Principles and Practices of Biosafety Environmental Health and Safety San Diego State University
BSC – Biosafety Cabinet BSL – Biosafety Level ABSL – Animal Biosafety Level BSO – Biosafety Officer BUA – Biological Use Authorization CA – California CDC – Center for Disease Control and Prevention DGR – Dangerous Goods Regulations DOT – Department of Transportation EHS – Environmental Health and Safety EPA – Environmental Protection Agency HMR – Hazardous Materials Regulations IACUC – Institutional Animal Care and Use Committee IATA – International Air Transport Association IBC – Institutional Biosafety Committee IRB – Institutional Review Board NIH – National Institute of Health OSHA – Occupational Health and Safety Administration PI – Principal Investigator PP – Physical Plant PPE – Personal Protective Equipment PS – Pubic Safety SD – San Diego USPS – U.S. Postal Service Abbreviations
Introduction The management of biological hazards through the proper application of engineered containment and administrative controls is referred to as biosafety or biohazard control. Biosafety or biohazard control is a team effort involving the PI, research lab personnel, BSO, IBC, IRB, IACUC, EHS, PP and PS.
Regulatory Requirements and Guidelines • NIH Guidelines for Research Involving Recombinant DNA Molecules (April 2002) • CDC/NIH Biosafety in Microbiological and Biomedical Laboratories (May 1999) • CAL/OSHA Bloodborne Pathogen Standard • CA Medical Waste Management Act • DOT Transportation of Hazardous Materials
SDSU Biosafety Requirements and Guidelines • Policies established by the Institutional Biosafety Committee (IBC) that meet or exceed applicable guidelines and regulations for use of RG 2 and RG 3 biohazardous materials or agents as well as non-exempt NIH Recombinant DNA research. • Policies established by Environmental Health and Safety (EHS) that meet or exceed applicable regulations and guidelines for minimizing bloodborne pathogen exposure and disposal of biohazardous wastes.
Infectious microorganisms (bacteria, viruses, fungi, parasites, prions, rickettsiae, etc.) affecting humans and animals Diagnostic (clinical) specimens Recombinant DNA (viral vectors, gene therapy, cloning) Genetically Modified Microorganisms (transgenic plants and animals) Human and non-human primate cells, cell culture (primary and immortalized), tissues, blood (whole blood or any components) and body fluids Animal or plant cells, cell cultures, fluids, tissues or derived wastes which may contain pathogens Animals known to be reservoirs of zoonotic disease Biohazardous Material
Institutional Biosafety Committee and Biological Use Authorization
Biological Use Authorization (BUA) Application • BSL 1, ABSL 1 and NIH-Exempt • Reviewed and approved by BSO • BSL 2 or 3, ABSL 2 or 3 and NIH- Non-exempt • Reviewed and approved by IBC Currently, no facilities at SDSU meet the minimum criteria for BSL 4; thus this type of work is prohibited.
Submission of New BUA • New infectious agents • New cell lines • New vector system • Enhanced replication or infectivity • Expression of toxic products • Partial genomes increased to more than two-thirds of whole genome • New or altered procedures that pose increased risk (e.g., aerosol or other type of exposure) • Work with non-human systems being changed into work with human system
SDSU Biosafety Approval Institutional Biosafety Committee (IBC) • Protocol Review • Biological Use Authorization (BUA) with respect to containment level (biosafety level) • Training • Inspection
Principal Investigator • Develops specific protocols to ensure the safe use of biohazardous materials. • Submits a BUA application and obtains approval from IBC prior to commencement of work. • Complies with specific biosafety protocols, practices and procedures described in the biohazard control manual. • Ensures that all laboratory staff are appropriately trained on biosafety. • Reports any significant problems, violations of the policies, practices and procedures, or any significant research-related accidents and/or laboratory acquired infection to the BSO immediately.
Laboratory Staff • Comply with the specific biosafety protocols, practices and procedures described in the biohazard control manual. • Report to the PI or the lab manager all problems, spills or violations in procedure immediately.
Classification of Infective Agents by Risk Group • Pathogenicity • Infectious Dose • Mode of Transmission • Host Range • Availability of Effective Preventive Measures and Treatment
Classification of Containment by Biosafety Levels • Practices and Procedures • Containment Equipment (Primary Barriers) • Containment Facility (Secondary Barriers) Note: CDC/NIH has 4 biosafety level classifications currently in use. Each level is appropriate for: • Operations Performed • Routes of Transmission (ingestion, inoculation, inhalation, mucous membrane exposure) • Laboratory Function
Risk Group and Biosafety Level Classifications • Not appropriate to use risk group or biosafety level when assessing toxins. • Can be found in the American Biological Safety Association website: www.absa.org/resriskgroup • Risk Groups and Biosafety Levels are not always the same!
