General Biosafety Tassanee Eamkamon Biosafety Professional Armed Forces Research Institute of Medical Sciences 23 March 2010

Objectives Principle of Biosafety Overview of Biosafety Levels (BSL) and Risk Groups (RG) Biosafty Level 2 Requirement 2

A fundamental objective of any biosafety program is the containment of potentially harmful biological agents. The purpose of containment is to reduce or eliminate exposure of follows to potentially hazardous agents. - Laboratory workers - Other persons, - Outside environment Principle of Biosafety 3

The term "containment" is used in describing: - Safe methods (Practices) -Safety Equipment (Primary Barriers and Personal Protective Equipment) - Facilities Design and Construction (Secondary Barrier) Principles of Biosafety 4

Biosafety Level? CDC-NIH Overview of Biosafety Levels (BSL) and Risk Groups (RG)

Biosafety Level (BSL) Combination 4 Levels Each level is specifically appropriate for: - Operations performed -Risk Group and documented or suspected routes of transmission of the infectious agents 6 Safe Practice Facility Design Safety Equipment Overview of Biosafety Levels (BSL) and Risk Groups (RG)

7 Appropriate for:  Undergraduate and secondary educational training and teaching laboratories  Laboratories in which work is done with defined and characterized strains of viable microorganisms not known to consistently cause disease in normal, healthy humans - Bacillus subtilis - Vaccine strains that have undergone multiple in vivo passages BSL 1 Safe Practice Facility Design Safety Equipme nt

Appropriate for:  Clinical, diagnostic, teaching, and other laboratories  Lab work with a broad spectrum of indigenous moderate- risk agents that are present in the community and associated with human disease of varying severity  Lab work with any human-derived blood, body fluids, tissues, or primary human cell lines where the presence of an infectious agent may be unknown -Hepatitis B virus- P. faciparum -Salmonellae-H1N1 BSL 2 Safe Practice Facility Design Safety Equipme nt

9 Appropriate for:  Clinical, diagnostic, teaching, research, or production facilities  Lab work with indigenous or exotic agents: -with a known potential for aerosol transmission -may cause serious and potentially lethal infection -Mycobacterium tuberculosis -Chikungunya BSL 3 Safe Practice Facility Design Safety Equipme nt

10 Appropriate for:  Lab work with dangerous and exotic agents which: -- pose a high individual risk of life-threatening disease -- may be transmitted via the aerosol route -- there is no available vaccine or therapy -Ebola virus - Hendra and Nipah virus BSL 4 Safe Practice Facility Design Safety Equipme nt

Risk Group? Overview of Biosafety Levels (BSL) and Risk Groups (RG)

Risk Group (RG) Risk classification based on: Pathogenicity Modes of transmission Host range Availability of effective preventive measures Availability of effective treatment Overview of Biosafety Levels (BSL) and Risk Groups (RG)

Risk Group (RG) 1. WHO (2004) 2. NIH Recombinant DNA Guidelines (USA, 2002) 3. Australian/New Zealand Standard (2002) 4. Canadian Laboratory Safety Guidelines (2004) 5. European Economic Community (2000)

RISK GROUP CLASSIFICATION NIH GUIDELINES FOR RESEARCH INVOLVING RECOMBINANT DNA MOLECULES 2002 WORLD HEALTH ORGANIZATION LABORATORY BIOSAFETY MANUAL 3RD EDITION 2004 Risk Group 1 Agents that are not associated with disease in healthy adult humans. Bacillus subtilis or Bacillus licheniformis A microorganism that is unlikely to cause human or animal disease. (No or low individual and community risk) Risk Group 2Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available. Campylobacter coli, Salmonella, P. falciparum, Hepatitis viruses 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 preventive measures are available and the risk of spread of infection is limited. (Moderate individual risk; low community risk) Overview of Biosafety Levels (BSL) and Risk Groups (RG)

RISK GROUP CLASSIFICATION NIH GUIDELINES FOR RESEARCH INVOLVING RECOMBINANT DNA MOLECULES 2002 WORLD HEALTH ORGANIZATION LABORATORY BIOSAFETY MANUAL 3RD EDITION 2004 Risk Group 3 Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available M. tuberculosis, R. tsutsugamushi, Japanese encephalitis virus, HIV 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. (High individual risk; low community risk) Risk Group 4Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available Ebola virus, Tick-borne encephalitis virus complex, Herpesvirus simiae (Herpes B or Monkey B virus) 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. (High individual and community risk) available. Overview of Biosafety Levels (BSL) and Risk Groups (RG)

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Biosafety Level & Risk Group Overview of Biosafety Levels (BSL) and Risk Groups (RG)

