receive specimens without pertinent information such as pa- tient history or clinical findings which may be sug- gestive of an infectious etiology. Furthermore, such specimens are often submitted with a broad request for microbiological examination for multiple agents (e.g., sputum samples submitted for"routine," acid- fast, and fungal cultures). It is the responsibility of the laboratory director to establish standard procedures in the laboratory which realistically address the issue of the infective
APPENDIX A hazard of clinical specimens. Except in extraordinary circumstances (e.g., suspected hemorrhagic fever) the initial processing of clinical specimens and iden- tification of isolates can be and are safely conducted using a combination of practices, facilities, and safety equipment described as Biosafety Level 2. Biologi- cal safety cabinets (Class I or II) should be used for the initial processing of clinical specimens when the nature of the test requested or other information is suggestive that an agent readily transmissible by in- fectious aerosols is likely to be present. Class II biological safety cabinets are also used to protect the integrity of the specimens or cultures by preventing contamination from the laboratory environment. Segregating clinical laboratory functions and limiting or restricting access to laboratory areas are the responsibility of the laboratory director. Importation' and interstate shipment of cer- tain biomedical materials. The importation of etio- logic agents and vectors of human diseases is subject to the requirements of the Public Health Service For- eign Quarantine regulations. Companion regulations of the Public Health Service and the Department of Transportation specify packaging, labeling, and ship- ping requirements for etiologic agents and diagnostic specimens shipped in interstate commerce (see Ap- pendix A.4~. The U.S. Department of Agriculture regulates the importation and interstate shipment of animal 89 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 Add. SECTION m. LABORATORY BIOSAFETY LEVEL CRITERIA The essential elements of the four Biosafety lev- els for activities involving infectious microorgan- isms and laboratory animals are summarized in Tables A.1 and A.2. The levels are designated in ascending order by degree of protection provided to personnel, the environment, and the community. Biosafety Level 1. Biosafety Level 1 is suitable for work involving agents of no known or minimal potential hazard to laboratory personnel and the en- vironment. The laboratory is not separated from the general traffic patterns in the building. Work is gen- erally conducted on open bench tops. Special con- tainment equipment is not required or generally used. Laboratory personnel have specific training in the procedures conducted in the laboratory and are su- pervised by a scientist with general training in micro- biology or a related science. The following standard and special practices, safety equipment, and facilities apply to agents as- signed to Biosafety Level 1. TABLE A.1 Summary of Recommended Biosafety Levels for Infectious Agents Biosafety level Practices and techniques Safety equipment None: primary containment provided by adherence to standard laboratory practices during open bench operations Facilities 1 Standard microbiological practices 2 3 Basic Level 1 practices plus: laboratory coats; decontamination of all infectious wastes; limited access; protective gloves and biohazard warning signs as indicated Level 2 practices plus: special labora- tory clothing; controlled access Partial containment equipment (i.e., Basic Class I or II Biological Safety Cabinets) used to conduct mechanical and manipulative procedures that have high aerosol potential which may in crease the risk of exposure to per sonnel Partial containment equipment used for Containment all manipulations of infectious material Level 3 practices plus: entrance Maximum containment equipment Maximum through change room where street clo- (i.e., Class III biological safety cabinet containment thing is removed and laboratory clo thing is put on; shower on exit; all wastes are decontaminated on exit from the facility ~ , ~ , _ _ . or partial containment equipment in combination with full-body, air- supplied, positive-pressure personnel suit) used for all procedures and activi- ties
Do APPENDIX A TABLE A.2 Summary of Recommended Biosafety Levels for Activities in Which Experimentally or Naturally Infected Vertebrate Animals Are Used Biosafety level Practices and techniques Safety equipment Facilities Basic 1 Standard animal care and management None practices Laboratory coats; decontamination of all infectious wastes and of animal cages prior to washing; limited access; protective gloves and hazard warning signs as indicated 3 Level 2 practices plus: special labora tory clothing; controlled access Level 3 practices plus: entrance through clothes change room where street clothing is removed and labora tory clothing is put on shower on exit; all wastes are decontaminated before removal from the facility Partial containment equipment and/or personal protective devices used for activities and manipulations of agents or infected animals that produce aero- sols Basic Partial containment equipment and/or Containment personal protective devices used for all activities and manipulations of agents or infected animals Maximum containment equipment Maximum (i.e., Class III biological safety cabinet containment or partial containment equipment in combination with full-body, air sup plied, positive-pressure personnel suit) used for all procedures and activities A. Standard?rucrobiologicalpractices 1. Access to the laboratory is limited or re stricted at the discretion of the laboratory direc tor when experiments are in progress. 2. Work surfaces are decontaminated once a day and after any spill of viable material. 3. All contaminated liquid or solid wastes are decontaminated before disposal. 4. Mechanical pipetting devices are used; mouth pipetting is prohibited. 5. Eating, drinking, smoking, and apply ing cosmetics are not permitted in the work area. Food may be stored in cabinets or refrigerators designated and used for this purpose only. Food storage cabinets or refrigerators should be lo cated outside of the work area. 6. Persons wash their hands after they handle viable materials and animals and before leaving the laboratory. 7. All procedures are performed carefully to minimize the creation of aerosols. 8. It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contami- washing. nation or soiling of street clothes. B. Specialpractices 1. Contaminated materials that are to be decontaminated at a site away from the labora tory are placed in a durable leakproof container which is closed before being removed from the laboratory. 2. An insect and rodent control program is in effect. C. Containment equipment Special containment equipment is generally not re- quired for manipulations of agents assigned to Bio- safety Level 1. D. Laboratoryfacilities 1. The laboratory is designed so that it can be easily cleaned. 2. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat. 3. Laboratory furniture is sturdy. Spaces between benches, cabinets, and equipment are accessible for cleaning. 4. Each laboratory contains a sink for hand 5. If the laboratory has windows that open, they are fitted with fly screens. Biosafety Level 2. Biosafety Level 2 is similar to Level 1 and is suitable for work involving agents
APPENDIX A of moderate potential hazard to personnel and the environment. It differs in that (1) laboratory person- nel have specific training in handling pathogenic agents and are directed by competent scientists, (2) access to the laboratory is limited when work is being conducted, and (3) certain procedures in which infectious aerosols are created are conducted in bio- logical 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~ucrobiologicalpractices 1. Access to the laboratory is limited or restricted by the laboratory director when work with infectious agents is in progress. 2. Work surfaces are decontaminated at least once a day and after any spill of viable material. 3. All infectious liquid or solid wastes are decontaminated before disposal. 4. Mechanical pipetting devices are used; mouth pipetting is prohibited. 5. Eating, drinking, smoking, and apply- ing cosmetics are not permitted in the work area. Food may be stored in cabinets or refrigerators designated and used for this purpose only. Food storage cabinets or refrigerators should be lo- cated outside of the work area. 6. Persons wash their hands after handling infectious materials and animals and when they leave the laboratory. 7. All procedures are performed carefully to minimize the creation of aerosols. B. Specialpractices 1. Contaminated materials that are to be decontaminated at a site away from the labora- tory are placed in a durable leakproof container which is closed before being removed from the laboratory. 2. The laboratory director limits access to the laboratory. In general, persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. The director has the final responsibility for assessing 91 each circumstance and determining who may enter or work in the laboratory. 3. The laboratory director establishes poli- cies and procedures whereby only persons who have been advised of the potential hazard and meet any specific entry requirements (e.g., im- munization) enter the ldboramry or animal rooms. 4. When the infectious magentas) in use in the laboratory require special provisions for en- try (e.g., vaccination), a hazard warning sign, incorporating the universal biohazard symbol, is posted on the access door to the laboratory work area The hazard warning sign identifies the in- fectious agent, lists the name and telephone number of the laboratory director or other re- sponsible personas), and indicates the special requirementts) for entering the laboratory. 5. An insect and rodent control program is in effect. 6. Laboratory coats, gowns, smocks, or uniforms are worn while in the laboratory. Be- fore leaving the laboratory for nonlaboratory areas (e.g., cafeteria, library, administrative of- f~ces), this protective clothing is removed and left in the laboratory or covered with a clean coat not used in the laboratory. 7. Animals not involved in the work being performed are not permitted in the laboratory. 8. Special care is taken to avoid skin con- tamination with infectious materials; gloves should be worn when handling infected animals and when skin contact with infectious materials is unavoidable. 9. All wastes from laboratories and animal rooms are appropriately decontaminated before disposal. 10. Hypodermic needles and syringes are used only for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or dispos- able syringe-needle units (i.e., needle is integral to the syringe) are used for the injection or aspi- ration of infectious fluids. Extreme caution should be used when handling needles and syringes to avoid autoinoculation and the generation of aero- sols during use and disposal. Needles should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontami
92 nated, preferably by autoclaving, before discard or reuse. 11. Spills and accidents which result in ovens exposures to infectious materials are immedi- ately reported to the laboratory director. Medi- cal evaluation, surveillance, and treatment are provided as appropriate, and written records are maintained. 12. When appropriate, considering the agentts) handled, base-line serum samples for laboratory and other at-risk personnel are col- lected periodically, depending on the agents handled or the function of the facility. 13. A Biosafety manual is prepared or adopted. Personnel are advised of special haz- ards and are required to read instructions on practices and procedures and to follow them. C. Containment equipment Biological safety cabinets (Class I or II) (see Appendix A.1) or other appropriate personal protec- tive or physical containment devices are used when- ever: 1. Procedures with a high potential for cre- ating infectious aerosols are conducted (821. These may include centrifuging, grinding, blend- ing, vigorous shaking or mixing, sonic disrup- tion, opening containers of infectious materials whose internal pressures may be different from ambient pressures, inoculating animals intrana- sally, and harvesting infected tissues from ani- mals or eggs. 2. High concentrations or large volumes of infectious agents are used. Such materials may be centrifuged in the open laboratory if sealed heads or centrifuge safety cups are used and if they are opened only in a biological safety cabi- net. D. Laboratoryfacilities 1. The laboratory is designed so that it can be easily cleaned. 2. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat. 3. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning. APPENDIX A 4. Each labo~auxy 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 in- fectious laboratory wastes is available. Biosafety Level 3. Biosafety Level 3 is appli- cable to clinical, diagnostic, teaching, research, or production facilities in which work is done with in- digenous or exotic agents which may cause serious or potentially lethal disease as a result of exposure by the inhalation route. Laboratory personnel have spe- cif~c training in handling pathogenic and potentially lethal agents and are supervised by competent scien- tists who are experienced in working with these agents. All procedures involving the manipulation of infectious material are conducted within biological safety cabinets or other physical containment de- vices or by personnel wearing appropriate personal protective clothing and devices. The laboratory has special engineering and design features. It is recog- nized, however, that many existing facilities may not have all the facility safeguards recommended for Biosafety Level 3 (e.g., access zone, sealed penetra- tions, directional airflow, etc.~. In these circumstances, acceptable safety may be achieved for routine or repetitive operations (e.g., diagnostic procedures in- volving the propagation of an agent for identifica- tion, typing, and susceptibility testing) in laborato- ries where facility features satisfy Biosafety Level 2 recommendations, provided the recommended "Stan- dard Microbiological Practices," "Special Practices," and "Containment Equipment" for Biosafety Level 3 are rigorously followed. The decision to implement this modification of Biosafety Level 3 recommenda- tions should be made only by the laboratory director. The following standard and special safety prac- tices, equipment, and facilities apply to agents as- signed to Biosafety Level 3. A. Standard microbiologicalpractices 1. Work surfaces are decontaminated at least once a day and after any spill of viable material. 2. All infectious liquid or solid wastes are decontaminated before disposal. 3. Mechanical pipetting devices are used; mouth pipetting is prohibited.
APPENDIX A 4. Eating, drinking, smoking, storing food, and applying cosmetics are not permitted in the work area 5. Persons wash their hands after handling infectious materials and animals and when they leave the laboratory. 6. All procedures are performed carefully to minimize the creation of aerosols. B. Specialpractices 1. Laboratory doors are kept closed when experiments are in progress. 2. Contaminated materials that are to be decontaminated at a site away from the labora- tory are placed in a durable leakproof container which is closed before being removed from the laboratory. 3. The laboratory director controls access to the laboratory and restricts access to persons whose presence is required for program or sup- port purposes. Persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. The director has the final responsibility for assessing each cir- cumstance and determining who may enter or work in the laboratory. 4. The laboratory director establishes poli- cies and procedures whereby only persons who have been advised of the potential biohazard, who meet any specific entry requirements (e.g., immunization), and who comply with all entry and exit procedures enter the laboratory or ani- mal rooms. 5. When infectious materials or infected animals are present in the laboratory or contain- ment module, a hazard warning sign, incorporat- ing the universal biohazard symbol, is posted on all laboratory and animal room access doors. The hazard warning sign identifies the agent, lists the name and telephone number of the labo- ratory director or other responsible personas), and indicates any special requirements for enter- ing the laboratory, such as the need for immuni- zations, respirators, or other personal protective measures. 6. All activities involving infectious mate- rials are conducted in biological safety cabinets or other physical containment devices within the 93 containment module. No work in open vessels is conducted on the open bench. 7. The work surfaces of biological safety cabinets and other containment equipment are decontaminated when work with infectious ma- terials is finished. Plastic-backed paper toweling used on nonperforated work surfaces within bio- logical safety cabinets facilitates cleanup. 8. An insect and rodent control program is in effect. 9. Laboratory clothing that protects street clothing (e.g., solid front or wrap-around gowns, scrub suits, coveralls) is worn in the laboratory. Laboratory clothing is not worn outside the labo- ratory, and it is decontaminated before being laundered. 10. Special care is taken to avoid skin con- tamination with infectious materials; gloves should be worn when handling infected animals and when skin contact with infectious materials is unavoidable. 11. Molded surgical masks or respirators are worn in rooms containing infected animals. 12. Animals and plants not related to the work being conducted are not permitted in the laboratory. 13. All wastes from laboratories and animal rooms are appropriately decontaminated before disposal. 14. Vacuum lines are protected with high efficiency particulate air (PAPA) filters and liq- uid disinfectant traps. 15. Hypodermic needles and syringes are used only for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or dispos- able syringe-needle units (i.e., needle is integral to Be syringe) are used for the injection or aspi- ration of infectious fluids. Extreme caution should be used when handling needles and syringes to avoid autoinoculation and the generation of aero- sols during use and disposal. Needles should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontami- nated, preferably by autoclaving, before discard or reuse. 16. Spills and accidents which result in overt or potential exposures to infectious materials are
94 i] immediately reported to the laboratory director. Appropriate medical evaluation, surveillance, and treatment are provided and written records are maintained. 17. Base-line serum samples for all labora- tory and other at-risk personnel should be col- lected and stored. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the laboratory. 18. A biosafety manual is prepared or adopted. Personnel are advised of special haz- ards and are required to read instructions on practices and procedures and to follow them. C. Contaminant equipment Biological safety cabinets (Class I, II, or III) (see Appendix A.1) or other appropriate combinations of personal protective or physical containment devices (e.g., special protective clothing, masks, gloves, res- pirators, centrifuge safety cups, sealed centrifuge ro- tors, and containment caging for animals) are used for all activities with infectious materials which pose a threat of aerosol exposure. These include: manipu- lation of cultures and of those clinical or environ- mental materials which may be a source of infectious aerosols; the aerosol challenge of experimental ani- mals; harvesting of tissues or fluids from infected animals and embryonated eggs; and necropsy of in- fected animals. D. Laboratoryfacilities 1. The laboratory is separated from areas which are open to unrestricted traffic flow within the building. Passage through two sets of doors is the basic requirement for entry into the labora- tory from access corridors or other contiguous areas. Physical separation of the high cont~n- ment laboratory from access corridors or other laboratories or activities may also be provided by a double-doored clothes change room (show- ers may be included), airlock, or other access facility which requires passage through two sets of doors before entering the laboratory. 2. The interior surfaces of walls, floors, and ceilings are water resistant so that they can be easily cleaned. Penetrations in these surfaces are sealed or capable of being sealed to facilitate decontaminating the area. APPENDIX A 3. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat. 4. Labolauxy furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning. 5. Each laboratory contains a sink for hand- washing. The sink is foot, elbow, or automati- cally 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 con- tainment module are self-closing. 8. An autoclave for decontaminating labo- ratory wastes is available, preferably within the laboratory. 9. A dueled exhaust air ventilation system is provided. This system creates directional air- flow that draws air into the laboratory through the entry area The exhaust air is not recirculated to any other area of the building, is discharged to the outside, and is dispersed away from occu- pied areas and air intakes. Personnel must verify that the direction of the airflow (into the labora- tory) is proper. The exhaust air from the labora- uxy room can be discharged to the outside with- out being filtered or otherwise treated. 10. The HEPA-filtered exhaust air from Class I or Class II biological safety cabinets is discharged directly to the outside or through the building exhaust system. Exhaust air from Class I or II biological safety cabinets may be recircu- lated within the laboratory if the cabinet is tested and certified at least every 12 months. If the PA-filtered exhaust air from Class I or II biological safety cabinets is to be discharged to the outside through the building exhaust air sys- tem, it is connected to this system in a manner (e.g., Nimble unit connection [801) that avoids any interference with the air balance of the cabi- nets or building exhaust system. Biosafety Level 4. Biosafety Level 4 is re- quired for work with dangerous and exotic agents which pose a high individual risk of life-threatening disease. Members of the laboratory staff have spe- ciD~c and thorough training in handling extremely hazardous infectious agents, and they understand the primary and secondary containment functions of the
