Transcript Basics of Biological Safety

BMBL and
BBP Overview
Environmental
Health & Safety
713-500-8100
Why Biological Safety
“An ounce of prevention is worth a pound of
cure.” Benjamin Franklin
•Harding & Byers (2000)
•1,267 overt LAIs
•22 deaths (5 were aborted fetuses)
•663 additional subclinical infections
Overview
• Definition & history of biological safety
• Regulations & guidelines
• Principles of biosafety
• Risk assessment (& biological agents)
• Containment
• Biological safety levels (BSL-1 thru BSL-4)
• Bloodborne pathogens (BBP)
Biological Safety
• The field of biosafety promotes safe
laboratory practices, procedures, along
with proper use of containment equipment
and facilities; also provides advice on
laboratory design.
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.
Biological Safety - History
• Laboratory-associated infections (LAIs) first
reported ~ start of 1900s
• Pike & Sulkin (1951) studies identified
• 4,079 LAIs reported (1930 – 1978)
• 168 deaths
• 10 most common causative agents
Source: BMBL, 5th Ed.
Biological Safety - History
• Next 20 yrs
• Harding & Byers (2000) worldwide literature
search
• 1,267 overt LAIs
• 22 deaths (5 were aborted fetuses)
• Most common causative agents
• 663 additional subclinical infections
Source: BMBL, 5th Ed.
Biological Safety - History
• Small number of specific incidents identified
• Non-specific:
• working with a microbiological agent
• being in or around the lab
• being around infected animals
• Possible contributing factors to reduced LAIs:
• improvements in containment equipment,
engineering controls, & greater emphasis on
safety training
Source: BMBL, 5th Ed.
Biological Safety - History
• US Army Biological Research Laboratories, Fort
Detrick (pioneering in biosafety, 1944 - 1969)
• Biosafety programs at USDA, National Animal
Research Center, DHHS, CDC, NIH
• 1974 – CDC & NIH publications (agent classification,
research safety)
• 1976 – NIH Guidelines 1st published
• 1984 – biosafety principles introduced in BMBL, 1st
Ed.
Source: BMBL, 5th Ed.
Regulations & Guidelines
• Biosafety in Microbiological
and Biomedical
th
Laboratories, 5 Edition, CDC/NIH (BMBL)
• NIH Guidelines for Research Involving Recombinant
DNA Molecules, April 2002 as amended
• 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)
Areas of Biosafety
• Laboratory Safety
• Bloodborne pathogens
(BBP) & other infectious
agents
• Respiratory Protection
(TB)
• Bioterrorism & select
agents/toxins
• Recombinant DNA
(rDNA)
• Mold & indoor air
quality
• Biological waste
disposal
• Occupational safety &
health in the use of
research animals
• Infectious substance
shipping
Biosafety in Microbiological and Biomedical
Laboratories (BMBL), 5th Edition (2007)
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Biological Risk Assessment (pg. 9)
Principles of Biosafety (pg. 22)
Laboratory Biosafety Level Criteria (pg.30)
Vertebrate Animal Biosafety Level Criteria
(pg. 60)
• Principles of Biosecurity (pg. 104)
• Occupational Health & Immunoprophylaxis
(pg. 114)
• Biological Agent Summary Statements(pg.
123)
Principles of Biosafety
• Containment
• microbiological practices, safety equipment
& facility safeguards that protect laboratory
workers, the environment & the public from
exposure to infectious agents that are
handled & stored in the laboratory
• Risk assessment
• process that enables the appropriate
selection of microbiological practices, safety
equipment & facility safeguards that can
prevent laboratory-associated infection (LAI)
Biological Risk Assessment
• Process used to identify:
• Hazardous characteristics of a known
infectious or potentially infectious agent or
material (agent hazards)
• Activities that can result in a person’s
exposure to an agent (lab procedure hazards
& capability to control hazards)
• Likelihood that such exposure will cause a
laboratory-associated infection (LAI)
• Probable consequences of such an infection
Biological Risk Assessment
• Principal hazardous characteristics of an
agent:
• Capability to infect & cause disease in
a susceptible human or animal host
• Virulence as measured by severity of
disease
• Availability of preventive measures &
effective treatments for the disease
Biological Risk Assessment
• Other hazardous characteristics of an
agent:
• Probable routes of transmission of
laboratory infection
• Infective dose
• Stability in the environment
• Host range
• Endemic nature
Hazard Classifications
of Microbial Agents
• Risk Group 1: Agents not associated with disease in
healthy adult humans (no or low individual risk).
• Risk Group 2: Agents associated with human disease
which is rarely serious & for which preventive or
therapeutic interventions are often available
(moderate individual risk).
• Risk Group 3: Agents associated with serious or lethal
human disease for which preventive or therapeutic
interventions may be available (high individual risk).
