Appendix 1: Definitions and Acronyms

Appendix 2: Forms

Appendix 3: Biological Safety Cabinets

Appendix 4: Decontamination, Sterilization, Disinfection and Antisepsis

Appendix 5: Biosafety in Microbiological and Biomedical Laboratories

Appendix 6: Agent Summary Statements Biohazard Safety level information

Appendix 7 Biohazard Spill Clean-up Procedures

Appendix 8 Shipping Infectious Agents

This Biological Safety Manual has been developed by the Office of Environmental Health and Safety (EHS) to reflect the policies of the Biological Safety Program. The Biological Safety Program is a part of the Hazardous Material Management Program, and as such, other EHS programs may also pertain to biological safety. Among these are Medical Waste Management, the Bloodborne Pathogen Policy, the Chemical Hygiene Plan and the Plan for the Prevention of the Transmission of Tuberculosis.

The goals of the Biological Safety Program are:

• To protect students and personnel from exposure to infectious agents.
• To prevent environmental contamination.
• To provide policies for high quality research while maintaining a safe work place.
• To comply with applicable federal, state and local requirements.

The Biological Safety Committee was initially established to meet requirements established by the NIH. This committee is now also involved in the oversight of all projects involving infectious agents.

In general the handling and manipulation of biological agents and recombinant DNA requires the use of various precautionary procedures and measures. This manual will provide assistance in the evaluation and control of biohazardous agents. It will also establish a framework for institutional monitoring. The success of the program is largely dependent upon the Laboratory Biological Safety Officer (LBSO); however, all employees and students must be active participants in biological safety to ensure the program goals are achieved.

Agents of no or minimal hazard under ordinary conditions of handling.

Class 1 agents require Biosafety Level 1 precautions. Biosafety Level 1 practices, safety equipment and facilities are appropriate for undergraduate and secondary educational labs in which work is done with defined and characterized strains of viable microorganisms not known to cause disease in healthy adult human beings. Bacillus subtillis, Naegleria gruberi, and infectious canine hepatitis virus are representative of microorganisms meeting these criteria. However, many agents not ordinarily associated with disease processes in humans are opportunistic pathogens and may cause infection in the young, the aged, immunocomprised individuals, and after accidental inoculation or exposure, such as a splash into the eyes.

Biosafety Level 1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for hand washing. A summary of the protection recommended for Biosafety Level 1 includes:

•  Discretionary limited access to the lab when experiments are in progress.
• Surfaces must be easily cleaned, impervious to water, and resistant to acid, alkali, organic solvents, and moderate heat.
• Work surfaces are decontaminated daily and after spills.
• Mechanical pipetting is required: mouth pipetting is prohibited.
• No eating, drinking or smoking is permitted.
• Hands must be washed before leaving the lab.
• Insect control and rodent control programs are to be in effect.
• Contaminated liquid or solid wastes must be decontaminated before disposal by an approved decontamination method, or treated as biohazardous waste to be removed by the Office of Environmental Health and Safety . (See waste disposal.)

Agents of ordinary potential hazard.

This class includes agents that may produce diseases of varying degrees of severity from accidental inoculation or injection or other means of cutaneous penetration but are contained by ordinary laboratory techniques. Class 2 agents require Biosafety Level 2 protection for lab workers.

Biosafety Level 2 practices, equipment and facilities are applicable to clinical, diagnostic and teaching laboratories. With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided the potential for producing splashes or aerosols is low. Hepatitis B virus, the Salmonellae and Toxoplasma spp. are representative of agents at this containment level. Biosafety level 2 is appropriate when work is done with any human-derived blood, body fluids or tissues where the presence of an infectious agent may be unknown. (Refer to Bloodborne Pathogen Policy for specific details.)

  Biosafety Level 2 requires the same guidelines as Biosafety level 1, but also:

• Access to the lab is limited when work is being conducted.
• Personnel are trained in handling pathogenic agents and are directed by competent scientists.
• Biological safety cabinets (BSC) are used when aerosols are generated. Otherwise, BSC are not required.
• A universal biohazard warning sign must be placed on the door of the lab identifying the infectious agent, giving specific precautions to be taken before entry and listing emergency phone numbers of persons to be contacted in case of an emergency.
• Gloves are to be worn when handling infected animals and when hands may contact infectious material, contaminated surfaces or equipment.
• Needle-locking syringes or disposable syringe-needle units are used.
• Spills and exposures must be reported. Medical treatment is provided when necessary and written records are maintained.
• An eyewash facility is readily available.
• Lab coats designated for lab use are worn while in the lab. This protective clothing is removed and left in the lab before leaving for non-laboratory areas.
• A biosafety manual must be prepared and available. Personnel are advised of special hazards and are required to read and to follow instructions. Document this requirement.

