Biosafety- working Safely in a BSL-3 Laboratory Parampal Deol, Ph.D, MBA, Director R&D Microbiology Anne Hutchins, Ph.D, Associate Staff Scientist

Agenda Biosafety: Principles/concepts Areas of Biosafety Biological Risk Groups Standards Types of Biosafety Laboratories Engineering controls Best practices Operational Controls Administrative controls BSL 3 laboratory in bMX Select Agents

Biological Safety Biosafety - the discipline addressing the safe handling & containment of infectious microorganisms & hazardous biological materials application of knowledge & the use of appropriate techniques & equipment prevent personal, laboratory & environmental exposure to potentially infectious agents or biohazards Source: BMBL, 5th Ed.

Areas of Biosafety Laboratory Safety Bloodborne pathogens (BBP) & other infectious agents Recombinant DNA (rDNA) Biological waste disposal Infectious substance shipping Respiratory Protection (TB) Bioterrorism & select agents/toxins Mold & indoor air quality Occupational safety & health in the use of research animals

Hazard Classifications of Microbial Agents Classification is based on Human Disease Risk Group 1: Agents not associated with disease K12 strain of E. coli Risk Group 2: Agents associated with disease rarely serious & preventive / therapeutic interventions are often available Salmonella species, HIV, Listeria monocytogenes Risk Group 3: Agents associated with serious or lethal disease preventive/ therapeutic interventions may be available Mycobacterium tuberculosis, Eastern Equine Encephalitis (EEE) Virus, Bacillus anthracis Risk Group 4: Agents likely to cause serious or lethal disease preventive or therapeutic interventions are not usually available Ebola virus, Lassa virus, Variola virus (smallpox)

Laboratory Design and Operational Practices Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th edition Centers for Disease Control and Prevention (CDC) National Institute of Health (NIH) Federal Select Agent Program (FSAP) World Health Organization (WHO) United States Department of Agriculture (USDA)

Regulations & Guidelines Biosafety in Microbiological and Biomedical Laboratories, 5th Edition, CDC/NIH (BMBL) NIH Guidelines for Research Involving Recombinant DNA Molecules, April 2002 as amended Occupational Safety and Health Administration (OSHA) Bloodborne Pathogens Standard 29 CFR 1910.1030 Respiratory Protection including Tuberculosis 29 CFR 1910.134 (independent standard 1910.139 repealed) State regulations concerning biological waste disposal IATA Dangerous Goods Regulations, DOT 49 CFR 171-185, & USPS 39 CFR Part 111 Select Agents and Toxins Regulations (HHS/CDC 42 CFR 73; USDA/APHIS 7 CFR 331 & 9 CFR 121)

Biosafety: Where to Start? Your laboratory wants to start working with an infectious agent and you need to have a lab space or renovate current lab. How do you decide what changes need to be made to the current space to safely handle the bacteria. What are the guidelines?

Biosafety Measures Illustrated Administrative Controls Training Operations Maintenance Risk Assessment Emergency Response Plans Lab Inspections Biosecurity Biosafety Manual SOPs Medical Surveillance Respiratory Protection Inventory Record Keeping Microbiological Practices Standard Special Safety Equipment (Primary Barriers) PPE BSCs Facility Design (Secondary Barriers)

Principles of Biosafety Containment microbiological practices safety equipment facility safeguards protect laboratory workers, the environment & the public from exposure to infectious agents that are handled & stored in the laboratory Risk assessment process enables the appropriate selection of microbiological practices, safety equipment & facility safeguards that can prevent laboratory-associated infection The backbone of the practice of biosafety is risk assessment WHO Biosafety Manual, 3rd ed.

Biological Containment Laboratory Practice & Technique Adherence to standard microbiological practices & techniques Awareness of hazards & training Biosafety or laboratory Standard Operating Procedures (SOPs) Safety Equipment (Primary Barriers) Biological safety cabinets (BSC) Safety centrifuge cups/sealed rotor heads Personal protective equipment (PPE) lab coats, gowns, gloves, safety glasses, face shields, shoe covers, respirators, Tyvek suit Facility Design & Construction (Secondary Barriers) Restricted Access from public Autoclave Directional airflow HEPA filter units Airlocks as lab entrances

Biological Containment Biological Safety Cabinets (BSC) Class I, Class II & Class III

Biological Safety Level - 1 (BSL-1) Standard microbiological practices: Hand washing sink No eating, drinking, smoking, applying cosmetics, handling contact lenses, or storing food No mouth pipetting Safe handling of sharps Decontaminate work surfaces after completion of work, & any spill or splash; any waste generated Biohazard symbol when infectious agents present Wear lab coats or gowns, protective eye wear, gloves Windows to exterior have screens WHO Biosafety Manual 3rd Ed., 2004

Biological Safety Level – 2 (BSL-2) All of BSL-1 practices plus: Access is restricted to trained personnel Lab doors should be self-closing & have locks BSC used when potential for aerosol production Sealed centrifuge cups/rotors Eyewash station must be readily available Method for decontaminating laboratory waste should be available in the facility WHO Biosafety Manual 3rd Ed., 2004

Biological Safety Level – 3 (BSL-3) All of BSL-2 practices plus: Lab is separated from unrestricted traffic flow Access to lab is restricted to entry through a series of 2 self-closing doors (ante-room) All manipulations must be conducted in a BSC, other containment, or when wearing proper PPE Protective clothing with a solid front (tie-back or wrap-around gowns, scrubs, coveralls) is worn Eye, face, & respiratory protection may be worn Hands-free sink near exit door WHO Biosafety Manual 3rd Ed., 2004

Biological Safety Level – 4 (BSL-4) All of BSL-3 practices plus: Generally a separate facility; dedicated mechanical, electrical, plumbing, HVAC Agents handled in Class III BSC or with one-piece positive pressure personnel suit Shower out practices Lab clothing decontaminated prior to laundering Liquid effluent decontaminated prior to release to sewer Redundant supply fans recommended, redundant exhaust fans required, interlocked supply & exhaust CDC workers in BSL-4 www.myajc.com

Biological Safety Level - 3 Laboratory Design Containment, ability to clean Full containment Controlled access Surfaces design for cleaning Sustained directional (negative) airflow Visual monitoring device at entry, gown in and prior to entrance in lab space Utilities Lab air supply and exhaust fans with HEPA HEPA filters certified and replaced as needed Utilities (exhaust) are separated Decontamination/Spill Management Waste disposal autoclave within lab Equipment (surface) Spill cleaning

BSL-3 in at bioMerieux in Durham Emergency Response Natural Disasters Tornado warnings Transport Import/export of cultures Operational SOPs/GMP All records electronic, if possible Medical Surveillance Sustainability and permits Annual certification

Select Agent Regulations What are the Select Agents? Biological agents /toxins pose a severe threat to human health 65 agents and toxins Reconstructed 1918 Influenza virus Ricin toxin Ebola virus * Bacillus anthracis* Francisella tularensis* * denotes Tier 1 Established to keep the agents/toxins out of possession of individuals who may misuse them USA Patriot Act Public Health Security and Bioterrorism Preparedness and Response Act of 2002 The Select Agent Regulations (7 CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73) www.selectagents.gov 2001 Anthrax Letter 22 illnesses 5 deaths Cost $23 million www.selectagent.gov

Who Can Possess Select Agents? Individuals FBI security clearance Entities Facility Design Requirements Biosafety Plan Security Plan Physical, control of select agents, inventory, and information system Incident Response Plan Natural Disaster events Failures- spills, equipment failures, unsecured containment, loss of power Need to Train and SA requires annual drills

Tier 1 Select Agents Greatest risk of deliberate misuse for mass casualties or devastating effect to the economy, critical infrastructure, or public confidence Requires additional measures such as: personnel suitability assessment occupational health program visitor procedures response time of security forces within 15 minutes incident response procedures annual training on reporting suspicious behavior > 3 physical security barriers intrusion detection system (IDS) with backup during power failure

Non-Tier 1 (Clinical laboratories) Can still test for and identify a Tier 1 agent Can not ‘possess’ a Tier 1 agent Have 7 days to transfer or destroy a Tier 1 agent State Public Health Laboratories Laboratory Response Network (LRN) LRN Network (www.cdc.gov)