Introduction to Biosafety

Transcription

1 Objectives Introduction to Biosafety Environmental Health and Safety Have a good working knowledge of risk groups and containment levels Recognize the risks associated with biohazards in the laboratory Use safe practices when handling biohazards Understand the strengths and limitations of autoclaves and disinfectants Know how to respond to accidental exposures and spills of biohazardous material Regulations & Guidelines Public Health Agency of Canada (PHAC) Guidelines Canadian Food Inspection Agency (CFIA) Standards Environment Canada New Substances Notification Transport Canada Transport of Dangerous Goods Workplace Hazardous Materials Information System (WHMIS) 1

2 Regulations P H A C Human Pathogens C F I A Animal Pathogens C F I A Prions Canadian Biosafety Standards and Guidelines CFIA Standards Containment Standards for Facilities Handling Plant Pests Containment Standards for Facilities Handling Aquatic Animal Pathogens (2010) Environment Canada New Substances Notification Regulations (Organisms) Reporting mandatory for manufactured/imported microorganisms if: Not on Domestic Substances List Research & development exemption unless: Introduced into experimental field study without containment WHMIS (Workplace Hazardous Materials Information System) CLASS AND SYMBOL CHARACTERISTICS PRECAUTIONS Class D Poisonous and Infectious Material Division 3: Biohazardous Infectious Materials Microbiological agents (e.g., bacteria, viruses, fungi and their toxins) that may cause illness or death Wear the recommended protective equipment and clothing Work with these materials in designated areas Disinfect area after handling Wash hands after handling 2

3 Where to find MSDS for infectious substances? Transport of Dangerous Goods Division 6.2 Biological Substances Transport Canada -TDG Regulations Division 6.2 Infectious Substances Packaging and labeling of biological agents or toxins transported to another entity Mandatory training for all who transport (shipping and/or receiving) 3

4 Definition: Biohazards Biohazards Bacteria Viruses Fungi Parasites Plants and algae Toxins of biological origin Biohazards Prions Human tissues, blood and body fluids Animals, animal tissues and carcasses Cell lines Nucleic acids Genetically modified strains & organisms Salmonella typhi Formaldehyde Mould spores Tetrodotoxin Ethidium bromide 4

5 Consequences of Lapses You or a colleague could be exposed... Personnel exposure Contamination of research Environmental release Negative public perception Unvaccinated student Not handled by victim Genetically modified strain Stored in freezer, not used for 5 years You or a colleague could be exposed... continued You or a colleague could be exposed students, 1 teacher infected Sheep anatomy experiment Lab disorganized, unsanitary Dean, party secretary relieved of duties Associate Professor Attenuated strain Cause of death: hemochromatosis -induced iron overload 5

6 ...your research could be exposed... as well as the environment... >600 animals culled Loss of 3.5 millions per day Lasted >20 days Cell culture contamination with mycoplasma Laboratory-Acquired Infections (LAI) LAI: Routes of Transmission Direct contact (e.g. splash) Inhalation Ingestion Parenteral inoculation Indirect contact Vectors: animal/insect bites 6

7 LAI: Routes of Transmission Inhalation - aerosols Infectious airborne particles Large droplets (100um) settle Small droplets respirable 5-10 microns: upper respiratory tract <5 microns: lungs LAI: Routes of Transmission Inhalation - aerosols Some aerosol generating procedures: homogenizing, blending, grinding vortexing, mixing, pipetting centrifugation opening snap-cap tubes animal cage changing, necropsy streaking agar plates spills LAI: U.S. and Canada What to do after accidental exposure? Under-reported (81 cases) 34 infections confirmed 3 deaths Most common causes: Inoculation (10) Skin & mucous membrane contamination (3) Unknown (21) 1. Ask for help 2. Serious injury: dial 911 and Security Downtown: Macdonald Campus: MNI: Needlesticks and cuts: wash with soap and water 4. Splashes: flush eyes, mouth, nose Public Health Agency of Canada

8 What to do after accidental exposure? What to do after accidental exposure? 5. If required, seek medical attention: Student Health Services Downtown M-F 9:00 16:00 Brown Bldg, Rm Macdonald Campus M/W/Th 9:00-15:30 Centennial Centre, Rm. CC1-124 T/F 13:00-15: Nearest hospital or clinic 6. Notify supervisor 7. Complete Accident, Incident & Occupational Disease Report Page 1: victim Page 2: supervisor/safety representative Submit to EHS (MNI Rm 778 if working at MNI) 8. Monitor for symptoms - most pathogens have an incubation period Accidental Exposure Bloodborne pathogens 8