D. CLASS 3 AGENTS

Agents that cause disease which may have serious or lethal consequences.

  Agents involving special hazards, or agents derived from outside the United States, that require a federal permit for importation, unless they are specified for higher classification. This class includes pathogens that require special conditions for containment. Mycobacterium tuberculosis, St. Louis encephalitis virus, and Coxiella burnetii are representative of microorganisms assigned to this level. These agents may cause a serious or potentially lethal disease as a result of exposure from inhalation and require Biosafety Level 3 work practices.

 Biosafety Level 3 includes all the same guidelines as Biosafety Level 1 and 2, but also:

• The lab area must be physically separated from access corridors and have self-closing, double door access.
• Lab doors must be closed when experiments are in progress.
• Access is highly restrictive, including a double set of doors separating the lab from other areas.
• Infectious work is done in a biological safety cabinet.
• Disposable plastic-backed matting is used on non-perforated work surfaces within biological safety cabinets.
• Respirators or surgical masks are worn in infected animal rooms.
• Vacuum lines are adapted with high efficiency particulate air (HEPA) filters and liquid disinfectant traps.
• Exhaust from biological safety cabinet is HEPA-filtered and discharged directly to the outside or through the building exhaust system if it is connected in a manner (e.g. thimble unit connection) that avoids any interference with the air balance of the cabinets or building exhaust system.
• Ducted exhaust air ventilation systems are used.
• Baseline serum samples are collected for all at-risk personnel.
• Lab personnel receive the appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g. hepatitis B vaccine or TB skin testing). Document this requirement.
• A biosafety manual must be prepared or adopted. Personnel must be advised of special hazards and are required to read and to follow instructions on practices and procedures. Document this requirement.
• The laboratory supervisor is responsible for insuring that, before working with organisms at Biosafety Level 3, all personnel demonstrate proficiency in standard microbiological practices. This might include prior experience in handling human pathogens or cell cultures or a specific training program. Document this requirement.
• All potentially contaminated waste materials (e.g. gloves, lab coats, etc.) from laboratories or animal rooms are decontaminated before disposal or reuse.

 E. CLASS 4 AGENTS

Agents that require the most stringent conditions for their containment because they are extremely hazardous to laboratory personnel or may cause serious epidemic diseases.

 These agents require Biosafety Level 4 which is maximum containment. Viruses such as Marburg or Congo-Crimean hemorrhagic fever are manipulated at Biosafety Level 4. Class 4 agents may not be used at the University of Alabama. Contact EHS for more information.

 See Table 1 in Appendix 5 for summary of recommended biosafety levels for infectious agents.

If experimental animals are used, institutional management must provide facilities and staff and establish practices which reasonably assure appropriate levels of environmental quality, safety and care. See Table 2 in Appendix 5 for a summary of recommended biosafety levels for activities in which experimentally or naturally infected animals are used.

Anytime animals are used in experimentation, prior approval by the Institutional Animal Care and Use Committee (IACUC) is required.

It is the responsibility of the lab supervisor to indicate how potentially infectious waste has been treated. If waste has been pretreated by autoclaving, it becomes "NORMAL WASTE’ and should be labeled to indicate it has been autoclaved. Autoclaved waste can be disposed of by routine housekeeping practices. However, if infectious waste is to be untreated, it must be placed in Biohazard bags or sharps containers and managed as "MEDICAL WASTE" in accordance with the Bloodborne Pathogens Guidelines.

Practices for the disposal of waste of potentially infectious material must be dictated by the Classification of Biological Agents on the basis of Hazard. For Class 1 and Class 2 agents, biohazardous waste may be handled either of two ways:

1.  Pretreated Waste - Decontamination (by autoclaving) before disposal. According to ADEM 335-13-7-08, infectious waste treated in the following manner may then be handled as ordinary trash.

1. The date, duration time, and operator for each cycle.
2. Approximate weight or volume of medical waste treated during each cycle.
3. The temperature and pressure maintained during each cycle.
4. Method used for confirmation of temperature and pressure.
5. Dates and results of calibration and maintenance.

2.  Untreated Waste - Non-autoclaved media that has utilized human body fluids or tissue or non-autoclaved discarded specimens of any potentially infectious material shall be collected in labeled biohazard bags and managed as medical waste. These procedures are described in the U of A Bloodborne Pathogen Policy.

For Class 3 and above agents, biohazardous waste must be pretreated by decontamination before disposal. Decontamination must be by an approved method such as autoclaving. All waste generated from Class 3 and above agents must be collected in biohazard bags or closed, leak-proof, labeled containers, to prevent spillage or protrusion of contents during handling or transport. Decontamination procedures must adhere to the guidelines for Pretreated Waste.

