Biosafety in Microbiological and Biomedical Laboratories S. Wongratanacheewin, Ph.D. Melioidosis Research Center, Faculty of Medicine, KKU. E-mail: sura_wng@kku.ac.th

September 11, 2001

ไทยรัฐ 20 กันยายน 2546 ไทยรัฐ 19 กันยายน 2546

What is bioterrorism The US president's 2006 budget requests more than $4 billion for biodefense activities at the Department of Health and Human Services (HHS). Within budget, approximately $1.7 billion will fund medical research and product development at the National Institute of Allergy and Infectious Diseases (NIAID), including Vaccines to immunize the public against diseases caused by bioterrorism agents Diagnostic tests to help first responders and other medical personnel rapidly detect exposure and provide treatment Therapies to help patients exposed to bioterrorism agents regain their health

Biosafety Personal protection Work place practices Administrative Engineering All play an equal role in protecting workers from occupational exposures in laboratories

Biosafety Personal protection Work place practices PPE, respirotors, gloves, booties, eye and ear protections Work place practices Entry and exit practices, decontamination, emergency procedure, handle sharp.

Biosafety Administrative Engineering Training Risk management Immunization Security, supervision Engineering System, Hepa filter, Accessible

CDC Bioterrorism Agents Category A Category B Category C

Category A Diseases/Agents can be easily disseminated or transmitted from person to person; result in high mortality rates and have the potential for major public health impact; might cause public panic and social disruption; and require special action for public health preparedness.

Category B Diseases/Agents Second highest priority agents include those that are moderately easy to disseminate; result in moderate morbidity rates and low mortality rates; and require specific enhancements of CDC's diagnostic capacity and enhanced disease surveillance.

Category C Diseases/Agents Third highest priority agents include emerging pathogens that could be engineered for mass dissemination in the future because of availability; ease of production and dissemination; and potential for high morbidity and mortality rates and major health impact.

The US National Institute of Allergy and Infectious Diseases (NIAID)

The US National Institute of Allergy and Infectious Diseases (NIAID)

Helen Quill & Maria Giovanni, Nature Immunology, 2004

Microbiological laboratories Microbiological laboratories are special, often unique work environments that may pose identifiable infectious disease risks to persons in or near them.

Containment Primary containment, the protection of personnel and the immediate laboratory environment from exposure to infectious agents, is provided by both good microbiological technique and the use of appropriate safety equipment. Secondary containment, the protection of the environment external to the laboratory from exposure to infectious materials,

Laboratory Practice and Technique Develop or adopt a biosafety or operations manual that identifies the hazards that will or may be encountered, and that specifies practices and procedures designed to minimize or eliminate exposures to these hazards.

Principles of Biosafety Laboratory personnel Safety practices, and techniques must be supplemented Appropriate facility design and engineering features Safety equipment, and management practices.

Safety Equipment (Primary Barriers) Biological safety cabinets (BSCs) Enclosed containers, Engineering controls designed to remove or minimize exposures to hazardous biological materials.

The biological safety cabinet (BSC) Device used to provide containment of infectious splashes or aerosols. Three types of biological safety cabinets (Class I, II, III) used in microbiological laboratories. Open-fronted Class I and Class II biological safety cabinets are primary barriers which offer significant levels of protection to laboratory personnel. Safety centrifuge cup, an enclosed container designed to prevent aerosols.

Personal Protection gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles.

Facility Design and Construction (Secondary Barriers) Depend on the risk of transmission of specific agents. Separation of the laboratory work area from public access, availability of a decontamination facility (e.g., autoclave), and handwashing facilities. If an infectious aerosol is present, higher levels of primary containment and multiple secondary barriers may become necessary to prevent infectious agents from escaping into the environment.

Biological Safety Biological Hazards Divided into 4 biosafety levels Levels define the lab requirements, protective equipment, and work practices 4 animal biosafety levels also

Biosafety Level 1 Definition Work with agents not known to cause disease in healthy adults Minimal potential hazard to personnel and environment

Biosafety Level 1 Requirements Wash hands before leaving lab No eating, drinking, etc. in lab Follow safe sharps procedures Decontaminate waste and work surfaces

Biosafety Level 1 Examples Escherichia coli K12 Klebsiella oxytoca Lactobacillus Saccharomyces

Biosafety Level 2 Definition Work with agents of moderate potential hazard to personnel and the environment. Agents associated with human disease which is rarely serious and for which preventative or therapeutic interventions are often available.