APPENDIX A standard and special practices, the containment equip- ment, and the laboratory design characteristics. They are supervised by competent scientists who are trained and experienced in working with these agents. Ac- cess to the laboratory is strictly controlled by the laboratory director. The facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building. A specific facility operations manual is prepared or adopted. Within work areas of the facility, all activities are confined to Class III biological safety cabinets or Class I or Class II biological safety cabinets used along with one-piece positive-pressure personnel suits ventilated by a life support system. The maximum containment laboratory has special engineering and design features to prevent microorganisms from being disseminated into the environment. The following standard and special safety prac- tices, equipment, and facilities apply to agents as- signed to Biosafety Level 4. A. Standard microbiological practices 1. Work surfaces are decontaminated at least once a day and immediately after any spill of viable material. 2. Only mechanical pipetting devices are used 3. Eating, drinking, smoking, storing food, and applying cosmetics are not permitted in the laboratory. 4. All procedures are performed carefully to minimize the creation of aerosols. B. Specialpractices 1. Biological materials to be removed from the Class III cabinet or from the maximum con- tainment laboratory in a viable or intact state are transferred to a nonbreakable, sealed primary container and then enclosed in a nonbreakable, sealed secondary container which is removed from the facility through a disinfectant dunk tank, fumigation chamber, or an airlock designed for this purpose. 2. No materials, except for biological ma- terials that are to remain in a viable or intact state, are removed from the maximum contain- ment laboratory unless they have been autoclaved 95 or decontaminated before they leave the facility. Equipment or material which might be damaged by high temperatures or steam is decontami- nated by gaseous or vapor methods in an airlock or chamber designed for this purpose. 3. Only persons whose presence in the fa- cility or individual laboratory rooms is required for program or support purposes are authorized to enter. Persons who may be at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. The supervisor has the fog responsibility for assessing each cir- cumstance and determining who may enter or work in the laboratory. Access to the facility is limited by means of secure, locked doors; acces- sibility is managed by the laboratory director, biohazards control officer, or other person re- sponsible for the physical security of the facility. Before entering, persons are advised of the pm tential biohazards and instructed as to appropri- ate safeguards for ensuring their safety. Author- ized persons comply with the instructions and all other applicable entry and exit procedures. A logbook, signed by all personnel, indicates the date and time of each entry and exit. Practical and effective protocols for emergency situations are established. 4. Personnel enter and leave the facility only through the clothing change and shower rooms. Personnel shower each time they leave the facility. Personnel use the airlocks to enter or leave the laboratory only in an emergency. S. Street clothing is removed in the outer clothing change room and kept there. Complete laboratory clothing, including undergarments, pants and shirts or jumpsuits, shoes, and gloves, is provided and used by all personnel entering the facility. Head covers are provided for per- sonnel who do not wash their hair during the exit shower. When leaving the laboratory and before propelling into the shower area, personnel re- move their laboratory clothing and store it in a locker or hamper in the inner change room. 6. When infectious materials or infected animals are present in the laboratory or animal rooms, a hazard warning sign, incorporating the universal biohazard symbol, is posted on all ac- cess doors. The sign identifies the agent, lists the name and telephone number of the laboratory
96 director or other responsible personts), and indi- cates any special requirements for entering the area (e.g., the need for immunizations or respira- tors). 7. Supplies and materials need in the fa- cility 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 open- ing the interior doors of the autoclave, fumi- gation chamber, or airlock. These doors are secured after materials are brought into the facil- ity. S. An insect and rodent control program is in effect. 9. Materials (e.g., plants, animals, and clothing) not related to the experiment being conducted are not permitted in the facility. 10. Hypodermic needles and syringes are used only for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking syringes or dispos- able syringe-needle units (i.e., needle is integral part of unit) are used for the injection or aspira- tion of infectious fluids. Needles should not be bent, sheared, replaced in the needle guard, or removed from the syringe following use. The needle and syringe should be placed in a punc- ture-resistant container and decontaminated, pref- erably by autoclaving, before discard or reuse. Whenever possible, cannulas are used instead of sharp needles (e.g., for Savage). 11. A system is set up for reporting labora- tory accidents and exposures and employee ab- senteeism, and for the medical surveillance of potential laboratory-associated illnesses. Writ- ten records are prepared and maintained. An essential adjunct to such a reporting-suneillance system is the availability of a facility for the quarantine, isolation, and medical care of per- sonnel with potential or known laboratory-asso- ciated illnesses. C. Containment equipment All procedures within the facility with agents assigned to Biosafety Level 4 are conducted in a Class III biological safety cabinet or in Class I or II biological safety cabinets used in conjunction with APPENDIX A one-piece positive-pressure personnel suits ventilated by a life support system. Activities with viral agents (e.g., Rift Valley fever virus) that require Biosafety Level 4 secondary containment capabilities and for which highly effective vaccines are available and used can be conducted within Class I or Class II biological safety cabinets within the facility without the one-piece positive-pressure personnel suit being used, if (1) the facility has been decontaminated; (2) no work is being conducted in the facility with other agents assigned to Biosafety Level 4; and (3) all other standards and special practices are followed. D. Laboratoryfacilities 1. The maximum containment facility con- sists of either a separate building or a clearly demarcated and isolated zone within a building. Outer and inner change rooms separated by a shower are provided for personnel entering and leaving the facility. A double-doored autoclave, fumigation chamber, or ventilated airlock is pro- vided for passage of those materials, supplies, or equipment which are not brought into the facil- ity through the change room. 2. Walls, floors, and ceilings of the facility are constructed to form a sealed internal shell which facilitates fumigation and is animal and insect proof. The internal surfaces of this shell are resistant to liquids and chemicals, thus facili- tating cleaning and decontamination of the area. All penetrations in these structures and surfaces are sealed. Any drains in the floors contain traps filed with a chemical disinfectant of demon- strated efficacy against the target agent, and they are connected directly to the liquid waste decon- tamination system. Sewer and other ventilation lines contain HEPA filters. 3. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize the horizontal surface area on which dust can settle. 4. Bench tops have seamless surfaces which are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat. 5. Laboratory furniture is of simple and sturdy construction, and spaces between benches, cabinets, and equipment are accessible for clean- ~ng. 6. A foot-, elbow-, or automatically oper
APPENDIX A ated handwashing sink is provided near the door of each laboratory room in the facility. 7. If there is a central vacuum system, it does not serve areas outside the facility. In-line HEPA filters are placed as near as practicable to each use point or service cock Filters are in- stalled to permit in-place decontamination and replacement. Other liquid and gas services to the facility are protected by devices that prevent backflow. S. If water fountains are provided, they are foot operated and are located in the facility cor- ridors outside the laboratory. The water service to the fountain is not connected to the backflow- protected distribution system supplying water to the laboratory areas. 9. Access doors to the laboratory are self- closing and lockable. 10. Any windows are breakage resistant. 11. A double-doored autoclave is provided for decontaminating materials passing out of the facility. The autoclave door which opens to the area external to the facility is sealed to the outer wall and automatically controlled so that the outside door can only be opened after the auto- clave "sterilization" cycle has been complete 12. A pass-through dunk tank, fumigation chamber, or an equivalent decontamination method is provided so that materials and equip- ment that cannot be decontaminated in the auto- clave can be safely removed from the facility. 13. Liquid effluents from laboratory sinks, biological safety cabinets, floors, and autoclave chambers are decontaminated by heat treatment before being released from the maximum con- tainment facility. Liquid wastes from shower rooms and toilets may be decontaminated with chemical disinfectants or by heat in the liquid waste decontamination system. The procedure used for heat decontamination of liquid wastes is evaluated mechanically and biologically by using a recording thermometer and an indicator microorganism with a defined heat susceptibil- ity pattern. If liquid wastes from the shower rooms are decontaminated with chemical disin- fectants, the chemical used is of demonstrated efficacy against the target or indicator microor- gan~sms. 14. An individual supply and exhaust air ventilation system is provided The system main 97 tains pressure differentials and directional air- flow as required to assure flows inward from areas outside of the facility toward areas of high- est potential risk within the facility. Manometers are used to sense pressure differentials between adjacent areas maintained at different pressure levels. If a system malfunctions, the manome- ters sound an alarm. The supply and exhaust airflow is interlocked to assure inward (or zero) airflow at all times. 15. The exhaust air from the facility is fil- tered through HEPA fetters and discharged to the outside so that it is dispersed away from occupied buildings and air intakes. Within the facility, the filters are located as near the labora- tories as practicable in order to reduce the length of potentially contaminated air ducts. The filter chambers are designed to allow in situ decon- tamination before filters are removed and to fa- cilitate cer~cation testing after they are replaced. Coarse filters and HEPA filters are provided to treat air supplied to the facility in order to in- crease the lifetime of the exhaust PAPA fetters and to protect the supply air system should air pressures become unbalanced in the laboratory. 16. The treated exhaust air from Class I and II biological safety cabinets can be discharged into the laboratory room environment or to the outside through the facility air exhaust system. If exhaust air from Class I or II biological safety cabinets is discharged into the laboratory, the cabinets are tested and certified at Month intervals. The treated exhaust air from Class III biological safety cabinets is discharged, without recirculation through two sets of HEPAfilters in series, via the facility exhaust air system. If the treated exhaust air from any of these cabinets is discharged to the outside through the facility exhaust air system, it is connected to this system in a manner (e.g., thimble unit connection [801) that avoids any interference with the air balance of the cabinets or the facility exhaust air system. 17. A specially designed suit area may be provided in the facility. Personnel who enter this area wear a one-piece positive-pressure suit that is ventilated by a life support system. The life support system includes alarms and emergency backup breathing air tanks. Entry to this area is through an airlock fitted with airtight doors. A chemical shower is provided to decontaminate
98 the surface of the suit before the worker leaves the area The exhaust air from the suit area is faltered by two sets of HEPA filters installed in series. A duplicate filtration unit, an exhaust fan, and an automatically starting emergency power source are provided. The air pressure within the suit area is lower than that of any adjacent area. Emergency lighting and communications sys- tems are provided. All penetrations into the internal shell of the suit area are sealed. A double- doored autoclave is provided for decontaminat- ing waste materials to be removed from the suit area SECTIONIV. VERTEBRATE ANIMAL BIOSAFETY LEVEL CRITERIA If experimental animals are used, institutional management must provide facilities and staff and establish practices which reasonably assure appro- priate levels of environmental quality, safety, and care. Laboratory animal facilities are extensions of the laboratory and in some situations are integral to and inseparable from the laboratory. As a general principle, the Biosafety Level (facilities, practices, and operational requirements) recommended for working with infectious agents in viva and in vitro are comparable. These recommendations presuppose that labora- tory animal facilities, operational practices, and qual- ity of animal care meet applicable standards and regulations and that appropriate species have been selected for animal experiments (e.g., Guide for the Care and Use of Laboratory Animals, HEW Publica- tion no. [NIH] 78-23, Rev. 197S, and Laboratory Animal Welfare Regulations, 9 CFR, Subchapter A, Parts 1, 2, and 3~. Ideally, facilities for laboratory animals used for studies of infectious or noninfectious disease should be physically separate from other activities such as animal production and quarantine, clinical laborato- ries, and especially from facilities that provide pa- tient care. Animal facilities should be designed and constructed to facilitate cleaning and housekeeping. A "clean halVdirty hall" layout is very useful in re- ducing cross contamination. Floor drains should be installed in animal facilities only on the basis of clearly defined needs. If floor drains are installed, the drain trap should always contain water. APPENDIX A These recommendations describe four combina tions of practices, safety equipment, and facilities for experiments on animals infected with agents which are known or believed to produce infections in hu- mans. These four combinations provide increasing levels of protection to personnel and to the environ- ment and are recommended as minimal standards for activities involving infected lab~xamry animals. These four combinations, designated Animal Biosafety Levels 1~, describe animal facilities and practices applicable to work on animals infected with agents assigned to corresponding Biosafety Levels 14. Facility standards and practices for invertebrate vectors and hosts are not specifically addressed in standards written for commonly used laboratory ani- mals. "Laboratory Safety for Arboviruses and Cer- tain Other Viruses of Vertebrates" (112), prepared by the Subcommittee on Arbovirus Laboratory Safety of the American Committee on Arthropod-Borne Viruses, serves as a useful reference in the design and operation of facilities using arthropods. Animal Biosafety Level 1 A. Standard practices 1. Doors to animal rooms open inward, are self-closing, and are kept closed when experi- mental animals are present. 2. Work surfaces are decontaminated after use or after any spill of viable materials. 3. Eating, drinking, smoking, and storing food for human use are not permitted in animal rooms. 4. Personnel wash their hands after han- dling 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. Bedding materials from animal cages are removed in such a manner as to minimize the creation of aerosols and disposed of in compli- ance with applicable institutional or local re- quirements.
APPENDIX A 2. Cages are washed manually or in a cage- washer. Temperature of final rinse water in a mechanical washer should be 180°F. 3. The wearing of laboratory coats, gowns, or uniforms in the animal room is recommended. It is further recommended that laboratory coats worn in the animal room not be worn in other areas. C. Containment equipment Special containment equipment is not required for animals infected with agents assigned to Bio- safety Level 1. D. Animalfacilities 1. The animal facility is designed and con- structed to facilitate cleaning and housekeeping. 2. A handwashing sink is available in the animal facility. 3. If the animal facility has windows that open, they are fitted with fly screens. 4. It is recommended, but not required, that the direction of airflow in the animal facility is inward and that exhaust air is discharged to the outside without being recirculated to other rooms. Animal Biosafety Level 2 A. Standard practices 1. Doors to animal rooms open inward, are self-closing, and are kept closed when infected animals are present. 2. Work surfaces are decontaminated after use or spills of viable materials. 3. Eating, drinking, smoking, and storing of food for human use are not permitted in ani- mal rooms. 4. Personnel wash their hands after han- dling 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. 99 B. Specialpractices 1. Cages are decontaminated, preferably by autoclaving, before they are clear and washed. 2. Surgical-Wpe masks are worn by all per- sonnel entering animal rooms housing nonhu- man primates. 3. Laboratory coats, gowns, or uniforms are worn while in the animal room. This protective clothing is removed before leaving the animal facility. 4. The laboratory or animal facility direc- tor limits access to the animal room to personnel who have been advised of the potential hazard and who need to enter the room for program or service purposes when work is in progress. In general, persons who may be at increased risk of acquiring infection or for whom infection might be unusually hazardous are not allowed in the animal room. 5. The laboratory or animal facility direc- tor establishes policies and procedures whereby only persons who have been advised of the per tential hazard and meet any specific requirements (e.g., for immunization) may enter the animal room. 6. When the infectious magentas) in use in the animal room requires special entry provi- sions (e.g., vaccination), a hazard warning sign, incorporating the universal biohazard symbol, is posted on the access door to the animal room. The hazard warning sign identifies the infectious agent, lists the name and telephone number of the animal facility supervisor or other respon- sible personas), and indicates the special requirements for entering the animal room. 7. Special care is taken to avoid skin con- tamination with infectious materials; gloves should be worn when handling infected animals and when skin contact with infectious materials is unavoidable. S. All wastes from the animal room are ap- propriately decontaminated, preferably by auto- claving, before disposal. Infected animal carcasses are incinerated after being transported from the animal room in leakproof, covered containers. 9. Hypodermic needles and syringes are used only for the parenteral injection or aspira- tion of fluids from laboratory animals and dia
100 poem bottles. Only needle-locking syringes or disposable needle-syringe units (i.e., the needle is integral to the syringe) are used for the injec- tion or aspiration of infectious fluids. Needles should not be bent, shamed, replaced in the sheath or guard, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and de- contaminated, preferably by autoclaving, before discard or reuse. 10. If floor drains are provided, the drain traps are always filed with water or a suitable disinfectant. 11. When appropriate, considering the agents handled, base-line serum samples from animal care and other at-risk personnel are col- lected and stored. Additional serum samples may be collected periodically, depending on the agents handled or the function of the facility. C. Containment equipment Biological safety cabinets, other physical con- tainment devices, and/or personal protective devices (e.g., respirators, face shields) are used whenever procedures with a high potential for creating aerosols are conducted (82~. These include necropsy of in- fected animals, harvesting of infected tissues or fluids from animals or eggs, intranasal inoculation of ani- mals, and manipulations 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 handwashing sink is available in the room where infected animals are housed. 3. If the animal facility has windows that open they are fitted with fly screens. 4. It is recommended, but not required, that the direction of airflow in the animal facility is inward and that exhaust air is discharged to the outside without being recirculated to other rooms. 5. An autoclave which can be used for de- contaminating infectious laboratory waste is available in the building with the animal facility. APPENDIX A Animal Biosafety Level 3 A. Standard practices 1. Doors to animals rooms open inward, are self-closing, and are kept closed when work with infected animals is in progress. 2. Work surfaces are decontaminated after use or spills of viable materials. 3. Eating, drinking, smoking, and storing of food for human use are not permitted in the animal room. 4. Personnel wash their hands after han- dling cultures and animals and before leaving the laboratory. 5. All procedures are carefully performed to minimize the creation of aerosols. 6. An insect and rodent control program is in effect. B. Specialpractices 1. Cages are autoclaved before bedding is removed and before they are cleaned and washed. 2. Surgical-Wpe masks or other respiratory protection devices (e.g., respirators) are worn by personnel entering rooms housing animals in- fected with agents assigned to Biosafety Level 3. Wrap-around or solid-front gowns or uniforms are worn by personnel entering the animal room. Front-button laboratory coats are unsuitable. Protective gowns must remain in the animal room and must be decontaminated be- fore being laundered. 4. The laboratory director or other respon- sible person restricts access to the animal room to personnel who have been advised of the po- tential 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 haz- ardous are not allowed in the animal room. 5. The laboratory director or other respon- sible person establishes policies and procedures whereby only persons who have been advised of the potential hazard and meet any specific re
APPENDIX A quirements (e.g., for immunization) may enter the animal room. 6. Hazard warning signs, incorporating the universal biohazard warning symbol, are posted on access doors to animal rooms containing ani- mals infected with agents assigned to Biosafety Level 3. The hazard warning sign should iden- tify the agates) in use, list the name and tele- phone number of the animal room supervisor or other responsible personas), and indicate any special conditions of entry into the animal room (e.g., the need for immunizations or respirators). 7. Personnel wear gloves when handling infected animals. Gloves are removed asepti- cally and autoclaved with other animal room wastes before being disposed of or reused. 8. All wastes from the animal room are autoclaved before disposal. All animal carcasses are incinerated. Dead animals are transported from the animal room to the incinerator in leakproof covered containers. 9. Hypodermic needles and syringes are used only for Savage or for parenteral injection or aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking sy- ringes or disposable needle-syringe units (e.g., the needle is integral to the syringe) are used. Needles should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before discard or reuse. Whenever possible, can- nulas should be used instead of sharp needles (e.g., for Ravage). 10. If floor drains are provided, the drain traps are always filled with water or a suitable disinfectant. 11. If vacuum lines are provided, they are protected with HEPA filters and liquid disinfec- tant traps. 12. Boots, shoe covers, or other protective footwear and disinfectant footbaths are avail- able and used when indicated. C. Containment equipment 1. Personal protective clothing and equip- ment and/or other physical containment devices 101 are used for all procedures and manipulations of infectious materials or infected animals. 2. The risk of infectious aerosols from in- fected animals or their bedding can be reduced if animals are housed in partial containment cag- ing systems, such as open cages placed in venti- lated enclosures (e.g., laminar flow cabinets), solid-wall and -bottom cages covered by filter bonnets, or other equivalent primary contain- ment systems. D. Animalfacilities 1. The animal facility is designed and con- structed to facilitate cleaning and housekeeping and is separated from areas which are open to unrestricted personnel traffic within the build- ing. Passage through two sets of doors is the basic requirement for entry into the animal room from access corridors or other contiguous areas. Physical separation of the animal room from access corridors or other activities may also be provided by a double-doored clothes change room (showers may be included), airlock, or other access facility which requires passage through two sets of doors before entering the animal room. 2. The interior surfaces of walls, floors, and ceilings are water resistant so that they may be easily cleaned. Penetrations in these surfaces are sealed or capable of being sealed to facilitate fumigation or space decontamination. 3. A foot-, elbow-, or automatically oper- ated handwashing sink is provided near each animal room exit door. 4. Windows in the animal room are closed and sealed. 5. Animal room doors are self-closing and are kept closed when infected animals are pres- ent. 6. An autoclave for decontaminating wastes is available, preferably within the animal room. Materials to be autoclaved outside the animal room are transported in a covered leakproof container. 7. An exhaust air ventilation system is pro- vided. This system creates directional airflow that draws air into the animal room through the entry area. The building exhaust can be used for