• Risk Group 4: Agents likely to cause serious or lethal
human disease for which preventive or therapeutic
interventions are not usually available (high individual
& community risk).
Biological Agents by Size
prions (smallest)
viruses (0.05 - 0.1 micrometer)
0.3 micrometer = HEPA filter testing particle size
bacteria (0.5 - 1.5 micrometers)
red blood cell (5 micrometers)
protozoa (1 - 300 micrometers)
fungi (largest)
Prions
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Proteinaceous infectious particles
Lack nucleic acid
Abnormal isoform of a cellular protein
PrPSc (protease-resistant protein, scrapie
isoform)
• Examples – Kuru, Creutzfeldt-Jakob disease
(CJD), Scrapies, Chronic Wasting Disease,
Bovine Spongiform Encephalopathy (BSE)
• Crosses blood-brain barrier
Prions
Viruses
• Structure - envelope,
capsid, core, genome
• Contain fundamental
information (DNA or RNA)
• Must utilize living cells
machinery & enzymes to
replicate or make copies
of themselves
Bacteria
• No membrane-bound nucleus or
organelles
• Circular DNA called plasmids
(transmit traits such as antibiotic resistance)
• Metabolically very diverse
• Ecological importance: nutrient
cycling, decomposition
• Shapes - cocci, bacilli, spirilla
• Cell wall - peptidoglycan
• Flagella - tail for movement
• Fimbriae - adherence
• Pili – transfer of bacterial DNA
Fungi
• Contain chitinous cell wall
• Most grow as multicellular filaments
called hyphae forming a mycelium, some
grow as single cells
• Obtain nutrients in an already
synthesized form
• They must live in or on their food
• Some parasitic fungi can absorb
nutrients directly from host
• Other parasitic fungi & all saprophytic
fungi must secrete enzymes to digest
surrounding material before absorbing it
Biological Containment
• Elements of Containment
– Laboratory Practice & Technique
– Safety Equipment (Primary Barriers &
Personal Protective Equipment)
– Facility Design & Construction
(Secondary Barriers)
Biological Containment
• Laboratory Practice & Technique
– Strict adherence to standard
microbiological practices & techniques
– Personnel must be aware of potential
hazards & must be trained
– Biosafety or laboratory Standard
Operating Procedures (SOPs)
Biological Containment
• 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
Biological Containment
Biological Safety Cabinets (BSC)
Class I, Class II & Class III
Biological Containment
• Facility Design & Construction (Secondary Barriers)
• Separation of lab from public access
• Controlled access zones
• Separate buildings or modules
• Decontamination facility (autoclave)
• Specialized ventilation systems (directional airflow)
• Air treatment systems to remove agents from
exhaust air (HEPA filter units)
• Airlocks as lab entrances
Biological Safety Levels (BSL)
• Biosafety levels are combinations of lab
practices, safety equipment and lab
facilities
• BSL-1 through BSL-4
• Ascending degree of protection provided
to personnel, the environment & the
community
• Increasing levels of containment
Biological Safety Level - 1 (BSL-1)
• Standard microbiological practices:
• Hand washing facility present
• No eating, drinking, smoking, applying cosmetics,
handling contact lenses, or storing food
• No mouth pipetting
• Safe handling of sharps policy implemented
• 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
Biological Safety Level – 2 (BSL-2)
• All of BSL-1 practices plus:
– Access is restricted to personnel trained to
handle pathogenic agents when work is being
conducted
– Lab doors should be self-closing & have locks
– BSC used when potential for aerosol production
– Sealed centrifuge cups or sealed rotors are
used
– Eyewash station must be readily available
– Method for decontaminating laboratory waste
should be available in the facility
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 must be worn in
rooms containing infected animals
• Hands-free sink near exit door
Biological Safety Level - 3 (cont.)
• Lab must be designed for easy cleaning & decon;
floors, walls & ceiling should be sealed; floors should
be slip resistant; windows must be sealed
• Vacuum lines must be protected with HEPA filters or
their equivalent, filters must be replaced as needed
• Method for decontaminating laboratory waste should
be available in the facility, preferably within the lab
• Sustained directional airflow (“clean” to “potentially
contaminated”), visual monitoring device at entrance
• Lab exhaust air must not re-circulate to other areas of
the building
Biological Safety Level – 3
(BSL-3) Lab
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
• Personnel enter via locked doors & clothing change
room; & exit via sequential inner (dirty) change room,
personal shower & outer (clean) change room
• 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
Appendices
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Appendix A – Primary Containment for Biohazards (BSC)
Appendix B – Decontamination and Disinfection
Appendix C – Transportation of Infectious Substances
Appendix D – Agriculture Pathogen Biosafety
Appendix E – Arthropod Containment Guidelines
Appendix F – Select Agents and Toxins
Appendix G – Integrated Pest Management
Appendix I – Toxins of Biological Origin
Appendix J – NIH Oversight of Research Involving
Recombinant Biosafety Issues
• Appendix K – Resources for Information
• Appendix L - Acronyms
Bloodborne Pathogens
•Hepatitis B virus (HBV) infection is the major infectious
bloodborne occupational hazard to healthcare workers.
