Attachment 8 Biosafety in Microbiological and Biomedical Laboratories (BMBL).pdf.pdf

Biosafety in Microbiological and Biomedical

Laboratories

6th Edition

Centers for Disease Control and Prevention

National Institutes of Health

Inside front cover

Biosafety in Microbiological and Biomedical

Laboratories

6th Edition

U.S. Department of Health and Human Services Public Health Service

Centers for Disease Control and Prevention

Revised June 2020 iiiForeword

Foreword Biosafety in Microbiological and Biomedical Laboratories (BMBL) has served as the cornerstone of biosafety practice in the United States since its initial release. We wish to emphasize that the sixth edition of BMBL remains an advisory document recommending best practices for the safe conduct of work in biomedical and clinical laboratories from a biosafety perspective. The BMBL is not intended to be a regulatory document although we recognize that some may use it in that way. The core principle of this document is protocol-driven risk assessment; it is not possible for a single document to identify all of the possible combinations of risks and mitigations feasible in biomedical and clinical labora-tories. The BMBL should be used as a tool in the assessment and proposed mitigation steps in biomedical and clinical laboratories.

This edition of BMBL includes revised sections, agent summary statements, and appendices. We harmonized the recommendations included in this edition with guidance issued and regulations promulgated by other organizations and federal agencies. Wherever possible, we clarified both the language and intent of the information provided. In order to serve the needs of our community better, this edition includes new appendices on the following topics: inactivation and verification; laboratory sustainability; large-scale biosafety; and clinical laboratory biosafety.

Over 200 of our scientific and professional colleagues contributed to the prepa-ration of the sixth edition through participation in technical working groups and serving as reviewers, guest editors, and subject matter experts. We wish to thank them all for their dedication and hard work. Without them, the sixth edition of BMBL would not be possible. We also recognize the hard work and contributions made by all who participated in preparation of the previous editions of BMBL; we have built on their solid work and commitment.

It would have been impossible to publish this revision without recognizing the visionary leadership of the previous BMBL editors—Drs. John Richardson, W. Emmett Barkley, Jonathan Richmond, Robert W. McKinney, Casey Chosewood, and Deborah Wilson—without whom the BMBL would not be the respected resource it is today. The Steering Committee members, Drs. Christy Myrick, Richard G. Baumann, Margy Lambert, Patricia Delarosa, and Theresa Lawrence, were instrumental in identifying authors, selecting additions to this edition, and reviewing submissions. Their significant contribution to this edition is sincerely appreciated.

We are truly grateful to Ms. Shaina Mangino and Dr. Mallory Pomales of Eagle Medical Services, LLC for their expertise and patience in assisting us with this undertaking. Their superb organizational and editing skills were critical in the creation of this document.

iv Biosafety in Microbiological and Biomedical Laboratories

We hope you find the sixth edition of Biosafety in Microbiological and Biomedical Laboratories complete, timely, and most of all, easy to use. Thank you for your patience and understanding during the long and comprehensive revision process.

Paul J. Meechan, PhD, MPH, RBP, CBSP(ABSA) Associate Director for Laboratory Safety Office of Laboratory Science and Safety Centers for Disease Control and Prevention Atlanta, GA

Jeffrey Potts, MPH, CBSP(ABSA) Chief, Biorisk Management Branch Division of Occupational Health and Safety

Bethesda, MD vForeword

Participants Senior Co-Editors Paul J. Meechan, PhD, MPH, RBP, CBSP(ABSA) Associate Director for Laboratory Safety Office of Laboratory Science and Safety Centers for Disease Control and Prevention

Jeffrey Potts, MPH, CBSP(ABSA) Chief, Biorisk Management Branch Division of Occupational Health and Safety National Institutes of Health

Steering Committee Richard G. Baumann, PhD, SM(NRCM) Biological Safety Officer Division of Occupational Health and Safety National Institutes of Health

Patricia Delarosa, PhD, CBSP(ABSA), CTM(ATAP) Health Scientist, Biosafety Office of Strategy, Policy, Planning, and Requirements Office of the Assistant Secretary for Preparedness and Response Department of Health and Human Services

Margy Lambert, PhD Health Scientist Office of Science and Technology Assessment Occupational Safety and Health Administration

