Laboratory Biosafety

DEPARTMENT OF HEALTH
RESEARCH INSTITUTE FOR TROPICAL
MEDICINE
LABORATORY BIOSAFETY IN
MOLECULAR DIAGNOSIS OF EMERGING
RESPIRATORY INFECTIOUS DISEASES
LEI LANNA M. DANCEL
Laboratory Manager
RITM – MOLECULAR BIOLOGY LABORATORY

Topics
2
The Changing Landscape of
Infectious Diseases
PCR-based Detection (MERS Co
Biosafety Considerations in PCR-based
Detection of Emerging Respiratory Viral
Infections (MERS-CoV)
Overview of Laboratory Biosafety
Principles of Biosafety

3RESEARCH INSTITUTE FOR TROPICAL MEDICINE
The Changing Landscape of Infectious
Diseases
3
2003
SARS
Coronavir
us
2009
Pandemic
Influenza
H1N1
2012
MERS
Coronavir
us
1975
Avian
Influenza
H7
1997
Avian
Influenza
H5
1999
Avian
Influenza
H9
Emerging and Re-Emerging Respiratory viruses
H7 H5 H9 SARS
CoV
PdmH10
9
MERS
CoV

Laboratory Response to Emerging
Respiratory Viral Infections
4
GLOBAL LAB
RESPONSE
NATIONAL LAB
RESPONSE
WHO Emerging and Dangerous
Pathogens Laboratory Network
 National Reference Lab at RITM
 DOH National-Subnational
Laboratory Network

Laboratory Biosafety
The safe handling of biological materials,
particularly infectious agents which are
classified on the basis of degree of risk to
humans
5
Safe
Handling
Contain
ment

6
Safe Handling and
Containment aim to
protect the :
Laboratory
worker
Pathogen
(Specime
n
Integrity)
Laboratory
Environment

7

laboratory
worker
Pathogen


laboratory
environment

8

laboratory
worker
Pathogen


laboratory
environment
BIOSAFETY BREACH

9RESEARCH INSTITUTE FOR TROPICAL MEDICINE 9
How to determine appropriate
containment and handling strategy
Risk
Assessment
Risk Group
Classification
Biosafety Level
Classification
Safe Handling
&
Containment

Risk Assessment
10
1 Identify key hazards and perform initial assessment
of the risk (capability to infect, severity of the
disease and availability of preventive measures and
effective treatment)
2 Identify the laboratory procedure hazards (aerosol
generation, use of sharps, use of experimental
animals)
3 Assign appropriate biosafety level and select
additional precautions
4 Evaluate the proficiencies of staff regarding safe
practices and integrity of safety equipment

Risk Group Classification (WHO)
11
 No or low individual and community risk
 Unlikely to cause human or animal disease
 Moderate individual risk, low community risk
 Can cause disease but unlikely to be a serious hazard
 Lab exposures may cause serious infection, but effective
treatment and preventive measures are available and risk of
spread of infection is limited
 High individual risk, low community risk
 Usually causes serious human or animal disease but
does not ordinarily spread. Effective treatment and
preventive measures are available.
 High individual and community risk
 Usually causes serious human or animal disease and
can be readily transmitted.
 Effective treatment and preventive measures are not
usually available

Biosafety Levels (WHO)
12
Suitable for work involving well-characterized agents not known
to cause disease in healthy adult humans and of minimal
potential hazard to laboratory personnel and the environment
Suitable for work involving agents of moderate potential
hazard to personnel and the environment
Suitable for work with infectious agents which may cause
serious or potentially lethal disease as a result of exposure
by the inhalation route
Suitable for work involving exotic infectious agents that
pose a high risk of life-threatening disease and can be
transmitted as an aerosol and for which there is no vaccine
or therapy

Safe Handling and
Containment
13
Facility
design
Lab
practices
Safety
Equipmt

14
Safe Lab Practices
Access Control
Use of appropriate
PPE
Waste Management
Practices
Decontamination/ Disinfection
Procedures
Good Personal Hygiene

15
Facility Design
Typical BSL1 Facility Typical BSL2 Facility

16
Facility Design
WHO, 2004. Laboratory Safety Manual 3rd Editio
Typical BSL3 Facility Typical BSL4 Facility

Molecular Detection of Emerging
Respiratory Infectious Diseases
17

Molecular Dx
18
Checks for the
+/-of a molecular
ID
Each pathogen may be
identified by
unique/signature sections
of their genome
Polymerase
Chain Reaction
(PCR)
simple method for making
virtually unlimited copies of
the DNA sequence of a
pathogen

19
Genomes
Comprised of the entire
genetic/hereditary
information of the
organism/agent

20
PCR Based Detection
Target
pathogen
suspended in
clinical
specimen
Break
apart the
virus
Isolate the
genetic
material
Select the unique
sequence
(molecular ID)
Amplify selected
sequence
Detection of
the amplified
sequence
Result
Analysis
Reporting of
Result
MERS-CoV
Positive

