Basic Biosafety Principles EH&S Academy Brenda J. Wong, UCSD Biosafety Officer October 2009

What is Biosafety? Safety from exposure to Infectious Agents Smallpox

Did the Plague Kill Illinois Scientist? What is Biosafety? Sunday, Sep. 20, 2009 Did the Plague Kill Illinois Scientist? By AP (AP / CHICAGO) — The University of Chicago Medical Center says the infection that killed a scientist may be connected to bacteria he researched that causes the plague. The university said Saturday that its researcher studied the genetics of harmful bacteria including Yersinia pestis, which causes the illness. He died Sept. 13. His name and age haven't been released The medical center says the bacteria he worked with was a weakened strain that isn't known to cause illness in healthy adults. The strain was approved by the Centers for Disease Control and Prevention for laboratory studies. An autopsy found no obvious cause of death but did find the presence of the bacteria. More tests are planned. No other illnesses have been reported.

Biosafety in Various Disciplines Biosafety is related to several fields ECOLOGY: referring to imported life forms not indigenous to the region (Reggie the alligator) AGRICULTURE: reducing the risk of alien viral or transgenic genes, or prions such as BSE/"MadCow“; reducing the risk of food bacterial contamination MEDICINE: referring to organs or tissues from biological origin, or genetic therapy products, virus; levels of lab containment protocols BSL-1, 2, 3, 4 in rising order of danger CHEMISTRY: i.e., nitrates in water, PCB levels affecting fertility EXOBIOLOGY: i.e., NASA's policy for containing alien microbes that may exist on space samples - sometimes called "biosafety level 5"

Biosafety in Academic Research Research Universities: Promoting safe laboratory practices, and procedures; proper use of containment equipment and facilities; provides advice on laboratory design and risk assesment of experiments involving infectious agents, rDNA in-vitro and in-vivo. Bottom Line: Risk & Containment

Biohazard Symbol Charles Baldwin at National Cancer Institute at NIH. Symbol to be “memorable but meaningless” so it could be learned. Blaze orange – most visible under harsh conditions

Biosafety Issues Laboratory Safety Bloodborne pathogens (BBP) Recombinant DNA (rDNA) Biological waste disposal Infectious substance and diagnostic specimen shipping

Biosafety Issues (con’t.) Respiratory Protection Bioterrorism and Select agents Mold and indoor air quality Occupational safety and health in the use of research animals Biohazards used in animal models

Biohazardous Materials Viruses Bacteria Fungi Chlamydiae/Rickettsiae Prions Recombinant DNA

Biohazardous materials Transgenic Plants, Animals and Insects          

Transgenic Insects

Biohazardous Materials Human and Primate Cells, Tissues, and Body Fluids Brain Tissue from Demented Patients Viral Vectors Replication deficient viruses

Biosafety Concepts Biosafety In Microbiological and Biomedical Laboratories “BMBL” (acronym) CDC/NIH Publication Safety “Guidelines” Regulations of Institution receives NIH funding Code of Practice and “Gold” Standard in Industry anl Gold Standard Clinical & Research Lab. Lab. Animal Facilities The 4th edition of the BMBL (1999) contained the first government writings about security concepts for Select Agents. By inclusion, these ideas can be used by all laboratories that store and use pathogens, particularly those that might be perceived as or used for terrorist purposes. HHS Publication No. (CDC) 93-8395

The New BMBL Early print edition…. Emphasis on “Risk & Containment”

Biosafety Concepts The BMBL The BMBL continues to be published by the CDC and the NIH 5th edition is now at the printers http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm

Are the NIH Guidelines Optional? “Guidelines” does not mean “optional” They are a term and condition of NIH funding for recombinant DNA research. Now although they are called ‘Guidelines’ we don’t want to give you the impression that they are optional. You can’t pick and choose which sections to follow based on what sounds like a good idea or seems to much trouble to deal with. The Guidelines are a term and condition of NIH funding for recombinant DNA research and failure to comply with them can affect the funding for recombinant DNA work at an institution. From Kathryn Harris, NIH, OBA

Biosafety Concepts from the BMBL Principles of Biosafety Practice and Procedures Standard Practices Special Practices & Considerations Safety Equipment Facility Design and Construction Increasing levels of protection

Principles of Biosafety Biosafety Levels 1-4 (BSL) Increasing levels of employee and environmental protection Guidelines for working safely in research & medical laboratory facilities Animal Biosafety Levels 1- 4 (ABSL) Laboratory animal facilities Animal models that support research Guidelines for working safely in animal research facilities

Biosafety Concepts The BMBL (1) Standard Microbiological Practices Most important concept / Strict adherence Aware of potential hazard Trained & proficient in techniques Supervisors responsible for: Appropriate Laboratory facilities Personnel & Training Special practices & precautions Occupational Health Programs

Biosafety Issues The BMBL (2) Safety Equipment Primary Containment Barrier Minimize exposure to hazard Prevent contact / Contain aerosols Engineering controls/ equipment Personal Protective Equipment (PPE) Gloves, gowns, Respirator, Face shield, Booties Biological Safety Cabinets Covered or ventilated animal cage systems

Biosafety Concepts The BMBL (3) Facility Design and Construction Secondary Barrier/ Engineering controls Contributes to worker protection Protects outside the laboratory Environment & Neighborhood Ex. Building & Lab design, Ventilation, Autoclaves, Cage wash facilities, etc.

Laboratory Design “Warehouse Type Lab”

Discussion What are some of the negatives and positives of this open lab concept?