The Institutional Biological Safety Committee (IBSC) was originally established in order to conform to NIH guidelines. The IBSC is now involved in the oversight of all projects involving infectious agents and recombinant DNA. The responsibilities of the IBSC are as follows:

• Review the use of Class 1 and 2 biohazardous agents.
• Review and approve the use of Class 3 agents and recombinant DNA.
• Review and approve the use of Select Agents.
• Review the Biological Safety Program annually to determine if the program is effective and compliant with regulations.
• Advise the Institutional Biological Safety Officer (ISBO) and support the biological safety program.
• Assist the IBSO with enforcement of regulations and guidelines.
• Advise the Vice President for Academic Affairs on matters concerning the use of biohazardous agents.

The Chair of the Biological Safety Committee shall be a University employee who is knowledgeable of and has experience with using biohazardous agents. The membership shall be appointed by the President. The Committee shall meet at least once during each six month period. To be considered an official meeting of the IBSC, at least three voting members must be present. A majority of the voting members present is required for committee action. The Chair shall moderate committee meetings. In the absence of the Chair, the IBSO, who is a voting member of the Committee, shall moderate. The Chair or a designated representative shall be responsible for keeping committee minutes and the distribution of these minutes.

The University shall appoint an Institutional Biological Safety Officer (IBSO) who shall serve as a voting member of the Institutional Biological Safety Committee and whose responsibilities shall include:

• To implement the Biological Safety Program.
• To investigate deviation from accepted practices.
• To implement and recommend corrective actions.
• To develop written policies and procedures as necessary.

All work involving Class 1 biohazardous agents shall be registered with the IBSC. The Class 1 registration form in Appendix 2 must be used.

All work involving Class 2 biohazardous agents shall be registered with the IBSC. The Class 2 registration form in Appendix 2 must also be accompanied by written protocols.

All work involving Class 3 biohazardous agents and recombinant DNA must be approved by the IBSC. The Class 3/r-DNA approval form is found in Appendix 2. All submittals must be complete to be considered for approval.

Once work is registered or approved, any changes must be described on the appropriate form and forwarded to the IBSC.

All registrations and approvals are valid until October 31 of each calendar year. Prior to this date information must be submitted to the IBSC. Each year all registrations and approvals must be verified. After November 1 of each calendar year, any Lab BSO who has not verified his/her registration will be removed from the Biological Safety Program.

Training of personnel and students is an integral part of any program. Laboratory Biological Safety Officers (LBSO's) who work with Class 2 or 3 biohazardous agents shall attend an annual information session. At these sessions changes in the program as well as specific problems will be discussed.

Persons who work with Class 2 or 3 biohazardous agents shall receive training from their LBSO. This shall be provided at the earliest opportunity and shall include emergency procedures, decontamination, biosafety principals and other information related to specific organisms or hazards which may be present in the lab. Information contained in the appendices of this manual may be used for training.

All persons who work in the lab shall be trained in emergency procedures by their LBSO. Basic emergency procedures are as follows:

• To evacuate the area if necessary.
• To notify the LBSO and IBSO as soon as possible.
• To assess the type of spill and hazard.
• To determine the most effective and least hazardous method of remediation.
• To decontaminate the area, waste and equipment utilized.

The class of biohazardous agent which is involved in the spill or accident will dictate the extent to which emergency procedures are utilized. An emergency involving Class 3 agents may require evacuation, extensive decontamination, and medical attention for exposed persons. EHS has equipment and personnel to assist during remediation.

It is the responsibility of the LBSO and all persons who work with biohazardous agents to follow the guidelines of the Biological Safety Program and to utilize safe procedures. Violations shall be documented by the IBSO or other EHS personnel. The responsible LBSO shall be informed in writing of the nature of the non compliance, ways to implement correction and results of failure to comply. The Chair of the IBSC and the responsible department head shall be provided a copy. In the event of a situation determined to be immediately hazardous, the IBSO may suspend the privileges of the responsible LBSO or take other actions necessary to protect the health of individuals or the safety of University facilities.

As the committee responsible for biological safety, the IBSC may suspend projects, revoke approvals or take other punitive actions deemed appropriate. Actions taken by the IBSC shall be reported to the responsible LBSO, Department Chair and Vice President. This situation could arise when a LBSO willingly or negligently violates University policies or established guidelines which govern the use of biohazardous agents.

N. SELECT AGENT REGISTRATION

Revised 3/15/03

On December 13, 2002 the Department of Health and Human Services and the Department of Agriculture published new interim rules governing the way select agents and toxins are to be managed within the United States. The interim rule is effective February 7, 2003, with a phase-in schedule for certain sections and now includes possession.

All registrations must be coordinated through the UA Office of EHS, which is responsible for the facility (entity) registration. Anyone planning to do research involving “select agents” must register with the UA Office of EHS before obtaining a select agent. No “select agent” may be received at or transferred from UA without prior registration.  All work with Select Agents must receive prior approval from the IBSC.