9 Risk Groups & Containment Levels Risk Groups Categorization of relative hazards of infective organisms (4 levels) Risk classification: Pathogenicity Infectious dose Mode of transmission Host range Effective prevention, e.g. vaccine Effective treatment, e.g. antibiotic, antiviral PEP Risk Groups Risk Groups Risk Group 1 Low individual risk Low community risk Unlikely to infect healthy humans & animals Risk Group 2 Moderate individual risk Low community risk Treatment/prevention available Standard molecular biology E. coli K12, Lactobacillus spp., Bacillus subtilis, Micrococcus spp., Aerococcus spp. Listeria spp., Salmonella spp., Leishmania spp., Toxoplasma spp., Ascaris spp., enteropathogenic E. coli, cell lines 9

10 Risk Groups Risk Groups Risk Group 3 High individual risk Low community risk Cause serious disease, treatment/prevention usually available Risk Group 4 High individual risk High community risk Cause serious or lethal disease by casual contact Treatment/prevention not usually available Hantavirus, Bacillus anthracis, Yersinia pestis, Histoplasma capsulatum, cultures of Hepatitis B or HIV Marburg virus, Ebola virus, Herpesvirus simiae, Crimean-Congo hemorrhagic fever Containment Levels Clostridium difficile Saccharomyces cerevisiae Minimum requirements for safe handling (4 levels) Operational practices Safe work practices Engineering, technical, physical: Location & access Surface finishes & casework HVAC Containment perimeter (windows, autoclave location, etc.) Services (water, drains, gas, electricity, equipment) Bovine Spongiform Encephalopathy 10

11 Risk Groups vs Containment levels Containment Level 1 Risk groups agent-specific Containment levels based on: Risk groups Potential for aerosol generation Quantity Concentration In vitro vs in vivo RG and CL not always the same Containment Level 1 Containment Level 2 Physical requirements: Basic design features Operational requirements: Work may be done on open bench top BSC not required, may be used for sterility Good microbiological practices (good hygiene) 11

12 Containment Level 2 Additional physical requirements, e.g. Limited access, signage, lockable doors Resistant, non-absorptive surfaces (for disinfection) Containment of aerosols, e.g.: BSC Centrifuges with sealed rotors or safety cups Minimize environmental contamination: Handwashing sinks Decontamination facilities (autoclaves) Containment Level 2+ Level 2 physical, Level 3 operational Mandatory Standard Operational Procedure (SOP) Entry, exit protocols Personal protective equipment Experimental procedures Effective use of BSC Safe use of lab equipment Emergency procedures Decontamination and waste disposal Specific training required, must be documented Containment Levels 3 and 4 Level 3 Respiratory protection HEPA filtration of lab exhaust Strictly controlled access Risk Assessment Contamination of research Level 4 Laboratory associated infection Biohazards Contamination of the environment Isolated facility with sealed perimeter Positive pressure suits or Class 3 BSC Effluent sterilization system Public perception 12

13 Human Blood and Body Fluids May contain bloodborne pathogens e.g. HBV, HCV, Treponema (Syphillis), HIV Use universal precautions HBV vaccination Cell Cultures Risks based on: Source of tissue Primary vs. characterized cell line ATCC screens for bacteria, fungi, mycoplasma only Presence of pathogens: Naturally Via contamination, recombination or transfection Cell Lines May contain viral DNA sequences: HEK 293: Adenovirus-5 COS-1, COS-7, SVEC4-10: Simian virus-40 Daudi: Epstein-Barr virus HS-5: Human papillomavirus-16 HeLa: Human papillomavirus RWPE-1: Human papillomavirus Cell Cultures Contaminants can affect: Growth of cells Cell metabolism Cell morphology and swelling Genome structure Recombination Yield of virus production Pauwels et al

14 Recombinant Organisms Case-by-case assessment based on Risks associated with recipient & donor Function of gene expressed in recombinant Host range alteration Replication capacity of recombinant Capability to revert to wild type Viability of host-vector system outside lab Viral Vectors Risk Group or 3 Virus Adeno-Associated virus Baculovirus (insect cells) Adenovirus Herpesvirus (EBV) Poxvirus (vaccinia, fowlpox) Lentivirus (HIV, SIV) Flavivirus Principles of Biosafety, PCIH 2004 Animals Risks based on type of work being conducted Animals can harbour infectious organisms (natural or introduced) Animal Use Protocols Occupational Health Program You are transfecting a containment level 1 cell line with a containment level 2 vector. What is the containment level of the transfected cell line? 14