CAPT Theresa Lawrence, PhD Senior Science Officer Office of the Associate Director for Preparedness and Response U.S. Department of Health and Human Services

Paul J. Meechan, PhD, MPH, RBP, CBSP(ABSA) Associate Director for Laboratory Safety Office of Laboratory Science and Safety vi Biosafety in Microbiological and Biomedical Laboratories

Christy Myrick, PhD, RBP(ABSA) Lead Auditor U.S. National Authority for Containment for Polioviruses Center for Preparedness and Response Centers for Disease Control and Prevention

Jeffrey Potts, MPH, CBSP(ABSA) Chief, Biorisk Management Branch Division of Occupational Health and Safety National Institutes of Health

Deborah E. Wilson, DrPH, CBSP(ABSA) RADM (ret.), U.S. Public Health Service, Division of Occupational Health and Safety—Director Office of Research Services National Institutes of Health

Technical Editors Shaina Mangino Senior Publications Editor Eagle Medical Services, LLC

Mallory J. Pomales, DO Senior Program Manager for Scientific Writers Senior Scientific Editor Eagle Medical Services, LLC

Primary Authors Matthew J. Arduino, MS, DrPH, FSHEA, M(ASCP)CM Sr. Adviser, Environmental Hygiene and Infection Prevention Office of the Director Division of Healthcare Quality Promotion Centers for Disease Control and Prevention

William D. Arndt Health Scientist—Biorisk Division of Laboratory Systems Centers for Disease Control and Prevention

Heike Bailin, MD Staff Physician, Occupational Medical Service Division of Occupational Health and Safety viiForeword

Richard G. Baumann, PhD, SM(NRCM) Biological Safety Officer Division of Occupational Health and Safety National Institutes of Health

Richard S. Bradbury, PhD, FFSc RCPA Division of Parasitic Diseases and Malaria Center for Global Health Centers for Disease Control and Prevention

Mary E. Brandt, PhD Office of Laboratory Science and Safety Centers for Disease Control and Prevention

Cristina Bressler, MBA Health Scientist Occupational Health and Safety Office Office of Safety, Security and Asset Management Centers for Disease Control and Prevention

Byron Caughey, PhD Senior Investigator Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories National Institute for Allergy and Infectious Diseases National Institutes of Health

Rear Admiral Terri R. Clark, DVM, DACLAM Director (ret.), Office of Animal Care and Use Assistant Surgeon General USPHS Commission Corps National Institutes of Health

Danielle Daniely, PhD, RBP(ABSA) Director, Research Safety Programs and High Containment Laboratories Office of Research Integrity Georgia State University

Patricia Delarosa, PhD, CBSP(ABSA), CTM(ATAP) Health Scientist, Biosafety Office of Strategy, Policy, Planning, and Requirements Office of the Assistant Secretary for Preparedness and Response Department of Health and Human Services viii Biosafety in Microbiological and Biomedical Laboratories

Stephen Denny, DVM, MS, DACLAM, DACVPM Acting Director, Office of Animal Care and Use Office of Intramural Research National Institutes of Health

Eileen Edmonson Transportation Regulations Specialist Standards and Rulemaking Division Pipeline and Hazardous Materials Safety Administration U.S. Department of Transportation

Samuel S. Edwin, PhD Director Division of Select Agents and Toxins Center for Preparedness and Response Centers for Disease Control and Prevention

Elizabeth (Zeba) Floyd, AIA, LEED AP BD+C ID+C Project Director Sustainable Design Consulting, LLC

Karen M. Frank, MD, PhD, D(ABMM) Chief, Department of Laboratory Medicine Clinical Center National Institutes of Health

Mark D. Gibson, MS, CIH Senior Industrial Hygienist Division of Occupational Health and Safety National Institutes of Health

Eduardo Gomez-Saladin, PhD, SM, RBP, CBSP(ABSA) Deputy Director Office of Laboratory Safety Centers for Disease Control and Prevention

Natasha K. Griffith, MS Chief, Quality and Safety Systems Branch Division of Laboratory Systems ixForeword

