Download Risk Assessment: Microorganisms and Materials Containing

09/06/58
Risk Assessment: Microorganisms and
Materials Containing Recombinant DNA
Srisin Khusmith
Professor, Department of Microbiology and
Immunology, Faculty of Tropical Medicine, Mahidol University
Committee, Technical Biosafety Committee, BIOTEC,
National Science and Technology Development Agency
Chair of MU-IBC, Mahidol University
Faculty of Tropical Medicine, Mahidol Univeristy
June 16-17, 2015
Biohazardous Materials
 Infectious microorganisms (bacteria, viruses, fungi,
parasites, prions, rickettsia, etc.) affecting humans and
animals
 Toxin agents
 Prions
 Recombinant DNA (viral vectors, gene therapy,
cloning)
 Diagnostic (clinical) specimens eg blood, serum,
secretions, or body fluids etc.
 Human and non-human primate cells, cell culture
(primary and immortalized), tissues, stem cells blood
(whole blood or any components) and body fluids
 Animal or plant cells, cell cultures, fluids, tissues or
derived wastes which may contain pathogens
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Laboratory Acquired Infections (LAI)
Laboratory Acquired Infections (LAI)
Bacterial:
 76% from clinical labs
 8% from research labs
Exposure:
60% acquired from inhalation
Virus
 16% from clinical labs
 70% from research labs
– 32% from animal related activities
Working Safely with Biological Materials, Central Michigan University
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
Only 20% causative or defined event
 80% of which are caused by human factors
 20% are caused by equipment failure

Top 4 accidents resulting in infection
 Spillages & splashes
 Needle and syringe
 Sharp object, broken glass
 Bite or scratch from animals or ecto-parasites
http://www.weizmann.ac.il/safety/bio2.html
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BioRisk Assessment Principles
What Are the Key Factors Needed to Conduct a
Risk Assessment for Infectious Microorganisms ?
There is always risk!
 What factors define likelihood?
The risk must be identified
 What factors define consequences?
What are the consequences of disease from accidental
exposure?
The risk is evaluated
What are the Agent factors?
The risk must be measured
Plan to minimize the risk
What are host range of agent/ vector?
What are the vectors (viral vector) / host / product?
What are the laboratory factors?
Risk Assessment
Source: http://www.austrac.gov.au/elearning_amlctf_programcourse/mod4/module_4_risk_16.html
Factors Should Be Considered in A Risk
Assessment for Pathogen
To determine the level of containment to handle a biohazardous
agent based on the following:
Pathogenicity- the ability of an organism to cause disease.
Virulence - the severity of disease.
Transmission route (route of exposure):
Inhalation: droplets or droplet nuclei (upper or lower
respiratory tract) (eg M. tuberculosis)
Ingestion: gastrointestinal tract (eg S. typhi
Contact: mucous membrane/ eye/ percutaneous, through
wound eg Staphylococcus
Direct injection: blood borne contact eg HIV-virus,
Hepatitis B,C
Parenteral Inoculation (needle stick)
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Factors Should Be Considered in A
Risk Assessment for Pathogen
Where are Blood-borne Pathogens Found?
Infectious dose: the dose required to cause infection in
humans or animals eg. M. tuberculosis (low) ; Francicella
tularensis etc.
 Agent stability: survival in environment or otherwise
prolonged viability (spore formation) eg. Rickettsia, B.
anthracis
 Host range
 Availability of effective preventive measure
(eg. vaccines)
 Availability of effective treatment (eg. antibiotics)
 Antibiotic resistance eg MDR-TB
Whether the pathogen is indigenous to the country
 Human blood: blood, blood component, blood
product
 Other Potentially infectious materials (OPIM):
 Unfixed human tissue or organ,
 Primary human tissue culture,
 Primary human cell lines (capability=20-70
passages),
 Human cell line (unlimited)
 Organ cultures
 Human body fluid eg. semen, vaginal secretion,
CSF, synovial fluid, pleural fluid, peritoneal
fluid, amniotic fluid, saliva etc.
 Infected experimental animals
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Blood Borne Pathogen:
Transmission Potential
 Contact with another
person’s blood or
bodily fluid that may
contain blood
 Mucous membranes:
eyes, mouth, nose
 Non-intact skin
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Facts for HBV, HCV and HIV
HBV/HCV can live on a dry surface for at least 7 days.