102 APPENDIX A this purpose if the exhaust air is not recirculated to any other area of the building, is discharged to the outside, and is dispersed away from occu- pied areas and air intakes. Personnel must verify that the direction of the airflow (into the animal room) is proper. The exhaust air from the animal room that does not pass through biological safety cabinets or other primary containment equip- ment can be discharged to the outside without being filtered or otherwise treated. X. The HEPA-filtered exhaust air from Class I or Class II biological safety cabinets or other primary containment devices is discharged directly to the outside or through the building exhaust system. Exhaust air from these primary containment devices may be recirculated within the animal room if the cabinet is tested and certified at least every 12 months. If the HEPA- f~tered exhaust air from Class I or Class II bio- logical safety cabinets is discharged to the out- side through the building exhaust system, it is connected to this system in a manner (e.g., thimble unit connection [801) that avoids any interference with the air balance of the cabinets or building exhaust system. Animal Biosafety Level 4 A. Standard practices 1. Doors to animal rooms open inward and are self-closing. 2. Work surfaces are decontaminated after use or spills of viable materials. 3. Eating, drinking, smoking, and storing of food for human use are not permitted in the animal room. 4. All procedures are carefully performed to minimize the creation of aerosols. 5. An insect and rodent control program is in effect. 6. Cages are autoclaved before bedding is removed and before they are cleaned and washed. B. Specialpractices 1. Only persons whose entry into the facil- ity or individual animal rooms is required for program or support purposes are authorized to enter. Persons who may be at increased risk of acquiring infection or for whom infection might be unusually hazardous are not allowed in the animal facility. Persons at increased risk may include children, pregnant women, and persons who are immunodef~cient or immunosuppressed. The supervisor has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory. Access to the facility is limited by secure, locked doors; accessibility is controlled by the animal facility supervisor, biohazards control officer, or other person responsible for the physical security of the facility. Before entering, persons are advised of the potential biohazards and instructed as to appropriate safeguards. Personnel comply with the instructions and all other applicable entry and exit procures. Practical and effective proto- cols for emergency situations are established. 2. Personnel enter and leave the facility only through the clothing change and shower rooms. Personnel shower each time they leave the facility. Head covers are provided to person- nel who do not wash their hair during the exit shower. Except in an emergency, personnel do not enter or leave the facility through the air- locks. 3. Street clothing is removed in the outer clothing change room and kept there. Complete laboratory clothing, including undergarments, pants and shirts or jumpsuits, shoes, and gloves, is provided and used by all personnel entering the facility. When exiting, personnel remove laboratory clothing and store it in a locker or hamper in the inner change room before enter- ing the shower area. 4. When infectious materials or infected animals are present in the animal rooms, a haz- ard warning sign, incorporating the universal biohazard symbol, is posted on all access doors. The sign identifies the agent, lists the name and telephone number of the animal facility supervi- sor or other responsible personas), and indicates any special conditions of entry into the area (e.g., the need for immunizations and respira- tors). 5. Supplies and materials to be taken into the facility enter by way of the double-door auto- clave, fumigation chamber, or airlock, which is appropriately decontaminated between each use. After securing the outer doors, personnel inside
APPENDIX A the facility retrieve the materials by opening the interior doors of the autoclave, fumigation cham- ber, or airlock. This inner door is secured after materials are brought into the facility. 6. Materials (e.g., plants, animals, cloth- ing) not related to the experiment are not permit- ted in the facility. 7. Hypodermic needles and syringes are used only for Savage or for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles. Only needle-locking sy- ringes or disposable syringe-needle units (i.e., needle is integral part of unit) are used. Needles should not be bent, sheared, replaced in the guard or sheath, or removed from the syringe follow- ing use. The needle and syringe should be promptly placed in a puncture-resistant container and decontaminated, preferably by autoclaving, before discard or reuse. Whenever possible, can- nulas should be used instead of sharp needles (e.g., for gavage). 8. A system is developed and is opera- tional for the reporting of animal facility acci- dents and exposures, employee absenteeism, and for the medical surveillance of potential labora- tory-associated illnesses. An essential adjunct to such a reporting-surveillance system is the availa- bility of a facility for the quarantine, isolation, and medical care of persons with potential or known laboratory-associated illnesses. 9. Base-line serum samples are collected and stored for all laboratory and other at-risk personnel. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the laboratory. C. Containment equipment Laboratory animals infected with agents assigned to Biosafety Level 4 are housed in a Class III biologi- cal safety cabinet or in parrial-containment caging systems (such as open cages placed in ventilated enclosures, solid-wall and -bottom cages covered with filter bonnets, or other equivalent primary contain- ment systems) in specially designed areas in which all personnel are required to wear one-piece positive- pressure suits ventilated with a life support system. Animal work with viral agents that require Biosafety Level 4 secondary containment and for which highly effective vaccines are available and used may be 103 conducted with partial-containment cages and with- out the one-piece positive-pressure personnel suit if the facility has been decontaminated, if no concur- rent experiments are being done in the facility which require Biosafety Level 4 primary and secondary containment, and if all other standard and special practices are followed. D. Animalfacility 1. The animal rooms are located in a sepa- rate building or in a clearly demarcated and iso- lated zone within a building. Outer and inner change rooms separated by a shower are pro- vided for personnel entering and leaving the fa- cility. A double-doored autoclave, fumigation chamber, or ventilated airlock is provided for passage of materials, supplies, or equipment which are not brought into the facility through the change room. 2. Walls, floors, and ceilings of the facility are constructed to form a sealed internal shell which facilitates fumigation and is animal and insect proof. The internal surfaces of this shell are resistant to liquids and chemicals, thus facili- tating cleaning and decontamination of the area. All penetrations in these structures and surfaces are sealed. 3. Internal facility appurtenances, such as light fixtures, air ducts, and utility pipes, are arranged to minimize the horizontal surface area on which dust can settle. 4. A foot-, elbow-, or automatically oper- ated handwashing sink is provided near the door of each animal room within the facility. 5. If there is a central vacuum system, it does not serve areas outside of the facility. The vacuum system has in-line HERA filters placed as near as practicable to each use point or serv- ice cock. Filters are installed to permit in-place decontamination and replacement. Other liquid and gas services for the facility are protected by devices that prevent backflow. 6. External animal facility doors are self- closing and self-locking. 7. Any windows must be resistant to break- age and sealed. 8. A double-doored autoclave is provided for decontaminating materials that leave the fa- cility. The autoclave door which opens to the
104 APPENDIX A area external to the facility is automatically con- trolled so that it can only be opened after the autoclave"sterilization" cycle is completed. 9. A pass-through dunk tank, fumigation chamber, or an equivalent decontamination method is provided so that materials and equip- ment that cannot be decontaminated in the auto- clave can be safely removed from the facility. 10. Liquid effluents from laboratory sinks, cabinets, floors, and autoclave chambers are decontaminated by heat treatment before being discharged. Liquid wastes from shower rooms and toilets may be decontaminated with chemi- cal disinfectants or by heat in the liquid waste decontamination system. The procedure used for heat decontamination of liquid wastes must be evaluated mechanically and biologically by us- ing a recording thermometer and an indicator microorganism with a defined heat susceptibil- ity pattern. If liquid wastes from the shower rooms are decontaminated with chemical disin- fectants, the chemicals used must have docu- mented efficacy against the target or indicator microorganisms. 11. An individual supply and exhaust air ventilation system is provided. The system main- tains pressure differentials, and directional air- flow is required to assure inflow from areas outside the facility toward areas of highest po- tential risk within the facility. Manometers are provided to sense pressure differentials between adjacent areas and are maintained at different pressure levels. The manometers sound an alarm when a system malfunctions. The supply and exhaust airflow is interlocked to assure inward (or zero) airflow at all times. 12. Air can be recirculated within an animal room if it is filtered through a HEPA filter. 13. The exhaust air from the facility is fil- tered by HEPA filters and discharged to the outside so that it is dispersed away from occu- pied buildings and air intakes. Within the facil- ity, the filters are located as near to the laborato- ries as practicable in order to reduce the length of potentially contaminated air ducts. The filter chambers are designed to allow in situ decon- tamination before filters are removed and to fa- cilitate certification testing after they are replaced. Coarse filters are provided for treatment of air supplied to the facility in order to increase the lifetime of the HEPA filters. 14. The treated exhaust air from Class I or Class II biological safety cabinets can be dis- charged into the animal room environment or to the outside through the facility air exhaust sys- tem. If exhaust air from Class I or II biological safety cabinets is discharged into the animal room, the cabinets are tested and certified at 6- month intervals. The treated exhaust air from Class III biological safety cabinets is discharged without recirculation via the facility exhaust air system. If the treated exhaust air from any of these cabinets is discharged to the outside through the facility exhaust air system, it is connected to this system in a manner that avoids any interfer- ence with the air balance of the cabinets or the facility exhaust air system. 15. A specially designed suit area may be provided in the facility. Personnel who enter this area wear a one-piece positive-pressure suit that is ventilated by a life support system. The life support system is provided with alarms and emergency backup breathing air tanks. Entry to this area is through an airlock fitted with airtight doors. A chemical shower is provided to decon- taminate the surface of the suit before the worker leaves the area. The exhaust air from the area in which the suit is used is filtered by two sets of PAPA filters installed in series. A duplicate fil- tration unit and exhaust fan are provided. An automatically starting emergency power source is provided. The air pressure within the suit area is lower than that of any adjacent area. Emer- gency lighting and communication systems are provided. All penetrations into the inner shell of the suit area are sealed. A double-doored auto- clave is provided for decontaminating waste materials to be removed from the suit area. SECTION V. RECOMMENDED BIOSAFETY LEVELS FOR INFECTIOUS AGENTS AND INFECTED ANIMALS Selection of an appropriate biosafety level for work with a particular agent or animal study depends upon a number of factors. Some of the most impor- tent are: the virulence, pathogenicity, biological sta- bility, route of spread, and communicability of the
APPENDIX A agent; the nature or function of the laboratory; the procedures and manipulations involving the agent; the quantity and concentration of the agent; the en- demicity of the agent; and the availability of effec- tive vaccines or therapeutic measures. Agent summary statements in this section pro- vide guidance for the selection of appropriate bio- safety levels. Specific information on laboratory hazards associated with a Ocular agent and rec- ommendations regarding practical safeguards that can significantly reduce the risk of laboratory-associated diseases are included. Agent summary statements are presented for agents which meet one or more of the following criteria: the agent is a proven hazard to laboratory personnel working with infectious materials (e.g., hepatitis B virus, tubercle bacilli); the potential for laboratory-associated infection is high even in the absence of previously documented laboratory-associated infections (e.g., exotic arboviruses); or the consequences of infection are grave (e.g., Creutzfeldt-Jakob disease, botulism). Recommendations for the use of vaccines and toxoids are included in agent summary statements when such products are available-either as licensed or Investigational New Drug ~ND) products. When applicable, recommendations for the use of these products are based on current recommendations of the Public Health Service Advisory Committee on Immunization Practice and are specifically targeted to at-risk laboratory personnel and others who must work in or enter laboratory areas. These specific recommendations should in no way preclude the rou- tine use of such products as diphtheria-tetanus toxoids, poliovirus vaccine, influenza vaccine, and others because of the potential risk of community expo- sures irrespective of any laboratory risks. Appropri- ate precautions should be taken in the administration of live attenuated virus vaccines in individuals with altered immunocompetence. Risk assessments and Biosafety Levels recom- mended in the agent summary statements presuppose a population of immunocompetent individuals. Those with altered immunocompetence may be at increased risk when exposed to infectious agents. Immunodefi- ciency may be hereditary, congenital, or induced by a number of neoplastic diseases, by therapy, or by radiation. The risk of becoming infected or the con- sequences of infection may also be influenced by such factors as age, sex, race, pregnancy, surgery 105 (e.g., splenectomy, gastrectomy), predisposing dis- eases (e.g., diabetes, lupus erythematosus), or altered physiological function. These and other variables must be considered in individualizing the generic risk as- sessments of the agent summary statements for spe- cific activities. The basic biosafety level assigned to an agent is based on the activities typically associated with the growth and manipulation of quantities and concen- trations of infectious agents required to accomplish identification of typing. If activities with clinical materials pose a lower risk to personnel than those activities associated with manipulation of cultures, a lower biosafety level is recommended. On the other hand, if the activities involve large volumes or highly concentrated preparations ("production quantities") or manipulations which are likely to produce aero- sols or which are otherwise intrinsically hazardous, additional personnel precautions and increased lev- els of primary and secondary containment may be indicated. "Production quantities" refers to large vol- umes or concentrations of infectious agents consid- erably in excess of those typically used in identifica- tion and typing activities. Propagation and concen- tration of infectious agents, as occurs in large-scale fermentations, antigen and vaccine production, and a variety of other commercial and research activities, clearly deal with significant masses of infectious agents that are reasonably considered "production quantities." However, in terms of potentially increased risk as a function of the mass of infectious agents, it is not possible to define "production quantities" in finite volumes or concentrations for any given agent. Therefore, the laboratory director must make a risk assessment of the activities conducted and select practices, containment equipment, and facilities ap- propriate to the risk, irrespective of the volume or concentration of agent involved. Occasions will arise when the laboratory direc- tor should select a biosafety level higher than that recommended. For example, a higher biosafety level may be indicated by the unique nature of the pro- posed activity (e.g., the need for special containment for experimentally generated aerosols for inhalation studies) or by the proximity of the laboratory to areas of special concern (e.g., a diagnostic laboratory located near patient care areas). Similarly, a rec- ommended biosafety level may be adapted to com- pensate for the absence of certain recommended
106 safeguards. For example, in those situations where Biosafety Level 3 is recommended, acceptable safety may be achieved for routine or repetitive operations (e.g., diagnostic procedures involving the propaga- tion of an agent for identification, typing, and sus- ceptibility testing) in laboratories where facility fea- tures satisfy Biosafety Level 2 recommendations, provided the recommended "Standard Microbiologi- cal Practices," "Special Practices," and "Contain- ment Equipment" for Biosafety Level 3 are rigor- ously followed. The decision to adapt Biosafety Level 3 recommendations in this manner should be made only by the laboratory director. This adaptation, however, is not suggested for agent production op- erations or activities where procedures are frequently changing. The laboratory director should also give special consideration to selecting appropriate safe- guards for materials that may contain a suspected agent. For example, sera of human origin may con- tain hepatitis B virus and should be handled under conditions which reasonably preclude cutaneous, mucous membrane, or parenteral exposure of per- sonnel, and sputa submitted to the laboratory for assay for tubercle bacilli should be handled under conditions which reasonably preclude the generation of aerosols or which contain any aerosols that may be generated during the manipulation of clinical ma- terials or cultures. The infectious agents which meet the previously stated criteria are listed by category of agent on the following pages. To use these summaries, fast locate the agent in the listing under the appropriate category of agent. Second, utilize the practices, safety equip- ment, and Me of facilities recommended for work- ing with clinical materials, cultures of infectious agents, or infected animals recommended in the agent - summary statement and described in Section V. The laboratory director is also responsible for appropriate risk assessment of agents not included in the Agent Summary Statements and for utilization of appropriate practices, containment equipment, and facilities for the agent used. Risk assessment. The risk assessment of labo ratory activities involving the use of infectious mi- croorganisms is ultimately a subjective process. Those risks associated with the agent, as well as with the activity to be conducted, must be considered in the assessment. The characteristics of infectious agents APPENDIX A and the primary laboratory hazards of working with the agent are described generically for agents in Bio- safety Levels 14 and specifically for individual agents or groups of agents on pages 87-88 and in Section V, respectively, of this Appendix. Hepatitis B virus (Kiev) is an appropriate model for illustrating the risk assessment process. Bv is among the most ubiquitous of human pathogens and most prevalent of laboratory-associated infections. The agent has been demonstrated in a variety of body secretions and excretions. Blood, saliva, and semen have been shown to be infectious. Natural transmis- sion is associated with parenteral inoculation or with contamination of the broken skin or of mucous mem- branes with infectious body fluids. There is no evi- dence of airborne or interpersonal spread through casual contact. Prophylactic measures include the use of a licensed vaccine in high-risk groups and the use of hepatitis B immune globulin following overt exposure. The primary risk of HBv infection in laboratory personnel is associated with accidental parenteral inoculation, exposure of the broken skin or mucous membranes of the eyes, nose, or mouth, or ingestion of infectious body fluids. These risks are typical of those described for Biosafety Level 2 agents and are addressed by using the recommended standard and special microbiological practices to minimize or elimi- nate these overt exposures. Hepatitis non-A non-B and AIDS acquired immune deficiency syndrome-pose similar infec- tion risks to laboratory personnel. The prudent prac- tices recommended for HBv are applicable to these two disease entities, as well as to the routine labora- tory manipulation of clinical materials of domestic . . Ongln. The described risk assessment process is also applicable to laboratory operations other than those involving the use of primary agents of human dis- ease. Microbiological studies of animal host-specific pathogens, soil, water, food, feeds, and other natural or manufactured materials, by comparison, pose sub- stantially lower risks of laboratory infection. Micro- biologists and other scientists working with such materials may nevertheless find the practices, con- tainment equipment, and facilities recommendations described in this publication of value in developing operational standards to meet their own assessed needs.