Investigators expecting to use a “select agent” are advised to contact EHS (4-5905). 

Since this list is subject to change, one may one may access CDC at web sites: http://www.cdc.gov/od/sap/index.htm and http://www.cdc.gov/od/sap/docs/salist.pdf for the most current information.

Except for exclusions listed in Appendix I, the viruses, bacteria, fungi, toxins, genetic elements, recombinant nucleic acids, and recombinant organisms specified in this list are Department of Health and Human Services (HHS) select agents and toxins, United States Department of Agriculture (USDA) high consequence livestock pathogens, or HHS/USDA overlap agents. Animal and Plant Heath Inspection Service (APHIS) regulated plant pathogens are listed in Appendix II.

Viruses:

African horse sickness virus ^a

African swine fever virus ^a

Akabane virus ^a

Avian influenza virus (highly pathogenic) ^a

Bluetongue virus (exotic) ^a

Camel pox virus ^a

Cercopithecine herpesvirus 1 (Herpes B virus) ^b

Classical swine fever virus ^a

Crimean-Congo haemorrhagic fever virus ^b

Eastern Equine Encephalitis virus ^c

Ebola viruses ^b

Foot-and-mouth disease virus ^a

Goat pox virus ^a

Hendra virus ^{c ‡}

Japanese encephalitis virus ^a

Lassa fever virus ^b

Lumpy skin disease virus ^a

Malignant catarrhal fever virus (exotic) ^a

Marburg virus ^b

Menangle virus ^a

Monkeypox virus ^b

Newcastle disease virus (exotic) ^a

Nipah virus ^{c ‡}

Peste des petits ruminants virus ^a

Rift Valley fever virus ^c

Rinderpest virus ^a

Sheep pox virus ^a

South American Haemorrhagic Fever viruses (Junin, Machupo, Sabia, Flexal, Guanarito) ^b

Swine vesicular disease virus ^a

Tick-borne encephalitis complex (flavi) viruses [Central European Tick-borne encephalitis, Far Eastern Tick-borne encephalitis (Russian Spring and Summer encephalitis, Kyasanur Forest disease, Omsk Hemorrhagic Fever)] ^b

Variola major virus (Smallpox virus) and Variola minor virus (Alastrim) ^b

Venezuelan Equine Encephalitis virus ^c

Vesicular stomatitis virus (exotic) ^a

Fungi:

Coccidioides immitis ^c

Coccidioides posadasii ^b

PRION

Bovine spongiform encephalopathy agent ^a

Bacteria:

·     Bacillus anthracis ^c

·     Brucella abortus ^c

·     Brucella melitensis ^c

·     Brucella suis ^c

·     Burkholderia mallei (formerly Pseudomonas mallei) ^c

·     Burkholderia pseudomallei (formerly Pseudomonas pseudomallei) ^c

·     Clostridium botulinum (and botulinum neurotoxin producing species of Clostridium) ^c

·     Cowdria ruminantium (Heartwater) ^a

·     Coxiella burnetii ^c

·     Francisella tularensis ^c

·     Mycoplasma capricolum/ M. F38/M. mycoides capri (contagious caprine pleuropneumonia) ^a

·     Mycoplasma mycoides mycoides (contagious bovine pleuropneumonia) ^a

·     Rickettsia prowazekii ^b

·     Rickettsia rickettsii ^b

·     Yersinia pestis ^b

Toxins:

 Abrin ^b

 Botulinum neurotoxin^c

 Clostridium perfringens epsilon toxin ^c

 Conotoxins ^b

 Diacetoxyscirpenol ^b

 Ricin ^b

 Saxitoxin ^b

 Shigatoxin ^c

 Shiga-like ribosome inactivating proteins ^b

 Staphylococcal enterotoxins ^c

 Tetrodotoxin ^b

 T-2 toxin ^c

^a USDA high consequence livestock pathogens

^b HHS select agents and toxins

^c HHS/USDA overlap agents

^‡ Nipah and Hendra Complex Viruses were previously listed as Equine Morbillivirus Virus

Genetic Elements, Recombinant Nucleic Acids, and Recombinant Organisms:

1. Viruses, bacteria, fungi, and toxins listed that have been genetically modified.  

2.  Select agent viral nucleic acids (synthetic or naturally derived, contiguous or fragmented, in host chromosomes or in expression vectors) that can encode infectious and/or replication competent forms of any of the select agent viruses.

3.  Nucleic acids (synthetic or naturally derived) that encode for the functional form(s) of any of the toxins listed if the nucleic acids:

• are in a vector or host chromosome;

• can be expressed in vivo or in vitro; or

• are in a vector or host chromosome and can be expressed in vivo or in vitro.

