2017

Nagasaki UniversityNagasaki UniversityEducation lectureEducation lecture

General course

Committee for biosafetyCommittee for biosafety生物災害等防止安全運営委員会

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Contents1.Rules and regulations for possession of pathogens

2 Properties of pathogens and Safety management2.Properties of pathogens and Safety management(Biosafety and Biosecurity)

3 Safety handling for pathogens3.Safety handling for pathogens

4.Basic operation in laboratory (BSL-2）

5.Other regulations

Rules of Biosafety management for Nagasaki University1.Properties of pathogens and Safety management2.Safety handling for pathogens according to BSL2.Safety handling for pathogens according to BSL3.BSL for experiments planed4.Measures against incidents and emergencies5.Physical and biological containmentsy g6.Safety management for experimental procedures7.Other concerns for biosafety

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1. Rules and regulations for possession of pathogens

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Rules and regulations for usage and possession of pathogens

Rules of Biosafety management for Nagasaki UniversityInstruction of Biosafety management for pathogen researchI.

II. Act on the Prevention of Infectious Diseases and Medical Care for Patients withInfectious Diseases (Japanese law: Kansenshouhou)

y g p g

Specified pathogens：Class I, II, III and IV PathogensInfectious Diseases (Japanese law: Kansenshouhou)

(The term "Specified Pathogens, etc." as used in this Act means Class I Pathogens, etc., Class II

III Domestic Animal Infectious Diseases Control Law

Pathogens, etc., Class III Pathogens, etc., and Class IV Pathogens, etc.）

III. Domestic Animal Infectious Diseases Control LawMonitoring infectious disease pathogens: Emphasis management Domestic Animal Infectiouspathogens, Management Domestic Animal Infectious pathogens, Notifiable diseases such as pathogens

・Act on the Conservation and Sustainable Use of Biological Diversitythrough Regulations on the Use of Living Modified OrganismsIV.

Organisms of class 2, 3, and 4

Regurations for animal experiments, radiation management, and so on 4

Check the BSL of the pathogens

Specified pathogensMonitoring infectious disease pathogens

Registration process

1. 2. 3.

BSL1A microorganism that is unlikely to cause human or animal disease. BCG vaccine strain,

p g disease pathogens

BSL2

attenuated live vaccine and Adeno-associated virus

Class IV pathogens, etcClass III pathogens, etcClass II pathogens, etc

Protozoa, Trematoda, Taneia, Caenorhabditis, Fungi such as A.fumigatus etc, Bacteria such as H.pylori, M.avium etc, Virus such as measles virus

N ifi bl VSV tDengue virus, Shiga toxin, C.Parvum (genotype I and II)etc

Monkeypox virus, Rabies virus (fixed virus)

C.botulinum,Botulinum toxinetcManagement

Notifiable VSV etc

SFTS virus,R.Japonica etc

West Nile virus,Serovar Typhi etc

BSL3SARS virus,B.Anthracis etcEmphasis management

Management

Fungi such as B.dermatitidis etc, Bacteria such as M.Africanum etc, Virus such as Chikungunya virus etc

Emphasis management

Class I pathogens, etcLassa virus, Zaire virus, Crimean-Congo Heamorrahgic virus etc

(BSL4)

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I.Registraion for Biosafety management for pathogen research

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I. Registration for Biosafety management for pathogen research

BSL1,2➡・Notification for usage and storageEnd report・End report

BSL3 ➡・Request for usaged tand storage

・Request for supply・End report

Specified pathogens, Monitoring infectious disease pathogens➡Registration process for specified➡Registration process for specifiedpathogens but not

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I. Registration for Biosafety management for pathogen research

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I. Registration for Biosafety management for pathogen research

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II. Registration for Rules of Biosafety management for Nagasaki University(Specified pathogens)

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Class I Class II Class III Class IV

Duty and penalty of a holder of specified pathogens

P i i

Designation by the Minister of Health, Labourand Welfare

Class I Class II Class III Class IV

Permission

NotificationPreparation of Infectious diseases prevention

Education and training

Assignment of supervisor

rule

Education and training

Record of usage and storage

Sterilization etc (in case of rescission of permission)