APPENDIX A AGENT SUMMARY STATEMENTS Parasitic Agents Agent: Nematode parasites of humans Laborawy-associated infections with Strongyloi- des spp. and hookworms have been reported (90~. Allergic reactions to various antigenic components of nematodes (e.g., aerosolized Ascaris antigens) may represent an individual risk to sensitized persons. Laboratory animal-associated infections (including arthropods) have not been reported, but infective larvae in the feces of nonhuman primates and of dogs infected with Strongyloides spp. are a potential in- fection hazard for laboratory and animal care person- nel. Laboratory hazards. Eggs and larvae in freshly passed feces of infected hosts are usually not infec- tive; development of the infective stages may take periods of 1 day to several weeks. Ingestion of the infective eggs or skin penetration of infective larvae are the primary hazards to laboratory and animal care personnel. Arthropods infected with fHarial parasites pose a potential hazard to laboratory personnel. In laboratory personnel with frequent exposure to aero- solized antigens of Ascaris spp., development of hypersensitivity is common. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities with infective stages of the parasites listed. Exposure to aerosolized sensitiz- ing antigens of Ascaris spp. should be avoided. Pri- mary containment (e.g., biological safety cabinet) may be required for work with these materials by hypersensitive individuals. Agent: Protozoal parasites of humans Laboratory-associated infections with Toxoplasma spp., Plasmodium spp. (including P. cynomologi), Trypanosoma spp., and Leishmania spp. have been reported (21, 49, 90, 100~. In addition, infections with Entamoeba histolytica, Giardia spp., and Coccidia spp. can result from ingestion of cysts in feces. Accidental laboratory infections as well as hu- man volunteer studies have proven the transmissibil- ity of P. cynomologi from nonhuman primates to humans via infected mosquitoes (403. Although labo 107 ratory animal-associated infections have not been reported, contact with lesion material from rodents with cutaneous leishmaniasis and with feces or blood of experimentally or naturally infected animals may be a direct source of infection for laboratory person- nel. Laboratory hazards. Infective stages may be present in blood, feces, lesion exudates, and infected arthropods. Depending on the parasite, accidental parenteral inoculation, transmission by arthropod vectors, skin penetration, and ingestion are the pri- mary laboratory hazards. Aerosol or droplet expo- sure of the mucous membranes of the eyes, nose, or mouth with trophozoites are potential hazards when working with cultures of Naegleria fawleri, Leish- mania spp., Trypanosoma cruzi, or tissue homoge- nates or blood containing hemoflagellates. Because of the grave consequence of toxoplasmosis in the developing fetus, women of childbearing age should be discouraged from working with viable Toxoplasma spp. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities with infective stages of the parasites listed. Infected arthropods should be maintained in facilities which reasonably preclude the exposure of personnel or their escape to the out- side. Primary containment (e.g., biological safety cabinet) or personal protection (e.g., face shield) may be indicated when working with cultures of T. cruzi, Leishmania, N. fawleri, or tissue homogenates or blood containing hemoflagellates. Gloves are recom- mended for activities where there is the likelihood of direct skin contact with infective stages of the para- sites listed. Agent: Trematode parasites of humans Laboratory-associated infections with Schis- tosoma spp. and Fasciola spp. have been reported none associated directly with laboratory animals (90~. Laboratory hazards. Infective stages of Schis- tosoma spp. (cercariae3 and Fasciola spp. (metacer- cariae) may be found, respectively, in the water or encysted on aquatic plants in laboratory aquaria used to maintain snail intermediate hosts. Skin penetration by schistosome cercariae and ingestion of fluke meta- cercariae are the primary laboratory hazards. Dissec- tion or crushing of schistosome-infected snails may
108 also result in exposure of skin or mucous membranes to cercariae-containing droplets. Additionally, meta- cercariae may be inadvertently transferred from hand to mouth by fingers or gloves following contact with contaminated aquatic vegetation or surfaces of aq- uaria. Most laboratory exposure to Schistosoma spp. would predictably result in low worm burdens with minimal disease potential. Safe and effective drugs are available for the treatment of schistosomiasis. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities with infective stages of the parasites listed. Gloves should be worn when there may be direct contact with water containing cercariae or vegetation containing metacercariae from naturally or experimentally infected snail intermedi- ate hosts. Snails and cercariae in the water of labora- tory aquaria should be killed by chemicals (e.g., hy- pochlorites, iodine) or heat before discharge to sew- ers. Agent: Cestode parasites of humans Although laboratory-associated infections with Ec hino coccus granulosus or Taenia so lium have not been reported, the consequences of such infections following the ingestion of infective eggs of T. solium or E. granulosus are potentially grave. Laboratory hazards. Infective eggs may be present in the feces of dogs or other canids (the definitive hosts of E. granulosus) or in the feces of humans (the definitive host of T. solium). Ingestion of infective eggs from these sources is the primary laboratory hard. Cysts and cyst fluids of E. gran- ulosus are not infectious for humans. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for work with infective stages of these parasites. Special attention should be given to per- sonal hygiene practices (e.g., handwashing) and avoid- ance of ingestion of infective eggs. Gloves are rec- ommended when there may be direct contact with feces or surfaces contaminated with fresh feces of dogs infected with E. granulosus or humans infected with T. solium adults. Fungal Agents Agent: Blastomyces dermatitidis Laboratory-associated local infections follow- ing accidental parenteral inoculation with infected APPENDIX A tissues or cultures containing yeast forms of B. der- matitidis (39, 54, 66, 103, 127) have been reported. A single pulmonary infection (asymptomatic) oc- curred following the presumed inhalation of conidia. Subsequently this individual developed an osteolytic lesion from which B. dermatitidis was cultured (30). Presumably, pulmonary infections are associated only with sporulating mold forms (conidia). Laboratory hazards. Yeast forms may be pres- ent in the tissues of infected animals and in clinical specimens. Parenteral (subcutaneous) inoculation of these materials may cause local granulomas. Mold form cultures of B. dermaizadis containing infectious conidia may pose a hazard of aerosol exposure. Recommended precautions. Biosafety Level 2 and Animal Biosafety Level 2 practices, containment equipment, and facilities are recommended for ac- tivities with clinical materials, animal tissues, and infected animals. Biosafety Level 3 practices, containment equip- ment, and facilities are recommended for processing mold cultures, soil, and other environmental materi- als known or likely to contain infectious conidia. Agent: Coccidioides immiNs Laboratory-associated coccidioidomycosis is a documented hazard (12, 28, 31, 32, 33, 64, 68, 79, 105, 106, 107~. Wilson et al. reported that 28 of 31 (90%) laboratory-associated infections in his institu- tion resulted in clinical disease, whereas more than half of infections acquired in nature were asympto- matic (128~. Laboratory hazards. Because of its size (2 to 5 ~m), the arthrospore is conducive to ready dispersal in air and retention in the deep pulmonary spaces. The much larger size of the spherule (30 to 60 ~m) considerably reduces the effectiveness of this form of the fungus as an airborne pathogen. Spherules of the fungus may be present in clini- cal specimens and animal tissues, and infectious arthrospores may be present in mold cultures and soil samples. Inhalation of althrospores from soil samples or mold cultures or following transformation from the spherule form in clinical materials is the primary laboratory hazard. Accidental percutaneous inocula- tion of the spherule form may result in local gran- uloma formation (118~. Disseminated disease may occur at a greater frequency in pregnant women, blacks, and Filipinos than in whites. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are
APPENDIX A recommended for handling and processing clinical specimens and animal tissues. Animal Biosafety Level 2 practices and facilities are recommended for ex- perimental animal studies when the route of chal- lenge is parenteral. Biosafety Level 3 practices and facilities are recommended for all activities with sporulating mold form cultures of C. i~rutis and for processing soil or other environmental materials known or likely to contain infectious arthrospores. Agent: Cryptococcus neoformans A single account of a laboratory exposure to C. neoformans as a result of a laceration by a scalpel blade heavily contaminated with encapsulated cells is reported (50~. This vigorous exposure, which did not result in local or systemic evidence of infection, suggests that the level of pathogenicity for normal immunocompetent adults is low. Respiratory infec- tions as a consequence of laboratory exposure have not been recorded. Laboratory hazards. Accidental parenteral in- oculation of cultures or other infectious materials represents a potential hazard to laboratory person- nel particularly to those that may be immunocom- promised. Bites by experimentally infected mice and manipulations of infectious environmental materials (e.g., pigeon droppings) may also represent a poten- tial hazard to laboratory personnel. Recommended precautions. Biosafety Level 2 and Animal Biosafety Level 2 practices, containment equipment, and facilities are recommended, respec- tively, for activities with known or potentially infec- tious clinical, environmental, or culture materials and with experimentally infected animals. The processing of soil or other environmental materials known or likely to contain infectious yeast cells should be conducted in a Class I or Class II biological safety cabinet. This precaution is also in- dicated for cultures of the perfect or sexual state of the agent. Agent: Histoplasma capsulatum Laboratory-as sociated his toplasmosis is a docu- mented hazard in facilities conducting diagnostic or investigative work (90, 91~. Pulmonary infections have resulted from handling mold form cultures (78~. Local infection has resulted from skin puncture dur- ing autopsy of an infected human (119) and from accidental needle inoculation of a viable culture (116~. 109 Collecting and processing soil samples from endemic areas have caused pulmonary infections in labora- tory workers. Spores are resistant to drying and may remain viable for long periods of time. The small size of the infective conidia (microconidia are less than 5 ~m) is conducive to airborne dispersal and intrapulmonary retention. Furcolow reported that 10 spores were almost as effective as a lethal inoculum in mice as 10,000 to 100,000 spores (45~. Laboratory hazards. The infective stage of this dimorphic fungus (conidia) is present in sporulating mold form cultures and in soil from endemic areas. The yeast form in tissues or fluids from infected animals may produce local infection following par- enteral inoculation. Recommended precautions. Biosafety Level 2 and Animal Biosafety Level 2 practices, containment equipment, and facilities are recommended for han- dling and processing clinical specimens and animal tissues and for experimental animal studies when the route of challenge is parenteral. Biosafety Level 3 practices and facilities are recommended for processing mold cultures, soil, or other environmental materials known or likely to contain infectious conidia. Agent: Sporothruc schencku S. schenclui has caused a substantial number of local skin or eye infections in laboratory personnel. Most cases have been associated with accidents and have involved splashing culture material into the eye (41, 125), scratching (13) or injecting (117) infected material into the skin, or being bitten by an experi- mentally infected animal (60, 61). Skin infections have resulted also from handling cultures (74, 81) or necropsy of animals (44) without a known break in technique. No pulmonary infections have been re- ported to result from laboratory exposure, although naturally occurring lung disease, albeit rare, is thought to result from inhalation. Recommended precautions. Biosafety Level 2 and Animal Biosafety Level 2 practices, containment equipment, and facilities are recommended for all laboratory and experimental animal activities with S. schenckii. Agents: Pathogenic members of the genera Ep- Mermophyton, Microsporum, and Trichophyton Although skin, hair, and nail infections by these dermatophytic molds are among the most prevalent
110 of human infections, the processing of clinical mate- rial has not been associated with laboratory infec- tions. Infections have been acquired through contacts with naturally or experimentally infected laboratory animals (mice, rabbits, guinea pigs, etc.) and, rarely, with handling cultures (71, 90, 51~. Laboratory hazards. Agents are present in the skin, hair, and nails of human and animal hosts. Contact with infected laboratory animals with inap- parent or apparent infections is the primary hazard to laboratory personnel. Cultures and clinical materials are not an important source of human infection. Recommended precautions. Biosafety Level 2 and Animal Biosafety Level 2 practices, containment equipment, and facilities are recommended for all laboratory and experimental animal activities with dermatophytes. Bacterial Agents Agent: Bacillus anthracis Forty (403 cases of laboratory-associated anthrax, occurring primarily at facilities conducting anthrax research, have been reported (38, 90~. No laboratory- associated cases of anthrax have been reported in the United States for more than 20 years. Naturally and experimentally infected animals pose a potential risk to laboratory and animal care personnel. Laboratory hazards. The agent may be present in blood, skin lesion exudates, and, rarely, in urine and feces. Direct and indirect contact of the intact and broken skin with cultures and contaminated labo- ratory surfaces, accidental parenteral inoculation, and, rarely, exposure to infectious aerosols are the pri- mary hazards to laboratory personnel. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities using clinical materials and diagnostic quantities of infectious cultures. Ani- mal Biosafety Level 2 practices and facilities are recommended for studies utilizing experimentally infected laboratory rodents. A licensed vaccine is available through the Centers for Disease Control; however, vaccination of laboratory personnel is not recommended unless frequent work with clinical specimens or diagnostic cultures is anticipated (e.g., animal disease diagnostic laboratory). Biosafety Level APPENDIX A 3 practices and facilities are recommended for work involving production volumes or concentrations of cultures and for activities which have a high poten- tial for aerosol production. In these facilities vacci- nation is recommended for all persons working with the agent, all persons working in the same laboratory room where Be cultures are handled, and persons working with infected animals. Agent: Brucella (B. abortus, B. cants, B. melitensis, B. suds) B. abortus, B. cants, B. melitensis, and B. suds have all caused illness in laboratory personnel (77, 90, 110~. Brucellosis is the most commonly reported laboratory-associated bacterial infection (903. Hy- persensitivity to Brucella antigens is also a hazard to laboratory personnel. Occasional cases have been attributed to expo- sure to experimentally and naturally infected animals or their tissues. Laboratory hazards. The agent may be present in blood, cerebrospinal fluid, semen, and occasion- ally urine. Most laboratory-associated cases have occurred in research facilities and have involved exposure to Brucella organisms being grown in large quantities. Direct skin contact with cultures or with infectious clinical specimens from animals (e.g., blood, uterine discharges) are also commonly impli- cated. Aerosols generated during laboratory proce- dures have caused large outbreaks (59~. Mouth pipet- ting, accidental parenteral inoculations, and sprays into eyes, nose, and mouth have also resulted in - intection. Recommended precautions. Biosafety Level 2 practices are recommended for activities with clini- cal materials of human or animal origin containing or potentially containing pathogenic Brucella spp. Bio- safety Level 3 and Animal Biosafety Level 3 prac- tices, containment equipment, and facilities are rec- ommended, respectively, for all manipulations of cultures of the pathogenic Brucella spp. listed in this summary and for experimental animal studies. Vac- cines are not available for use in humans. Agent: Chlamydia psittaci, C. trachomatis Infections with psittacosis, lymphogranuloma venereum (LGV), and trachoma are documented
APPENDIX A hazards and the fifth most commonly reported labm ratory-associated bacterial infection. The majority of cases were of psittacosis, occurred before 1955, and had the highest case-fatality rate of all groups of infectious agents (90~. Contact with and exposure to infectious aerosols in the handling, care, or necropsy of naturally or experimentally infected birds are the major sources of laboratory-associated psittacosis. Infected mice and eggs are less impmant sources of C. psittaci. Laboratory animals are not a reported source of human infection with C. trachomatis. Laboratory hazards. C. psittaci may be present in the tissues, feces, nasal secretions, and blood of infected birds and in blood, sputum, and tissues of infected humans. C. trachomatis may be present in genital, bubo, and conjunctival fluids of infected humans. Exposure to infectious aerosols and droplets created during the handling of infected birds and tissues is the primary hazard to laboratory personnel working with psittacosis. The primary laboratory hazards of C. trachomatis are accidental parenteral inoculation and direct and indirect exposure of mu- cous membranes of the eyes, nose, and mouth to genital, bubo, or conjunctival fluids, cell culture ma- terials, and fluids from infected eggs. Infectious aero- sols may also pose a potential source of infection. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities involving the necropsy of infected birds and the diagnostic examination of tissues of cultures known to be infected or poten- tially infected with C. psittaci or C. trachomatis. Wetting the feathers of infected birds with a deter- gent-disinfectant prior to necropsy can appreciably reduce the risk of aerosols of infected feces and nasal secretions on the feathers and external surfaces of the bird. Animal Biosafety Level 2 practices and facili- ties and respiratory protection are recommended for personnel working with caged birds naturally or ex- perimentally infected. Gloves are recommended for the necropsy of birds and mice, the opening of inocu- lated eggs, and when there is the likelihood of direct skin contact with infected tissues, bubo fluids, and other clinical materials. Additional primary contain- ment and personnel precautions, such as those rec- ommended for Biosafety Level 3, may be indicated for activities with high potential for droplet or aero- sol production and for activities involving produc- tion quantities or concentrations of infectious materi- als. Vaccines are not available for use in humans. Agent: Clostridium botulinum While there are no reported cases of botulism associated with the handling of the agent or toxin in the laboratory or working with naturally or experi- mentally infected animals, the consequences of such intoxications would be grave. Laboratory hazards. C. botulinum or its toxin may be present in a variety of food products, clinical materials (serum, feces), and environmental samples (soil, surface water). Exposure to the toxin of C. botulinum is the primary laboratory hazard. The toxin may be absorbed after ingestion or following contact with the skin, eyes, or mucous membranes, including the respiratory tract. Accidental parenteral inocula- tion may also represent a significant exposure to toxin. Broth cultures grown under conditions of opti- mal toxin production may contain 2 x 106 mouse LDSo per ml (111~. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities with materials known to contain or potentially containing the toxin. A pen- tavalent (ABCDE) botulism toxoid is available through the Centers for Disease Control as an Inves- tigational New Drug (IND). This toxoid is recom- mended for personnel working with cultures of C. botulinum or its toxins. Solutions of sodium hydrox- ide (0.1 N) readily inactivate the toxin and are rec- ommended for decontaminating work surfaces and spills of cultures or toxin. Additional primary con- tainment and personnel precautions, such as those recommended for Biosafety Level 3, may be indi- cated for activities with a high potential for aerosol or droplet production, those involving production quantities of toxin, and those involving purif~ed tox- ins. Animal Biosafety Level 2 practices and facilities are recommended for diagnostic studies and titration ot tox~n. Agent: Clostridium tetani Although the risk of infection to laboratory per- sonnel is negligible, Pike (90) has recorded five inci- dents related to exposure of personnel during ma- nipulation of the toxin. Laboratory hazards. Accidental parenteral in- oculation and ingestion of the toxin are the primary hazards to laboratory personnel. Since tetanus toxin is poorly absorbed through mucous membranes, aero