•It’s estimated that 8,700 infections occur each year in
HCW according to The Hepatitis Branch of the Centers for
Disease Control (CDC)
•For HCW these HBV infections cause:
•over 2,100 cases of clinical acute hepatitis
•400-440 hospitalizations
•approximately 200 deaths each year in healthcare
workers.
Bloodborne Pathogens
•Death may result from both acute and chronic hepatitis.
•One milliliter of HBsAg positive blood may contain 100
million infectious doses of virus; thus, exposure to
extremely small inocula of HBV-positive blood may
transmit infection.
•In different studies, 7% to 30% of susceptible healthcare
workers sustaining needlestick puncture injuries from
HBsAg positive patients became infected if they did not
receive post-exposure prophylaxis
Bloodborne Pathogens (BBP)
• OSHA Bloodborne Pathogen Standard 29 CFR
1910.1030
http://www.osha.gov/SLTC/bloodbornepathogens/index.html
• Originally issued in 1991
• In 2001 the Standard was revised with the
Needlestick Reduction Act which includes:
• Education and selection of sharps injury reduction
devices (e.g., self-sheathing needles)
• Maintenance of a contaminated sharps injury log
Bloodborne Pathogens (BBP)
• Texas Department of State Health Services
Bloodborne Pathogen Control
• Prevention of contaminated sharps injuries,
needlesticks
• Exposure control plan designed to minimize
exposure of governmental entity employees to
bloodborne pathogens
http://www.dshs.state.tx.us/idcu/health/bloodborne_pathogens/
pathogen_control/
Appendix H of BMBL
• Guidelines for Work with Human, NHP,
and Other Mammalian Cells and Tissues
• “Recommended Practices. Human and
other primate cells should be handled
using Biosafety Level 2 practices and
containment.”
Bloodborne Pathogens (BBP)
• Definition:
• Pathogenic microorganisms that are
present in human blood or other
potentially infectious material (OPIM),
and can infect and cause disease in
humans. These microorganisms can be
transmitted through contact with
contaminated blood, body fluids or OPIM.
Bloodborne Pathogens (BBP)
• Examples:
• HIV
• HBV
• HCV
• T. pallidum
• Herpes Virus
• M. tuberculosis (typically an aerosol hazard)
• Human T-Lymphotropic Virus Type I (HTLV-I)
Bloodborne Pathogens (BBP)
• Body fluids that can harbor BBP:
• Blood
• Semen & vaginal secretions
• Saliva involved in dental procedures
• Synovial fluid
• Cerebrospinal fluid
• Human tissue & cell cultures
• All body fluids containing blood
Bloodborne Pathogens (BBP)
Occupational Exposure*
• Mucous membrane contact
• Splash to the eyes, nose or mouth
• Percutaneous inoculation
• Misuse of sharps (broken glass, needles,
scalpels)
• Exposure to broken / damaged skin
• Risk increases if contact involves a large
area of broken / damaged skin or if contact
is prolonged
* Risk increases with high titer levels in the source
Preventive Measures
• Risk of exposure can be minimized or
eliminated by using the following:
• Engineering controls
• Personal protective equipment (PPE)
• Administrative controls
• Work place practices
Engineering Controls
• Leakproof containers
• Use for storage & transport of ALL BBP
material
• Sharps containers
• Fill no greater than ¾ full
• Needleless devices
• Use retractable syringes, self-sheathing
needles
• Biosafety cabinet (BSC)
• Directional air flow
• High efficiency particulate air (HEPA) filtration
• Access control
Personal Protective Equipment
• Face protection
• Goggles or safety glasses with side shields
• Clothing
• Lab coats, scrubs, disposable gowns (long
pants only and no open toed shoes)
• Replaced immediately when contaminated
• Lab clothing restricted to work area
• Gloves
• Replaced immediately if torn
• Not worn outside the lab area!
Administrative Controls
• Medical surveillance
• TB skin test (PPD), Quantiferon test
• Immunizations
• Hepatitis B series
• Training
• Management of staff (SOP compliance)
• Background checks, security clearance
Good Work Place Practices
• Practice Universal / Standard
Precautions
• Treat all human blood &
body fluids as if known to be
infectious for HIV, HBV or
other potentially infectious
material (OPIM) ! !
Good Work Place Practices (cont.)
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NEVER recap needles!