PCR Detection of MERS
Coronavirus

Virology of MERS CoV
22
http://www.cdc.gov/coronavirus/mers/
EM : spherical particles
within the cytoplasm of an
infected cell
Positive-sense, single-stranded RNA virus
Newest member of the Betacoronavirus group (lineage C)
Genome is most similar to bat coronaviruses

PCR Detection of MERS CoV
23
Clinical Specimen
Specimen
Inactivation
RNA Extraction
1 2 3

Mol Dx of MERS-CoV
24
PCR Setup PCR Run Results Analysis
4 5 6

Mol Dx of MERS-CoV
25
Results Validation Results Reporting
7 8
Sequence Analysis
(if applicable)

Biosafety Considerations in the Molecular
Diagnosis of Respiratory Viral Diseases
26
Target
pathogen
suspended in
clinical
specimen
Break
apart the
virus
Isolate the
genetic
material
Select the unique
sequence
(molecular ID)
Amplify selected
sequence
Detection of
the amplified
sequence
Result
Analysis
Reporting of
Result

27
MERS CoV Risk Group Classification : RG3
 High individual risk, low community risk
 Usually causes serious human or animal disease but
does not ordinarily spread. Effective treatment and
preventive measures are available.

28
Laboratory Activities involved in
MERS CoV detection (Canadian Biosafety
Standards)
Activity
BSL2
Facility
BSL3
Facility
Processing specimens for
packaging and distribution to
laboratories
Diagnostic Testing activities
(excluding culture)
Molecular diagnostic testing
Culture of specimens
Preparatory work for in vivo
activities
Processing positive cultures for

Step1
Obtain
appropri
ate
specime
n
 Appropriate container :
sterile, leak-proof)
 PPE during collection :
respiratory and barrier protection
(respirators, gloves, disp. lab gown)
 Transport : properly-labeled, no
spillage, triple-packaged
 Safe Practices :
• manner which minimizes risk for
inadvertent exposure
• Hand hygiene

30
MERS CoV Detection : Appropriate
Specimen Types
(WHO & CDC)
SEROLOGY
Multiple specimen
types recommended :
and/or
Acute serum
Convalescent
serum
LRT
URT
serum
rRT-PCR &/or
SEQUENCING

31
 BAL
 Tracheal aspirate
 Pleural fluid
 sputum
LRT
Appropriate Specimen Types
(WHO & CDC)
Refrigerate specimen at 2-8°C up to 72 hours; if exceeding
72 hours, freeze at -70°C and ship on dry ice

32
 NPS and OPS in
VTM/UTM
 Nasopharyngeal
wash/aspirate
 Nasal aspirate
URT
Refrigerate specimen at 2-8°C up to 72 hours; if exceeding
72 hours, freeze at -70°C and ship on dry ice
Appropriate Specimen Types
(WHO & CDC)

Step1
Obtain
appropri
ate
specime
n
 Acceptable respiratory
protection devices for
MERS CoV specimen
collection :
 Properly-fit tested
 N95 or higher level

Step2
Specimen
Inactivatio
n
 Containment :
BSL-2 Facility, BSC Class II
 PPE during inactivation
Properly fit-tested respirator
(N95 with face shield or
PAPR)
Coveralls, gloves
 Safe practices :
Limit aerosol-generating activities
(vortexing, shaking of specimen)
Hand Hygiene

Biosafety Level 2 Facility (WHO)
35

Step3
RNA
Extraction
 Containment :
BSL-2 Facility, BSC Class II
 PPE during RNA
Extraction
Properly fit-tested respirator
(N95)
Solid-front, cuffed sleeve,
disp lab gown
Gloves
 Safe practices :
Limit aerosol-generating activities
(vortexing, shaking of specimen)
Hand Hygiene

(MERS CoV)
38
Target
pathogen
suspended in
clinical
specimen
Break
apart the
virus
Isolate the
genetic
material
Select the unique
sequence
(molecular ID)
Amplify selected
sequence
Detection of
the amplified
sequence
Result
Analysis
Reporting of
Result
BSL2 Facility but with BSL practices
BSL 1

RITM Access Control
Limited or restricted access of laboratory
areas to staff, as required by activity
Laboratory personnel informed of routine
and emergency procedures
LRD POLICY
Laboratory “police-ing” on set institutional policies on
access control
Policies on access control for laboratory trainees and
observers

40
Door to the laboratory must be closed,
[preferably] with self-closing mechanism
Appropriate warning sign where infectious
microorganisms are handled
LRD POLICY
LRD Standard Access Control Information and
Biohazard Warning (if applicable) signage for each
laboratory
RITM Access Control

RITM Access Control
41
BSL classification,
agents identification
Required PPE
Admittance information
Emergency information

RITM Access Control
42
Employees must inform supervisor of changes
in health status or other medical issues
Supervisor must reevaluate access control
measures when risk potential changes
Certain conditions can result in altering
susceptibility to infection
Laboratory supervisor is responsible for ensuring
that employees are not at risk

Summary
43
 It is in the best interest of our country and its people to
invest in Biosafety
 To achieve a safe laboratory environment, Laboratory
Biosafety principles have to be ingrained in the
Institutional culture
 Each has a role in the successful implementation of
Biosafety

Laboratory Biosafety

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