laboratory biosafety

DEPARTMENT OF HEALTH

RESEARCH INSTITUTE FOR TROPICAL

MEDICINE

LABORATORY BIOSAFETY IN

MOLECULAR DIAGNOSIS OF EMERGING

RESPIRATORY INFECTIOUS DISEASES

LEI LANNA M. DANCELLaboratory Manager

RITM – MOLECULAR BIOLOGY LABORATORY

Topics

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The Changing Landscape of

Infectious Diseases

PCR-based Detection (MERS CoV

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

9MERS

CoV


Laboratory Response to Emerging

Respiratory Viral Infections

4

GLOBAL LAB

RESPONSENATIONAL 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)

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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)

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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

PracticesDecontamination/ Disinfection

Procedures

Good Personal Hygiene

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Facility Design

Typical BSL1 Facility Typical BSL2 Facility

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Facility Design

WHO, 2004. Laboratory Safety Manual 3rd Edition.

Typical BSL3 Facility Typical BSL4 Facility

Molecular Detection of Emerging

Respiratory Infectious Diseases

17

Molecular Dx

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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

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Genomes

Comprised of the entire

genetic/hereditary

information of the

organism/agent

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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

AnalysisReporting of

Result

MERS-CoV

Positive

PCR Detection of MERS

Coronavirus


Virology of MERS CoV

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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 SpecimenSpecimen

InactivationRNA 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


Result

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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.

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Laboratory Activities involved in

MERS CoV detection (Canadian Biosafety

Standards)

ActivityBSL2

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

packaging and distribution to labs




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

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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

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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

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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 inactivationProperly 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)

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Biosafety

Cabinet

Class II

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Step3

RNA

Extraction

Containment : BSL-2 Facility, BSC Class II

PPE during RNA

ExtractionProperly 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


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

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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

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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

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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

DEPARTMENT OF HEALTH

RESEARCH INSTITUTE FOR TROPICAL

MEDICINE

LABORATORY BIOSAFETY IN

MOLECULAR DIAGNOSIS OF EMERGING

RESPIRATORY INFECTIOUS DISEASES

LEI LANNA M. DANCELLaboratory Manager

RITM – MOLECULAR BIOLOGY LABORATORY

laboratory biosafety

Healthcare