Biosafety Level-1 Concepts of Biosafety Biosafety Level-1 (BSL-1 or ABSL-1) Well characterized agents Agents not known to cause disease (in healthy human adults; now healthy immunocompetent adults) Prophylactic treatment available Open bench procedures Animals in open cage system or open environment (outdoors) Good laboratory practices

Risk Group 1 Agents E.coli K-12 Transgenic Plants Plasmids Fungi Mold Yeast

BSL-1 Practices Bench-top work allowed Daily Decontamination Manual pipetting Required Handwashing Red bag waste Bio cabinet not required (unless creating aerosols) 2˚ containment

Risk Group 2 Agents Human or Primate Cells Herpes Simplex Virus Replication Incompetent Attenuated Human Immunodeficiency Virus Patient specimens

BSL-2 Practices Concepts of Biosafety Practices & Procedures Agents associated w/ human disease Treatment for disease available Agent poses moderate hazard to personnel and environment Direct contact or exposure Percutaneous exposure Scratch, Puncture, Needle stick Mucus membrane exposure Eyes, Mouth, open cut

BSL-2 Practices Limited access to lab when work in progress Daily decontamination Mechanical pipetting Labcoat, safety glasses and gloves required Red bag & sharps containers required

BSL-2 Practices (con’t) Biohaz. Sign posted at entrance to lab Label all equipment (incubators, freezers, etc.) TC room – negative air flow Documented training Baseline serology or pre-vaccination may be required

Risk Group 3 Agents Human Immunodeficiency Virus Mycobacterium tuberculosis Coxiella burnetii

Biosafety Level 3 Working in High Containment Biosafety Level-3 (BSL-3 or ABSL-3) Indigenous or exotic agents Aerosol transmission Serious health effects Treatment may or may not exist

BSL-3 Practices Public access NOT permitted Daily decontamination after spill and upon completion of experiment Autoclave required and waste is disposed at the end of day Required foot activated handwashing sink and controls No sharps unless absolutely necessary

BSL-3 Practices (con’t) Aerosol minimization procedures required Wrap around disposable clothing is required. Specialized equipment may be required depending upon procedures Biohaz. Signs and labels posted Air flow from low hazard to high hazard “Pressure Mapping”

BSL-3 Practices (con’t) Bench top work not permitted Documented training and personnel competency certification (for BSL-3 procedures) Baseline serology Spills – report immediately and treat accordingly Vaccinations/post exposure protocols and SOP’s, Biosafety Manual, Biosafety Officer

UCSD’s BSL-3

Biosafety Level-4 Working in High Containment Builds on BSL-3/ ABSL-3 practices Maximum containment facilities Pressurized Containment Suite BSL-3 + Class III Biosafety Cabinet Chemical decontamination showers Liquid effluent collection / decontamination No BSL-4 labs exist at UCSD

Biosafety Level 4 Lassa Fever Virus Ebola Hemmorrhagic Fever Virus Marburg Virus Herpes B Virus

Biosafety Concepts Working in High Containment Biosafety Level-4 (BSL-4 or ABSL-4) Dangerous/exotic agents Life threatening disease Aerosol transmission Agents of unknown risk of transmission or health affects No known treatment

Animal Biosafety Level-4 Working in High Containment

General Good Lab Technique Hygienic Practices No Smoking, Eating, Applying cosmetics, lip balm, contacts Wash hands after procedures Decontaminate lab bench before and after work

General Operational Practices Proper attire Minimum – lab coat, safety glasses, gloves Plan your work Know in advance what you are working with Read available resources (MSDS) http://www.hc-sc.gc.ca/pphb-dgspsp/msds-ftss/index.html

Animal Containment Points CDC - 1957 CDC & UCSD - 2005 Courtesy of Paul Vinson, CDC

Discussion # 2 Based on what you know about Biosafety Levels, Practices and Operational Controls, what are some discussion issues for conducting Biohazard risk assessments? How do you approach risks when addressing a particular organism?

Risk Assessment In-Vitro Human Clinical Trial In-Vivo

Addressing Risk Assessments What is the organism? Is it Wild-type, attenuated, irradiated, or chemically treated? Look at kill data or kill curves. What is the max. concentration, volume, infectious dose? What is the work space like? Aerosolizing procedures? How do they contain their aerosols?

Risk Assessment, con’t Tom Pugh Are personnel trained? Do personnel understand the organism, infectious dose and symptoms? What are their experimental procedures? Will they be transporting the material? Shipping intra, inter-state or international? Are they doing tissue culture? Do they have adequate containment equipment?

Risk Assessment, Con’t Are they doing this work in-vivo? Have you consulted and discussed this with the Vets and IACUC to determine special needs and housing? Waste issues addressed? Pregnancy issues with the organisms?

Risk Assessment, con’t Do they share their Tissue Culture room? Do they have more than 1 Biosafety Cabinet? Occupational Health informed and set up to receive patient or offer counseling?

Accidental Spills Evacuate area, alert personnel and cordon off so that aerosols may settle Don PPE; Cover with paper towels and apply bleach (1 part bleach : 9 parts water Allow 15 – 20 min contact time Wipe up working towards center Use tongs if broken glass is involved Is Recombinant DNA involved?

First Aid Measures Splash to Eye or Needlestick Injury Rinse thoroughly for 15 minutes at the eyewash or sink Call Occupational Medicine  619 471-9210 Call EH&S to report exposure – 858 534-5366

What Helps?

Resources UCSD Biosafety: http://blink.ucsd.edu/safety/research-lab/biosafety/ Biological Safety MSDS: http://www.phac-aspc.gc.ca/msds-ftss/index-eng.php NIH BMBL: http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm See Handout for additional resources..

Questions? Brenda Wong Bwong@ucsd.edu 858 534-6059 Cell: 858 583-3277 Additional Resources: Biosafety Resources.doc