Record of usage and storage

Standard of facility

Standard of storage

Notification of transfer

Accident report

g

Coping with disaster

Duty and penalty Order to improvement12

II. Registration for Rules of Biosafety management for Nagasaki University(Specified pathogens)

RegistrationClass II, III and IV pathogens, etc➡ Request for the facility,

End report for the facilityEnd report for the facility,Request for usage,Request for sterilization,Request for supply,Request for supply,Request for transport

Registration and notification to Ministry of Health, Labour

d W lfand Welfare

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Request for usageRequest for the facility

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Request for supply Request for transport

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III. Registration for Domestic Animal Infectious Diseases Control Law

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III. Registration for Domestic Animal Infectious Diseases Control Law

Registration

Emphasis management Domestic Animal Infectious pathogens,Management Domestic AnimalManagement Domestic Animal Infectious pathogens,Notifiable diseases such as pathogens ➡Request for the facility,Request for the facility,

End report for the facility,Request for usage,Request for sterilization,q ,Request for supply,Request for transport

Registration and notification toMinistry of Agriculture, Forestry

d Fi h iand Fisheries

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Nagasaki UniversityOrganization for storage and usage of pathogens in Nagasaki University

・Pathogens Supervisor

Nagasaki University

・Holder of specified pathoigens (Dean)

・Safety officer

Pathogens SupervisorDepartment

Safety officerLaboratory for Specified pathogens and Monitoring infectious disease pathogens

Laboratory for pathogens

・Researcher (lab member)・Responsible party

infectious disease pathogens

R h (l b b )・Responsible party

・Researcher (lab member)

P ll d t t・Researcher (lab member)

・Researcher (lab member)・Researcher (lab member)・Person allowed to enter

・Person allowed to enter・Person allowed to enter

・Person allowed to enterPerson allowed to enter

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Responsibility of Responsible party

・Registration and notification for usage storage and

・Preparation of manuals and standard operation

・Registration and notification for usage, storage and transfer of pathogens Experimental

planp p

procedures (SOP)

Education,・Practical education for operations in each laboratory・Practical training in each laboratory・Education for a person to laboratory temporarily

training

Facility,E i t

・Record of pathogen usage, storage and transfer・Maintenance for safety equipment

record

Equipment

・Preservation of records for five years・Record of entrance and exit

M f i id t d i

record

Incidents, ・Measures for incidents and emergencies・Others according to Rules of Biosafety management for Nagasaki University,Instruction of Biosafety management for pathogen research

emergencies

Safety officer

y g p g

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2.Properties of pathogens and Safety management

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PathogensParasites BacteriaParasites Bacteria

Toxin

FungiVirus

Toxin

Prion

・病原体等（長崎大学生物災害等防止安全管理規則）病原微生物及び動植物が産出する毒性物質等，生物学的相互作用を通して人体や環境に災害を及ぼす可能性のある物質

・特定病原体等、監視伝染病病原体（長崎大学生物災害等防止安全管理規則）病原体等のうち 感染症法 家畜伝染病予防法施行規則に規定する病原体

・病原微生物（研究用微生物安全管理要領）哺乳動物等に対して病原性を持つ微生物（原核生物、真菌、ウイルス、ウイロイド、原虫、寄生虫及びプリオン）

病原体等のうち、感染症法、家畜伝染病予防法施行規則に規定する病原体

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Route of infection

Droplet infectionDroplet nuclei infection (aerosol transmission)

Contact infectionPhysical contact with the person suffering the disease

Respiratory infection

Droplet nuclei infection (aerosol transmission)gCasual contact with their secretions or objects touched by them or airborne route among other routes

Food, water etcM i i k

Oral infectionVector-borne infection

Mosquito, tick, etc

Perinatal Blood-borne infection

Vertical infection

through contamination by

Conditions of infection

through contamination by blood and other body fluids

・Route of infection・Immune status （sensitivity, resistance）

Conditions of infection・Types of pathogens ・Infectious dose (quantities)

