BIOSAFETY TRAINING. NH IN-BRE May 22, Brenda Petrella, PhD Dartmouth College Biosafety Officer. DARTMOUTH COLLEGE Environmental Health & Safety

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1 BIOSAFETY TRAINING NH IN-BRE May 22, 2014 Brenda Petrella, PhD Dartmouth College Biosafety Officer

2 Objectives Biosafety overview Background Principles Containment Risk Assessment Bloodborne Pathogens Best Practices Laboratory Inspections rdna overview What is recombinant or synthetic DNA Safety considerations of rdna experiments

3 What is Biosafety? The principles, practices, procedures, and containment measures designed to prevent the accidental exposure to or release of biological agents and toxins. What is Biosecurity? The protection, control, and accountability measures implemented to prevent the loss, theft, misuse, diversion or intentional release of biological agents and toxins.

4 What is a Biohazard? An agent or material of biological origin that has the capacity to produce deleterious effects in humans, animals, or the environment: Viruses, bacteria, fungi, parasites, prions Blood, body fluids, cells, and tissues from humans or animals Biological toxins, allergens, venoms Recombinant DNA

5 Principles of Biosafety

6 Why Biosafety? Laboratory Acquired Infections (LAIs) Route Inhalation Inoculation Practice Aerosol generation pipetting, vortexing, centrifuging, sonication Needlestick accidents; sharps; animal or insect bites scratches Mucous Membrane Spills/splashes into eyes, mouth, nose; aerosols Ingestion Skin Contamination Splashes into mouth; contaminated fingers near or in mouth; eating in lab Spills/splashes without PPE; contaminated surfaces or equipment Sewell, DL. Clinical Microb. Rev., July 1995, p

7 Why Biosafety? Laboratory Acquired Infections (LAIs) 4,079 LAIs 168 deaths Update: 1,141 ( ) LAIs 2033 LAIs 37 deaths 24 deaths and counting 43 yrs yrs Pike, R.M Annu. Rev. Mocrobiol. 33: Harding L.H. and Byers, K.B Epidemiology of laboratory-acquired infections. Biological Safety Principles and Practices.

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9 Outbreak of Salmonella Typhimurium LAIs Teaching and Clinical Microbiology Labs BSL2 agent 109 LAIs 4 were in children

10 Containment Primary goal of biosafety: reduce or eliminate human and environmental exposure to potentially harmful agents Biological Containment Inactivating mutations Reducing virulence or pathogenicity Primary Containment Protects lab personnel and the immediate lab environment Biological Safety Cabinet (BSC) Good microbiological techniques (PPE, decontamination, etc.) Safety centrifuge cups Secondary Containment Protects environment external to the lab Facility design and operations lab doors and/or anterooms specialized air handling systems for contamination control autoclaves hand washing, eyewash, shower stations

11 Clean bench vs Fume hood vs BSC Clean bench Protects sample ONLY Fume Hood Protects user from volatile compounds Does not protect against infectious agents Biological Safety Cabinet Protects user, sample, and environment from biologics Does not provide chemical protection

12 Risk Groups No/low individual risk No/low community risk Moderate individual risk Low community risk High individual risk Low community risk High individual risk High community risk

13 What are Biosafety Levels (BSLs)? The primary risks that determine levels of containment are: Risk Group category Infectivity Severity of disease Transmissibility Nature of the work conducted Route of exposure

14 Biosafety Level 1 (BSL1) Risk Group 1 Agents: E.coli K-12 Transgenic plants Fungi Mold Yeast Lab Practice Standard microbiological practices Open bench work ok unless aerosols generated, then use BSC Daily decontamination Required hand-washing Autoclave waste Safety Equipment PPE gloves, lab coat, eye protection as needed BSC if generating aerosols Facility Sink available for hand washing Door on lab with appropriate signage

15 Biosafety Level 2 (BSL2) Builds on BSL1 requirements Risk Group 2 Agents: Human or primate cells Herpes Simplex Virus (HSV) Replication-incompetent attenuated HIV Patient specimens Staphylococcus aureus Lab Practice Access to the laboratory is restricted when work is being conducted PPE = lab coats, gloves, eye protection, face shields or masks as needed. Decontamination after each procedure Autoclave waste Document training

16 BSL2 cont. Safety Equipment Biosafety cabinet (BSC) Sharps containers Autoclave waste bags/containers Facility Self-closing doors with BSL2 signage Autoclave Sink and eyewash station readily available Label all equipment in contact with biohazard

17 Biosafety Level 3 (BSL3) Builds on BSL2 requirements Risk Group Agents: Respiratory transmission Mycobacterium tuberculosis HIV (wild-type) Coxiella burnetii Lab Practice Medical surveillance, baseline serology Immunizations available or required Autoclave all waste at end of day Avoid use of sharps No bench top all work in BSC PPE double gloves, PAPR, wrap around disposable gown

18 BSL3 cont. Safety Equipment All work with microbes must be performed within an appropriate BSC Facility A hands-free sink and eyewash Exhaust air cannot be recirculated Sustained directional airflow by drawing air into the lab from clean areas towards potentially contaminated areas Entrance to the lab is through two sets of self-closing and locking doors Access to the laboratory is restricted and controlled at all times.