WHO 2004: Laboratory Biosafety Manual

BSLAGENTSPRACTICES PRIMARY BARRIERS AND SAFETY EQUIPMENT FACILITIES (SECONDARY BARRIERS) 1 Not known to consistently cause diseases in healthy adults Standard Microbiological Practices None required Laboratory bench and sink required 2 Agents associated with human disease Routes of transmission include percutaneous injury, ingestion, mucous membrane exposure BSL-1 practice plus: Limited access Biohazard warning signs “Sharps” precautions Biosafety manual defining any needed waste decontamination or medical surveillance policies Primary barriers: Class I or II BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials PPEs: Laboratory coats; gloves;face protection as needed BSL-1 plus: Autoclave available Summary of Recommended Biosafety Levels for Infectious Agents Overview of Biosafety Levels (BSL) and Risk Groups (RG) CDC-NIH 2007: Biosafety in Microbiological and Biomedical Laboratories (BMBL)

BSLAGENTSPRACTICES PRIMARY BARRIERS AND SAFETY EQUIPMENT FACILITIES (SECONDARY BARRIERS) 3 Indigenous or exotic agents with potential for aerosol transmission Disease may have serious or lethal consequences BSL-2 practice plus: Controlled access Decontamination of all waste Decontamination of laboratory clothing before laundering Baseline serum Primary barriers: Class I or II BSCs or other physical containment devices used for all open manipulation of agents PPEs: Protective laboratory clothing; gloves; respiratory protection as needed BSL-2 plus: Physical separation from access corridors Self-closing, double-door access Exhaust air not recirculated Negative airflow into laboratory Summary of Recommended Biosafety Levels for Infectious Agents Overview of Biosafety Levels (BSL) and Risk Groups (RG) CDC-NIH 2007: BMBL

BSLAGENTSPRACTICES PRIMARY BARRIERS AND SAFETY EQUIPMENT FACILITIES (SECONDARY BARRIERS) 4 Dangerous/exotic agents which pose high risk of life threatening disease Aerosol-transmitted laboratory infections have occurred; or related agents with unknown risk of transmission BSL-3 practices plus: Clothing change before entering Shower on exit All material decontaminated on exit from facility Primary barriers: All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, air- supplied, positive pressure personnel suit BSL-3 plus: Separate building or isolated zone Dedicated supply and exhaust, vacuum, and decontamination systems Other requirements outlined in the text Summary of Recommended Biosafety Levels for Infectious Agents Biosafty Level 2 Requirement

Biological risk assessments Provide a guide for the selection of appropriate biosafety levels and microbiological practices, safety equipment, and facility safeguards that can prevent LAIs Responsibility: Directors and principal investigators of, Institutional biosafety committees (IBC), Animal care and use committees, Biological safety professionals, and Laboratory animal veterinarians Overview of Biosafety Levels (BSL) and Risk Groups (RG)

Risk Criteria for Establishing Ascending Levels of Containment Infectivity Severity of disease Transmissibility Nature of the work being conducted Origin of the agent: Indigenous or Exotic Overview of Biosafety Levels (BSL) and Risk Groups (RG) caribbean.scielo.org/scielo.php%...26nrm%3D Safe Practice Facility Design Safety Equipme nt

Reduce/eliminate risk of infection BSL ? Agent Virulence Exposure Susceptibility 1. Identify Hazards Environmental and Procedural 2. Assess Hazards 3. Develop Controls Make Decisions 4. Implement Controls 5. Supervise and Evaluate

27 BSL 1 BSL 2 BSL 3

Biosafty Level 2 Standard Microbiological Practices Special Practices Safety Equipment Laboratory Facilities Safe Practice Facility Design Safety Equipment

Biosafety Level 2: Standard Microbiological Practices Control access to the laboratory. Wash their hands after working and before leaving the laboratory. Safely handle of sharps. Perform all procedures to minimize the creation of splashes and/or aerosols.

Biosafety Level 2: Standard Microbiological Practices Decontaminate work surfaces after completion of work and after any spill or splash. Decontaminate all cultures, stocks, and other potentially infectious materials before disposal. Implement an effective integrated pest management program. skitterbot.com/blog/%3Ftag%3Drobotics bhrcivicgroup.blogspot.com/ 2009/...hem.html Disinfectant

Biosafety Level 2: Standard Microbiological Practices Laboratory supervisor must ensure that laboratory personnel receive appropriate training:  Duties  Necessary precautions to prevent exposures  Exposure evaluation procedures. Personnel must receive annual updates or additional training.

Biosafety Level 2: Standard Microbiological Practices Personal health status may impact an individual’s susceptibility to infection, ability to receive immunizations or prophylactic interventions.

