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Biocontainment Practices Inside the Animal Lab

Guglielmo Vismara

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Topics

- Biological Risk

- Primary and Secondary Barriers

- Biosafety Cabinets and Decon

- Layouts

Biocontainment

- Biological risk: risk associated to biological agentsMicroorganisms, 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

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ABSL-1 Defined organisms Not known to cause disease in healthy adults

ABSL-2 Moderate-risk agents present Disease of varying severity

ABSL-3 Indigenous or exotic agents, aerosol transmission Serious and potentially lethal infection

ABSL-4Dangerous or exotic high risk agents, Aerosol transmission Serious and potentially lethal infection

Basic Facility

Containment Facility

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(Animal) Biological Safety Levels

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

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Risk Assesment and Management

Risk assessment

Risk management

Evaluation of the risk

Managment of the risk

Validation and surveillance

Corrective actions

Multiple Factors

Risk Assesment – examples of factors

1) 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, …..

2) Exposition to the biological agent: skin contact, eyes contact, inhalation,

ingestion, inoculation. Splashes – Aerosols – Puntures – Animal bites and scratches,…

3) 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

BiologicalAgent 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

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Primary barriers

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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

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- 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

Personal Protective Equipments

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The design, engineering and construction of the facility provides protection for the laboratory workers’ & provides a barrier to protect persons outside the laboratory.

Depend on the transmission risk of agents used.

Secondary barriers

ABSL3 vivarium: what’s “HOT”???

HEPA AUTOCLAVE

Cage

s on

Rack

Classic Changing station

MICROISOLATORS?OPEN CAGES?IVCs’?

HEPA AUTOCLAVE

Cage

s on

Rack

Laminar Flow

MICROISOLATORS?OPEN CAGES?IVCs’?

- Area fully contaminated- PPE

Risk assessment

BSL3 vivarium: what’s “HOT”???

HEPA AUTOCLAVE

Laminar Flow

Biocontainment at cage level

BSL3 vivarium: what’s “HOT”???

HEPA AUTOCLAVE

Contamination at enclosures levelPPEBut different risk assessment

BSL3 vivarium: what’s “HOT”???

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%)

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 treatmentChemical or physical treatment

Huge choice of chemicals available on the market Don’t be disoriented!

Decontamination

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!

Decontamination by gas

Some BSL3 Layouts 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

Some BSL3 LayoutsBSL 3

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Guidelines

BMBL 5th Edition (CDC/NIH)

Guidelines

Thanks for your attention!