Introduction • California Code of Regulations, Title 8, Sec 5193 • Applies to all employees who could “reasonably anticipate” as a result of performing their job tasks contact with blood and other potentially infectious material (OPIM) i.e., body fluids, unfixed tissues or organs • Limit occupational exposure to blood and OPIM which could result in transmission of bloodborne pathogens i.e., Hepatitis B virus (HBV), HIV • Requires a written Exposure Control Plan
Elements of an Exposure Control Plan • Exposure Determination • Method of Compliance • HIV, HBV and HCV Research Laboratories • Hepatitis B Vaccination and Post-Exposure Evaluation and Follow-Up • Hazard Communication • Record Keeping • Evaluation of Exposure Incidents • Sharps Injury Log
Exposure Determination • Exposure evaluation based upon the job description. • Exposure evaluation based upon reasonably anticipated contact (skin, eye, mucous membrane, parenteral contact, etc.) with blood or other potentially infectious materials resulting from performing the assigned tasks.
Method of Compliance • Universal Precaution – treating all human blood and certain human body fluids as if infectious for bloodborne pathogens • Engineering and Work Practice Controls • Needleless system or needles/sharps with engineered sharps injury protection • Needles and other sharps shall not be recapped, bent or broken • Needles and other sharps shall be disposed in rigid, puncture-proof, leak resistant and properly labeled sharps container • Sharps container shall be closed immediately prior to removal or replacement to prevent spillage or protrusion of contents during handling or transport • Specimens of blood or OPIM shall be placed in a closable, leakproof, properly labeled red bag prior to handling, collection or transport • Personal Protective Clothing and Equipment
Hepatitis B Vaccination and Post-Exposure Follow-Ups • Hepatitis B vaccination shall be provided at no cost to employee who has a potential for becoming exposed to blood or OPIM. • Post-exposure follow-ups shall be provided if an occupational exposure occurs.
Hazard Communication • Signs and Labels • Signs shall be posted at the entrance to the work areas which shall bear: • Name of infectious agent • International symbol for biohazard in fluorescent orange-red • Special requirements for entering the area • Name and telephone number of lab director or other responsible person • Warning labels shall be affixed to containers of infectious wastes, refrigerators and freezers containing blood or OPIM, or other containers used to store or transport blood or OPIM. • Labels shall have the international symbol for biohazard in fluorescent orange-red • Training
Bacterial Laboratory Acquired Infections • 76% of exposures occurred in clinical labs; 6% in vaccine manufacturing facilities; 8% in research labs. • Exposure modes: 60% inhalation, other exposure modes included ingestion (intentional, poor technique such as mouth pipetting, smoking and eating in the lab) and secondary transmission.
Viral Laboratory Acquired Infections • >70% associated with research labs; 32% of all viral LAIs associated with animals • 18% of total were Hantavirus; of these, 8% were working with known infectious material or rodents - others thought they were working with uninfected rodents • 16% were in clinical labs; rest were in production or field work • Major exposure modes: inhalation, percutaneous (especially from animals)
Rickettsial Laboratory Acquired Infections • All rickettsial LAIs were associated with research laboratories • 95% of overt infections were by Coxiella burnetii; remainder were Murine typhus • All Q fever cases by inhalation; all infected staff worked with or were in close proximity to sheep • Remaining cases were by percutaneous, inhalation, mucous membrane or unknown exposure modes
It’s a Matter of Perspective • The investigators who submit IBC protocols want to perform their experiments safely. • However, their perception of the risks involved will not necessarily be the same as that of a biosafety professional.
Risk Assessment The following risk assessment will identify the biological containment system to be used: • Properties of the donor organism • Nature of the DNA sequences that will be transferred • Properties of the recipient organism • Properties of the environment
Biological Expression System Most routine genetic engineering experiments can be performed safely in E. coli K12/pUC18 at BSL 1 provided the inserted foreign DNA sequences do not require a higher BSL.
Donor Organism and Cloned DNA Insertion of well-characterized DNA sequences that are unlikely to be involved in pathogenicity may not require additional safety measures. In cases where these sequences are not characterized, a situation that is typically encountered when a library of genomic DNA of an organism is being established, a higher BSL will be required. Cloning of genes coding for proteins that have potential pharmacological activity such as toxins may therefore require higher BSL.