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Biosafety and Biosecurity

Bi h d Bi t i

Bio-riskBiohazard Bioterrorism

A threat to the health of humans and other living organisms

Intentional release or dissemination of biologicaland other living organisms dissemination of biological agents

Risk control

Biosafety Biosecurity

Risk control

Biosafety BiosecurityPrevention humans and environments

t f th

Prevention pathogens from hetc from pathogens humans

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Examples of Bio-risk

・Handling pathogens not using safety cabinet

・Inadequate wearing PPE

・Careless administration of controlled area

・Careless administration of pathogens

Handling pathogens not using safety cabinet

・Mistake and inadequate handling of pathogens ・Inadequate storage and handling of pathogens

Careless administration of controlled area

・Spills of pathogens from broken container

・Wrong use of syringe needle, pipette ・Malicious user

・Unauthorized experiments

BioterrorismBiohazard

・Needle-stick injury, bite

・Contact infection through a wound

・Theft and loss of pathogens

・Discrepancy of pathogens inventory

・contact and droplet infection through a mucosa

・Inhalation of contaminated aerosol

・Invasion of suspicious person

・Dual use

・Frowing out of contaminant ・Wrongful action of researchers

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Risk-control

Containment is crucial for risk-control.

Biosafety laboratoryEntrance f b ildi

yPrevention of infection in workers,Prevention of flowing out from laboratory

Bi it

Freezer etcSafety cabinet

of building

E t f l bTube etcBiosecurityControl of access to pathogens Controlled

area

Entrance of labTube etc

Hardware and softwarehardwareBuilding equipment

Before and after measure

beforePreliminary measuresBuilding, equipment, Preliminary measures

afterPreparation of measures for restoration

softwareAdministration of safety and security

According to pathogens

p

According to pathogens

Biosafety level （BSL）-1, BSL-2, BSL-3, BSL-425

Biosafety Biosecurity

Examples of bio-risk control

Set up of safety cabinet

Set up of safety equipment

Locking mechanism for the building

Designation of a Locking mechanism for the laboratory

Isolator for animal husbandry Locking device for storage freezer

Designation of a controlled area

g y

Air-conditioning unit with HEPA filterAccess control system for

hard

wa

Sterilization equipment

Display of evacuation route

laboratory

Record of laboratory entry and exit

are

Wearing PPE

Record of inventory for pathogensUse safety cabinet!

Periodic inspection of inventory

Training

Adequate disinfection and sterilization

Restriction of sharpsCorrect knowledge and act of pathogen research

Manuals

softw

are

Usage of safety equipment

Health care for workers SOPs

Adequate disinfection and sterilization

Report of theft and loss

Preparation for emergency

Appropriate coping with accidents

Correct knowledge and act of dual use

Measures are according to biosafety levels26

3.Safety handling for pathogens

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Containment of laboratory according to biosafety level

BSL 1BSL-2

Safety cabinetA tocla eBSL-1 Autoclave

BSL-3 BSL-4（suit type）HEPA filter

HEPA filter

l b t

Changing roomPersonal shower Suit room

Chemical shower

anteroom

HEPA filter

laboratoryAir Air laboratoryAir flow

Air flow

Keep negative pressure in the roomKeep negative pressure in the room

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Confirm the standards of structure and equipment according to biosafety level in Rules of Biosafety management for Nagasaki University

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Examples of BSL-2 laboratory

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Personal Protective Equipment (PPE)

BSL-2 BSL-3

Surgical maskFace guard

N95 k

Gloves

N95 mask

Overall, apron

Lab coat Double gloves

Boots (shoe covers)

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Safety cabinet（primary containment）

Class II（a, b, c） Uase a class II type safety cabinet for pathogens!!

Class I Class III Clean bench

Don’t use for pathogen or any clinical specimen!

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Safety equipment

Centrifuge machines

Isolator for animal housing PipetmanPipetman, pipet aid

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DisinfectantA ti i bi l t t d t i i th t li i th bj tAntimicrobial agents to destroy microorganisms that are living on the objects.Disinfection does not necessarily kill all microorganisms.