112 sots and droplets probably represent minimal haz- ards. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities involving the manipula- tion of cultures or toxin. While the ris`; of laboratory- associated tetanus is low, the administration of an adult diphtheria-tetanus toxoid at 10-year intervals may further reduce the risk to laboratory and animal care personnel of toxin exposures and wound con- tamination (24~. Agent: Corynebacterium diphthenae Laboratory-associated infections with C. diphth- eriae are documented. Pike (90) lists 33 cases re- ported in the world literature. Laboratory animal-associated infections have not been reported. Laboratory hazards. The agent may be present in exudates or secretions of the nose, throat (tonsil), pharynx, larynx, and wounds, in blood, and on the skin. Inhalation, accidental parenteral inoculation, and ingestion are the primary laboratory hazards. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infected clinical materials or cultures. Animal Biosafety Level 2 facilities are recommended for studies utilizing infected laboratory animals. While the risk of laboratory-associated diphtheria is low, the administration of an adult diphtheria-tetanus toxoid at 10-year intervals may further reduce the risk to laboratory and animal care personnel of toxin exposures and work with infectious materials (24~. Agent: Francisella tularensis Tularemia is the third most commonly reported laboratory-associated bacterial infection (90~. Almost all cases occurred at facilities involved in tularemia research. Occasional cases have been related to work with naturally or experimentally infected animals or their ectoparasites. Laboratory hazards. The agent may be present in lesion exudate, respiratory secretions, cerebrospi- nal fluid, blood, urine, tissues from infected animals, and fluids from infected arthropods. Direct contact of skin or mucous membranes with infectious mate- nals, accidental parenteral inoculation, ingestion, and APPENDIX A exposure to aerosols and infectious droplets have resulted in infection. Cultures have been more com- monly associated with infection than have clinical materials and infected animals. The human 25-50% infectious dose is on the order of 10 organisms by the respiratory route (121~. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities with clinical materials of human or animal origin containing or potentially containing F. tularensis. Biosafety Level 3 and Ani- mal Biosafety Level 3 practices and facilities are recommended, respectively, for all manipulations of cultures and for experimental animal studies. An in- vestigational live attenuated vaccine (103 is available through the Centers for Disease Control and is rec- ommended for persons working with the agent or with infected animals and for persons working in or entering the laboratory or animal room where cul- tures or infected animals are maintained. Agent: Leptospira interrogans all serovars Leptospirosis is a well-documented laboratory hazard. Sixty-seven laboratory-associated infections and 10 deaths have been reported (903. An experimentally infected rabbit was identified as the source of an infection with L. interrogans serovar icterohemorrhagiae (97~. Direct and indirect contact with fluids and tissues of experimentally or naturally infected mammals during handling, care, or necropsy are potential sources of infection. In ani- mals with chronic kidney infections, the agent is shed in the urine in enormous numbers for long peri- ods of time. Laboratory hazards. The agent may be present in urine, blood, and tissues of infected animals and humans. Ingestion, accidental parenteral inoculation, and direct and indirect contact of skin or mucous membranes with cultures or infected tissues or body fluids-especially urine-are the primary laboratory hazards. The importance of aerosol exposure is not known. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities involving the use or manipulation of known or potentially infectious tis- sues, body fluids, and cultures and for the housing of infected animals. Gloves are recommended for the handling and necropsy of infected animals and when
APPENDIX A there is the likelihood of direct skin contact with infectious materials. Vaccines are not available for use in humans. Agent: Legio)lella pneumophila; other Legionella- like agents A single documented nonfatal laboratory-asso- ciated case of legionellosis due to presumed aerosol or droplet exposure during animal challenge studies with Pontiac fever agent (L. pneumophila) is recorded (16~. Human-to-human spread has not been docu- mented. Experimental infections are readily produced in guinea pigs and embryonated chicken eggs (72~. Challenged rabbits develop antibodies but not clini- cal disease. Mice are refractory to parenteral expo- sure. Unpublished studies by Kaufmann, Feeley, and others at the Centers for Disease Control have shown that animal-to-animal transmission did not occur in a variety of experimentally infected mammalian and · - avlan species. Laboratory hazards. The agent may be present in pleural fluids, tissue, sputa, and environmental sources (e.g., cooling tower water). Since the natural mode of transmission appears to be airborne, the greatest potential hazard is the generation of aerosols during the manipulation of cultures or of other con centrations of infectious materials (e.g., infected yolk sacs and tissues.) Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities involving the use or manipulation of known or potentially infectious clini cal materials or cultures and for the housing of in fected animals. Primary containment devices and equipment (e.g., biological safety cabinets, centri fuge safety cups) should be used for activities likely to generate potentially infectious aerosols. Vaccines are not available for use in humans. Agent: Mycobacterium leprae Inadvertent parenteral human-to-human trans mission of leprosy following an accidental needle stick in a surgeon (69) and the use of a presumably contaminated tattoo needle (87) have been reported. There are no cases reported as a result of working in a laboratory with biopsy or other clinical materials of human or animal origin. While naturally occurring ~3 leprosy or leprosy-like diseases have been reported in armadillos (120) and in nonhuman primates (35, 76), humans are the only known important reservoir of this disease. Laboratory hazards. The infectious agent may be present in tissues and exudates from lesions of infected humans and experimentally or naturally infected animals. Direct contact of the skin and mu- cous membranes with infectious materials and acci- dental parenteral inoculation are the primary labora- tory hazards associated with handling infectious clinical materials. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities with known or poten- tially infectious clinical materials from infected humans and animals. Extraordinary care should be taken to avoid accidental parenteral inoculation with contaminated sharp instruments. Animal Biosafety Level 2 practices and.facilities are recommended for animal studies utilizing rodents, armadillos, and nonhuman primates. Agent: Mycobacterium spp. other than M. tubercu- losis, M. bovis, or M. Ieprae Pike reported 40 cases of nonpulmonary "tuber- culosis" thought to be related to accidents or inci- dents in the laboratory or autopsy room (90~. Pre- sumably these infections were due to mycobacteria other than M. tuberculosis or M. bovis. A number of mycobacteria which are ubiquitous in nature are as- sociated with diseases other than tuberculosis or lep- rosy in humans, domestic animals, and wildlife. Char- acteristically, these organisms are infectious but not contagious. Clinically, the diseases associated with infections by these `'atypical" mycobacteria can be divided into three general categories: 1. Pulmonary diseases resembling tubercu- losis which may be associated with infection with M. kansasii, M. avium complex, and, rarely, with M. xenopi, M. malmoense, M. asiaticum, M. simiae, and M. szalgai 2. Lymphadenitis which may be associated with infection with M. scrofulaceum, M. avium complex, and, rarely, with M. fortuitum and M. kan- sasii 3. Skin ulcers and soft tissue wound infec- tions which may be associated with infection with
114 M. ulcerans, M. marinum, M. fortuitum, and M. che- lonei Laboratory hazards. The agents may be pres- ent in sputa, exudates from lesions, tissues, and envi- ronmental samples (e.g., soil and water). Direct con- tact of skin or mucous membranes with infectious materials, ingestion, and accidental parenteral inocu- lation are the primary laboratory hazards associated with clinical materials and cultures. Infectious aero- sols created during the manipulation of broth cul- tures or tissue homogenates of these organisms asso- ciated with pulmonary disease also pose a potential infection hazard to laboratory personnel. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities with clinical materials and cultures of Mycobacterium spp. other than M. tuberculosis or M. bovis. Animal Biosafety Level 2 practices and facilities are recommended for animal studies with the mycobacteria other than M. tubercu- losis, M. bovis, or M. Ieprae. Agent: Mycobacterium Tuberculosis, M. bovis M. tuberculosis and M. bovis infections are a proven hazard to laboratory personnel as well as to others who may be exposed to infectious aerosols in the laboratory (90, 93~. The incidence of tuberculosis in laboratory workers working with M. tuberculosis is three times higher than that of laboratorians not working with the agent (95~. Naturally or experimen- tally infected nonhuman primates are a proven source of human infection (e.g., the annual tuberculin con- version rate in personnel working with infected nonhu- man primates is about 70/10,000 compared with less than 3/10,000 in the general population (62~. Experi- mentally infected guinea pigs or mice do not pose the same problem, since droplet nuclei are not produced by coughing in these species; however, litter from infected animals may become contaminated and serve as a source of infectious aerosols. Laboratory hazards. Tubercle bacilli may be present in sputum, gastric ravage fluids, cerebrospi- nal fluid, urine, and lesions from a variety of tissues (3~. Exposure to laboratory-generated aerosols is the most important hazard encountered. Tubercle bacilli may survive in heat-fixed smears (1) and may be aerosolized in the preparation of frozen sections and during manipulation of liquid cultures. Because of APPENDIX A the low infectious dose of M. tuberculosis for hu- mans (i.e., 50% infectious dose equals <10 bacilli) (98, 99) and in some laboratories a high rate of isolation of acid-fast organisms from clinical speci- mens (~10%) (47), sputa and other clinical speci- mens from suspected or known cases of tuberculosis must be considered potentially infectious and handled with appropriate precautions. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities (see American Thoracic Society laboratory service levels I and II) (2, 65) are recommended for preparing acid- fast smears and for culturing sputa or other clinical specimens, provided that aerosol-generating manipu- lations of such specimens are conducted in a Class I or II biological safety cabinet. Liquification and con- centration of sputa for acid-fast staining may also be conducted on the open bench at Biosafety Level 2 by first treating the specimen with an equal volume of 5% sodium hypochlorite solution (undiluted house- hold bleach) and waiting 15 min before centrifuga- tion (85, 108~. Biosafety Level 3 practices, containment equip- ment, and facilities (see American Thoracic Society laboratory service level II~ (2, 65) are recommended for activities involving the propagation and manipu- lation of cultures of M. tuberculosis or M. bovis and for animal studies utilizing nonhuman primates ex- perimentally or naturally infected with M. tuberculo- sis or M. bovis. Animal studies utilizing guinea pigs or mice can be conducted at Animal Biosafety Level 2. Skin testing with purified protein derivative (PPD) of previously skin-tested-negative laboratory person- nel can be used as a surveillance procedure. A li- censed attenuated live vaccine (BCG) is available but is not routinely used in laboratory personnel. Agent: Neisseria gonorrhoeae Four cases of laboratory-associated gonorrhea have been reported in the United States (34, 90~. Laboratory hazards. The agent may be present in conjunctival, urethral, and cervical exudates, syno- vial fluid, urine, feces, and cerebrospinal fluid. Acci- dental parenteral inoculation and direct or indirect contact of mucous membranes with infectious clini- cal materials are the primary laboratory hazards. The importance of aerosols is not determined. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are
APPENDIX A recommended for all activities involving the use or manipulation of clinical materials or cultures. Gloves should be worn when handling infected laboratory animals and when there is the likelihood of direct skin contact with infectious materials. Additional primary containment and personnel precautions, such as those described for Biosafety Level 3, may be indicated for aerosol or droplet production and for activities involving production quantities or concen- trations of infectious materials. Vaccines are not avail- able for use in humans. Agent: Neissena meningitis Meningococcal meningitis is a demonstrated but rare hazard to laboratory workers (4, 92~. Laboratory hazards. The agent may be present in pharyngeal exudates, cerebrospinal fluid, blood, and saliva. Parenteral inoculation, droplet exposure of mucous membranes, and infectious aerosol and ingestion are the primary hazards to laboratory per- sonnel. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious body fluids and tissues. Addi- tional primary containment and personnel precau- tions, such as those described for Biosafety Level 3, may be indicated for activities with high potential for droplet or aerosol production and for activities in- volving production quantities or concentrations of infectious materials. The use of licensed polysaccha- ride vaccines (19) should be considered for person- nel regularly working with large volumes or high concentrations of infectious materials. Agent: Pseudomonas pseudomallei Two laboratory-associated cases of melioidosis are reported, one associated with a massive aerosol and skin contact exposure (48), the second resulting from an aerosol created during the open-flask sonica- tion of a culture presumed to be Pseudomonas ce- pacia (102~. Laboratory hazards. The agent may be present in sputa, blood, wound exudates, and various tissues, depending on site of localization of the infection. Direct contact with cultures and infectious materials from humans, animals, or the environment, inges- tion, autoinoculation, and exposure to infectious aero ~5 sots and droplets are the primary laboratory hazards. The agent has been demonstrated in blood, sputum, and abscess materials and may be present in soil and water samples from endemic areas. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious body fluids and tissues. Gloves should be worn when handling, and during necropsy of, infected animals and when there is the likelihood of direct skin contact with infectious materials. Ad- ditional primary containment and personnel precau- tions, such as those described for Biosafety Level 3, may be indicated for activities with a high potential for aerosol or droplet production and the activities involving production quantities or concentrations of infectious materials. Agent: Salmonella cholera-suds, S. enteritiJdis all serotypes Salmonellosis is a documented hazard to labora- tory personnel (90~. Primary reservoir hosts include a broad spectrum of domestic and wild animals in- cluding birds, mammals, and reptiles, all of which may serve as a source of infection to laboratory per- sonnel. Laboratory hazards. The agent may be present in feces, blood, and urine and in food, feed, and environmental materials. Ingestion or parenteral in- oculation are the primary laboratory hazards. The importance of aerosol exposure is not known. Natu- rally or experimentally infected animals are a poten- tial source of infection for laboratory and animal care personnel and for other animals. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities with clinical materials known to contain or potentially containing the agents. Animal Biosafety Level 2 practices and facilities are recommended for activities with experimentally or naturally infected animals. Agent: Salmonella Phi Typhoid fever is a demonstrated hazard to labo- ratory personnel (7, 92~. Laboratory hazards. The agent may be present in feces, blood, gallbladder (bile), and urine. Humans are the only known reservoir of infection. Ingestion
116 and parenteral inoculation of the organism represent the primary laboratory hazards. The importance of aerosol exposure is not known. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious clinical materials and cultures. Licensed vaccines, which have been shown to protect 7~90% of recipients, may be a valuable ad- junct to good safety practices in personnel regularly working with cultures or clinical materials which may contain S. Phi (7~. Agent: Shigella spp. Shigellosis is a demonstrated hazard to labora- tory personnel, with 49 cases reported in the United States (90~. While outbreaks have occurred in captive nonhuman primates, humans are the only significant reservoir of infection. Experimentally infected guinea pigs, other rodents, and nonhuman primates are a proven source of infection. Laboratory hazards. The agent may be present in feces, and, rarely, in blood of infected humans or animals. Ingestion and parenteral inoculation of the agent are the primary laboratory hazards. The oral 25-50% infectious dose of S. fle~cneri for humans is on the order of 200 organisms (122~. The importance of aerosol exposure is not known. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious clinical materials or cultures. Animal Biosafety Level 2 facilities and practices are recommended for activities with experimentally or naturally infected animals. Vaccines are not avail- able for use in humans. Agent: Treponema pallidum Syphilis is a documented hazard to laboratory personnel who handle or collect clinical material from cutaneous lesions. Pike lists 20 cases of laboratory- associated infection (90~. Humans are the only known natural reservoir of the agent. No cases of laboratory animal-associated infec- tions are reported; however, rabbit-adapted strains of T. pallidum (Nichols and possibly others) retain their virulence for humans. APPENDIX A Laboratory hazards. The agent may be present in materials collected from primary and secondary cutaneous lesions and in blood. Accidental paren- teral inoculation and contact of mucous membranes or broken skin with infectious clinical materials (and, perhaps, infectious aerosols) are the primary hazards to laboratory personnel. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities involving the use or manipulation of blood or lesion materials from hu- mans or infected rabbits. Gloves should be worn when there is a likelihood of direct skin contact with lesion materials. Periodic serological monitoring should be considered in personnel regularly working with infectious materials. Vaccines are not available for use in humans. Agent: Vibrionic enteritis (Campylobacter fetus subsp. jejuni, Vibrio cholerae, V. parahaemoly- ticus) Vibrionic enteritis due to C. fetus jejuni; V. ch~l- erae, or V. parahaemolyticus is a documented but rare cause of laboratory-associated illnesses (92~. Naturally and experimentally infected animals are a potential source of infection (94~. Laboratory hazards. All pathogenic vibrios may occur in feces. C. fetus may also be present in blood, exudates from abscesses, tissue, and sputa. Ingestion of V. cholerae and ingestion or parenteral inocula- tion of other vibrios constitute the primary labora- tory hazard. The human oral infecting dose of V. cholerae in healthy non-achlorhydric individuals is of the order of 108 organisms (94~. The importance of aerosol exposure is not known. The risk of infec- tion following oral exposure may he Increased In achlorhydric individuals. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities with cultures or poten- tially infectious clinical materials. Animal Biosafety Level 2 practices and facilities are recommended for activities with naturally or experimentally infected animals. Although vaccines have been shown to pro- vide partial protection of short duration (3-6 months) to nonimmune individuals in highly endemic areas (7), the routine use of cholera vaccine in laboratory staff is not recommended.