Viral Vectors for Gene Transfer Although viral vectors used in gene therapy or gene transfer are replication-defective, they should be handled at the same BSL as the parent viral vector from which they are derived since the virus stocks may be contaminated with replication-competent viruses, which are generated by rare spontaneous recombination events in the complementing cell line.
Transgenic and “Knock-Out” Animals Animals carrying foreign genetic information (transgenic animals) should be handled in the containment level appropriate to the characteristics of the products of the foreign genes. For each new line of transgenic animal, the routes by which the animals can be infected, the inoculum size required for infection, and the extent of the virus shedding by the infected animal must be determined. Animals with targeted deletions of specific genes (“knock-out” animals) do not generally present particular biological hazards.
Blood and blood products Vaginal secretions Semen Amniotic fluid Unfixed tissues Cerebrospinal, synovial, pleural, pericardial and peritoneal fluids Cell cultures Saliva Urine Tears Sputum Feces Vomit Other excretions and secretions Human Source Material Second column not covered in Bloodborne Pathogen Standard, possibly not occupationally related.
Human Source Material • May transmit infectious agents • Imperfect knowledge of infectious status • Incubation period (asymptomatic) • No test for every pathogen • Most tissues and body fluids • Bloodborne Pathogens (HBV, HCV, HIV, HTLV-1) • Pathogens causing Malaria, Syphilis, Babesiosis, Brucellosis, Leptospirosis, Arboviral infections, Relapsing fever, Creutzfeldt-Jacob disease and viral hemorrhagic fever
Cell Culture Risks • Contaminating pathogenic agents • natural (often zoonotic) or inadvertent • ~20 LAIs from primary cultures in last 30 years • e.g., Herpes B (CHV-1), prions • Oncogenic potential • could be oncogene or oncogenic agent • e.g., HPV-18, MPMV genomes in HeLa cells • Unexpected (adventitious) agents • e.g., SIV, STLV, SV5 in primate cells, HHV-8 in BCBL-1 cells • Hazardous chemicals added to culture medium
Cell Culture under Bloodborne Pathogen Standard • ABSA requested OSHA’s interpretation in 1994: • Do human cell cultures fall under the Bloodborne Pathogen (BBP) Standard? • Response: • All primary human cell cultures (explants) and subsequent in vitro passages fall under the BBP Standard • To be exempted from the BBP requirements, cell strains and lines must undergo testing and characterization (documented) for bloodborne pathogens (not just HBV, HCV and HIV)
Cell Culture Safety • Extend Universal/Standard Precautions to all human and animal cell cultures • Consider working at BSL 2 (most work there already to protect the cell cultures) • Handle all cultures in a biosafety cabinet • If human origin and not demonstrated to be free of human bloodborne pathogens, adhere to requirements of the BBP Standard • Wear PPE appropriate to human source material
Human Source Materials May be regulated Can be biohazardous Use Universal Precautions at all times Visible blood means increased risk Don’t consider “normal” source Human and Non-human Primate Cell Cultures Treat human cultures as possible biohazards Beware of non-human primate cells Beware of CNS, corneal, pituitary cells Some cells may be OK at BSL 1 Summary
Risk Assessment for Work with Research Animals • Risks associated with the research agent used in the animal • chemical, physical, biological • Risks associated with the species of animal used • zoonotic agents • Risks associated with animal maintenance • ergonomic factors, bites, scratches, allergens
Risks Associated with the Agent Used • Chemical agents • carcinogens, mutagens • toxic chemicals • anesthetics • Physical agents • radiation • heat • sound
Risks Associated with the Agent Used • Potentially biohazardous agents • deliberate use of an infectious agent in animals for research purposes • maintenance of infected animal for duration of experiment • sacrifice, necropsy and harvesting of agent or infected tissue
Transmission of Biohazards During Work with Animals Airborne • Release of infectious aerosols by animal by sneezing, coughing • Release during nasal infection or aerosol challenge • Aerosolization from bedding and excreta • During surgical procedures • During birthing
Transmission of Biohazards During Work with Animals Direct Inoculation • Needlesticks during injection/inoculation process • Bites and scratches from infected animal
Transmission of Biohazards During Work with Animals Direct exposure of mucous membranes (by splash or splatter) • During surgical procedures • During injection • During necropsy
Transmission of Biohazards During Work with Animals Indirect transmission and ingestion • From contaminated hands or gloves to mouth • Facial contamination directly from animal • Transfer of parasites by animal handling Indirect transmission with eye or mucous membrane exposure • Dust from bedding • Splash during cage washing • “Dirty” environment