15 Definition: Biosecurity You are culturing small volumes of recombinant E. coli K-12 expressing constitutively a mammalian oncogene for injection in mice. What factors increase the risk of this project? Prevention of theft, misuse, intentional release Specific for facility Includes: physical protection (e.g. card access, locks) personnel suitability/reliability pathogen accountability (e.g. inventory & tracking) incident reporting, emergency response Definition: Biosafety Engineering Controls Measures employed when handling biohazardous materials to avoid infecting oneself, others or the environment Engineering controls Reduce or eliminate hazards at source First and best strategy Administrative controls Examples: Handwashing facilities Autoclave for waste decontamination Cleanable surfaces Adequate lighting Biological safety cabinets 15

16 Engineering Controls Examples: Filters for flasks, pipette tips & vacuum lines Plasticware substituted for glassware Gasketed blenders, homogenizers Sealed centrifuge rotors, safety cups Microincinerators Administrative Controls Examples: Limited access Safety manual available and use encouraged Insect/rodent control Record-keeping Medical surveillance Training Spill response plan Inventory Administrative Controls Inventory Description (cell lines, organisms, vectors, etc.) Supplier name, if applicable Quantity and volume Location Date in storage Date out of storage Initials Occupational illnesses in the US BioWarfare Program ( ) Era Years Case by million man hours of lab work Pre-BSC BSC Vaccines & BSC s Wedum AG. J Amer Biol Safty Assn 1996;1:

17 Standard Microbiological Practices Standard Microbiological Practices Eating, drinking, applying cosmetics, inserting and removing contact lenses, storing food and utensils not permitted Minimize splashes and aerosols Mouth pipetting prohibited Cover open wounds, cuts, scratches with waterproof dressing Work on plastic-backed absorbent mat Transport biohazards in leak-proof containers Use 4-sided cart Decontaminate work surfaces daily Decontaminate waste Standard Microbiological Practices Handwashing Effective in preventing infection When to wash? Before starting any manipulations When hands are obviously soiled Whenever gloves are removed Whenever leaving the lab Standard Microbiological Practices Safe use of sharps Use blunt-end needles if possible Do not bend, shear, recap needles Discard immediately after use Separate contaminated and noncontaminated Use puncture-proof waste containers 17

18 CFU recovered from operator s gloves Run Before pouring After pouring into centrifuge tubes Average Standard Microbiological Practices Safe pipetting Use mechanical pipetting devices Avoid mixing by vigorous suction & expulsion Discharge liquid down side of container Deliver as close as possible to contents Never blow out last drop Suspension poured contained 10 9 /ml. Flavobacterium (Burnett et al. 2005) Personal Protective Equipment McGill PPE Policy No shorts, open shoes, etc. Use personal protective equipment Fastened lab coat Gloves Eye protection Respirators only if fit-tested Contact EHS 18

19 Sterilization & Disinfection Definitions Sterilization Destruction of all microorganisms, including spores Disinfection Destruction of microorganisms may not kill spores Decontamination Destruction or reduction of pathogens to safe level Microbial Resistance Prions Bacterial spores, protozoan cysts (Bacillus subtilis, Clostridium sporogenes) Mycobacteria (Tubercle bacterium) Non-lipid or small viruses (non-enveloped) (Poliovirus, Coxsackievirus, Rhinovirus) Fungi (Trichophyton, Cryptococcus, Candida, Aspergillus) Vegetative bacteria (P. aeruginosa, S. aureus, S. choleraesuis) Lipid or medium-size viruses (enveloped) (Herpes simplex, HBV, HIV, CMV) Sterilization & Disinfection Physical Methods Heat: dry, moist UV light Infrared radiation (small metal & glass items) Microwaves (biowaste, liquids, nonmetallics) Filtration (heat- or chemical- sensitive liquids) Gamma irradiation (chemical-, heat-, pressuresensitive items) 19

20 Sterilization & Disinfection Chemical Agents Alcohols (70-85%) Aldehydes (glutaraldehyde, formaldehyde) Phenolic compounds (HG7) Halogens (iodine, chlorine) Quaternary ammonium compounds ( Quats ) Oxidizing agents (hydrogen peroxide, ethylene oxide gas) Selection Criteria Chemical Agents Number and nature of microbes Type of item to be treated Purpose (disinfection vs sterilization) Interactions with other chemicals Amount of soil/organic matter Selection Criteria Chemical Agents Required contact time Toxicity to cultures, humans, etc ph, temperature, hardness of dilution water Cost Organic Load Ex: Blood, sputum, milk, bedding, feed, manure Proteins physically protect and stabilize many microorganisms Disinfectant Log reduction of Listeria monocytogenes TSB* Serum Milk 70% Ethanol Sodium hypochlorite Glutaraldehyde *TSB: trypticase soy broth (soybean casein) Gauggel et al