You have a 6 to 30% greater chance of contracting
HBV/HCV from a needle stick versus a 1% chance of
contracting HIV from a needle stick.
There IS a vaccine for HBV.
There is NO vaccine for Hepatitis C.
Medical personnel have 0.4% chance of exposure to HIV
from a needle stick.
There is NO vaccine for HIV
 Contaminated sharps/needles
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Classification of Infectious Microorganism by Risk Group
Risk
Group 1
no or low
individual and
community risk
A microorganism that is unlikely to cause human disease or animal
disease eg. non-pathogenic organisms , non-pathogenic E. coli-K12,
B. subtilis; well established human cell line- history of safe etc.
Risk
Group 2
moderate
individual risk,
low community
risk
A pathogen that can cause human or animal disease but is unlikely
to be a serious hazard to laboratory workers, the community,
livestock or the environment. Laboratory exposures may cause
serious infection, but effective treatment and preventative measures
are available and the risk of spread of infection is limited.
Eg. Bacillus cereus, Clostridium spp, Campylobacter, Most wild type E.
coli eg E.coli O157 , Hepatitis B virus, Adeno virus, CMV, Vaccinia
virus, human blood & blood products, clinical samples etc.
Risk
Group 3
high individual
risk, low
community risk
A pathogen that usually causes serious human or animal disease but
does not ordinarily spread from one infected individual to another.
Effective treatment and preventive measures are available.
Serious respiratory agents eg. Bacillus anthrasis , Mycobacterium
tuberculosis, Rickettsia spp, HIV, Yersenia pestis (resistant strains),
prion etc.
Risk
Group 4
high individual
and community
risk
A pathogen that usually causes serious human or animal disease and
that can be readily transmitted from one individual to another,
directly or indirectly. Effective treatment and preventive measures
are not usually available.
Eg. Ebola virus, Marburg virus, Crimean-Congo hemorrhagic fever
Fungal Agents vs BSL
 Infections are not communicable, but require common
exposure from a point source.
 Some fungi have dimorphic: yeast form and mold form
 Yeast forms may be present in the tissues of infected
animals and in clinical specimens, identifying isolates, and
processing animal tissues.
 Mold form cultures containing infectious conidia or spore
may pose a hazard of aerosol exposure.
 BSL-2 and ABSL-2 practices, containment equipment, and
facilities are recommended for activities with clinical
materials, animal tissues, yeast-form cultures, and infected
animals eg. Crytococcus neoformans, yeast form of
Blastomyces dermatitidis.
 BSL-3 practices, containment equipment, and facilities are
required for or propagating and manipulating sporulating
mold-form cultures eg. Blastomyces dermatitidis, eg.
Coccidioides spp, Histoplasma capsulatum etc.
BMBL 5th Edition (2010)
WHO Laboratory Biosafety Manual 3rd Edition (2004)
External factors to be considered in a risk
assessment include…………….
Risk Assessment:
Large Volumes and High Concentrations
 Titer/volume of material used - titer may increase
several orders of magnitude compared to levels in
clinical samples, upon culturing.
 What are the laboratory factors? (Lab procedures
with risk of exposure: e.g., potential for splashes,
vortexing, centrifugation, sharps, needles or injection,
animals; skills and training level of investigators.
 Health status of investigator - e.g., immune state,
pregnancy, vaccination status.

Scaling up a process can change
BSL-1, bench-top experiment to
BSL-2, PPE required experiment

Small or bench scale: < 10 liters

Large scale: > 10 liters

High Concentrations: 103 versus
1010
have different requirements
http://ehs.unc.edu/training/self_study/bsl2_dla
m/container.php?page=66&x=15&y=13
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Relation of Risk Group to Biosafety Level, Practices and
Equipment
Risk management
Appropriate Biosafety Level
Risk
Biosafety
Lab Type
Lab Practices
Group
Level
Risk
Basic –
Basic teaching, GMT
Group Biosafety Level research
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1
Agent Risk Group + Risk Assessment =
Appropriate Biosafety Level


Risk of activity – same agent can have different
containment levels
Protection should match the risk
Safety Equipment
None; open bench
Biosafety work, sink, eye
shower
Risk
Group
2
Basic Primary health
Biosafety Level services;
diagnostic
2
services,
research
GMT plus protective
clothing, biohazard
sign
Open bench plus BSC for
potential aerosols
Risk
Group
3
Containment Special
Biosafety Level diagnostic
services,
3
research
As Level 2 plus
special clothing
controlled access
directional airflow
BSC and/or other primary
devices for all activities
Risk
Group
4
Maximum
Dangerous
containment –
pathogen units
Biosafety Level
4
As Level 3 plus
airlock entry, shower
exit, special waste
disposal
Class III BSC, or positive
pressure suits in
conjunction with Class II
BSCs, double-ended
autoclave (through the
wall), filtered air
WHO Laboratory Biosafety Manual 3rd Edition (2004)
Potential Harzadous Human Cell Lines
Risk Asessment
Cells, Tissues and Cell Lines
Potential laboratory hazards associated with human cells and
tissues include
Blood borne pathogens HBV, HIV, HCV, EBV, HPV and CMV
M. tuberculosis that may be present in human lung tissue. Other
primate cells and tissues also present risks to laboratory workers.