Ted Hackstadt, PhD Chief, Host-Parasite Interactions Section Laboratory of Bacteriology Rocky Mountain Laboratories National Institute for Allergy and Infectious Diseases National Institutes of Health

Susan B. Harper, DVM, MS, DACLAM, DACVPM, RBP(ABSA) Office of National Programs USDA Agricultural Research Service

Kathryn L. Harris PhD, RBP(ABSA) Senior Outreach and Education Specialist Biosafety, Biosecurity and Emerging Biotechnology Policy Division Office of Science Policy National Institutes of Health

Mark L. Hemphill, MS Deputy Director, Division of Select Agents and Toxins Center for Preparedness and Response Centers for Disease Control and Prevention

Barbara L. Herwaldt, MD, MPH Medical Epidemiologist, Division of Parasitic Diseases and Malaria Centers for Disease Control and Prevention

Nancy P. Hoe, PhD, CBSP(ABSA) Biosafety Officer/Responsible Official for Rocky Mountain Laboratories Division of Occupational Health and Safety Office of the Director, National Institutes of Health

Joseph P. Kozlovac, MS, RBP, CBSP(ABSA), SM(NRCM) Agency Biosafety Officer USDA Agricultural Research Service Office of National Programs

Margy Lambert, PhD Health Scientist Office of Science and Technology Assessment Occupational Safety and Health Administration

George W. Lathrop Veterinary Medical Officer National Center for Emerging and Zoonotic Infectious Diseases x Biosafety in Microbiological and Biomedical Laboratories

Susan Lawrence, PhD Branch Chief Microbiology Branch Environmental Protection Agency

Susan A. Lippold, MD, MPH Medical Director Occupational Health Clinic Centers for Disease Control and Prevention

R. Trevor Lubbert Board Certified Senior Staff Entomologist Community Health Branch Division of Occupational Health and Safety Office of Research Safety National Institutes of Health

Carolina Lúquez, PhD National Botulism and Enteric Toxins Team Enteric Diseases Laboratory Branch Division of Foodborne, Waterborne, and Environmental Diseases National Center for Emerging and Zoonotic Infectious Diseases Centers for Disease Control and Prevention

Patrick M. McNutt, PhD Principal Investigator Department of Neuroscience U.S. Army Medical Research Institute of Chemical Defense

Paul J. Meechan, PhD, MPH, RBP, CBSP(ABSA) Associate Director for Laboratory Safety Office of Laboratory Science and Safety Centers for Disease Control and Prevention

Barbara Owen, MPH, CBSP(ABSA), RBP, NRCM, CMM Director, Global Safety and Environment Corporate Biosafety Officer Merck & Co., Inc.

Steven Piguet, AIA, LEED Fellow, Fitwel Ambassador Associate Principal Sustainable Design Consulting, LLC xiForeword

Segaran P. Pillai, PhD, SM(NRCM), SM(ASCP), FAAM Director, Office of Laboratory Science and Safety Office of the Commissioner Food and Drug Administration

Jeffrey Potts, MPH, CBSP(ABSA) Chief, Biorisk Management Branch Division of Occupational Health and Safety National Institutes of Health

Nathaniel Powell Jr., DVM, MS Chief, Comparative Medicine Branch Division of Scientific Resources National Center for Emerging and Zoonotic Infectious Diseases Centers for Disease Control and Prevention

Ann M. Powers, PhD Lead, Virology Team Arboviral Diseases Branch Division of Vector-Borne Diseases Centers for Disease Control and Prevention

Reynolds M. Salerno, PhD Director, Division of Laboratory Systems Centers for Disease Control and Prevention

Dr. Martin L. Sanders, PhD, CSP Director, Safety, Emergency, and Environmental Compliance Department of Health and Human Services

James M. Schmitt, MD, MS Medical Director Occupational Medical Service Division of Occupational Health and Safety National Institutes of Health

Stephen Tomasino, PhD Senior Scientist Environmental Protection Agency

Elizabeth G. Weirich, MS, SM(NRCM), CBSP(ABSA) Division of Laboratory Systems xii Biosafety in Microbiological and Biomedical Laboratories

David M. White, DVM, PhD, RBP(ABSA), DACVM Safety & Security Unit Lead National Centers for Animal Health USDA Animal and Plant Health Inspection Service