APPENDIX A Agent: Yersinia pestis Plague is a proven but rare laboratory hazard. Four cases have been reported in the United States (1 1, 90~. Laboratory hazards. The agent may be present in bubo fluid, blood, sputum, cerebrospinal fluid, feces, and urine from humans, depending on the clini- cal form and stage of the disease. Direct contact with cultures and infectious materials from humans or rodents, infectious aerosols or droplets generated during the manipulation of cultures and infected tis- sues and in the necropsy of rodents, accidental auto- inoculation, ingestion, and bites by infected fleas collected from rodents are the primary hazards to laboratory personnel. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities involving the han- dling of potentially infectious clinical materials and cultures. Special care should be taken to avoid the generation of aerosols of infectious materials and during the necropsy of naturally or experimentally infected rodents. Gloves should be worn when han- dling field-collected or infected laboratory rodents and when there is the likelihood of direct skin con- tact with infectious materials. Necropsy of rodents is ideally conducted in a biological safety cabinet. A1- though field trials have not been conducted to deter- mine the efficacy of licensed inactivated vaccines, experience with these products has been favorable (14~. Immunization is recommended for personnel working regularly with cultures of Y. pestis or in- fected rodents (26~. Additional primary containment and personnel precautions, such as those described for Biosafety Level 3, are recommended for activities with high potential for droplet or aerosol production, for work with antibiotic-resistant strains, and for activities involving production quantities or concentrations of infectious materials. Rickettsial Agents Agent: Coxiella burned) Pike's summary indicates that Q fever is the second most commonly reported laboratory-associ- ated infection, with outbreaks involving 15 or more ~7 persons recorded in several institutions (903. A broad range of domestic and wild mammals are natural hosts for Q fever and may serve as potential sources of infection for laboratory and animal care person- nel. Exposure to naturally infected and often asymp- tomatic sheep and to their birth products is a docu- mented hazard to personnel (20, 109~. The agent is remarkably resistant to drying and is stable under a variety of environmental conditions (121~. Laboratory hazards. The agent may be present in infected arthropods and in the blood, urine, feces, milk, or tissues of infected animal or human hosts. The placenta of infected sheep may contain as many as 109 organisms per gram of tissue, and milts may contain 10S organisms per gram. Parenteral inocula- tion and exposure to infectious aerosols and droplets are the most likely sources of infection for laboratory and animal care personnel. The estimated human 25- 50% infectious dose (inhalation) for Q fever is 10 organisms (122~. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for nonpropagative laboratory proce- dures, including serological examinations and stain- ing of impression smears. Biosafety Level 3 prac- tices and facilities are recommended for activities involving the inoculation, incubation, and harvesting of embryonated eggs or tissue cultures, the necropsy of infected animals, and the manipulation of infected tissues. Since infected guinea pigs and other rodents may shed the organisms in urine or feces (90), ex- perimentally infected rodents should be maintained under Animal Biosafety Level 3. Recommended pre- cautions for facilities using sheep as experimental animals are described by Spinelli (109) and by Ber- nard (63. An Investigational New Phase-1 Q fever vaccine (IND) is available from theU.S. Army Medi- cal Research Institute for Infectious Diseases, Fort Detrick, Maryland. The use of this vaccine should be limited to those at high risk of exposure who have no demonstrated sensitivity to Q fever antigen. Agent Rickettsia Atari, Rochalimaea quintana, and Rochalimaea vinsonu Based on the experience of laboratories actively working with Rickettsia akari, it is likely that the five cases of rickettsialpox recorded by Pike (90) were associated with exposure to bites of infected mites
118 rather than aerosol or contact exposure to infected tissues. There are no recorded cases of laboratory- associated infections with trench fever (Rochalimaea quintana) or vole rickettsia (Rochalimaea vinsonii). Laboratory hazards. The agent of nckettsialpax may be present in blood and other tissues of infected house mice or humans and in the mite vector Lipo- nyssoides sanguineus. Exposure to naturally or ex- perimentally infected mites and accidental parenteral inoculation are the most likely sources of human infection with rickettsialpox. The agent of trench fever may be present in the blood and tissues of infected humans and in the body fluids and feces of infected human body lice (Pediculus h. hurnanus). Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for propagation and animal studies with Rickettsia akari, Rochalimaea vinsonii, and Rochalimaea quintana. Appropriate precautions should be taken to avoid exposure of personnel to infected mites that are maintained In the laboratory or that may be present on naturally infected house mice. Agent: Rickettsia pro wazekii, Rickettsia O?phi (R. mooseri), Rickettsia tsutsugamushi, Rickettsia Canada, and Spotted Fever Group agents of hu- man disease other than Rickettsia rickettsii and Rickettsia Atari Pike reported 57 cases of laboratory-associated typhus (type not specified), 56 cases of epidemic APPENDIX A recommended for nonpropagative laboratory proce- dures, including serological and fluorescent-antibody procedures, and for the staining of impression smears. Biosafety Level 3 practices and facilities are recom- mended for all other manipulations of known or po- tentially infectious materials, including necropsy of experimentally infected animals and tnturation of their tissues and inoculation, incubation, and har- vesting of embryonated eggs or tissue cultures. Ani- mal Biosafety Level 2 practices and facilities are recommended for activities with infected mammals other than flying squirrels or arthropods. Vaccines are not currently available for use in humans. Be- cause the mode of transmission of R. prowazekii from flying squirrels to humans is not defined, Ani- mal Biosafety Level 3 practices and facilities are recommended for animal studies with flying squir- rels naturally or experimentally infected with R. . · ~ prowaze~u. Agent: Rickettsia rickettsii Rocky Mountain spotted fever is a documented hazard to laboratory personnel. Pike (90) reported 63 laboratory-associated cases, 11 of which were fatal. Oster (86) reported nine cases occurring over a ~ year period in one laboratory which were believed to have been acquired as a result of exposure to infec tious aerosols. Laboratory hazards. Accidental parenteral in oculation and exposure to infectious aerosols are the most likely sources of laboratory-associated infec typhus with three deaths, and 68 cases of murine lion (57~. Successful aerosol transmission has been typhus (90~. More recently, three cases of murine experimentally documented in nonhuman primates typhus were reported from a research facility (18~. (101~. Naturally and experimentally infected mam Two of these three cases were associated with work with infectious materials on the open bench; the third case resulted from an accidental parenteral inocula tion. These three cases represented an attack rate of 20% in personnel working with infectious materials. Laboratory hazards. Accidental parenteral in oculation and exposure to infectious aerosols are the most likely sources of laboratory-associated infec tions. Naturally or experimentally infected lice, fleas, and flying squirrels (GIaucomys spp.) (9) may also be a direct source of infection to laboratory person nel. The organisms are relatively unstable under ambient environmental conditions. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are mars, their ectoparasites, and their infected tissues are sources of human infection. The organism is relatively unstable under ambient environmental conditions. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all nonpropagative laboratory procedures, including serological and fluorescent- antibody tests, and staining of impression smears. Biosafety Level 3 practices and facilities are recom- mended for all other manipulations of Mown or potentially infectious materials, including necropsy of experimentally infected animals and Hituration of their tissues and inoculation, incubation, and har- vesting of embryonated eggs or tissue cultures. Ani
APPENDIX A mat Biosafety Level 2 practices and facilities are recommended for holding of experimentally infected rodents; however, necropsy and any subsequent manipulation of tissues from infected animals should be conducted at Biosafety Level 3. Because of the proven value of antibiotic ther- apy in the early stages of infection, it is essential that laboratories working with R. ric/`ettsii have an effec- tive system for reporting febrile illnesses in labora- tory personnel, medical evaluation of potential cases, and, when indicated, institution of appropriate anti- biotic therapy. Vaccines are not currently available for use in humans (see Appendix A.3~. Viral Agents Agent: Hepatitis A virus Laboratory-associated infections with hepatitis A virus do not appear to be an important occupa- tional risk among laboratory personnel. However, the disease is a documented hazard in animal han- dlers and others working with chimpanzees which are naturally or experimentally infected (92~. Laboratory hazards. The agent may be present in feces of infected humans and chimpanzees. Inges- tion of feces, stool suspensions, and other contami- nated materials is the primary hazard to laboratory personnel. The importance of aerosol exposure has not been demonstrated. Attenuated or avirulent strains have not been fully defined but appear to result from serial passage in tissue culture. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities with known or poten- tially infected feces from humans or chimpanzees. Animal Biosafety Level 2 practices and facilities are recommended for activities using naturally or ex- perimentally infected chimpanzees. Animal care per- sonnel should wear gloves and take other appropriate precautions to avoid possible fecal-oral exposure. - . vaccines are not available for use in humans, but are in the developmental stages. Agent: Hepatitis B. hepatitis non-A non-B Pike concluded that hepatitis B is currently the most frequently occurring laboratory-associated in- fection (90~. The incidence in some categories of laboratory workers is seven times greater than that of ~9 the general population (104~. Epidemiological evi- dence indicates that hepatitis non-A non-B is a blood- borne disease similar to hepatitis B. Laboratory hazards. The agent of hepatitis B may be present in blood and blood products of hu- man origin and in urine, semen, cerebrospinal fluid, and saliva. Parenteral inoculation, droplet exposure of mucous membranes, and contact exposure of bro- ken skin are the primary laboratory hazards. The virus may be stable in dried blood or blood compo- nents for several days. Attenuated or avirulent strains are not defined. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious body fluids and tissues. Addi- tional primary containment and personnel precau- tions, such as those described for Biosafety Level 3, may be indicated for activities with high potential for droplet or aerosol production and for activities in- volving production quantities or concentrations of infectious materials. Animal Biosafety Level 2 prac- tices, containment equipment, and facilities are rec- ommended for activities utilizing naturally or experi- mentally infected chimpanzees or other nonhuman primates. Gloves should be worn when working with infected animals and when there is the likelihood of skin contact with infectious materials. A licensed inactivated vaccine is available and is recommended for laboratory personnel, who are at substantially greater risk of acquiring infection than is the general population (27~. Agent: Herpesvirus sundae (B-virus) Although B-virus presents a potential hazard to laboratory personnel working with the agent, labora- tory-associated human infections with B-virus have, with rare exceptions, been limited to personnel hav- ing direct contact with living Old World monkeys (29, 56, 89~. Exposure to in vitro monkey tissues (i.e., primary rhesus monkey kidney) has been asso- ciated with a single documented case (29~. B-virus is an indigenous chronic and/or recur- rent infection of macaques and possibly other Old World monkeys and is a frequent enzootic infection of captive Macaca mulatta. Laboratory personnel handling Old World mon- keys run the risk of acquiring B-virus from a bite or contamination of broken skin or mucous membranes
120 by an infected monkey. Fifteen fatal cases of human infections with B-virus have been reported (29~. Laboratory hazards. The agent may be present in oral secretions, thoracic and abdominal viscera, and central nervous system tissues of naturally in- fected macaques. Bites from monkeys with oral her- pes lesions are the greatest hazard to laboratory and animal care personnel. Exposures of broken skin or mucous membranes to oral secretions or to infec- tious culture fluids are also potential hazards. The importance of aerosol exposure is not known. At- tenuated or avirulent strains have not been defined. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities involving the use or manipulation of tissues, body fluids, and primary tissue culture materials from macaques. Additional containment and personnel precautions, such as those recommended for Biosafety Level 3, are recom- mended for activities involving the use or manipula- tion of any material known to contain H. simiae. Biosafety Level 4 practices, containment equip- ment, and facilities are recommended for activities involving the propagation of H. simiae, manipula- tions of production quantities or concentrations of H. sirruae, and housing vertebrate animals with proven natural or induced infection with the agent. The wearing of gloves, masks, and laboratory coats is recommended for all personnel working with nonhuman primates-especially macaques and other Old World species-and for all persons entering ani- mal rooms where nonhuman primates are housed. Vaccines are not available for use in humans. Agent: Herpesviruses The herpesviruses are ubiquitous human patho- gens and are commonly present in a variety of clini- cal materials submitted for virus isolation. While these viruses are not demonstrated causes of labora- tory-associated infections, they are primary as well as opportunistic pathogens, especially in immuno- compromised hosts. Nonpolio enteroviruses, ade- noviruses, and cytomegalovirus pose similar low potential infection risks to laboratory personnel. A1- though this diverse group of indigenous viral agents does not meet the criteria for inclusion in agent- specific summary statements (i.e., demonstrated or high potential hazard for laboratory-associated infec- tion; grave consequences should infection occur), the APPENDIX A frequency of their presence in clinical materials and their common use in research warrants their inclu- sion in this publication. Laboratory hazards. Clinical materials and isolates of herpesviruses, nonpolio enteroviruses, and other indigenous pathogens may pose a risk of infec- tion following ingestion, accidental parenteral inocu- lation, droplet exposure of the mucous membranes of the eyes, nose, or mouth, or inhalation of concen- ~ated aerosolized materials. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities utilizing known or po- tentially infectious clinical materials or cultures of indigenous viral agents which are associated with or identified as a primary pathogen of human disease. Although there is no definitive evidence that infec- tious aerosols are a significant source of laboratory- associated infections, it is prudent to avoid the gen- eration of aerosols during the handling of clinical materials and isolates or during the necropsy of ani- mals. Primary containment devices (e.g., biological safety cabinets) constitute the basic barrier protect- ing personnel from exposure to infectious aerosols. Agent: Influenza virus Laboratory-associated infections with influenza are not normally documented in the literature but are known to occur by informal accounts and punished reports, particularly when new strains showing anti- genic drift or shift are introduced into a laboratory for diagnostic or research purposes (36~. Laboratory animal-associated infections are not reported; however there is a high possibility of hu- man infection from infected ferrets and vice versa. Laboratory hazards. The agent may be present in respiratory tissues or secretions of man or most infected animals and in the cloaca of many infected avian species. The virus may be disseminated in multiple organs in some infected animal species. Inhalation of virus from aerosols generated by aspirating, dispensing, or mixing virus-infected samples or by infected animals is the primary labora- tory hazard. Genetic manipulation of virus has un- known potential for altering host range and patho- genicity or for introducing into man transmissible . . . . viruses wit ~ nove antigenic composition. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are
APPENDIX A recommended when receiving and inoculating rou- tine laboratory diagnostic specimens. Autopsy mate- rial should be handled in a biological safety cabinet using Biosafety Level 2 procedures. Activities utilizing noncontemporary virus strains. Biosafety considerations should take into account the available information about infectious- ness of the strains being used and the potential for harm to the individual or society in the event that laboratory-acquired infection and subsequent trans- mission occur. Research or production activities util- izing contemporary strains may be safely performed using Biosafety Level 2 containment practices. Sus- ceptibility to infection with older noncontemporary human strains, with recombinants, or with animal isolates warrants the use of Biosafety Level 2 con- tainment procedures. Current experience suggests, however, that there is no evidence for laboratory- acquired infection with reference strains A/PR/8/34 and A/WS/33 or their commonly used neurotropic variants. Agent: Lymphocytic choriomeningitis (L CM) virus Laboratory-associated infections with lympho- cytic choriomeningitis virus are well documented in facilities where infections occur in laboratory ro- dents, especially mice and hamsters (S. 90~. Tissue cultures which have inadvertently become infected represent a potential source of infection and dissemi- nation of the agent. Natural infections are occasion- ally found in nonhuman primates, swine, and dogs. Laboratory hazards. The agent may be present in blood, cerebrospinal fluid, urine, secretions of the nasopharynx, feces, and tissues of infected humans and other animal hosts. Parenteral inoculation, inha- lation, and contamination of mucous membranes or broken skin with infectious tissues or fluids from infected animals are common hazards. Aerosol trans- mission is well documented (8~. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious body fluids or tissues and for tissue culture passage of mouse brain-passaged strains. All manipulations of known or potentially infectious passage and clinical materials should be conducted in a biological safety cabinet. Additional primary containment and personnel precautions such as those 121 described for Biosafety Level 3 may be indicated for activities with high potential for aerosol production and for activities involving production quantities or concentrations of infectious materials. Animal Bio- safety Level 2 practices and facilities are recom- mended for studies in adult mice with mouse brain- passaged strains. Animal Biosafety Level 3 practices and facilities are recommended for work with in- fected hamsters. Vaccines are not available for use in humans. Agent: Poliovirus Laboratory-associated infections with polio- viruses are uncommon and are generally limited to unvaccinated laboratory persons working directly with the agent. Twelve cases have been reported in the world literature (90~. Laboratory animal-associated infections have not been reported (73~; however, naturally or experimen- tally infected nonhuman primates could provide a source of infection to exposed unvaccinated persons. Laboratory hazards. The agent may be found in the feces and in throat secretions. Ingestion and parenteral inoculation of infectious tissues or fluids by unimmunized personnel are the primary hazards to laboratory personnel. The importance of aerosol exposure is not known. Laboratory exposures pose negligible risk to appropriately immunized persons. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious culture fluids and specimen materials. All laboratory personnel working directly with the agent must have documented polio vaccina- tion or demonstrated serologic evidence of immunity to all three poliovirus types (25~. Agent: Poxviruses Sporadic cases of laboratory-associated poxvirus infections have been reported. Pike lists 24 cases of yaba and tanapox vines infection and 18 vaccinia and smallpox infections (90~. Epidemiological evidence suggests that transmission of monkeypox virus from nonhuman primates or rodents to humans may have occurred in nature but not in the laboratory setting. Naturally or experimentally infected laboratory ani- mals are a potential source of infection to exposed unvaccinated laboratory personnel.
