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

DARTMOUTH COLLEGE Environmental Health & Safety

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


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.

DARTMOUTH COLLEGE Environmental Health & Safety http://www.cdc.gov/biosafety/publications/bmbl5/

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


Principles of Biosafety


Why Biosafety? Laboratory Acquired Infections (LAIs)


Route Practice Inhalation Aerosol generation – pipetting,

vortexing, centrifuging, sonication Inoculation Needlestick accidents; sharps;

animal or insect bites scratches Mucous Membrane Spills/splashes into eyes, mouth,

nose; aerosols Ingestion Splashes into mouth;

contaminated fingers near or in mouth; eating in lab

Skin Contamination

Spills/splashes without PPE; contaminated surfaces or equipment

Sewell, DL. Clinical Microb. Rev., July 1995, p. 389–405. http://wwwnc.cdc.gov/eid/article/12/1/05-1126_article.htm

Why Biosafety? Laboratory Acquired Infections (LAIs)

Pike, R.M. 1979. Annu. Rev. Mocrobiol. 33:41-66. Harding L.H. and Byers, K.B. 2006. Epidemiology of laboratory-acquired infections. Biological Safety Principles and Practices.


4,079 LAIs

168 deaths

1935 1978 43 yrs

1,141 LAIs

24 deaths

1979 2005 26 yrs

Update: (1979-2013)

2033 LAIs 37 deaths

and counting

Outbreak of Salmonella Typhimurium LAIs Teaching and Clinical Microbiology Labs

http://www.cdc.gov/salmonella/typhimurium-laboratory/011712/index.html

BSL2 agent 109 LAIs 4 were in children

2010-2011


Containment Primary goal of biosafety: reduce or eliminate human and environmental exposure to potentially harmful agents


Primary Containment •  Protects lab personnel and the immediate lab environment •  Biological Safety Cabinet (BSC) •  Good microbiological techniques (PPE, decontamination, etc.) •  Safety centrifuge cups

Biological Containment •  Inactivating mutations •  Reducing virulence or pathogenicity

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

Clean bench vs Fume hood vs BSC

Biological Safety Cabinet •  Protects user, sample, and

environment from biologics •  Does not provide chemical

protection

Fume Hood •  Protects user from volatile

compounds •  Does not protect against

infectious agents

Clean bench Protects sample ONLY

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


Risk Groups

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


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 DARTMOUTH COLLEGE

Environmental Health & Safety

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


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


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


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.


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 DARTMOUTH COLLEGE Environmental Health & Safety

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) DARTMOUTH COLLEGE Environmental Health & Safety

RG1 RG2 RG3 RG4

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.

BSL1

What PPE should be worn? Can this work be conducted on the bench top?

At what BSL level should this work be conducted?


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.

BSL2

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?


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

Routes of exposure •  Needle-sticks •  Other sharps •  Skin scratches •  Mucous membrane

Who is at risk? •  Lab personnel •  Custodial staff

Bloodborne Pathogens

HIV


Bloodborne Pathogens OSHA Standard (29 CFR 1910.1030)

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 post-

exposure monitoring DARTMOUTH COLLEGE Environmental Health & Safety

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


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


Best Practices - Vacuum Traps


Trap flask w/ wescodyne HEPA

filter 2° container

Tube from media to top inlet of flask

(part a)

Tube from side of flask to

house vac line (part b)

Direction of vac flow Working end of tube

used to aspirate biological waste

with pipette

Rubber stopper w/pipette

House vac port

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

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


rDNA OVERVIEW


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.


What is DNA? DNA = information storage molecule


What is recombinant DNA (rDNA)? Recombination = creation of a new DNA molecule from two different DNA molecules

Why does it work? Genetic code is universal!

rDNA

bacterial plasmid carrying human insulin gene


human insulin gene

bacterial plasmid

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 DARTMOUTH COLLEGE Environmental Health & Safety

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


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)

Biosafety Summary

Biohazard recognition

Risk Assessment

Biohazard mitigation


Resources •  BMBL: http://www.cdc.gov/biosafety/publications/bmbl5/

•  NIH Guidelines:http://osp.od.nih.gov/office-biotechnology-activities/biosafety/nih-guidelines

•  ABSA: http://www.absa.org/

•  WHO Biosafety Manual: •  http://www.who.int/csr/resources/publications/biosafety/

WHO_CDS_CSR_LYO_2004_11/en/

•  OSHA Bloodborne Pathogens Standard: https://www.osha.gov/dte/grant_materials/fy09/sh-18796-09/bloodbornepathogens.pdf

•  Public Health Agency of Canada – pathogen safety sheets: •  http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/pseudomonas-spp-eng.php


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