O' Doblhoff IAM

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How Safe is Biosafe
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Overview

• Biological Hazards
• Transmission of Infectious Substances
• Safety Measures
• Relevant Guidelines and Laws

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Biological Hazards

• Bacteria
• Fungi
• Viruses
• Protozoa and helminths
• Unconventional infectious agents (prions)
• Toxins
• Allergens

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Risk Groups

• 1
• unlikely to cause human disease

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Risk Groups

• 2
• can cause human disease and might be a hazard to
  workers it is unlikely to spread to the
  community there is usually effective prophylaxis
  or treatment available

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Risk Groups

• 3
• can cause severe human disease and present a
  serious hazard to workers it may present a risk
  of spreading to the community, but there is
  usually effective prophylaxis or treatment
  available

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Risk Groups

• 4
• causes severe human disease and is a serious
  hazard to workers it may present a high risk of
  spreading to the community there is usually no
  effective prophylaxis or treatment available

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Group 1

• E.Coli K12
• Lactobacilli used in food processing
• many thermophile bacteria
• Saccharomyces cerevisiae
• Lambda Phages
• Tabaccomosaic virus
• Vaccine strains

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Group 2

• Streptococcus sp.
• Legionella sp.
• Clostridium tetani
• Trichophyton mentagrophytes (skin myc.)
• Entamoeba histolytica
• Mumps
• Hep-A

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Group 3

• Brucella abortus
• Mycobacterium tuberculosis
• Yersinia pestis
• Histoplasma capsulatum (fungal lung inf.)
• Plasmodium falciparum (Malaria)
• HIV
• Hepatitis-C-Virus

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Group 4

• Lassa
• Junin
• Ebola
• Marburg

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Risks Associated with Animals, Animal Cells and
Tissues

• viruses (including latent viruses)
• mycoplasma
• bacteria, yeast, fungi
• other pathogenic substances such as prions
• Oncogenic DNA and other oncogenic materials

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Risk assesment of retroviral vectors

• Viral particle generation
• Viral sequences
• Transforming sequences
• No re-creation of particles
• Subgenomic non-pathogenic sequences

http//www.rki.de/GENTEC/GENTEC.HTM
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Blood Born Pathogens

• Viruses often in latent phase gt no symptoms
  gt no warning
• Other biological agents during septicemic stage
  (normally associated with symptoms gt warning)

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Occupationally acquired infections - history

• 1949 Serum hepatitis in a blood bank (Leibowitz
  et al., 1949)
• Health care laboratory workers had sevenfold
  higher hepatitis incidence than normal population
  (Skinhoj and Soeby 1981)
• 55 documented cases of HIV-1 infections in the US
  (CDC, 1999)
• Hepatitis C 1 - 2.8 as compared to 0.3 - 1.5
  in general population (Lanphear et al., 1994)

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Other cases

• Lassa (Frame et al., 1970)
• Marburg (Smith et al., 1970)
• .....
• Bacteria
• Borrelia (Felsenfeld, 1971)
• ......
• Parasites, Rickettsia

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Risk of non-human blood and serum

• Rule of thumb
• The closer the relationship between hosts the
  higher the risk

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Transmission of blood born pathogens

• Needle sticks
• Cuts
• Splashing
• Aerosols

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Transmission from animals

• Biting
• Scratching
• Direct contact (hand --gt eye, ingestion)
• Aerosols

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Transmission

• Inhalation
• Accidental injection
• Ingestion

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Precautionary Principle

• Treat all material from unknown sources as
  infectious
• Even when handling certified group 1 organisms
  use Good Microbiological Practice
• Always avoid aerosol formation (allergies!)

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Principles

• Risk Assessment
• Work Procedures and Containment

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Containment

• Routine Operations
• Accidents

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Containment of operations

• Work Practices
• Personal Hygiene
• Protective Clothing
• Biological Safety Cabinets (primary barriers)
• Waste treatment (solids, effluents)

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Containment for the Case of Accidents

• Facility Design
• Ventilation (HEPA, air changes, inward
  directional air flow)
• Lock Systems
• Decontamination Procedures
• Personal Protective Equipment (e.g. ventilated
  suits)

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Working Together

• Risk Assessment
• Biological Agents
• Work Procedures
• Engineering
• Necessary Primary Barriers
• Necessary Secondary Barriers
• Facility design
• Validation, Maintainance

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Operational procedures - Level 1

• Good Microbiological Practice
• Access limitation
• No eating and drinking, smoking, applying
  cosmetics, handling contact lenses
• Wash hands after work
• No mouth pipetting

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Operational procedures - Level 1

• Good Microbiological Practice ...
• Avoid aerosols
• Clean work surfaces
• Inactivate waste

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Containment - Level 1

• Access control
• Protective clothing
• Handwash sink
• Suitable lab furniture
• Cleanable lab