122 Laboratory hazards. The agents may be pres- ent in lesion fluids or crusts, respiratory secretions, or tissues of infected hosts. Ingestion, parenteral in- oculation, and droplet or aerosol exposure of mucous membranes or broken skin to infectious fluids or tissues are the primary hazards to laboratory and animal care personnel. Some poxviruses are stable at ambient temperature when dried and may be trans- mitted by fomites. Recommended precautions. The possession and use of variola viruses are restricted to the World Health Organization Collaborating Center for Small- pox and Other Poxvirus Infections located at the Centers for Disease Control, Atlanta, Georgia. Bio- safety Level 2 practices, containment equipment, and facilities are recommended for all activities involv- ing the use or manipulation of poxviruses other than variola that pose an infection hazard to humans. All persons working in or entering laboratory or animal care areas where activities with vaccinia, monkeypod, or cowpox viruses are being conducted should have documented evidence of satisfactory vaccination within the preceding 3 years (23~. Activities with vaccinia, cowpox, or monkeypox viruses in quanti- ties or concentrations greater than those present in diagnostic cultures may also be conducted by immu- nized personnel at Biosafety Level 2, provided that all manipulations of viable materials are conducted in Class I or II biological safety cabinets or other primary containment equipment. Agent: Rabies virus Laboratory-associated rabies infections are ex- tremely rare. Two have been documented. Both resulted from presumed exposure to high-titered in- fectious aerosols generated in a vaccine production facility (129) and a research facility (17~. Naturally or experimentally infected animals, their tissues, and their excretions are a potential source of exposure to laboratory and animal care personnel. Laboratory hazards. The agent may be present in all tissues of infected animals. Highest titers are demonstrated in central nervous system tissue, sali- vary glands, and saliva. Accidental parenteral inocu- lation, cuts, or sticks with contaminated laboratory equipment, bites by infected animals, and exposure of mucous membranes or broken skin to infectious droplets of tissue or fluids are the most likely source of exposure for laboratory and animal care person APPENDIX A net. Infectious aerosols have not been a demonstrated hazard to personnel working with clinical materials and conducting diagnostic examinations. Fixed and attenuated strains of virus are presumed to be less hazardous, but the only two recorded cases of labora- tory-associated rabies resulted from exposure to a fixed challenge virus standard (CVS) derived from SAD (Street Alabama Dufferin) strain (17, 129~. Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious materials. Preexposure immu- nization is recommended for all individuals working with rabies virus or infected animals or engaged in diagnostic, production, or research activities with rabies virus. Preexposure immunization is also rec- ommended for all individuals entering or working in the same room where rabies virus or infected animals are used. When it is not feasible to open the skull or remove the brain within a biological safety cabinet, it is pertinent to wear heavy protective gloves to avoid cuts or sticks from cutting instruments or bone frag- ments, and to wear a face shield to protect the mu- cous membranes of the eyes, nose, and mouth from exposure to infectious droplets or tissue fragments. If a Stryker saw is used to open the skull, avoid striking the brain with the blade of the saw. Additional pri- mary containment and personnel precautions, such as those described for Biosafety Level 3, may be indicated for activities with a high potential for drop- let or aerosol production and for activities involving production quantities or concentrations of infectious materials. Agents: Transmissible spongiform encephalopa- thies (Creutzfeldt-Jakob and kuru agents) Laboratory-associated infections with the trans- missible spongiform encephalopathies have not been documented. The consequences of infection are grave, however, and there is evidence that Creutzfeldt-Jakob disease has been transmitted to patients by comeal transplant and by contaminated electroencephalo- graphic electrodes. There is no known nonhuman reservoir for Creutzfeldt-Jakob disease or kuru. Nonhuman primates and other laboratory animals have been infected by inoculation, but there is no evidence of secondary transmission. Laboratory hazards. High titers of a transmis- sible agent have been demonstrated in the brain and
APPENDIX A spinal cord of persons with kuru. In persons with Creutzfeldt-Jakob disease, a transmissible agent has been demonstrated in the brain, spleen, liver, lymph nodes, lungs, spinal cord, kidneys, cornea, and lens. Accidental parenteral inoculation, especially with neural tissues, and including Formalin-f~ed speci- mens, is extremely hazardous. Although nonneural tissues are less often infective, all tissues of humans and animals infected with these agents should be considered potentially hazardous. The risk of infec- tion from aerosols and droplets and from exposure to intact skin and gastric and mucous membranes is not known; however, there is no evidence of contact or aerosol transmission. These agents are characterized by extreme resistance to conventional inactivation procedures, including irradiation, boiling, and chemi- cals (Formalin, betapropiolactone, alcohols). Recommended precautions. Biosafety Level 2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious tissues and fluids from natu- rally infected humans and from experimentally in- fected animals. Extreme care must be taken to avoid accidental autoinoculation or other traumatic paren- teral inoculations of infectious tissues and fluids (463. Although there is no evidence to suggest that aerosol transmission occurs in the natural disease, it is pru- dent to avoid the generation of aerosols or droplets during the manipulation of tissues and fluids and during the necropsy of experimental animals. It is further recommended that gloves should be worn for activities which provide the opportunity for skin contact with infectious tissues and fluids. Vaccines are not available for use in humans. Agent: Vesicular stomatitis virus (VSV) Forty-six laboratory-associated infections with indigenous strains of VSV have been reported (112~. Laboratory activities with indigenous strains of VSV present two different levels of risk to laboratory per- sonnel and are related, at least in part, to the passage history of the strains utilized. Activities utilizing in- fected livestock, their infected tissues, and virulent isolates from these sources are a demonstrated haz- ard to laboratory and animal care personnel (52, 88~. Seroconversion and clinical illness rates in personnel working with these materials are high (88~. Similar risks may be associated with exotic strains such as Piry (112~. 123 In contrast, anecdotal information indicates that activities with less virulent laboratory-adapted strains (e.g., VSV-Indiana [San Juan and Glasgow]) are rarely associated with seroconversion or illness. Such strains are commonly used by molecular biologists, often in large volumes and high concentrations, under condi- tions of minimal or no primary containment. Experi- mentally infected mice have not been a documented source of human infection. Laboratory hazards. The agent may be present in vesicular fluid, tissues, and blood of infected ani- mals and in blood and throat secretions of infected humans. Exposure to infectious aerosols and infected droplets, direct skin and mucous membrane contact with infectious tissues and fluids, and accidental auto- inoculation are the primary laboratory hazards asso- ciated with virulent isolates. Accidental parenteral inoculation and exposure to infectious aerosols rep- resent potential risks to personnel working with less virulent laboratory-adapted strains. Recommended precautions. Biosafety Level 3 practices, containment equipment, and facilities are recommended for activities involving the use or manipulation of infected tissues and of virulent iso- lates from naturally or experimentally infected live- stock. Gloves and respiratory protection are recom- mended for the necropsy and handling of infected animals. Biosafety Level 2 practices and facilities are recommended for activities utilizing laboratory- adapted strains of demonstrated low virulence. Vac- cines are not available for use in humans. ARBOVIRUSES Arboviruses Assigned to Biosafety Level 2 The American Committee on Arthropod-Borne Viruses (ACAV) registered 424 arboviruses as of December 31, 1979. The ACAV's Subcommittee on Arbovirus Laboratory Safety (SALS) has categorized each of these 424 agents into one of four recom- mended levels of practice and containment which parallel the recommended practices, safety equip- ment, and facilities described in this publication as Biosafety Levels 1 - 4 (112~. It is the intent of SALS to periodically update the 1980 publication by pro- viding a supplemental listing and recommended lev- els of practice and containment for arboviruses regis- tered since 1979. SALS categorizations were based on risk assessments from information provided by a
124 worldwide survey of SXS laboratories working with arboviruses. SALS recommended that work with the majority of these agents should be conducted at the equivalent of Biosafety Level 2. These viruses are listed alphabetically on pages 124-126 and include the following agents which are the reported cause of laboratory-associated infections (53, 90, 112~. Virus Cases (SALS) Vesicular stomatitis Colorado tick fever Dengue Pichinde Western equine encephalomyelitis Rio Bravo Kunjin Catu Caraparu Ross River Bunyamwera Eastern equine encephalomyelitis Zika Apeu Mmituba Tacaribe Muructucu O'nyong nyong Modoc Oriboca Ossa Keystone Bebaru Bluetongue 46 16 11 17 7 (2 deaths) 7 6 6 s s 4 4 4 2 2 2 The list of arboviruses in Biosafety Level 2 includes yellow fever virus (17D strain) and Venezuelan equine encephalomyelitis (VEE) virus (TCX3 strain), pro- vided that personnel working with these vaccine strains are immunized. The results of the SALS survey clearly indicate that the suspected source of the laboratory-associ- ated infections listed above was other than exposure to infectious aerosols. Recommendations that work with these 334 arboviruses should be conducted at Biosafety Level 2 were based on the existence of adequate historical laboratory experience to assess risks for the virus which indicated that: (a) no overt APPENDIX A laboratory-associated infections are reported; or (b) infections resulted from exposures other than to in- fectious aerosols; or (c) if aerosol exposures are docu- mented they represent an uncommon route of expo- sure. Laboratory hazards. Agents listed in this group may be present in blood, cerebrospinal fluid, central nervous system and other tissues, and infected ar- thropods, depending on the agent and the stage of infection. While the primary laboratory hazards are accidental parenteral inoculation, contact of the virus with broken skin or mucous membranes, and bites by infected laboratory rodents or arthropods, infectious aerosols may also be a potential source of infection. Arboviruses Assigned to Biosafety Level 2 Abu Hammad Acado Acara Aguacate Alfuy Almpiwar Amapari Anhanga Anhembi Anopheles A Anopheles B Apeu Apoi Aride Arkonam Aruac Arumowot Aura Avalon Bagaza Bahig Bakau Baku Bandia Bangoran Bangui Banzi Barur Batai Batu Bauline Bebaru Belmont Bertioga Bimiti Birao Bluetongue (indigenous) Boraceia Botambi Boteke Bouboui Bujaru Bunyamwera Burg el Arab Bushbush Bussuquara Buttonwillow Bwamba Cacao Cache Valley Caimito Califomia encephalitis Calovo Candiru Cape Wrath Capim Caraparu Carey Island Catu Chaco Chagres Chandipura Changuinola Charleville
APPENDIX A 125 Chenuda Irituia Lokem Pacui Chilibre Isfahan Lone Star Pahayokee Chobar Itaporanga Lukuni Palyam Gorge Itaqui M'poko Parana CloMor Jamestown Madrid Pata Colorado Tick Canyon Maguan Pathum Thani Fever Japanaut Mahogany Patois Comparta Jerry Slough Hammock Phnom-Penh bat Cotia Johnston Atoll Main Drain Pichinde Cowbone Ridge Joinjakak ~Malakal Pixuna D'Aguilar Juan Diaz Manawa Pongola Dakar Bat Jugra Manzanilla Pretoria Dengue-1 Jurona Mapputa Puchong Dengue-2 Jutiapa Maprik Punta Salinas Dengue-3 Kadam M~o Punta Toro Dengue4 Kaeng khoi Marituba Qalyub Dera Ghazi Khan Kaikalur Matariya Quaranfil Eastern equine Kaisodi Matruh Restan encephalomyeli- Kamese Matucare Rio Bravo tis Kammamavanpet- Melao Rio Grande Edge Hill tai Mermet Ross River Entebbe Bat Kannamangalam Minatitlan Royal Farm Epizootic Kao shuan Mima1 Sabo hemorrhagic Karimabad Mirim Saboya disease Karshi Mitchell River Saint Floris Eubenangee Kasba Modoc Sakhalin Eyach Kemervo Moju Salehabad Flanders Kem Canyon Mono Lake San Angelo Fort Morgan Ketapang Mont. myotis leuk. Sandfly F. (Naples) Frijoles Keterah Moriche Sandfly F. (Sicilian) Gamboa Keuraliba Mossuril Sandjimba Gomoka Keystone Mount Elgon bat Sathuperi Gossas Klamath Murutucu Sawgrass Grand Arbaud Kokobera Navarro Sebokele Great Island Kolongo Nepuyo Seletar Guajara Koongol Ngaingan Sembalam Guama Kowanyama Nique Shamonda Guaroa Kunjim Nkolbisson Shark River Gumbo limbo Kununurra Nola Shuni Hart Park Kwatta Ntaya Silverwater Hazara La Crosse Nugget Simbu Huacho Lagos bat Nyamanini Simian hemorrhagic Hughes La Joya Nyando fever Icoaraci Landjia O'nyong-nyong Sindbis Ieri Langat Okhotskiy Sixgun City Iheus Lanjan Okola Snowshoe hare Ilesha Latino Olifantsvlei Sokuluk Ingwavuma Lebombo Oriboca Soldado Inkoo Le Dantec Ossa Sororoca Ippy Lipovnik Pacora Stratford
26 APPENDIX A Sunday Canyon Thottapalayam Urucuri Whamro Tacaiuma Timbo Usutu Witwatersrand Tacaribe Toure Uukuniemi Wongal Taggert Tribec Vellore Wongorr Tahyna Triniti Venezuelan equine Wyeomyia Tamiami Trivittatus encephalomyelitis Yaquina Head Tanga Trubanaman =-83) Yata Tanjong Rabok Tsuruse Venkatapuram Yellow fever Tataguine Turlock Vesicular stomatitis (17D) Tembe Tyuleniy (seep. 123) Yogue Tembusu Uganda S Wad Medani Zaliv Terpeniya Tensaw Umatilla Wallal Zegla Tete Umbre Wanowrie Zika Tettnang Una Warrego Zingilamo Thimiri Upolu Western equine Zirqa encephalomyelitis Recommended precautions. Biosafety Level 2 practices, safety equipment, and facilities are recom- mended for activities with potentially infectious clini- cal materials and arthropods and for manipulations of infected tissue cultures, embryonated eggs, and rodents. Infection of newly hatched chickens with eastern and western equine encephalomyelitis viruses is especially hazardous and should be undertaken under Biosafety Level 3 conditions by immunized personnel. Investigational vaccines ~ND) against eastern equine encephalomyelitis and western equine encephalomyelitis viruses are available th~ough the Centers for Disease Control and the U.S. Army Medi- cal Research Institute for Infectious Diseases, re- spectively. The use of these vaccines is recommended for personnel who work directly and regularly with these two agents in the laboratory. Western equine encephalomyelitis immune globulin (human) is also available from the Centers for Disease Control. The eff~cacy of this product has not been established. Arboviruses and Arenaviruses Assigned to Bio- safety Level 3 SALS has recommended that work with the ar- boviruses included in the alphabetical listing on page 127 should be conducted at the equivalent of Bio- safety Level 3 practices, safety equipment, and fa- cilities. These recommendations are based on one of the following criteria: overt laboratory-associated in- fections with these agents have occurred by aerosol route if protective vaccines are not used or are un available, or laboratory experience with the agent is inadequate to assess risk and the natural disease in humans is potentially severe or life threatening or causes residual damage. Hantaan virus, which was not included in the SALS publication, has been placed at Biosafety Level 3 based on documented labora- tory-associated infections. Rift Valley fever virus, which was classified by SALS at Containment Level 3 (i.e., HEPA filtration required for all air exhausted from the laboratory), was placed in Biosafety Level 3 provided that all personnel entering the laboratory or animal care area where work with this virus is being conducted are vaccinated. Laboratory or labo- ratory animal-associated infections have been reported wi~ the following agents (53, 90, 112, 124~. virus Cases (SALS) Venezuelan equine encephalitis Rift Valley fever Chikungunya Yellow fever Japanese encephalitis Louping ill West Nile Lymphocytic choriomeningitis Orungo P~' Wesselsbron Mucambo Oropouche Germiston 150 (1 death) 47 (1 death) 39 38 22 22 18 15 13 13 13 10 7 6 (8 dea~s)
APPENDIX A Bhanja Hantaan (Korean hemorrhagic fever) Mayaro Spondweni St. Louis encephalitis Murray Valley encephalitis Semliki Forest Powassan Dugbe Issyk-kul Koutango 6 6 5 4 4 3 3 (1 death) 2 2 1 1 Large quantities and high concentrations of Semliki Forest virus are commonly used or manipu- lated by molecular biologists under conditions of moderate or low containment. Although antibodies have been demonstrated in individuals working with this virus, the first overt (and fatal) laboratory-asso- ciated infection with this virus was reported in 1979 (1263. Because this infection may have been influ- enced by a compromised host, an unusual route of exposure or high dosage, or a mutated strain of the virus, this case and its outcome may not be typical. Since exposure to an infectious aerosol was not indi- cated as the probable mode of transmission in this case, it is suggested that most activities with Semliki Forest disease virus can be safely conducted at Bio- safety Level 2. Some viruses (e.g., Ibaraki, Israel turkey menin- goencephalitis) are listed by SALS in Level 3, not because they pose a threat to human health, but be- cause they are exotic diseases of domestic livestock or poultry. Laboratory hazards. The agents listed in this group may be present in blood, cerebrospinal fluid, urine, and exudates, depending on the specific agent and stage of disease. The primary laboratory hazards are exposure to aerosols of infectious solutions and animal bedding, accidental parenteral inoculation, and broken skin contact. Some of these agents (e.g., Vene- zuelan equine encephalitis EVEE]) may be relatively stable in dried blood or exudates. Attenuated strains are identified in a number of the agents listed (e.g., yellow fever 17D strain and VEE TCS3 strain). Recommended precautions. Biosafety Level 3 practices, containment equipment, and facilities are recommended for activities using potentially infec- tious clinical materials and infected tissue cultures, animals, or arthropods. |27 A licensed attenuated live virus is available for immunization against yellow fever and is recom- mended for all personnel who work with this agent or with infected animals and for those who enter rooms where He agents or infected animals are pres- ent. An investigational vaccine (IND) is available for immunization against VEE and is recommended for all personnel working with VEE (and the related Ev- erglades, Mucambo, Tonate, and Cabassou viruses) or infected animals or entering rooms where these agents or infected animals are present. Work with Hantaan (Korean hemorrhagic fever) virus in rats, voles, and other laboratory rodents should be con- ducted with special caution (Biosafety Level 4~. An inactivated, investigational new Rift Valley fever vaccine ~ND) is available from the U.S. Army Medi- cal Research Institute for Infectious Diseases and recommended for all laboratory and animal care per- sonnel working with the agent or infected animals and for all personnel entering laboratories or animal rooms when the agent is in use. Arboviruses Assigned to Biosafety Level 3 Aino Akabane Araguali Batama Batken Bhanja Bimbo Bluetongue (exotic)a Bobaya Bobia Buenaventura CabassouC ChikungunyaC Chim Cocal Dhori Dugbe EvergladesC Garba GermistonC Getah Gordil Guaratuba Ibaraki Ihangapi . · - 1nm1 Israel turkey meningoenceph- alitis Issyk-kul Itaituba Japanese encepha- litis Kairi Khasan Korean hemor- rhagic fever (Hantaan) Koutango Kyzlagach Louping illa Lymphocytic choriom . . . emngltls Mayaro Middelburg Mosqueiro MucamboC Murray Valley encephalitis Nariva Ndumu