Wear & utilize appropriate PPE
Follow SOPs
Survey work area
• Note locations of all necessary equipment,
waste containers, disinfectants, soaps
• Establish & maintain clean & dirty zones
• DO NOT eat, drink, or apply cosmetics in
the work area
Good Work Place Practices (cont.)
• Decontaminate work surfaces
• At start and end of procedures
• Immediately after spill
• Before removal of equipment
• Dispose of waste properly
• Label containers - hazard communication
• Chemical & biological working stocks
• Transport containers
• Wash hands frequently & always before
leaving work area (e.g., lab)!
Routine Surface Decontamination
Cleaning agents/disinfectants you will
encounter:
•Bleach – what concentration is the most
effective?
•10% solution of household bleach
•Alcohol
•Cidex
Start from perimeter and work your way in!
Blood (or OPIM) Spill Clean Up
• Wear appropriate PPE
• Clean all blood (or OPIM) spills with a 10% bleach
solution or another EPA-registered disinfectant
• Apply disinfectant to spill perimeter 1st, working
inward
• Allow > 15 minutes of disinfectant contact time
• BE CAREFUL OF ANY SHARPS! Remove using
tongs or dust pan with brush
• Wipe up spill by slowly proceeding inward
• Discard all materials used to clean up spill
• (e.g., towels, gloves) in a biohazard bag
Biological Waste Disposal
• Place red biohazard bags inside
hard-walled, leakproof secondary
containment
• Do not over fill !
• Place sharps in an appropriate
hard-walled, leakproof sharps
container
• Do not recap needles!
• Do not over fill
• (no more than ¾ full)!
Proper Hand Washing
• Wet hands with warm (not hot) water
• Apply soap on hands
• Liquid soap is better (germs can live on wet soap bars)
• Rub hands together for at least 15 seconds
• Wash longer if there is visible dirt on hands
• Cover all surfaces of hands and fingers - including
between fingers, backs of hands, thumbs, under
fingernails
• Rinse hands thoroughly with warm water
• Dry hands thoroughly
• If using blow dryer, push button with elbow
• If available, use towel to turn off water
• What song is about 15 seconds long….
Source: Hand Hygiene Resource Center http://www.handhygiene.org/
Hand Hygiene
• Alcohol sanitizers
• 62% ethyl alcohol
• Accepted as effective under
certain conditions
• Should not be used when
there is visible dirt or grime!
Needlestick Injury / Possible Exposure
• Apply routine first aid immediately
• Clean site of injury with soap & flush
with warm water for at least 15
minutes
• Flush mucous membranes with water
or saline for at least 15 minutes
• Notify supervisor
• Seek medical attention
Medical Surveillance
• Baseline labs
• HIV antibody (with consent)
• RPR (Syphilis)
• Hepatitis B surface antibody
• HCV antibody
• If source is known to be Hepatitis C+, also obtain
liver function & HCV RNA tests
• CBC with differential and platelets, chemistry profile,
urine pregnancy test if source is known HIV+ and if
exposed personnel chooses to utilize post-exposure
prophylaxis
• Hepatitis B vaccination
• Tuberculosis skin test, Quantiferon test
Your Rights
In the event of a possible exposure to
bloodborne pathogens, the person is
entitled to:
• Confidential medical evaluation and follow-up
• Documentation of routes of exposure
• Identification, documentation, testing & results of
the source individual
• Source blood draw
• Texas Department of State Health Services
• Counseling
CDC Exposure Risk
• Percutaneous injury transmission rates
with blood or blood products:
• Hepatitis B
2 - 40%
• Hepatitis C
3 - 10%
• HIV
0.2 - 0.5%
Most Cited Sources of
Needlestick Injuries
• Improper sharps disposal
• Overfilled sharps containers
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Uncooperative subjects
Improper lighting
Not familiar with device
Improper handling
• Improper passing of sharps to other personnel
• Improper techniques
Recombinant DNA (rDNA) Guidelines
• The following must be reported to EHS:
• Significant problems, significant research-related
accidents or illnesses involving rDNA
• Needlestick containing rDNA; transgenic animal
escape; spill of high-risk recombinant materials
outside of BSC
• Overt exposure to RG2 agent, & overt or potential
exposure to RG-3 agent must be reported
immediately!
• Non-compliance with the NIH Guidelines
• Upon initial report, EHS will investigate & report
to NIH OBA, & IBC, if required, within 30 days
References
• Biosafety in Microbiological and Biomedical
Laboratories, 5th Edition, CDC/NIH (BMBL)
• NIH Guidelines for Research Involving
Recombinant DNA Molecules, April 2002
• OSHA Bloodborne Pathogens Standard 29 CFR
1910.1030
• DSHS - state regulations concerning bloodborne
pathogen control, biological waste disposal, &
hazard communication
• Biological Safety Program (713-500-4193)
• www.uth.tmc.edu/safety
• EHS (713-500-8100)
Questions?