21 Contact Time Extending contact time may increase effectiveness Disinfectant Log reduction of Mycobacteria spp. 1 min 10 min 20 min 70% Ethanol Sodium hypochlorite Glutaraldehyde Gauggel et al Autoclaves Autoclaves Hazards: Steam Heat High chamber pressure Potential exposure to biohazards 21

22 Autoclaves Mode of action: coagulation of proteins Effective conditions: Gravity displacement:121 o C/15 psi, mins Pre-vacuum: 132 o C/27 psi for 4-20 mins Steam must contact material For heat and moisture-stable material Autoclaves Gravity: Longer sterilization time, lower temp and pressure than pre-vac For dry materials (tubes, tips, pipettes, etc.) and liquids Pre-vacuum Creates vacuum to pull steam into chamber, removes air pockets Shorter cycle, higher temp and pressure than gravity Not all items can withstand temp or pressure Autoclave Primary & Secondary Containers Do not autoclave: Chemicals (solvents, corrosives) Radioactive materials Bleach and other chlorinated products Plastic and glass not rated for autoclave use Use only approved autoclave bags Use secondary containers to contain potential spillage Ensure that they are autoclavable 22

23 Primary & Secondary Containers Best: Polypropylene (PP) and polycarbonate (PC) Borosilicate glass (Pyrex) Stainless steel Poor: Polystyrene (PS), polyethylene (PE) and high density polyethylene plastics (HDPE) Regular glassware. Packing & Loading Solids: pack for steam penetration, do not close bags/containers tightly Do not overpack autoclave bags (max 75%) Liquids: Loosen screw caps or use self-venting caps to avoid rupture Do not fill more than 2/3 full Cap open containers loosely with foil Packing & Loading Clean drain screen Avoid crowding or stacking Items must not touch top or sides Do not place items on autoclave floor - use racks or secondary trays Know which cycle to use (e.g. liquid vs dry) Unloading Wear insulated gloves or mitts Ensure chamber pressure gauge reads 0 psi Crack open door, wait 5-10 mins Liquids Wear rubber apron in addition Beware of bubbling (explosion hazard) Remove load, allow to cool before handling 23

24 Temperature Sensitive Tape Biological Indicators Indicates autoclaving temperature reached Markings appear at ~121 C Markings do not prove successful sterilization of contents Use biological indicators, e.g. Geobacillus stearothermophilus, regularly (weekly) to prove that effective cycle in use Indicator spores killed after 15 mins at 121 o C Contact EHS for more information Virginia Tech Environmental Health & Safety 2009 Spill Kit Spill Response Disposable protective clothing Absorbent paper Autoclavable container or bags (if using autoclave) Appropriate disinfectant (fresh) Autoclavable (or disposable) forceps, dustpan Don t panic Remove contaminated clothing Assess degree of contamination & action needed Allow aerosols to settle 24

25 Spill Response Spill Response Don protective clothing Cover spill with disinfectantsoaked towel Gently pour disinfectant around perimeter Work disinfectant toward centre Let sit 30 minutes Wipe down walls, equipment, etc. Pick up broken sharps with forceps, place in sharps container Pick up (forceps, dustpan) absorbent materials and dispose appropriately Spill in BSC Video Leave BSC running Follow spill response procedure If material has gone through grill: Pour disinfectant through grill Allow sufficient contact time Open drain valve, collect liquid for disposal 25

26 Biohazards Applications McGill Biohazards Policy* ``Prior to beginning work with biohazardous materials, the responsible person must complete and submit an Application to Use Biohazardous Materials to Environmental Health & Safety for review and approval.`` Biohazards Applications Completed by PI/lab supervisor prior to starting project Required for research, teaching, diagnostic activities using biohazards Required for all activities, including Level 1 Submit to EHS for approval (~1 week) *University Laboratory Safety Committee September 24, 2007 Biohazards Applications General requirements for Master s and Doctoral theses* Research involving...microorganisms, living cells, other biohazards... must have had the appropriate compliance certification. Copies of any certificates of compliance must be provided to the Thesis Office at the time of submission Environmental Health & Safety Contacts Telephone: Fax: ehs@mcgill.ca Website: *Graduate and Postdoctoral Studies: 26