OTHER RESTRICTIONS:

1. Experiments utilizing recombinant DNA that involve the deliberate transfer of a drug resistance trait to the listed agents that are not known to acquire the trait naturally, if such acquisition could compromise the use of the drug to control disease agents in humans, eternity medicine, or agriculture.

2. Experiments involving deliberate formation of recombinant DNA containing genes for the biosynthesis of listed toxin lethal for vertebrates at an LD50 <100 ng/kg body weight.

APPENDIX A

Exclusions:

      1.  Any select agent or toxin that is in its naturally occurring environment provided it has not been intentionally introduced, cultivated, collected, or otherwise extracted from its natural source. 

2.  Non-viable select agent organisms or nonfunctional toxins. 

3.  Fixed tissues that bear or contain select agents or toxins.*

4.  Genetic elements or sub-units of agents or toxins, if the genetic elements or sub-units are not capable of causing disease.*

5.  The vaccine strain of Junin virus (Candid #1).

6.   The vaccine strain of Rift Valley fever virus (MP-12).

7.   Venezuelan Equine encephalitis virus vaccine strain TC-83. 

8.   The medical use of toxins for patient treatment.

9.  The following toxins (in the purified form or in combinations of pure and impure forms) if the aggregate amount under the control of a principal investigator does not, at any time, exceed the amount specified:

• 100 mg of Abrin

• 0.5 mg of Botulinum neurotoxins

• 100 mg of Clostridium perfringens epsilon toxin

• 100 mg of Conotoxins

• 1,000 mg of Diacetoxyscirpenol

• 100 mg of Ricin

• 100 mg of Saxitoxin

• 100 mg of Shigatoxin

• 100 mg of Shiga-like ribosome inactivating proteins

• 5 mg of Staphylococcal enterotoxins

• 100 mg of Tetrodotoxin

• 1,000 mg of T-2 toxin

The administrator may exclude from this list attenuated strains of HHS select agents or toxins upon a determination that they do not pose a severe threat to the public health and safety.

* The importation and interstate movements of these nonviable agents, fixed tissues, and genetic elements or subunits are still subject to the permit requirements under 9 CFR part 122.

Viruses:

 Plum pox potyvirus

Fungi:

 Peronosclerospora philippinensis

 Phakopsora pachyrhizi

 Sclerophthora rayssiae var. zeae

 Synchytrium endobioticum

Bacteria:

·      Liberobacter africanus

·      Liberobacter asiaticus 

·      Ralstonia solanacearum, race 3, biovar 2

·      Xanthomonas oryzae pv. oryzicola

·      Xylella fastidiosa (citrus variegated chlorosis strain)

 TRANSPORT OF BIOLOGICAL AGENTS

The transfer of biological agents to or from campus may be subject to the regulatory requirements of EPA, TSCA, DOT, CDC or USDA or possibly some other federal or state regulatory agency.  Before transferring any biological agent, contact the Office of EHS so that transport compliance can be satisfied.  Refer to Appendix 8 for additional information.

(Revised 3/15/03) Biological agents that require permits, are SA’s or are BSL2’s or above must have prior approval and be shipped to EHS at 15 Research Drive.

ABSL - Animal Biosafety Level (See Biosafety Level below).

ACF - Animal Care Facility

ADEM - Alabama Department of Environmental Management

Antisepsis - The application of a liquid antimicrobial chemical to living tissue to prevent growth or destroy potentially infections organisms.

BBP - (See Blood-borne Pathogen).

Biohazard - (See Biohazardous Materials).

Biohazardous Materials - Hazardous biological materials and organisms, including: a) infectious organisms (bacteria, fungi, parasites, prions, rickettsias, viruses, etc), which can cause disease in healthy humans and/or significant environmental or agricultural impact; b) human or primate tissues, fluids, cells, or cell culture; c) recombinant DNA; and d) animals known to be vectors of zoonotic diseases.

Biosafety Cabinet - (Biological Safety Cabinet) - A devise enclosed (except for necessary exhaust purposes) on three sides and top and bottom, designed to draw air inward by mean of mechanical ventilation, operated with insertion of only the hands and arms of the user, and in which virulent pathogens are used. Biosafety Cabinets are classified as: Class I - Provides personnel and environmental protection but no product protection, the exhaust is HEPA filtered; Class II - Provides personnel, product and environmental protection; Class III - Totally enclosed (glove box) ventilated cabinet with gas-tight construction.

Biosafety Level - Combinations of laboratory practices and techniques, safety equipment, and laboratory facilities appropriate for the operations performed and are based on the potential hazards imposed by the agents used and for the laboratory function and activity. Biosafety Level I provides the least stringent containment conditions and Biosafety Level 4 the most stringent.