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Operational procedures - Level 2

• Good Microbiological practice
• Immuncompromised persons not allowed
• Biohazard Sign
• Training
• Removal of sharps (or special precautions)
• Laboratory desinfection
• Handle and report spills and accidents

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Containment - Level 2

• Procedures creating aerosols - closed equipment
  or handle in biological safety cabinet
• Face protection where necessary (splashes)
• Protective clothing - remove before leaving - do
  not take home
• Gloves when handling infectious material

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Guidelines and Laws

• COUNCIL DIRECTIVE 90/679/EEC on the protection of
  workers from risks related to exposure to
  biological agents at work ( adaptions and
  amendments)
• COUNCIL DIRECTIVE 90/219/EEC on the contained use
  of genetically modified micro-organisms
• COUNCIL DIRECTIVE 90/220/EEC on the deliberate
  release of genetically modified organisms

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Austrian Regulations on Gene-Technology

• Austrian Gene Technology Act (Gentechnikgesetz,
  BGBl. Nr. 510/1994, updated BGBl. I Nr. 73/1998)
  entered into force in January 1995, amended in
  1998
• Ordinance on Work with GMOs in Contained Use
  (Systemverordnung, BGBl. Nr.116/1996)
• Ordinance on Deliberate Release of GMOs into the
  Environment (Freisetzungsverordnung, BGBl. II
  Nr.49/1997)

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Criteria for Risk assessment

• 1 Characteristics of donor and recipient
• 2 Characteristics of the vector
• 3 Characteristics of the modified micro-organism
  and the gene products
• Health considerations
• Environmental considerations

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Donor and recipient

• Pathogenicity
• Virulence
• Toxigenicity
• Allergenicity
• Mutagneicity

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Structure

• Responsible Manager of Site
• Biological Safety Officer
• Biological Safety Committee
• Project Leader
• Ministry of Science / Ministry of Health
• Gene Technology Advisory Commission
• Scientific Subcommittees

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Applications under the Gene Law
Audit
Biosafety Officer
Applicant
Biosafety Committee
Apply
Approve / Reject / Safety Measures
Site Management
Approve / Reject
Audit
Minister
Ministry
Advise
Gene Technology Advisory Committee
Scientific Subcommittees
Advise
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Approvals
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Type A / Type B
Small Scale RG 1 - 300 liter RG 2 - 50
liter RG 3 - 10 liter RG 4 - 10 liter Type A
Small scale for teaching, and non
industrial or non-commercial research or
development Type B all other work !! will
change soon !!
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Prions
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Pathogenesis - the Prusiner hypothesis

• 1. Conformational change
• Conversion of normal cellular prion protein
  (PrPc) to abnormal conformation such PrP (e.g. as
  in scrapie PrPsc).
• 2. Neuronal degeneration
• PrPsc builds up --gt
• Neuronal death
• Microscopically visible spongiform changes in the
  brain
• 3. Amyloid deposition
• amyloid plaques formation
• neurofibrillary tangles (in some forms of prion
  disease)

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Conformational changes in PrP
Graphics Prusiner S.B., Scientific American,
1995, 272, 1, 30-37
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Human Prion Diseases

• Creutzfeld Jacob disease (CJD)
• sporadic (1921)
• familial (1924)
• iatrogenic (1974)
• variant (1996)
• Kuru (1957)
• Gerstmann-Straussler-Scheinker disease (1936)
• Fatal insomnia (1986)

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Animal Prion Diseases

• Scrapie
• Bovine spongiform encephalopathy
• Transmissible mink encephalopathy
• Chronic wasting disease (mule deer, elk)
• Feline spongiform encephalopathy
• Zoological spongiform encephalopathy (eland,
  nyala, oryx, kudu, gemsbok, cheetah, puma,
  ocelot, ostrich)

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Clinical features of CJD like human prion diseases

• Neurodegenerative disorders affecting the CNS.
• Characteristic EEG abnormality consisting of
  generalised repetitive triphasic periodic
  complexes.
• Symptoms
• Dementia
• Movement disorders (not in kuru)
• Neurological deficits
• Depression
• Insomnia

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variant CJD

• From October 1996 to early November 2000
• 85 cases United Kingdom (UK)
• 3 cases France
• 1 Republic of Ireland

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Decontamination

• Immerse in sodium hydroxide 1 N NaOH heat in a
  gravity displacement autoclave at 121C for 30
  min clean and subject to routine sterilization.
• porous load (134C) autoclave for 1 hr. clean
  and subject to routine sterilization.
• Autoclave at 134C for 18 minutes
• others, see WHO report
• WHO Infection Control Guidelines for
  Transmissible Spongiform Encephalopathies
  (WHO/CDS/CSR/APH/2000.3)
• http//www.who.int/emc-documents/tse/docs/whocdscs
  raph20003.pdf