128 Negishi New Minto Nodamura Northway OropoucheC Orungo Ouango Oubangui Paramushir Pirate Ponteves Powassan Razdan Rift Valley fevered C Rochambeau RocioC Sagiyama Sakpa Salanga Santa Rosa Saumarez Reef Semliki Forest Sepik Serra do Navio Slovakia Spondweni St. Louis encephalitis Tamdy Telok Forest Thogoto Tlacotalpan TonateC Venezuelan equine encephalomye- 1itisc VSV-Alagoas Wesselsbrona C West Nile Yellow fevef zingaa~b~c a lhe importation, possession, or use of this agent is re- stricted by USDA regulation or administrative policy. See Ap- pendi~c A.5. b Zinga ~us is now recognized as being identical to Rift Valley fever wrus. c SALS mcommends that woric with this agent should be conducted only in Biosafety Level 3 facilities which provide for HEPA filtration of all exhaust air pr~or to discharge from the laborato~y. All persons wodting with agents for which a vaccine is available dhould be immw~zed. Arboviruses, Arenaviruses, or Filoviruses Assigned to Biosafety Level 4 SALS has recommended that work with the ar- boviruses, arenaviruses, or filoviruses (63) included in the listing that follows should be conducted at the equivalent of Biosafety Level 4 practices, safety equipment, and facilities. These recommendations are based on documented cases of severe and fre- quently fatal naturally occurring human infections and aerosol-transmitted laboratory infections. Addi- tionally, SALS recommended that certain agents with a close or identical antigenic relationship to the Bio- safety Level 4 agents (e.g., Absettarov and Kumlinge viruses) also be handled at this level until sufficient laboratory experience is obtained to retain these agents at this level or to work with them at a lower level. APPENDIX A Laboratory or laboratory animal-associamd infections have been reported with the following agents (37, 53, SX, 67, 90, 1 12, 12~. Virus Cases (SALS) Kyasanur Forest disease Hypr Junin Marburg Russian spring-summer encephalitis Congo-Crimean hemorrhagic fever Omsk hemmorrhagic fever Lassa Machupo Ebola 133 37 (2 deaths) 21 (1 death) 25 (5 deaths) 8 X (1 death) 2 (1 death) 1 (1 death) Rodents are natural reservoirs of Lassa fever vws (Mastomys natalensis), Junin and Machupo vwses (Calomys spp.), and perhaps other viruses assigned to Biosafety Level 4. Nonhuman primates were associated with the initial outbreaks of Kyas- anur Forest disease (Presbytis spp.) and Marburg disease (Cercopithecus spp.), and arthropods are the natural vectors of the tick-borne encephalitis com- plex agents. Work with or exposure to rodents, nonhu- man primates, or vectors naturally or experimentally infected with these agents represents a potential source of human infection. Laboratory hazards. The infectious agents may be present in blood, urine, respiratory and throat secretions, semen, and tissues from human or animal hosts and in arthropods, rodents, and nonhuman pri- mates. Respiratory exposure to infectious aerosols, mucous membrane exposure to infectious droplets, and accidental parenteral inoculation are the primary hazards to laboratory or animal care personnel (67, 124~. Recommended precautions. Biosafety Level 4 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious materials of human, animal, or arthropod origin. Clinical specimens from persons suspected of being infected with one of the agents listed in this summary should be submitted to a labo- ratory with a Biosafety Level 4 maximum contain- ment facility (22~.
APPENDIX A Arboviruses, Arenaviruses, and Filoviruses Assigned to Biosafety Level 4 Congo-Crimean hemorrhagic fever Marburg Tick-borne encephalitis virus complex (Absettarov, Ebola Hanzalova, Hypr, Kumlinge, Junin Kyasanur Forest disease, Lassa Omsk hemorrhagic fever, and Machupo Russian spring-summer encephalitis) APPENDIX A.1. BIOLOGICAL SAFETY CABINETS Biological safety cabinets are among the most effective, as well as the most commonly used, pri- mary containment devices in laboratories working with infectious agents. Each of the three types Class I, II, and III has performance characteristics which are described below. In addition to the design, construction, and performance standards for vertical laminar flow biological safety cabinets (Class III), the National Sanitation Foundation has also devel- oped a list of such products which meet the reference standard. Utilization of this standard (80) and list should be the first step in selection and procurement of a biological safety cabinet. Class I and II biological safety cabinets, when used in conjunction with good microbiological tech- niques, provide an effective partial containment sys- tem for safe manipulation of moderate and high-risk microorganisms (i.e., Biosafety Level 2 and 3 agents). Both Class I and II biological safety cabinets have comparable inward face velocities (75 linear ft/min) and provide comparable levels of containment in protecting the laboratory worker and the immediate laboratory environment from infectious aerosols generated within the cabinet. It is imperative that Class I and II biological safety cabinets are tested and certified in situ at the time of installation within the laboratory, at any time the cabinet is moved, and at least annually thereafter. Certification at locations other than the final site may attest to the performance capability of the individual cabinet or model but does not supersede the critical certification prior to use in the laboratory. As with any other piece of laboratory equip 129 meet, personnel must be trained in the proper use of the biological safety cabinets. The slide-sound train- ing firm developed by NIH (Effective Use of the Laminar Flow Biological Safety Cabinet) provides a thorough training and orientation guide. Of par~cu- lar note are those activities which may disrupt the inward directional airflow through the work opening of Class I and II cabinets. Repeated insertion and withdrawal of the workers' arms in and from the work chamber, opening and closing doors to the laboratory or isolation cubicle, improper placement or operation of materials or equipment within the work chamber, or briskly walking past the biological safety cabinet while it is in use are demonstrated causes of He escape of aerosolized particles from within the cabinet. Strict adherence to recommended practices for the use of biological safety cabinets is as important in attaining the maximum containment capability of the equipment as is the mechanical per- formance of the equipment itself. Horizontal laminar flow "clean benches" are present in a number of clinical, pharmacy, and labo- ratory facilities. These "clean benches" provide a high-quality environment within the work chamber for manipulation of nonhazardous materials. Cau- tion: Since the operator sits in the immediate down- stream exhaust from the "clean bench," this equip- ment must never be used for the handling of toxic, infectious, or sensitizing materials. Class I. The Class I biological safety cabinet (Figure A.1) is an open-fronted, negative-pressure, ventilated cabinet with a minimum inward face ve- locity at the work opening of at least 75 ft/min. The exhaust air from the cabinet may be used in three operational modes: with a full-width open front, with an installed front closure panel not equipped with gloves, and with an installed front closure panel equipped with arm-length rubber gloves. Class II. The Class II vertical laminar-flow bio- logical cabinet (Figure A.2) is an open-fronted, ven- tilated cabinet with an average inward face velocity at the work opening of at least 75 ft/min. This cabinet provides a HEPA-filtered, recirculated mass airflow within the work space. The exhaust air from the cabinet is also filtered by PAPA filters. Design, con- struction, and performance standards for Class II cabinets have been developed by and are available from the National Sanitation Foundation, Ann Ar- bor, Michigan (80).
130 1~ ~-11 ~4J~ ~ ~ FIGURE A.1 Class I biological safety cabinet 1~ 1 rim ~ ~ ~ ~ 114~1 \ / ~ 1': Lit ~.~ Type A 1 1 ~-~,1 At\ \N 11 ...1- 1... ,: ~. ~'' ~ A. ~ I, ~ ~ ../.. ,..../ .::....'. . NEW li ~ ~ t ~ ;~1 \ ~\ ~ \\ IL I I I I ~ 11411 ~ , W~ ll Type B FIGURE A.2 Class II biological safety cabinets APPENDIX A Class III. The Class III cabinet (Figure A.3) is a totally enclosed ventilated cabinet of gas-tight con- s~uction. Operations within the Class III cabinet are conducted through attached nabber gloves. When in use, the Class III cabinet is maintained under nega- tive air pressure of at least 0.5 in. water gauge. Sup- ply air is drawn into the cabinet through HEPA fil- ters. The cabinet exhaust air is filtered by two HEPA falters, installed in series, before discharge outside of the facility. The exhaust fan for the Class III cabinet is generally separate from the exhaust fans of the facility's ventilation system. Use of cabinets. Personnel protection provided by Class I and Class II cabinets is dependent on the inward airflow. Since the face velocities are similar, they generally provide an equivalent level of person- nel protection. The use of these cabinets alone, however, is not appropriate for containment of highest-risk infectious agents because aerosols may accidentally escape through the open front. The use of a Class II cabinet in the microbiologi- cal laboratory offers the additional capability and advantage of protecting materials contained within it from extraneous airborne contaminants. This capa- bility is provided by the HEPA-filtered, recirculated mass airflow within the work space. The Class III cabinet provides the highest level of personnel and product protection. This protection is provided by the physical isolation of the space in which the infectious agent is maintained. When these cabinets are required, all procedures involving infec 1 7W 5Z5Z l Was 11 ~ \ f TV d, itt O O O O FIGURE AN Class m biological safety cabinet
APPENDIX A tious agents are contained within them. Several Class III cabinets are therefore typically set up as an inter- connected system. All equipment required by the laboratory activity, such as incubators, refrigerators, and centrifuges, must be an integral part of the cabi- net system. Double-doored autoclaves and chemical dunk tanks are also attached to the cabinet system to allow supplies and equipment to be safely introduced and removed. Personnel protection equivalent to that provided by Class III cabinets can also be obtained with a personnel suit area and Class I or Class II cabinets. This area is one in which the laboratory worker is protected from a potentially contaminated environ- ment by a one-piece positive-pressure suit ventilated by a life-support system. This area is entered through an airlock fitted with airtight doors. A chemical shower is provided to decontaminate the surfaces of the suit as the worker leaves the area. The exhaust air from the suit area is filtered by two HEPA units installed in series. APPENDIX A.2. IMMUNOPROPHYLAXIS An additional level of protection for at-risk per- sonnel may be achieved with appropriate prophylactic vaccinations. A written organizational policy which defines at-risk personnel, which specifies risks as well as benefits of specific vaccines, and which dis- tinguishes between required and recommended vaccines is essential. In developing such an organiza- tional policy, these recommendations and require- ments should be specifically targeted at infectious diseases known or likely to be encountered in a par- ticular facility. Vaccines for which the benefits (levels of antibody considered to be protective) clearly exceed the risks (local or systemic reactions) should be re- quired for all clearly identified at-risk personnel. Examples of such preparations include vaccines against yellow fever, rabies, and poliomyelitis. Rec- ommendations for giving less efficacious vaccines, those associated with high rates of local or systemic reactions, or those that produce increasingly severe reactions with repeated use should be carefully con- sidered. Products with these characteristics (e.g., cholera, tularemia, and typhoid vaccines) may be recommended but should not ordinarily be required 131 for employment. A complete record of vaccines re- ceived on the basis of occupational requirements or recommendations should be maintained in the em- ployee's permanent medical file. Recommendations for the use of vaccines, adapted from those of the Public Health Service Advisory Committee on Immunization Practices, are included in the agent summary statements in Section V. APPENDIX Am. SURVEILLANCE OF PERSONNEL FOR LABORATORY ASSOCIATED RICKETTSIAL INFECTIONS Under natural circumstances, the severity of dis- ease caused by rickettsial agents varies considerably. In the laboratory, very large inocula which might produce unusual and perhaps very serious responses are possible. Surveillance of personnel for labora- tory-associated infections with rickettsial agents can dramatically reduce the risk of serious consequences of disease. Recent experience indicates that infections treated adequately with specific anti-rickettsial chemother- apy on the first day of disease do not generally pres- ent serious problems. Delay in instituting appropriate chemotherapy, however, may result in debilitating or severe acute disease ranging from increased periods of convalescence in typhus and scrub typhus to death in R. rickettsii infections. The key to reducing the severity of disease from laboratory-associated infec- tions is a reliable surveillance system which includes (1) round-the-clock availability of an experienced medical officer, (2) indoctrination of all personnel into the potential hazards of working with rickettsial agents and advantages of early therapy, (3) a report- ing system for all recognized overt exposures and accidents, (4) the reporting of all febrile illnesses, especially those associated with headache, malaise, prostration, when no other certain cause exists, and (5) a nonpunitive atmosphere that encourages report- ing of any febrile illness. Rickettsial agents can be handled in the labora- tory with minimal real danger to life when an ade- quate surveillance system complements a staff who are knowledgeable about the hazards of rickettsial infections and who put to use the safeguards recom- mended in the agent summary statements.
132 APPENDIX A.4. IMPORTATION AND INTERSTATE SHIPMENT OF HUMAN PATHOGENS AND RELATED MATERIALS The importation or subsequent receipt of etio- logic agents and vectors of human disease is subject to the Public Health Service Foreign Quarantine Regulations (42 C:FR, Section 71.56~. Permits au- thorizing the importation or receipt of regulated ma- terials and specifying conditions under which the agent or vector is shipped, handled, and used are issued by the Centers for Disease Control. The interstate shipment of indigenous etiologic agents, diagnostic specimens, and biological prod- ucts is subject to applicable packaging, labeling, and shipping requirements of the Interstate Shipment of Etiologic Agents (42 CFR, Part 72~. Packaging and labeling requirements for interstate shipment of etio- logic agents are summarized and illustrated in Figure A.4. Additional information on the importation and interstate shipment of etiologic agents of human dis- ease and other related materials may be obtained by writing to: Centers for Disease Control Attention: Office of Biosafety 1600 Clifton Road, N.E. Atlanta, GA 30333 Telephone: (404) 329-3883 FTS: 23~3883 APPENDIX A.5. RESTRICTED ANIMAL PATHOGENS Nonindigenous pathogens of domestic livestock and poultry may require special laboratory design, operation, and containment features not generally addressed in this publication. The importation, pos- session, or use of the following agents is prohibited or restricted by law or by U.S. Department of Agri- culture regulations or administrative policies: African horse sickness virus African swine fever virus Besnoitia besnoia Boma disease virus APPENDIX A Bovine ephemeral fever Bovine infectious petechial fever agent Camelpax virus Foot and mouth disease virus Fowl plague virus Histoplasma (Zymonema) ~ . . larc~m~nosum Hog cholera virus Louping ill virus Lumpy skin disease virus Mycoplasma agalactiae Mycoplasma mycoides Nairobi sheep disease virus (Ganjam virus) Newcastle disease virus (velogenic strains) Pseudomonas mallet Rickettsia ruminantium Rift Valley fever virus Rinde~pest virus Swine vesicular disease virus Teschen disease virus Theileria annulata Theileria bovis Theileria hirci Theileria lawrencei ~. 1 rypanosoma Evans Trypanosoma vivax Vesicular exanthema virus Wesselsbron disease virus The importation, possession, use, or interstate shipment of animal pathogens other than those listed above may also be subject to regulations of the U.S. Deponent of Agriculture. Additional information may be obtained by writ- ing to: Chief Staff Veterinarian Organisms and Vectors Veterinary Services Animal and Plant Health Inspection Service U.S. Department of Agriculture Hyattsville, MO 20782 Telephone: (301) 436-8017 FTS 436-8017
APPENDIX A APPENDIX A.6. RESOURCES FOR INFORMATION Resources for information, consultation, and advice on biohazard control, decontamination proce- dures, and other aspects of laboratory safety manage- ment include: Centers for Disease Control Attention: Office of Biosafety Atlanta, GA 30333 Telephone: (404) 329-3883 FTS 236-3883 133 National Institutes of Health Attention: Division of Safety Bethesda, MD 20892 Telephone: (301) 496-1357 FTS 496-1357 National Animal Disease Center U.S. Department of Agriculture Ames, IA 50010 Telephone: (515) 862-8258 FTS 862-8258
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