Deborah E. Wilson, DrPH, CBSP(ABSA) RADM (ret.), U.S. Public Health Service, Division of Occupational Health and Safety—Director Office of Research Services National Institutes of Health

Liz E. York, FAIA Chief Sustainability Officer Office of the Chief Operating Officer xiiiForeword xiiiForeword

Contributors Michael Adler

Karen Anderson

Rebecca V. Anderson

Matthew J. Arduino

David Asher

John Balog

Shawn A. Bean

David E. Bentzel

Cary R. Binder

Brad Blitvich

Kathryn Board

William P. Bozza

Megan Morgan Brose

Robert Bull

Cara Burns

Sheldon Campbell

Cynthia Cary

Nick Chaplinski

Mark Chappell

Konstantin Chumakov

Jeffrey Cohen

Eugene Cole

Nancy Cornish

Whitni Davidson

C. Todd Davis

Sabrina Debose

Johnathan R. Deeds

Thomas Denagamage

Beverly Dickson

Gerhard Dobler

Mike Drebot

Edward Dubovi

Eilyn Fabregas

Chadi Filfili

Betty A. Forbes

Marshall Gayton

Sarah Genzer

Christopher Good

Andrew Haddow

Vibeke (Vips) Halkjaer-Knudsen

Alexander Hamberg

Glen Hansen

David Harbourt

Kathryn L. Harris

Courtney Harrison

Michael W. Hart

Robert Hawley

Henry Hays

John Henneman

Stephen Higgs

Julia Hilliard

Michael Holbrook

Bill Homovec

Romney Humphries

Debra Hunt

Freeda Isaac

Eddie L. Jackson

Peter Jahrling

Robert Jambou

Eric Jeppesen

Barbara Johnson

Crystal Johnson

Eric A. Johnson

Julie Johnson

Estella Z. Jones

Joanne Jones-Meehan

Gerardo Kaplan

Subhashinie Kariyawasam

Jaqueline Katz

Arifa Khan

Lydia Kibiuk

Chris Kiley

Manley Kiser

Rajen Koshy

Laura Kramer

Philip Krause

Jens Kuhn

Anna Llewellyn

Maria Lorenzo

Luis Lugo-Roman

Nicole Lukovsky- Akhsanov

Marian Major

Monear Makvandi

Alison Mawle

Erin McElvania

Thomas A. McKeon

David Scott McVey

Thomas P. Monath

Rashida Moore

David Morens

Eric C. Mossel xiv Biosafety in Microbiological and Biomedical Laboratoriesxiv Biosafety in Microbiological and Biomedical Laboratories

Krista Murray

Christy Myrick

Brandy Nelson

Joseph Newsome

Stuart Nichol

Kenneth E. Nusbaum

Steve Oberste

Patricia Olinger

Lillian Orciari

Eugene O’Reilly

Mark Pagala

Subbian Satheshkumar (Sathesh) Panayampalli

Eun-Chung Park

Amar Patil

Michael A. Pentella

William Peters

Brett Petersen

Brian R. Petuch

Susan C. Piguet

Ewan Plant

Kristin Prentice

Suzette Priola

Amy Pullman

Richard Rebar

Yvonne Reed

Ryan F. Relich

Pierre Rollin

Eugene Rosenthal

Scott Rusk

Janice Rusnak

Arick P. Sabin

Mo D. Salman

Lawrence B.

Schonberger

Lynne M. Sehulster

Brianna Skinner

Halley Smith

James Synder

Marisa Elkins St. Claire

James Stevens

Molly Stitt-Fischer

James R. Swearengen

William M. Switzer

Sandra Tallent

Cassandra Tansey

Robert Tesh

Anil J. Thachil

Eileen L. Thacker

Natalie J. Thornburg

William H. Tolleson

John Tonkiss

Althea C. Treacy

Anita Trichel

Jessica Tucker

Terrence Tumpey

Timothy Uyeki

Francisco A. Uzal

Nikos Vasilakis

Linfa (Lin-Fa) Wang

David Warnock

Scott Weaver

Zachary Weiner

Rebecca Weingarten

Robbin Weyant

Mark Whary

Diana Whipple

Temeri Wilder-Kofie

Axel Wolff

Zhiping Ye

Kyoung-Jin Yoon

Edward You

Jessica Young

Baolin Zhang xvContents

Contents Foreword ............................................................................................................ iii Participants ......................................................................................................... v

Senior Co-Editors ........................................................................................... v Steering Committee ....................................................................................... v Technical Editors ............................................................................................vi Primary Authors .............................................................................................vi

Section I—Introduction The Occurrence of Laboratory-associated infections Evolution of National Biosafety Guidelines Risk Criteria for Establishing Ascending Levels of Containment Agent Summary Statements Laboratory Biosecurity Using Biosafety in Microbiological and Biomedical Laboratories Looking Ahead References

Section II—Biological Risk Assessment The Risk Management Process Risk Communication Facilitating a Culture of Safety through Risk Assessment Conclusion References

Section III—Principles of Biosafety Safety Equipment (Primary Barriers) Personal Protective Equipment Facility Design and Construction (Secondary Barriers) Facility Practices and Procedures Biosafety Levels Animal Facilities Clinical Laboratories Laboratory Biosecurity References

Section IV—Laboratory Biosafety Level Criteria Biosafety Level 1

A. Standard Microbiological Practices B. Special Practices xvi Biosafety in Microbiological and Biomedical Laboratories

C. Safety Equipment (Primary Barriers and Personal Protective Equipment)

D. Laboratory Facilities (Secondary Barriers) Biosafety Level 2

A. Standard Microbiological Practices B. Special Practices C. Safety Equipment (Primary Barriers and Personal

Protective Equipment) D. Laboratory Facilities (Secondary Barriers)

Biosafety Level 3 A. Standard Microbiological Practices B. Special Practices C. Safety Equipment (Primary Barriers and Personal

Protective Equipment) D. Laboratory Facilities (Secondary Barriers)

Biosafety Level 4 A. Standard Microbiological Practices B. Special Practices C. Safety Equipment (Primary Barriers and Personal

Protective Equipment) D. Laboratory Facilities (Secondary Barriers)

Section V—Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities

Animal Biosafety Level 1 A. Standard Microbiological Practices B. Special Practices C. Safety Equipment (Primary Barriers and Personal

Protective Equipment) D. Animal Facilities (Secondary Barriers)

Animal Biosafety Level 2 A. Standard Microbiological Practices B. Special Practices C. Safety Equipment (Primary Barriers and Personal

Protective Equipment) D. Animal Facilities (Secondary Barriers)

Animal Biosafety Level 3 A. Standard Microbiological Practices B. Special Practices xviiContents

C. Safety Equipment (Primary Barriers and Personal Protective Equipment)

D. Animal Facilities (Secondary Barriers) Animal Biosafety Level 4

A. Standard Microbiological Practices B. Special Practices C. Safety Equipment (Primary Barriers and Personal

Protective Equipment) D. Animal Facilities (Secondary Barriers)

References Section VI—Principles of Laboratory Biosecurity

Biosafety and Laboratory Biosecurity Developing a Laboratory Biosecurity Program Example Guidance: A Laboratory Biosecurity Risk Assessment and Management Process Elements of a Laboratory Biosecurity Program References

Section VII—Occupational Health Support for Biomedical Research Framework for Occupational Health Support of Biomedical

Research Elements of an Occupational Health Program Supporting

Biomedical Research Conclusion References

Section VIII—Agent Summary Statements References

Section VIII-A: Bacterial Agents Bacillus anthracis Bordetella pertussis Brucella species Burkholderia mallei Burkholderia pseudomallei Campylobacter species Chlamydia psittaci, C. trachomatis, C. pneumoniae Clostridium botulinum and neurotoxin-producing species of Clostridia Clostridioides (formerly Clostridium) difficile xviii Biosafety in Microbiological and Biomedical Laboratories

Clostridium tetani and Tetanus toxin Corynebacterium diphtheriae Francisella tularensis Helicobacter species Legionella pneumophila and other Legionella spp Leptospira Listeria monocytogenes Mycobacterium leprae Mycobacterium tuberculosis complex Mycobacterium spp. other than M. tuberculosis complex and M. leprae Neisseria gonorrhoeae Neisseria meningitidis Salmonella serotypes, other than S. enterica serotype