Blood-borne Pathogen (BBP) - Microorganisms that are present in human/primate blood, tissues or fluids and can cause disease in humans. These pathogens include (but are not limited to) hepatitis B virus (HBV) and human immunodeficiency virus (HIV).

BSC - (See Biosafety Cabinet).

BSL - Biosafety Level.

BSO - Biological Safety Officer

CDC - Centers for Disease Control and Prevention.

Containment - Safe methods for managing infectious agents in the laboratory environment where they are being handled or maintained. The purpose of containment is to reduce or eliminate exposure to laboratory workers, other persons and the environment to potentially hazardous agents.

Decontamination - Routinely required step of destroying/inactivating microorganisms in microbiological laboratories to protect laboratory workers and prevent contamination of their work. This is the destruction or removal of microorganisms to some lower level, but not necessarily total destruction. Sterilization, disinfection and antisepsis are all forms of decontamination.

Disinfection - Implies the use of antimicrobial agents to inanimate objects (e.g., work surfaces, equipment, etc) to destroy all organisms that could pose a potential hazard to humans or compromise the integrity of the experiment.

DNA - Deoxyribonucleic Acid.

DOT - Department of Transportation.

Exposure Control Plan (ECP) - A written document of practices and procedures, required equipment and facilities designed to eliminate or minimize employee exposure to infectious agents or biohazardous materials.

EHS - Office of Environmental Health and Safety

Etiologic Agent - Biological agent or agents that causes disease in humans or animals.

Gene Therapy - The delivery of exogenous DNA to mammalian cells to cause the expression of this material thereby altering the cells phenotypically.

HBV - Hepatitis B Virus.

HEPA - High Efficiency Particulate Air Filter has an efficiency of 99.97% for particles of 0.3 microns or larger. Biological Safety Cabinets filter air through one of more sets of HEPA filters.

HIV - Human Immunodeficiency Virus.

IACUC - Institutional Animal Care and Use Committee

Infectious Agents - Microorganisms capable of producing an infection and disease in a healthy human.

Infectious Substance - (Same as Etiologic agent).

IBSC - Institutional Biological Safety Committee.

IBSO - Institutional Biological Safety Officer.

Laminar Flow Hoods - Cabinets which are designed to protect only the product. These cabinets blow air into the face of the worker. Use of these cabinets for any biological organism is discouraged.

LBSO - Laboratory Biological Safety Officer - The faculty member primarily responsible for the space in which work is being performed.

NIH - National Institutes of Health.

NIOSH - National Institute for Occupational Safety and Health.

OSHA - Occupational Safety and Health Administration.

Pathogen - Microorganism or substance capable of producing disease.

PPE - Personal Protective Equipment.

LBSO - Laboratory Biological Safety Officer

Recombinant DNA (rDNA) - a) molecules that are constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell, or b) molecules that result from replication of those described in a) above.

RNA - Ribonucleic Acid.

Sterilization - The process of destroying or removing all living organisms and viruses, usually by steam or gas autoclaving.

Universal Precautions - Refers to the infection-control method in which all human/primate blood, tissues, and/or fluids are treated as if they are known to be infectious.

USDA - United States Department of Agriculture.

Zoonotic Diseases - Diseases communicable from animals to humans under natural conditions.

1. There are three basic types of biological safety cabinets:

• Class I
• Class II
• Class III

2. Class I cabinets offer personnel protection but no product protection. Characteristics and limitations of Class I cabinets includes:

• The front opening should be approximately 8 inches.
• Face velocity should be a minimum of 75 linear fpm.
• Use for class 2 biohazardous agents.

3. Class II cabinets are laminar flow which is designed to protect the worker, the product and the environment. Characteristics and limitations of Class II cabinets includes:

4. Characteristics and limitations of class III cabinets includes:

5. Biological safety cabinets should be certified:

6. Class II cabinets are used most effectively if the following procedures are observed.

Editors Diane O. Fleming et al. American Society for Microbiology Press, p 219-237

Editors Diane O. Fleming et al. American Society for Microbiology Press, p 293-354

SECTION VII*

Agent Summary Statements

PARASITIC AGENTS

Nematode Parasites of Humans

Protozoal Parasites of Humans

Trematode Parasites of Humans

Cestode Parasites of Humans

AGENT: Nematode Parasites of Humans

Laboratory-associated infections with Ascaris spp.; Strongyloides spp.; hookworms; and Enterobius spp. have been reported . 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 infected with Strongyloides spp. are a potential infection hazard for laboratory and animal care personnel.

LABORATORY HAZARDS: Eggs and larvae in freshly passed feces of infected hosts are usually not infective; development to the infective stages may take periods of one day to several weeks. Trichinella is of concern since fresh or digested tissue may contain larvae and would be infective if ingested. Ingestion of the infective eggs or skin penetration of infective larvae are the primary hazards to laboratory and animal care personnel. Arthropods infected with filarial parasites pose a potential hazard to laboratory personnel. In laboratory personnel with frequent exposure to aerosolized antigens of Ascaris spp. development of hypersensitivity is common.