Extreme physical and/or chemical process

Liquid chemicals Classification of Chemical Disinfection

p y p

Disninfectant Bacteria Tuberculosis

Spore Fungi Non-enveloped virus

Enveloped virus

High-level disinfectionComplete elimination of all

Glutaral ＋ ＋ ＋ ＋ ＋ ＋

Peracetic acid ＋ ＋ ＋ ＋ ＋ ＋microorganisms in or on an instrument, except for small numbers of bacterial spores.

Peracetic acid ＋ ＋ ＋ ＋ ＋ ＋

Phtaral ＋ ＋ ＋ ＋ ＋ ＋

Intermediate-level disinfection

Sodium hypochlorite ＋ ＋ ＋ ＋ ± ＋disinfectionElimination of most vegetative bacteria, some fungi, and some viruses in a practical period of time (<10 minutes)

Alcohols ＋ ＋ - ＋ ± ＋

Povidone iodine ＋ ＋ - ＋ ± ＋

Cresol soap ＋ ＋ - ± ± ＋

Low-level disinfectionElimination of most vegetative bacteria, some fungi, and some viruses in a practical

i d f ti ( 10 i t )

Quaternary Ammonium Compounds ＋ - - ± - ＋

Chlorhexidine ＋ - - ± - ＋period of time (<10 minutes) Amphoteric surface

active agents ＋ ± - ± - ＋

＋：Effect －：No effect ±：require high concentration and/or prolonged time34

SterilizationProcess that eliminates, removes, kills, or deactivates

Autoclave Dry heat

Process that eliminates, removes, kills, or deactivates all forms of life and other biological agents

Chemical indicator

Gas

・121℃, longer than 15 min

・Confirm that sterilization process successfully done by indicator・steam ➡ effective on the surface ➡ need validation for animal carcasses

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Storage of pathogens

・Store in storage container such as freezers etc with locking device. Containers have to be registered.

・Control the area equipped storage containers.

・For stock of pathogens in freeze, use cryotubes but not the regular tubes.

・Lock the door of freezer.

・Manage the key by responsible party

・ Record inventory of pathogens

D ’t i di t th th th t・Don’t indicate the pathogen names on the storage containers

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RecordExample of specified class II pathogenExample of specified class II pathogen

・Transfer and Receipt of pathogens（the types and quantities if they are Toxins ）・Year month and date of Transfer and Receipt of pathogens・Year, month and date of Transfer and Receipt of pathogens・Means and places of storage・Type of pathogens for sterilization etc・Year, month and, means and place for sterilization of pathogens andcontaminants

・Personal full name who transferred and received pathogensPersonal full name who transferred and received pathogens・Personal full name who entered in and exited from laboratory・Year, month and of enter and exit・Personal full name who used pathogens・Personal full name who sterilized・Year month and date of inspection of facility and equipment for specified class・Year, month and date of inspection of facility and equipment for specified class

II pathogen, results of inspections and the contents of treatment, personal fullname who inspected and treatedY th d d t f d ti d t i i f th i d h・ Year, month and date of education and training for authorized persons who usespecified class II pathogen, the contents and full names of trainees

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1. Basic Responsibilities and attitude of ScientistsScientists shall recognize that they are responsible for assuring the quality of the specialized

Code of conduct for scientists

Scientists shall recognize that they are responsible for assuring the quality of the specialized knowledge and skills that they themselves create, and for using their expert knowledge, skills and experience to contribute to the health and welfare of humankind, the safety and security of society and the sustainability of the global environment. Scientists shall always make judgments and act with honesty and integrity endeavoring to maintain and improve their own expense abilities and skills andhonesty and integrity, endeavoring to maintain and improve their own expense, abilities and skills, and shall make the utmost effort to scientifically and objectively demonstrate the accuracy and validity of the knowledge they create through scientific research.