Biosafety Level 2: Standard Microbiological Practices Post a sign incorporating the universal biohazard symbol at the entrance to the laboratory. Information must include:  Biosafety level  Supervisor’s name (or other responsible personnel)  Telephone number,  Required procedures for entering and exiting the laboratory  Agent information

Biosafety Level 2: Standard Microbiological Practices Do Not Eat, drink, smoke, handle contact lenses, apply cosmetics, and store food for human consumption in laboratory areas. Store food outside the laboratory area. Mouth pipette.

Biosafety Level 2:. Special Practices Advised all persons entering the laboratory of the potential hazards and meet specific entry/exit requirements. Prepare and adopt a laboratory-specific biosafety manual as policy. The manual is available and accessible.

1.Protect employees, the surrounding community and the environment during activities involving potentially hazardous biological agents. 2.Provide minimum technical safety and occupational health requirements and guidelines for developing and control process for operation involving etiologic agents. 3.Provide an environment for high quality research while maintaining a safe work place. 4.Comply with applicable federal, state and local requirements. Biological Safety Manual Goals

Biosafety Level 2:. Special Practices Laboratory supervisor must ensure that laboratory personnel demonstrate proficiency in standard and special microbiological practices before working. AFRIMS Department of Enteric Disease Training 2005

Biosafety Level 2:. Special Practices Provided Laboratory personnel medical surveillance and offer appropriate immunizations. Store a baseline serum sample, when appropriate. radiologyinthai.blogspot.com/200...ive.html

Biosafety Level 2:. Special Practices Place potentially infectious materials in a durable, leak proof container during collection, handling, processing, storage, or transport within a facility. Decontaminate laboratory equipment routinely. Decontaminate equipment before repair, maintenance, or removal from the laboratory.

Biosafety Level 2:. Special Practices Contain, decontaminate, and clean up spills by staff properly trained.

Biosafety Level 2:. Special Practices Immediately evaluated and treated incidents. Report all such incidents to the laboratory supervisor. Provide medical evaluation, surveillance, and treatment and maintain appropriate records.

Biosafety Level 2:. Special Practices Conduct all procedures that may generate an aerosol in a BSC or other physical containment devices. Do not permit animals and plants not associated with the work being performed in the laboratory.

Biosafety Level 2: Safety Equipment (Primary Barriers and Personal Protective Equipment) Use properly maintained BSCs (preferably Class II) Appropriate PPEs Other physical containment. otinc.com

Laminar Flow (LF): - Product protection (no personnel protection) - Not for biohazard agents or chemical fumes Fume Hood: - Removes toxic chemical (ducting sys./ductless) - No HEPA filter -> not for biohazard agents Biohazard Safety Cabinet (BSC) - Product and personnel protection (not Class 1) web.princeton.edu/.../labsafetymanual/bsc1.jpg ateam.lbl.gov/.../su99/vogel/hoodsketch.jpg

I III BSC Class Protection SampleΧ Operator√ Environment√ Application with Chemicals Nonvolatile Toxic Chem Radionuclide √ Volatile Toxic Radionuclide √* Protection Sample√ Operator√ Environment√ Application with Chemicals Nonvolatile Toxic Chem Radionuclide √ Volatile Toxic Chem Radionuclide √ (Small amount) Airflow pattern In at front Exhausted through HEPA to the outside* or into the room. Airflow pattern Supply air inlets and hard-duct exhausted to outside through two HEPA filters in series. BMBL 5 th

II A1 Protection Sample√ Operator√ Environment√ Application with Chemicals Nonvolatile Toxic Chem Radionuclide √ Volatile Toxic Chem Radionuclide Χ Airflow pattern 70% recirculated to the cabinet work area through HEPA Exhaust air recirculate to the laboratory or discharged from the building via a canopy connection. II A2 (A/B3) Protection Sample√ Operator√ Environment√ Application with Chemicals Nonvolatile Toxic Chem Radionuclide √ Volatile Toxic Chem Radionuclide √* (minute amounts ) Airflow pattern Same as II, A1 Exhaust air recirculate to the laboratory or discharged from the building via a canopy connection*. BSC Class BMBL 5 th

Types of BSC ESCO

Biosafety Level 2: Safety Equipment (Primary Barriers and Personal Protective Equipment) Remove protective clothing before leaving for non- laboratory Dispose of or deposit for laundering protective clothing appropriately. Persons who wear contact lenses in laboratories should wear eye protection.

Biosafety Level 2: Safety Equipment (Primary Barriers and Personal Protective Equipment) Select glove base on an appropriate risk assessment. Gloves must not be worn outside the laboratory.  Remove gloves and wash hands when work with hazardous materials has been completed and before leaving the laboratory.  Do not wash or reuse disposable gloves.  Dispose of used gloves with other contaminated laboratory waste.