RECOMMENDED PRECAUTIONS: Biosafety Level 2 practices and facilities are recommended for activities with infective stages listed. Exposure to aerosolized sensitizing antigens of Ascaris spp. should be avoided. Primary containment (e.g., biological safety cabinet) may be required for work with these materials by hypersensitive individuals. Appropriate treatment for most nematode infections exists, and information on dosage, source of drugs, etc. is available.

AGENT: Protozoal Parasites of Humans

Laboratory-associated infections with Toxoplasma spp.; Plasmodium spp. (including P. cynomologi); Trypanosoma spp.; Entamoeba spp.; Coccidia spp.; Giardia spp.; Leishmania spp.; Sarcocystis spp.; and Cryptosporidia spp. have been reported . In addition, no laboratory infections with Babesia spp. or Microsporidia spp. have been reported but could result from accidental needlestick or ingestion of cysts, oocysts, or spores in feces.

Although laboratory animal-associated infections have not been reported, a direct source of infection for laboratory personnel may be contact with lesion material from rodents with cutaneous leishmaniasis and with feces or blood of experimentally or naturally infected animals.

Laboratory-related infections with Cryptosporidia have occurred with regularity in almost every laboratory working with this agent, especially those in which calves are utilized as the source of oocysts. Other experimentally-infected animals pose potential risks as well. There is circumstantial evidence that airborne transmission of oocysts of this small organism may occur. Rigid adherence to protocol should reduce the occurrence in laboratory and animal care personnel.

LABORATORY HAZARDS: Infective stages may be present in blood, feces, lesion exudates, and infected arthropods. Depending on the parasite, ingestion, skin penetration through wounds or microabrasions, accidental parenteral inoculation, and transmission by arthropod vectors are the primary laboratory hazards. Aerosol or droplet exposure of the mucous membranes of the eyes, nose, or mouth with trophozoites are potential hazards when working with cultures of Naegleria fowleri, Leishmania spp., T. cruzi, or with tissue homogenates or blood containing hemoflagellates. Immunocompromised individuals should avoid working with live organisms. Because of the grave consequences of toxoplasmosis in the developing fetus, serologically negative women of childbearing age who might become pregnant should not work with Toxoplasma in the same laboratory room where these materials are handled.

RECOMMENDED PRECAUTIONS: Biosafety Level 2 practices 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 outside. Primary containment (e.g., biological safety cabinet) or personal protection (e.g., face shield) may be indicated when working with cultures of Naegleria fowleri, Leishmania spp., T. cruzi or with tissue homogenates or blood containing hemoflagellates. Gloves are recommended for activities where there is the likelihood of direct skin contact with infective stages of the parasites listed. Appropriate treatment for most protozoal infections exists, and information on dosage, source of drugs, etc., is available.

AGENT: Trematode Parasites of Humans (Schistosoma spp. and Fasciola spp.) Laboratory-associated infections with Schistosoma spp. and Fasciola spp. have been reported, none associated directly with laboratory animals.

LABORATORY HAZARDS: Infective stages of Schistosoma spp. (cercariae) and Fasciola spp. (metacercaria) 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 metacercaria are the primary laboratory hazards. Dissection or crushing of schistosome-infected snails may also result in exposure of skin or mucous membrane to cercariae-containing droplets. Additionally, metacercaria may be inadvertently transferred from hand to mouth by fingers or gloves following contact with contaminated aquatic vegetation or surfaces of aquaria. Most laboratory exposures 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 and facilities are recommended for activities with infective stages of the parasites listed. Gloves should be worn when there may be direct contact with a water containing cercariae, or vegetation containing metacercaria from naturally or experimentally infected snail intermediate hosts. Long-sleeved laboratory coats or other protective garb should be worn when working arouquaria or other water sources that may contain schistosome cercariae. Snails and cercariae in the water of laboratory aquaria should be killed by chemicals (e.g., hypochlorites, iodine) or heat before discharge to sewers. Appropriate treatment for most trematode infections exists, and information on source of drugs, dosage, etc. is available .

AGENT: Cestode Parasites of Humans - Echinococcus granulosus, Taenia solium (cysticercus cellulosae) and Hymenolepsis nana.

Although no laboratory-associated infections with either E. granulosus or T. solium have been reported, the consequences of such infections following the ingestion of infective eggs of E. granulosus or T. solium are potentially grave. H. nana is a very cosmopolitan parasite, does not require an intermediate host, and is directly transmissible by ingestion of feces of infected humans or rodents.