2 Attitude of Scientists2. Attitude of ScientistsScientists shall always make judgments and act with honesty and integrity, endeavoring to maintain

and improve their own expense, abilities and skills, and shall make the utmost effort to scientifically and objectively demonstrate the accuracy and validity of the knowledge they create through scientific research Scientists shall recognize that scientific autonomy is upheld by public trust and the mandateresearch. Scientists shall recognize that scientific autonomy is upheld by public trust and the mandate of the people, understand the relationships between science, technology, society, and the natural environment from a wide-ranging perspective, and act in an appropriate manner.

3 Accountability and Disclosure3. Accountability and DisclosureScientists shall strive to disclose and actively explain the roles and significance of their own research,

evaluate the possible effects of their research on people, society and the environment as well as the changes that their research might engender, neutrally and objectively disclose the results of this evaluation, and build a constructive dialogue with society.

4. Dual Use of Scientific Research OutcomesScientists shall recognize that there exist possibilities that their research results, contrary to their

own intentions, may be used for destructive actions, and shall select appropriate means and methods as allowed by society in conducting research and publicizing the results.

Science Council of Japan January 201338

Emergency response

Prepare manuals and measures for incidents and accidents such as disaster, occurrence of abnormity of an electric facility, illness of workers Invasion of suspicious person

Prepare and indicate an emergency contact list in the laboratory

workers, Invasion of suspicious person.

If you have an accident during pathogen experiment,Don’t panicDon’t panicStop experimentsI f dInform the staff immediatelyInform everyone around

Evacuate immediately if necessary

I f l th k d fi t i tIn case of a large earthquake and fire, stop experiments, secure the pathogens, and evacuate

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Transport of pathogensTransport of infectious and potentially infectious materials is subject to strict national

Laboratory personnel must ship infectious substances according to applicable

Transport of infectious and potentially infectious materials is subject to strict national and international regulations. These regulations describe the proper use of packaging materials, as well as other shipping requirements.

Laboratory personnel must ship infectious substances according to applicabletransport regulations. Compliance with the rules will:1. Reduce the likelihood that packages will be damaged and leak, and thereby2. Reduce the exposures resulting in possible infections3 I th ffi i f k d li

container

3.Improve the efficiency of package delivery.

Container for Category A or Category B pathogens

pathogen（primary container）

lid

seal Dry-ice Label indicating the contents

absorber

Shock absorber seal

Seconday container

container made of styrofoam

Don’t put in dry-ice into this box!!40

Responsible party should create rules a set of rules for laboratories.Practice proper biosafety and biosecurity.

Prepare manuals and SOPs, train researches and other staffs.41

Controlled area

・Designate a controlled area including laboratory andstock rooms for pathogens, room for sterilizationprocess, air-conditioning machine room and so on.

・Only authorized persons can enter in the controlled area.

・The international biohazard warning symbol and sign mustbe displayed on the doors of the rooms (controlled area)p y ( )

1. BSL2. Name and phone number of responsible party3. The international biohazard warning symbol3. The international biohazard warning symbol

Don’t indicate the type of pathogens and pathogen names!!

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Inspection of safety equipment

Annually inspection is required for the safety equipment to use specified pathogens and Monitoring infectious disease pathogens

・BSL-3 facility

・safety cabinet

・Autoclave

・Storage freezer and refrigeratorg g

Centrifuge machine etc・Other equipment if neccessary

Centrifuge machine etc

Should be inspected by responsible party and confirmed by safetyShould be inspected by responsible party, and confirmed by safety officer

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4. Basic operation in laboratory (BSL-2）

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Only authorized person can enterOnly authorized person can enter BSL-2 room. Administrate with key or ID card system etc.

Change your shoes to slippers.If hIf you use your own shoes, prepare one which completely covers your toes.

Don’t open the door for a long time. Security guard comes if door remainsSecurity guard comes if door remains opened for more than 90s.

Don’t escort non‐authorized persons without permission

Don’t open the window during experimentsDon t open the window during experimentsDon’t keep opend the door。

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Wash you hands before doing experiment.

Wear white lab coat when working with infectiousworking with infectious pathogens.