Biosafety Level 2 Requirements All requirements for BSL1 plus: Training to handle organisms Personal protective equipment (PPE)- lab coats, eyewear, and gloves Minimize aerosol generation Biohazard sign posted on door

Biosafety Level 2 Examples Pathogenic E. coli Salmonella sp. Streptococcus sp. Hepatitis B and C Viruses

Biosafety Level 3 Definition Agents associated with serious or lethal disease for which preventative or therapeutic intervention may be available. High individual risk, low community risk. Often risk is by inhalation route.

Biosafety Level 3 Requirements All requirements for BSL2 plus: Limited lab access 2 doors in series to access lab Able to wash entire lab Special exhaust ventilation- not recirculated

Biosafety Level 3 Requirements All requirements for BSL2 plus: High level of training Personnel receive vaccinations if available Work in biosafety cabinets

Biosafety Level 3 Examples HIV Mycobacterium tuberculosis

BSL 3 cabinet

HEPA Filters Class I High Efficiency Particulate Air filter Removes over 99.95% of particles 0.3 mm in diameter or larger

Class II A Class II B1

Class II B2 Connection to building exhaust system required.

Class II B3 Connection to building exhaust system required.

Biosafety Level 4 Definition Agents likely to cause serious or lethal disease for which preventative or therapeutic intervention is usually NOT available. Dangerous or exotic agents.

Biosafety Level 4 Requirements All requirements for BSL3 plus: Class III biosafety cabinet or positive pressure suits Shower/change rooms - clothing autoclaved before laundering Air locks

Biosafety Level 4 Examples No Bacteria or Fungi Ebola Virus Monkey B Virus Marburg Virus

Safe Work Practices Wash hands after work; before leaving lab; when removing gloves No eating, drinking, applying cosmetics, handling contact lenses in lab Maintain labs in clean, orderly fashion Limit access to lab when work with organisms is in progress Use good microbiological techniques No mouth pipetting When possible use plastic instead of glass

Personal Protective Equipment Contaminated personal protective equipment (PPE) should remain in the work area- do not wear in any "clean" areas PPE that is contaminated must be discarded as infectious waste or disinfected prior to routine laundering

Laminar Flow Equipment Biosafety cabinets (BSCs) Contain infectious agents to protect personnel and the environment Laminar flow clean benches (LFBs) Non-hazardous work only Protect work from contamination

Laminar Flow Benches DO NOT protect personnel or the environment Discharges HEPA filtered air across work surface toward user DO NOT use biohazardous, radioactive, chemical, toxic, mutagenic or carcinogenic agents in a LFB Should have a pink sticker stating this

Biosafety Cabinets 3 Classes All exhaust is HEPA filtered before leaving cabinet Class I- do not protect the work from contamination Air entering cabinet is not filtered

Biosafety Cabinets Class II- 4 types- A, B1, B2, B3 Each type recirculates different amount of air Some are hard-ducted, some exhaust into room Class III Totally enclosed, ventilated cabinets Work through portals with attached gloves

Use of Equipment Minimize airflow disturbances (moving in/out of cabinet, people walking by, opening doors, blocking grilles with equipment) Wipe down surfaces with alcohol before and after work. Let run 10 minutes to clean it Minimize use of flames in cabinet Try not to use chemicals in cabinets- only use in Class I or II hoods that are ducted- contact DOHS Do not store excess equipment in cabinet

Centrifuge Safety Aerosol generation from broken tubes, opening tubes, decanting supernatant, resuspending materials Balance load Use sealed tubes, safety buckets or rotors when possible When possible fill & open centrifuge tubes or buckets in BSC

Centrifuge Accidents If a tube breaks- Turn off centrifuge Leave the lab Call Officers If you find a tube broke when you open the centrifuge- Close the lid