19 Biosafety Level 4 (BSL4) Maximum containment facility - builds on BSL3 requirements Risk Group Agents high risk of aerosol-transmitted infections exotic, frequently fatal examples: Ebola and Marburg viruses Lab Practice Change clothing before entering Shower upon exiting Decontaminate all materials before exiting Safety Equipment All work performed in Class III, wearing a full body, air-supplied, positive pressure suit. Facility separate building or restricted zone dedicated supply and exhaust air, and vacuum lines and decontamination systems

20 Biosafety Risk Assessment 1. Identify the hazard 2. Assess potential risk to personnel and/or environment: Identify Risk Group What is the scale of work volume, concentration What are the procedures/manipulations aerosol generating? Use of sharps? Determine pathogenicity is it wild-type or attenuated? What is the infectious dose? What is the route of entry? Are prophylaxis treatments available? Are personnel trained? Any risk to immuno-compromised or pregnant personnel? 3. Determine appropriate containment/biosafety level (BSL) RG1 RG2 RG3 RG4

21 Question 1 An undergraduate is studying the regulation of circadian rhythm genes using the mold, Neurospora crassa (RG1), as a model system. Methodology will be limited to quantitative PCR and western blotting to assess gene regulation and expression. What PPE should be worn? Can this work be conducted on the bench top? At what BSL level should this work be conducted? BSL1

22 Question 2 A principal investigator just received IN-BRE funding to study antibiotic resistance of Pseudomonas aeruginosa (RG2) in cystic fibrosis. He will be using a human lung cell line model system for infectivity studies using various clinical isolates of the bacteria. What PPE should be worn? Can this work be conducted on the bench top? What types of containment should be considered? At what BSL level should this work be conducted? BSL2

23 Bloodborne Pathogens What are they? Microorganisms that are transmitted through the bloodstream. HIV Hep B Hep C Syphilis Malaria Where are they found? Blood or other bodily fluid Human cell lines, fresh tissues HIV Routes of exposure Needle-sticks Other sharps Skin scratches Mucous membrane Who is at risk? Lab personnel Custodial staff

24 Bloodborne Pathogens OSHA Standard (29 CFR ) ü Establish exposure control plan, updated annually ü Implement use of Universal Precautions ü Use engineering controls sharps disposal containers self-sheathing needles or other engineered sharps-injury protection reduce the need for needles ü Identify work practice controls change the way a task is performed increasing containment proper labeling ü Provide appropriate PPE ü Provide training ü Availability of Hepatitis B vaccine and postexposure monitoring

25 Best Practices - Programmatic Biosafety Manual Exposure Control Plan Training IBC oversight NIH Guideline oversight, reporting, (rdna, toxins, biohazardous agents) Standard Operating Procedures (SOPs) Injury and Accident Reports Lab Inspections Occupational Health Program

26 Best Practices Sharps Bloodborne pathogens Use safety-engineered sharps Use biohazard sharps containers Use plastic pipets instead of glass pasteur pipets for aspiration Never: Re-cap needles Bend or break needles Re-use needles or syringes Let sharps container get too full Put hands into sharps container

27 Best Practices - Vacuum Traps House vac port Tube from media to top inlet of flask (part a) Working end of tube used to aspirate biological waste with pipette Direction of vac flow Tube from side of flask to house vac line (part b) Rubber stopper w/pipette Trap flask w/ wescodyne HEPA filter 2 container

28 Best Practices Prevention of Aerosols Perform manipulations inside BSC Pipetting Vortexing Sonication Opening of centrifuge tubes Aspiration rather than decanting Use safety cups with o-rings for centrifugation

29 Biosafety Inspection Checklist PPE, handwashing supplies Eyewash stations Sharps eliminated where feasible Biohazard waste bags (autoclave bags) no sharps! Biohazard sharps containers No recapping of needles Waste containers not overfilled Proper disinfection, spill supplies, disposal procedures Vacuum traps Equipment (BSC certification, aerosol resistant centrifuges) Autoclaving waste - verification

30 rdna OVERVIEW

31 What is recombinant DNA (rdna)? According to the NIH Guidelines, recombinant and synthetic nucleic acids are defined as: (i) molecules that a) are constructed by joining nucleic acid molecules b) that can replicate in a living cell, i.e., recombinant nucleic acids (ii) nucleic acid molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules, i.e., synthetic nucleic acids (iii) molecules that result from the replication of those described in (i) or (ii) above.

32 What is DNA? DNA = information storage molecule

33 What is recombinant DNA (rdna)? Recombination = creation of a new DNA molecule from two different DNA molecules human insulin gene rdna bacterial plasmid Why does it work? Genetic code is universal! bacterial plasmid carrying human insulin gene

34 Common ways to make rdna Molecular cloning Arts & Crafts: Cut/paste pieces of DNA together; grow up new DNA in bacteria (bugs replicate new DNA for us) PCR (polymerase chain reaction) Xerox machine: exponentially duplicate same finite sequence of DNA Chemical synthesis ( synthetic DNA ) Mad scientist Generation of artificial DNA molecules that do not exist in nature (no starting sequence) Able to recombine with naturally occurring nucleic acid molecules

35 Common lab uses of rdna Overexpression of a gene in cells, animals, or plants Knockdown of a gene in cells, animals, or plants Copying or mutating a gene Sequencing DNA Measuring gene expression

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37 Safety Considerations for rdna work Increase pathogenicity? Change host range? (tropism) Inactivate a tumor suppressor gene or activate an oncogene? What are the potential downstream effects of release? Does it make an organism resistant to treatment or change their growth/development? GMOs Antibiotic resistant bacteria Recombinant bovine growth hormone (rbgh)

38 Biosafety Summary Biohazard recognition Biohazard mitigation Risk Assessment

39 Resources BMBL: NIH Guidelines: ABSA: WHO Biosafety Manual: WHO_CDS_CSR_LYO_2004_11/en/ OSHA Bloodborne Pathogens Standard: bloodbornepathogens.pdf Public Health Agency of Canada pathogen safety sheets: