Biocontainment Practices Inside the Animal Lab Guglielmo Vismara

Topics Biological Risk Primary and Secondary Barriers Biosafety Cabinets and Decon Layouts

Biocontainment Microorganisms, Animals manipulation, Allergens… Biological risk: risk associated to biological agents Microorganisms, Animals manipulation, Allergens… Invisible risk, it could be underestimated It must be assessed by experienced and responsible people (biosafety officer), for evaluation of the optimal working conditions Risk = probability of accident x consequence of accident The risk is always present. It can be minimized but never eliminated at all Pathogens associated with living animals increase the level of biological risk

(Animal) Biological Safety Levels ABSL-1 Defined organisms Not known to cause disease in healthy adults Basic Facility ABSL-2 Moderate-risk agents present Disease of varying severity ABSL-3 Indigenous or exotic agents, aerosol transmission Serious and potentially lethal infection Containment Facility ABSL-4 Dangerous or exotic high risk agents, Aerosol transmission Serious and potentially lethal infection

The importance to know what pathogen is in use Micobacterium Tuberculosis: level 3 (CDC-NIH) Avian Influenzae H5N1: level 2/3 (CDC), level 3 (NIH) Human adherent cancer cells: generally level 2 - Herpes, Papilloma, Citomegalovirus: level 2

Risk Assesment and Management Evaluation of the risk Multiple Factors Managment of the risk Risk assessment Risk management Validation and surveillance Corrective actions

Risk Assesment – examples of factors Evaluation of exposed personnel: who is exposed to animals, who enters the vivarium, who do the service, who clean the rooms. P.I. – Animal care takers – Technicians, ….. Exposition to the biological agent: skin contact, eyes contact, inhalation, ingestion, inoculation. Splashes – Aerosols – Puntures – Animal bites and scratches,… Evaluation of the vivarium rooms layout and all the available equipment (barriers)

Assesment/Management - examples Example: contaminated ferret urine spilled on the floor Is the microorganism in the urine? Can the urine be infective? What is the volume of contaminant? Choice of the absorbent material What waste container to be used What personal protective equipment to wear What tools to use to remove the waste How to decontaminate the floor surface

Managing the Biological Risk To involve all the aspects in the lab activities Equipment choice, and best use Organization of the room layouts Organization of logistic in the room Evaluate the people and material flow in the vivarium Write and “publish” the SOPs Train of people All this process is DYNAMIC and subjected to revisions Biological Agent Host Environment Risk

Enclosure for the Biological Agents Primary barrier: first protective barrier from biohazard to the operators to protect people and environment close to the source of contamination physical shield Biosafety cabinet, animal ventilated/sealed cage, tube, centrifuge… Every equipment must be associated with SOPs to be an effective barrier

When working with an infected animal, it is the cage Primary barriers When working with an infected animal, it is the cage

Features of a Good Primary Barrier Tight Sealed, no air leakage Filtration by HEPA filters – high efficiency, biological filters Autoclavable Compatible with disinfectants and chemicals Compatible with the size of a Biosafety Cabinet Ventilation controlled by pressure Air proof – Water proof

Personal Protective Equipments - If animals are housed in open cages, PPEs are the primary barrier - In case of sealed primary enclosures, PPEs can be downgraded, anyway always represent an important protection PPEs: Gloves Coats/Gowns Respirators Goggles Face shields Shoe covers

Secondary barriers Depend on the transmission risk of agents used. The design, engineering and construction of the facility provides protection for the laboratory workers’ & provides a barrier to protect persons outside the laboratory.

ABSL3 vivarium: what’s “HOT”??? HEPA AUTOCLAVE Cages on Rack Classic Changing station MICROISOLATORS? OPEN CAGES? IVCs’?

BSL3 vivarium: what’s “HOT”??? HEPA AUTOCLAVE MICROISOLATORS? OPEN CAGES? IVCs’? Area fully contaminated PPE Risk assessment Cages on Rack Laminar Flow

BSL3 vivarium: what’s “HOT”??? HEPA AUTOCLAVE Biocontainment at cage level Laminar Flow

BSL3 vivarium: what’s “HOT”??? HEPA AUTOCLAVE Contamination at enclosures level PPE But different risk assessment

Some peculiarities of a Biocontainment Lab Equipment Changing station = Biosafety Cabinet (Class II, Biohazard…) Animal cage = sealed containment cage PPE = very well defined, often respirators Rooms Very restricted access, locked doors Negative pressure, constant monitoring Decontaminate all is taken out Anteroom, gradients of pressure, showers Strong Procedures

Biosafety Cabinets Very important equipment for procedures/animal cage change/biological agents manipulation Mandatory for BSL and ABSL > 1 Certified equipment, requires training and ability to use Laminar Flow, protects product, people and environment under specific certifications

Biosafety Cabinets Class II Cabinet – EN12469, NSF49 Frontal aperture, the operator can manipulate the product inside the cabinet Frontal air barrier to protect the operator Laminar flow from the top to protect the product HEPA filtered exhausted air to protect the environment Partial recirculation of air (60-70%)

Decontamination Different words around this topic Sanitization: reduction of the microorganisms up to a level considered safe for public health Disinfection: chemical or physical process to eliminate a biological agent Sterilization: eliminates-inactivates all the biological agents In a Biocontainment lab/facility the decon equipment is on the barrier

Decontamination What is the microorganism (virus, bacteria, spore) Need effective decontamination What is the media: surface/liquid waste/room Right concentration and volume Is the material resistant or not to the treatment Chemical or physical treatment Huge choice of chemicals available on the market Don’t be disoriented!

Decontamination by gas Cold sterilization: useful for what cannot be treated by heat or liquid Rooms: vaporization and ventilation, routine vivarium shutdown Equipment: bulky, not removable from the room, or sensitive (electrics) Performed in decon-locks: flexible system Peroxide – Formaldehide – Clorine Dioxide MUST KNOW: NOT EFFECTIVE BELOW THE SURFACE!

Some BSL3 Layouts Similar to BSL-2 personal protective equipment Primary barriers: Similar to BSL-2 personal protective equipment Respiratory equipment if risk of infection through inhalation Secondary barriers: Autoclave in the barrier Corridors separated from direct access to lab Access through self-closing double doors, locked Air handling systems to ensure negative air flow (air flows into the lab) BSC II or BSC III

BSL 3 Some BSL3 Layouts

Guidelines BMBL 5th Edition (CDC/NIH)

Guidelines

Thanks for your attention!