Shipping/ Transporting Who regulate transportation of biologicals???? Includes infectious materials, diagnostic specimens, dry ice Program being developed- to include mandatory training

Disinfection All equipment, environmental, and working surfaces must be cleaned and decontaminated after work with infectious materials Broken glass or sharps shall be cleaned up using mechanical means- NOT by hand Different levels of cleaning- decontamination, sanitization, disinfection, sterilization

Decontamination Use appropriate disinfectant for agents in use Follow instructions, paying attention to dilution, shelf life, and contact time to assure effective kill Care must be used to ensure mixing of incompatible materials does not occur

Infectious Waste What is infectious waste Procedures for disposal Solid waste Liquid waste Sharps Autoclaving waste Containers and pick-ups Mixed waste

Emergencies Safety equipment in labs Showers Eye washes

BSL2 Spills Small spills- Wipe up with disinfectant-soaked paper towel then clean surface with suitable disinfectant Large BSL2 Spill in BSC Keep cabinet running during and 10 minutes after cleanup Don PPE Cover spill with disinfectant- let set- wipe up Clean catch basin under work surface if spill ran there Remove PPE when done and wash Leave the lab, close door, post lab off-limits Decon people if necessary After 30 minutes, put on PPE and assemble supplies

Bacteria Virus Parasite Fungus Agents Lab Animals B. anthracis, Bordetella pertussis, N. gonorrhoeae, N. meningitidis, B. pseudomallei, L. pneumophila, S. typhi, Y. pestis Brucella sp, F. tularensis, Campylobacter spp, C. diphtheriae, E. coli (cytotixin), H. pyroli, L. interrogans, L. monocytogenes, M. leprae, Salmonella spp., Shigella spp., T. pallidum, V. cholera M. tuberculosis BSL 2, BSL 3 BSL 2 BSL 3 Virus Prion, Retrovirus Rabies virus Rickettsial agents, LCM, Hepatitis B, C, D Herpes virus, Hepatitis A, E, influenza, Poliovirus, Poxvirus, Other zoonosis virus Parasite All pathogenic parasites Fungus Blastomyces dermatitidis, C.neoformans, Histoplasma capsulatum, Sporothrix schenckii, Miscellaneous Molds Coccidioides immitis

Biosafety Agents Practices Safety Equipment Facilities Level BSL-1 not generally associated with disease ·    Good microbiological practice Hand washing No eating, drinking Pipeting devices- mouth pipeting is prohibited BSL-2 These agents are associated with human disease ·    Limited lab access. Most work may be performed on a bench top Biohazard warning. Biosafety manual defining any needed waste decontamination or medical surveillance policies ·    Class I or II Biological Safety Cabinets. Lab coats, gloves, face protection, as needed ·    Open bench-top sink for hand washing is required Autoclave

Agents Practices Safety Equipment Facilities Level Agents Practices Safety Equipment Facilities BSL-3 ·    associated with human disease and cause illness by spreading through the air diseases that may have serious or lethal BSL-2 practice plus ·    Controlled access ·    Decontamination of all waste Decontamination of lab clothing before laundering ·    Class I or II Biological Safety Cabinets (BSCs) or other physical containment devices Protective lab clothing, gloves, respiratory protection as needed BSL-2 plus ·    Physical separation from access corridors ·    Self-closing, double-door access ·    Exhaust air is not recirculated ·    Negative airflow into laboratory Design includes back up/redundant systems BSL-4 ·    BSL-3 or have an unknown cause of transmission Cause diseases that are usually life-threatening BSL-3 practices plus ·    Clothing change before entering ·    Shower on exit All material decontaminated on exit from facility 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 ·    Design includes back-up/redundant systems

Thanks you Biosafety in the Microbiological and Biomedical Laboratory More information Biosafety in the Microbiological and Biomedical Laboratory http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm

Acknowledgements T2 (Thailand Tropical Diseases Research Program). TRF (Thailand Research Fund). NSTDA ONSITE, Emory University Faculty of Science, MU The Virology Association of Thailand Faculty of Medicine, KKU.