Cells immortalized with viral agents such as SV-40, EBV
adenovirus or HPV, as well as cells carrying viral genomic
material also present potential hazards to laboratory workers.
Tumorigenic human cells also are potential hazards as a result of
self-inoculation. There has been one reported case of
development of a tumor from an accidental needle-stick.
Laboratory workers should never handle autologous cells or
tissues, blood, lymphoid and neural tissues should always be
considered potentially hazardous
The origin of the cells or tissues (species and
tissue type)
The source (recently isolated or wellcharacterized).
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Primary Cell Culture/Cell Lines vs BSL
BMBL 5 th edition (CDC) recommended that human and
other primate cells should be handled using BSL2 practice and
containment. All work should be performed in a BSC
 Primary cultures of insect or mammalian cells from a source
where infection status is UNKNOWN (or known to be
infected) must be treated as though they are contaminated
with infectious agents and utilize BSL-2 practices and
procedures.
 All primary human cell cultures (explants) and subsequent in
vitro passages fall under the Blood borne pathogen standard
(BSL2)
Human cell lines are considered to be potentially infectious
unless the specific cell line has been characterized to be free
of hepatitis viruses, HIV, Epstein-Barr virus, human
papilloma viruses and other recognized blood borne
pathogens (BSL2)
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Research Involving Recombinant DNA
Molecules
Molecules constructed outside living cells by joining natural
or synthetic DNA segments to DNA molecules that can
replicate in a living cell
Synthetic DNA segments, likely to yield a potentially harmful
polynucleotide or polypeptide, are considered equivalent to
their natural DNA counterpart.
 Examples:
 Human gene transfer
 Cloning plasmids
 Viral or bacterial vectors
 Transgenic animals
 Genetically modified cell lines
Primary Cell Culture/Cell Lines vs BSL
 Transformed mammalian cell lines – Risk Group 2
-HeLa (Human - contains papovavirus)
 Any injections with human cell lines into animals will be
conducted in a BSC and handled at Animal Biosafety Level
2 (ABSL-2).
 When in doubt, all human, primate, mammalian, or insect
cell lines obtained from an outside source (e.g. repositories
such as the American Type Culture Collection, other
institutions, or investigators) must be treated using biosafety
levels used by that source.
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RISK ASSESSMENT: Recombinant DNA
The risk assessment for recombinant DNA should include:
 Source of the DNA to be transferred (host)
 Ability of vector to survive outside the laboratory (vector)
 Interaction between transferred gene and host (product)
When assessing the risk group and containment level for a
genetic engineered protocol, if one of the components is
potentially hazardous, a risk level appropriate to the known
hazard is assigned.
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Factors Should Be Considered in a Risk
Assessment for Recombinant Research
What are the vectors (viral vector) / host / product?
1. Host /recipient system:
 Intrinsic characteristics of host/recipient (risk group)
 Source of DNA, nature of insert gene
- Is the gene or sequence (including synthetic) from RG-2,
RG-3, or RG-4 agent, biological toxin, or select agent?
- Is there any risks associated w/ the gene or sequence
such as: up-regulation/silencing expression, regain of
function, oncogenes, virulence factors, toxins, or
expanded host range?
- Does the gene or sequence change sensitivity to
antibiotics, herbicides, pesticides, or insecticides that
would be used to control the host?