Prepare your own lab coat for BSL-2 room use only.D ’t l b t t idDon’t wear lab coat outside laboratory

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Wear disposable gloves when using infectious pathogens.

T k ff lTake off gloves outside laboratory

When wearing gloves, don’t touch door knob and PC.

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Book or record your experiments in the sheet (name, pathogen, time) on top of the safety cabinet

Confirm to turn on the light, fan and

Look for the waste bottle If it’s full

gas burner.

Look for the waste bottle. If it s full, autoclave it. Get another bottle for use.

Look for the a tocla ed steel can If it’sLook for the autoclaved steel can. If it’s full, autoclave it and get another one.

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Wait 15 min after turn onWait 15 min after turn on the cabinet fan to stabilize the air flow

Place minimum number ofPlace minimum number of equipment in the cabinet.

Don’t leave any stuff on the air inlet

K l ki

air inlet.

Keep a large working space for easy handling.

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Spray ethanol on your glove, p y y g ,when you touch something outside of the cabinet.

For spinning 15 or 50 ml tube, b k tuse bucket.

You can open the bucket only in a safety cabinet.

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Prepare disinfectant solutions such as 70% ethanolPrepare disinfectant solutions such as 70% ethanol besides on the safety cabinet.

If ill i f ti t b t lIf you spill infectious agents, cover by paper towel, pour disinfectant gentry, place longer than 10 min, and retrieve paper towel, and then wipe them off with cotton or tissuepaper towel, and then wipe them off with cotton or tissue paper.

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After use, take out waste chips, tubes and pipettes Put them in the steel can and thenpipettes. Put them in the steel can and then autoclave.

For pipettes, use double autoclave bags. When full take the inside bag and replaceWhen full, take the inside bag and replace with a new bag.

Discard used gloves in the steel can and then autoclave.

If autoclave machine is busy, let the can remain near the safety cabinet and autoclave later.

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After use spray 70% ethanol toAfter use, spray 70% ethanol to equipment and wipe with cotton or tissue paper.

Rinse tubes of aspirator withRinse tubes of aspirator with disinfectant

Keep working cabinet fan 10 min after work by turn off

Keep used equipment and other

y

laboratory items under UV for at least 10 min, and then take out from the safety cabinet.y

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Keep micropipettes and tubes in the small movable cabinet. Don’t keep inside the safet cabinetDon’t keep inside the safety cabinet.

Plug in pipette aid. Keep minimum stuff inside safety cabinet.

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Discard used ice in a sink and place ice container under a sink.

D ’t f t t l thDon’t forget to close the stop cock of gas.

Return tube racks to the assigned shelf.

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Confirm the correct water level of autoclave machine before use.

Program is already set at 121°CProgram is already set at 121 C, 20min. Lock the door and push start button.

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Check indicator tape and discard autoclaved stuffdiscard autoclaved stuff separately as burnable, plastic, or aluminum foil.

Put used clinical items such as blood needles and hematocritblood, needles and hematocrit tubes inside a medical box.

Clinical samples include thoseClinical samples include those from humans and animals.

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5.Other regulations

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Recombinant gene experiments(Act on the Conservation and Sustainable Use of Biological Diversity through R l ti th U f Li i M difi d O i )Regulations on the Use of Living Modified Organisms)

１．Class 2, 3 and 4 organism include pathogensTh l ifi ti diff t f th f K h h

Living modified organism including pathogen genes have to be used in P2 or P3 according to the levels

These classifications are different from those of Kansenshouhou

２．Laboratory containment levels are called P1 to 3, but

used in P2 or P3 according to the levels.

not BSL-1 to 3Required registrations of each laboratory for recombinant gene experiments

３．Minister confirmation experiments

experiments

① in case the host of Living modified organism is “virus”② in case the Living modified organism includes class 4 organism③ in case an unlisted organism is used for the recombinant gene

experiments.59

Summary

・Complete required registrations before experimentsComplete required registrations before experiments

・Practice proper biosafety and biosecurity

・Decide rules for each laboratory and train their staffs

・Please contact with Kenkyu-kikakuka if you havequestions

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