Biosafety Level 2: Laboratory Facilities (Secondary Barriers) Laboratory doors should be self-closing and have locks. Laboratories must have a sink for hand washing. The laboratory should be designed so that it can be easily cleaned and decontaminated. Carpets and rugs in laboratories are not permitted.

Laboratory furniture must be capable of supporting anticipated loads and uses. Bench tops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals. Chairs used in laboratory work must be covered with a non-porous material. Biosafety Level 2: Laboratory Facilities (Secondary Barriers)

Laboratory windows that open to the exterior are not recommended. Otherwise, must be fitted with screens. Vacuum lines should be protected with HEPA filters, or their equivalent. Liquid disinfectant traps may be required. Biosafety Level 2: Laboratory Facilities (Secondary Barriers) flickr.com/photos/ %40N

BSCs must be installed so that fluctuations of the room air supply and exhaust do not interfere. Biosafety Level 2: Laboratory Facilities (Secondary Barriers)

An eyewash station must be readily available. Mechanical ventilation systems provide an inward flow of air without recirculation to spaces outside of the laboratory. Biosafety Level 2: Laboratory Facilities (Secondary Barriers)

HEPA filtered exhaust air from a Class II BSC can be safely re-circulated back into the laboratory environment if the cabinet is tested and certified at least annually. Biosafety Level 2: Laboratory Facilities (Secondary Barriers) web.princeton.edu/.../labsafetymanual/bsc1.jpg

A method for decontaminating all laboratory wastes should be available in the facility (e.g., autoclave, chemical disinfection, incineration, or other validated decontamination method). Biosafety Level 2: Laboratory Facilities (Secondary Barriers) Disinfectant

Autoclave What should not be autoclaved? Corrosive chemicals Flammable chemicals Combustible Explosive Radioactive materials

Animal Biosafty Level 2 Standard Microbiological Practices Special Practices Safety Equipment Laboratory Facilities Safe Practice Facility Design Safety Equipment

Animal Biosafety Level 2: Standard Microbiological Practices Prior to beginning a study animal protocols must be reviewed and approved by the IACUC and the Institutional Biosafety Committee. A safety manual specific to the animal facility is prepared or adopted The need for an animal allergy prevention program should be considered.

The animal facility is separated from areas that are open to unrestricted personnel traffic within the building. External facility doors are self-closing and self-locking. Doors to areas where infectious materials and/or animals are housed, open inward, are self-closing. Animal Biosafety Level 2: Laboratory Facilities (Secondary Barriers)

Animal Biosafety Level 2: Laboratory Facilities (Secondary Barriers) Sink traps are filled with water, and/or appropriate liquid to prevent the migration of vermin and gases. Floor drains must be maintained and filled with water, and/or appropriate disinfectant to prevent the migration of vermin and gases. home.howstuffworks.com/home-impr...ains.htm

Animal Biosafety Level 2: Laboratory Facilities (Secondary Barriers) External windows are not recommended; if present, windows should be sealed and must be resistant to breakage. Cages should be autoclaved or otherwise decontaminated prior to washing. Mechanical cage washer should have a final rinse temperature of at least 180°F.

Biosafety Program Implementation

Implementation Process Personnel are charged with responsibilities to accomplish the mission.

Responsibilities: 1.The Commander/Director assumes responsibility for communicating this program to affected personal, for providing procedures to ensure its observance and enforce policy. 2.Biosafety Committee will review research activities involving potential biohazard agent to ensure that are IAW the biological safety manual. Implementation Process

Responsibilities: 3.Department Chiefs are responsible for providing the support to researchers which ensures that appropriate facilities are available to control biological hazard and to enable PIs to comply with pertinent policies. 4.PIs and supervisors are directly and primarily responsible for the compliance of all laboratory personnel with all institute biosafety policies and procedures and for the safe operation of the laboratory. Implementation Process

Responsibilities: 5.Laboratory personnel will follow all institute biosafety policy and applicable standard operating procedure. 6.Department Safety Officer, responsible for providing assistance during laboratory inspections, reporting any deficiencies within department to the Safety Office. Implementation Process

Responsibilities: 7.Biosafety Officer and Biosafety Professional will coordinate and manage the daily operations of the institute Biosafety Program, report any biosafety deficiency to the Safety Office and provide abatement plan in order to conduct research in a safe manner. 8.Safety Officer and Safety Professional responsible for directing all institute safety activities including the Biosafety Program. Implementation Process

Responsibilities: 9.Occupational Health Officer and Occupational Health Nurse will assist and support Biosafety Program with the medical evaluations and follow all SOP. 10.Biosurety Officer will ensure the integration of Biosurety and Biosafety Program to comply with Biosurety regulations and policies. Implementation Process

References American Biosafety Association CDC-NIH, Biosafety in Microbiological and Biomedical Laboratories (Fifth Edition). WHO, Laboratory Biosafety Manual.

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