WHO Laboratory Biosafety Manual 3rd Edition (2004); Section III‐D of the NIH Guidelines Viral Vector Considerations
1. Route of Transmission
 Blood-borne
 Lentivirus
 Direct contact
 Vaccinia
 Respiratory
 Adenovirus, Adeno-associated virus (>150 pfu
intranasal)
 Influenza virus (790 pfu (nasopharyncheal)
WHO Laboratory Biosafety Manual 3rd Edition (2004)
Factors Should Be Considered in a Risk
Assessment for Recombinant Research
2. Vector system
 Intrinsic characteristics (risk group)
 Replication competence
 Residual viral gene expression
Expression vectors: Increased risk over vector alone
3 End product/Gene product effects:
 Expression of a foreign gene
 Possible introduction or increase of virulence
 Toxicity (Gene code for toxin)
 Increased ability to evade immune system
 Allergenicity
 Pharmaceutical activity eg. antibiotic resistance
WHO Laboratory Biosafety Manual 3rd Edition (2004); Section III‐D of the NIH Guidelines
Viral Vector Considerations
2. Host Range
 Based on human pathogens
 Replication incompetent eg Lentivirus
 Potential for oncogenesis through insertional
mutagenesis eg Lentivirus
 Viral gene products in vector
 Non-pathogenic viruses
 Adeno-associated virus
 Based on non-human pathogens
 Reduced pathogenicity (Vaccinia, Avipox)
 Non-pathogenic (Baculovirus)
 Tropism and host range
 Change in cell type or species affected
WHO Laboratory Biosafety Manual 3rd Edition (2004)
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Classification of Recombinant DNA Activities by Risk Group
Viral Vector Considerations
Risk
Group 1
-rDNA activities holding no or a negligible risk eg. rDNA
no or low
individual and activities using such non-pathogenic organisms as hosts for
community risk the expression of genes eg E. coli K-12, Saccharomyces
cerevisiae, vector from commercial supplier, molecular
technique which did not involve organism eg. PCR,
northern &southern blotting except DNA coding for
oncogenes etc.
Risk
Group 2
moderate
individual risk,
low community
risk
Risk
Group 3
high individual Recombinant DNA activities using genetic material from BSL3 organisms or such organisms as host cells, cell lines modified
risk, low
community risk using DNA from Risk Group 3, cell lines contains a toxin with
an LD50 of less than 100 nanograms per kg/kg body weight
Risk
Group 4
high individual Recombinant DNA activities using genetic material from
and community BSL-3 organisms or such organisms as host cells, cell lines
modified using DNA from Risk Group 4
risk
3. Contamination
 Viral vectors, e.g. adenovirus may be contaminated with
replication-competent viruses, generated by rare
spontaneous recombination events in the propagating
cell lines, or may derive from insufficient purification.
These vectors should be handled at the same
biosafety level as the parent adenovirus from which
they are derived.
-recombinant DNA activities using viral vector systems such as
Adenoviruses and some Retroviral vectors, particularly
Lentiviral vectors, and expression of recombinant DNA in
BSL-2 organisms.
WHO Laboratory Biosafety Manual 3rd Edition (2004)
WHO Laboratory Biosafety Manual 3rd Edition (2004)
Experiments that are exempt but still require
registration:
1. Experiments that use synthetic nucleic acids that can neither
replicate nor generate nucleic acids capable of replicating in any
living cell; are not designed to integrate into DNA, and do not
poduce a toxin that is lethal for vertebrates at an LD50 of <100
ng/kg body weight.
2. Cloning of all other DNA in E. coli K12 Host-Vector systems, or
derivatives of E. coli K-12; Saccharomyces Host-Vector Systems eg.
S. Cerevisiae and S. uvarum ; and B. subtilis or Bacillus subtilis or
Bacillus licheniformis Host-Vector Systems host-vector systems
(with the exception of DNA from Risk Group 3 or 4 pathogens).
3. Introduction into cultured cells of any recombinant DNA containing
less than half of a eukaryotic viral genome (with the exception of
Risk Group 3 or 4 pathogens).
4. Breeding experiments to generate transgenic rodents that may be
housed under BSL1,with the exception of those listed in Section IIIE.
Section III-F NIH Guidelines 31
Experiments that require IBC approval
PRIOR to initiation
 Experiments using RG 2-4 agents as host/vector system
 Deliberate transfer of a drug resistance trait to microorganisms
that are not known to acquire the trait naturally if such
acquisition could compromise the use of the drug to control
disease.
 Use of viral vectors (e.g. lentivirus, adenovirus, pox virus, vectors
for gene delivery to living systems)
 III-B-1: Experiments involving the cloning of toxin molecules with
LD50 of less than 100 ng per kg body weight
 Experiments involving the use of infectious DNA or RNA viruses
or defective DNA or RNA viruses in the presence of helper virus
in tissue culture systems
 Transfer of rDNA to human research participants
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Section III-A,B,C, D NIH Guidelines
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Examples of Experiments That Require IBC
Approval PRIOR to Initiation
 Experiments involving the use of infectious DNA or RNA
viruses or defective DNA or RNA viruses in the presence of
helper virus in tissue culture systems
 Transfer of rDNA to human research participants eg.
e.g., in vivo human gene therapy)
 Experiments involving viable rDNA-modified microorganisms
tested on whole animals.
 Experiments involving more than 10 liters of
culture.
RG2 and RG3 Recombinant DNA Technology
Most vaccine experiments will fall into one of the following
categories:
 Experiments using Risk Group 2 or Risk Group 3 agents as
Host-Vector systems.
 Experiments in which DNA from Risk Group 2 or Risk
Group 3 agents is cloned into nonpathogenic prokaryotic or
lower eukaryotic Host-Vector systems.
Many vaccine projects, as well as other studies involving
animals
 Experiments involving whole animal work.
NIH Guidelines
Section III-D NIH Guidelines33
Recombinant DNA Experiments
Section III-A of the NIH Guidelines
Deliberate transfer of a drug resistant trait to
microorganisms that could compromise medicine or
agriculture in its control.
Creation of a multi-drug resistant virus or bacteria that
would compromise medicine, agriculture or public
health, e.g., a recombinant HBV virus that was would
render the current HBV vaccine series as ineffective.
These experiments require Institutional Biosafety
Committee Approval Before Initiation and is
considered a Major Action under the NIH Guidelines.
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Recombinant DNA Experiments
Section III-B of the NIH Guidelines
The deliberate formation of recombinant DNA containing
genes for the biosynthesis of toxin molecules lethal at an
LD50 of less than 100 nanograms per kilogram of body
weight.
These experiments involve the potential manipulation of
sequences from microbial toxins such as the Botulinum
toxins, tetanus toxin, diphtheria toxin, and Shigella
dysenteriae neurotoxin, e.g., creation of recombinant
organism, such as E-coli K12 with a sequence to synthesize
Clostridium botulinum toxin
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Recombinant DNA Experiments
Section III-B of the NIH Guidelines
Biosafety Guidelines for Infectious Microorganisms /
Recombinant DNA Technology
 WHO Laboratory Biosafety Manual
3rd Edition (2004)
Biosafety in Microbiological and Biomedical
Laboratories (BMBL) 5th Edition (2010)
jointly published by NIH and CDC
The deliberate transfer of recombinant DNA or RNA
derived from recombinant DNA into one or more human
research participants (e.g., in vivo human gene therapy)
 NIH Guidelines for Research
Involving Recombinant DNA
Molecules (2013)
These experiments often involve in-vivo human gene
therapy but may also include vaccine studies, e.g., an
adenoviral mediated gene transfer experiment
These experiments require Institutional Biosafety
Committee and EC (IRB) Before Research Participant
Enrollment
Canadian Laboratory Safety 
Guidelines (2004)
Australian/New Zealand Standard Safety in laboratories 
Part 3: Microbiological safety and containment
National Guidelines for Pathogens
Guidelines from Department of Medical Science, Ministry of
Public Health
National Guidelines for Biotechnology
1. Biosafety Guidelines in Genetic Engineering and Biotechnology for
Laboratory Work (1992)
2. Biosafety Guidelines in Genetic Engineering and Biotechnology for
Field Work and Planned Release (1992)
3. Biosafety Guidelines for Field Trials of Transgenic Plants (1994)
4. Biosafety Guidelines in Safety Assessment of Genetically Modified
Foods(2001)
5. Biosafety Guidelines for Contained Use of Genetically Modified
Microorganisms at Pilot and Industrial Scales
6. Biosafety Guidelines for Work Related to Modern Biotechnology or
Genetic Engineering (revised from 2 and 6) (2004, 2009, 2011)
2004
2009
2011
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Package of MU Safety Guidelines
Q&A
Package: distributed to Faculty/Institution
MU Staff /student: download from Mahidol University website (COSHEM)
Thank you
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