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 are the primary laboratory hazard. Cysts and cyst fluids of E. granulosus are not infectious for humans. Ingestion of cysts containing the larval stage of T. solium (Cysticercus cellulosae) readily produces human infection with the adult tapeworm. With either parasite, the ingestion of a single infective egg from the feces of the definitive host could potentially result in serious disease. Ingestion of the eggs of H. nana in the feces of the definitive host could result in intestinal infection.

RECOMMENDED PRECAUTIONS: Biosafety Level 2 practices and facilities are recommended for work with infective stages of these parasites. Special attention should be given to personal hygiene practices (e.g., handwashing) and avoidance of ingestion of infective eggs. Gloves are recommended when there may be direct contact with feces or surfaces contaminated with fresh feces of dogs infected with E. granulosus, humans infected with T. solium adults, or humans or rodents infected with H. nana. Appropriate treatment for many cestode infections exists, and information concerning source of drugs, dosage, etc., is available.

SECTION VII

Agent Summary Statements

FUNGAL AGENTS

Blastomyces dermatitidis

Coccidiodes immitis

Cryptococcus neoformans

Histoplasma capsulatum

Sporothrix schenckii

Pathogenic Members of the Genera Epidermophyton, Microsporum and Trichophyton

Miscellaneous Molds

AGENT: Blastomyces dermatitidis

Laboratory-associated local infections following accidental parenteral inoculation with infected tissues or cultures containing yeast forms of B. dermatitidis have been reported. Pulmonary infections have occurred following the presumed inhalation of conidia; two developed pneumonia and one had an osteolytic lesion from which B. dermatitidis was cultured. Presumably, pulmonary infections are associated only with sporulating mold forms (conidia).

LABORATORY HAZARDS: Yeast forms may be present 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. dermatitidis containing infectious conidia may pose a hazard of aerosol exposure.

RECOMMENDED PRECAUTIONS: Biosafety Level 2 and Animal Biosafety Level 2 practices and facilities are recommended for activities with clinical materials, animal tissues, cultures, and infected animals.

AGENT: Coccidiodes immitis

Laboratory-associated coccidioidomycosis is a documented hazard. Smith reported that 28 of 31 (90%) laboratory-associated infections in his institution resulted in clinical disease, whereas more than half of infections acquired in nature were asymptomatic.

LABORATORY HAZARDS: Because of the size (2-5 millimicrons), the arthroconidia are conducive to ready dispersal in air and retention in the deep pulmonary spaces. The much larger size of the spherule (30-60 millimicrons) considerably reduces the effectiveness of this form of the fungus as an airborne pathogen. Spherules of the fungus may be present in clinical specimens and animal tissues, and infectious arthroconidia in mold cultures and soil samples. Inhalation of arthroconidia from soil samples, mold cultures, or following transformation from the spherule form in clinical materials, is the primary laboratory hazard. Accidental percutaneous inoculation of the spherule form may result in local granuloma formation. Disseminated disease occurs at a much greater frequency in blacks and Filipinos than whites.

RECOMMENDED PRECAUTIONS: Biosafety Level 2 practices and facilities are recommended for handling and processing clinical specimens, identifying isolates, and processing animal tissues. Animal Biosafety Level 2 practices and facilities are recommended for experimental animal studies when the route of challenge is parenteral.

Biosafety Level 3 practices and facilities are recommended for propagating and manipulating sporulating cultures already identified as C. immitis and for processing soil or other environmental materials known or likely to contain infectious arthroconidia.

AGENT: Cryptococcus neoformans

A single account is reported of a laboratory exposure to Cryptococcus neoformans as a result of a laceration by a scalpel blade heavily contaminated with encapsulated cells. 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 infections as a consequence of laboratory exposure have not been recorded.

LABORATORY HAZARDS: Accidental parenteral inoculation of cultures or other infectious materials represents a potential hazard to laboratory personnel -- particularly to those that may be immunocompromised. Bites by experimentally infected mice and manipulations of infectious environmental materials (e.g., pigeon droppings) may also represent a potential hazard to laboratory personnel.

RECOMMENDED PRECAUTIONS: Biosafety Level 2 and Animal Biosafety Level 2 practices and facilities are recommended, respectively, for activities with known or potentially infectious 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 indicated for culture of the perfect or sexual state of the agent.

AGENT: Histoplasma capsulatum

Laboratory-associated histoplasmosis is a documented hazard in facilities conducting diagnostic or investigative work. Pulmonary infections have resulted from handling mold form cultures. Local infection has resulted from skin puncture during autopsy of an infected human and from accidental needle inoculation of a viable culture. Collecting and processing soil samples from endemic areas has caused pulmonary infections in laboratory workers. Encapsulated spores are resistant to drying and may remain viable for long periods of time. The small size of the infective conidia (less than 5 microns) is conducive to airborne dispersal and intrapulm