Typhi (S. Typhi) Salmonella enterica serotype Typhi (S. Typhi) Shiga toxin (Verocytotoxin)-producing Escherichia coli Shigella Staphylococcus aureus (Methicillin-Resistant, Vancomycin-

Resistant, or Vancomycin-Intermediate) Treponema pallidum Vibrio species Yersinia pestis References

Section VIII-B: Fungal Agents Blastomyces dermatitidis and Blastomyces gilchristii Coccidioides immitis and Coccidioides posadasii Histoplasma capsulatum Sporothrix schenckii species complex Miscellaneous Yeast and mold organisms causing human infection References

Section VIII-C: Parasitic Agents General Issues Blood and Tissue Protozoal Parasites Intestinal Protozoal Parasites xixContents

Cestode Parasites Trematode Parasites Nematode Parasites References

Section VIII-D: Rickettsial Agents Coxiella burnetii Rickettsia species and Orientia tsutsugamushi References

Section VIII-E: Viral Agents Hantaviruses Hendra Virus (formerly known as Equine Morbillivirus) and

Nipah Virus Hepatitis A Virus, Hepatitis E Virus Hepatitis B Virus, Hepatitis C Virus, Hepatitis D Virus Macacine alphaherpevirus 1 (Herpesvirus Simiae, Cerocopithecine herpesvirus I, Herpes B Virus) Human Herpes Virus Influenza Viruses Lymphocytic Choriomeningitis Virus Poliovirus Poxviruses Rabies Virus and related lyssaviruses Retroviruses, including Human and Simian Immunodeficiency

Viruses (HIV and SIV) Severe Acute Respiratory Syndrome (SARS) and Middle East

Respiratory Syndrome (MERS) Coronaviruses References

Section VIII-F: Arboviruses and Related Zoonotic Viruses Risk Group 2 Viruses with BSL-2 Containment Recommended Risk Group 3 Viruses with BSL-3 Containment Recommended West Nile Virus (WNV) Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine

Encephalitis Virus (VEEV), and Western Equine Encephalitis Virus (WEEV)

Rift Valley Fever Virus (RVFV) References xx Biosafety in Microbiological and Biomedical Laboratories

Section VIII-G: Toxin Agents Botulinum Neurotoxin Staphylococcal Enterotoxins (SE) Ricin Selected Low Molecular Weight (LMW) Toxins References

Section VIII-H: Prion Diseases Bovine Spongiform Encephalopathy Handling and processing of tissues from patients with suspected prion disease Handling and processing of multiple human prion tissue samples References

Appendix A—Primary Containment for Biohazards: Selection, Installation, and Use of Biological Safety Cabinets

Part 1—Introduction Part 2—High-Efficiency Particulate Air (HEPA) Filters and the

Development of Biological Containment Devices Part 3—Biological Safety Cabinets Part 4—Other Laboratory Hazards and Risk Assessment Part 5—BSC Use by the Investigator: Work Practices and Procedures Part 6—Facility and Engineering Requirements Part 7—Certification of BSCs Acknowledgments References

Appendix B—Decontamination and Disinfection of Laboratory Surfaces and Items

Purpose and Scope Antimicrobial Products—U.S. Regulations Environmentally-Mediated Transmission of Infection Principles of Cleaning, Disinfection, and Sterilization Decontamination References

Appendix C—Transportation of Infectious Substances International Harmonization of Shipping and Transport Regulations Transportation Regulations Select Agents Regulations xxiContents

Importation and Transfers Transfer of USDA Plant Pests DOT Packaging of Infectious Substances Intrafacility Specimen and Sample Transfers References

Appendix D—Biosafety and Biocontainment for Pathogens Affecting Agricultural Animals and Animals that are Loose-Housed or in Open Penning

Introduction Potential Enhancements for BSL-2 and ABSL-2 Facilities for

Conducting Work with Pathogens Affecting Agricultural Animals Potential Enhancements for BSL-3 and ABSL-3 Facilities for

Conducting Work with Pathogens Affecting Agricultural Animals BSL-4 and ABSL-4 Facilities that Work with Pathogens Affecting

Agricultural Animals Potential Enhancements for Animal Biosafety Level 2-Agriculture

(ABSL-2Ag) Facilities for Conducting Work with Animals that are Loose-Housed or in Open Penning

Animal Biosafety Level 3-Agriculture (ABSL-3Ag) Facilities required for activities involving the use of hazardous biological agents designated as High-Consequence Foreign Animal Diseases and Pests by USDA APHIS in animals that are loose-housed or in open penning

Animal Biosafety Level 4-Agriculture (ABSL-4Ag) Facilities for Conducting Work with Animals that are Loose-Housed or in Open Penning

References Appendix E—Arthropod Containment Guidelines (ACG)

References Appendix F—Select Agents and Toxins Appendix G—Integrated Pest Management (IPM)

References Appendix H—Working with Human, Non-Human Primate (NHP), and Other Mammalian Cells and Tissues

Bloodborne pathogens and risk assessment related to material source and type

Risk Mitigation References xxii Biosafety in Microbiological and Biomedical Laboratories

Appendix I—Guidelines for Work with Toxins of Biological Origin General Considerations for Toxin Use Training and Laboratory Planning Safety Equipment and Containment Inadvertent Toxin Aerosols Mechanical Injuries Additional Precautions Decontamination and Spills Select Toxins References

Appendix J—NIH Oversight of Research Involving Recombinant Biosafety Issues Appendix K—Inactivation and Verification

Background Filtration and Centrifugation Development of Inactivation Procedures Validation of Inactivation Procedures Alternative Strategies Attenuation Methods Process Verification Institutional Verification Tracking of and Communication about Inactivated Samples Ongoing Review and Oversight of Inactivation and Verification

Procedures Other Important Considerations Conclusion References

Appendix L—Sustainability Introduction and Issues Strategies for Existing Laboratories and Operations Strategies for New and Renovated Laboratories References

Appendix M—Large-Scale Biosafety Introduction Risk Assessment Conclusion References xxiiiContents

Appendix N—Clinical Laboratories Clinical Laboratory Biosafety Conducting Risk Assessments in a Clinical Laboratory Environment Implementing Mitigation Measures in the Clinical Laboratory

Environment Challenges in a Clinical Laboratory Environment Implementing Performance Management in a Clinical Laboratory

Environment Risk Ethics in a Clinical Laboratory Environment Summary References

Appendix O—Acronyms Glossary Index Accessibility Descriptions of Figures

Appendix A—Primary Containment for Biohazards Appendix C—Transportation of Infectious Substances xxiv Biosafety in Microbiological and Biomedical Laboratories

Tables Section IV—Laboratory Biosafety Level Criteria

Table 1. Summary of Laboratory Biosafety Levels (BSLs) Section VIII-B: Fungal Agents

Table 1. Miscellaneous Yeast and Mold Section VIII-E: Viral Agents

Table 1. Viruses currently included in the genus Lyssavirus Section VIII-F: Arboviruses and Related Zoonotic Viruses

Table 1. Vaccine Strains of Specific Viruses that May Be Handled at BSL-2 Table 2. Explanation of Symbols Used in Tables 3 and 4 to Define Basis for

Assignment of Viruses to Biosafety Levels Table 3. Alphabetic Listing of Arboviruses and Hemorrhagic Fever Viruses* Table 4. Alphabetic Listing of Arboviruses and Hemorrhagic Fever Viruses* Table 5. Laboratories working with the viruses at BSL-3 listed below are recommended to HEPA filter the exhaust air Section VIII-H: Prion Diseases

Table 1. Human Prion Diseases Table 2. Animal Prion Diseases Table 3. Tissue Preparation for Human CJD and Related Diseases Table 4. Prion Inactivation Methods for Reusable Instruments and Surfaces

Appendix A—Primary Containment for Biohazards: Selection, Installation, and Use of Biological Safety Cabinets

Table 1. Selection of a Safety Cabinet through Risk Assessment Table 2. Comparison of Biosafety Cabinet Characteristics

Appendix B—Decontamination and Disinfection of Laboratory Surfaces and Items

Table 1. Activity Levels of Selected Liquid Chemical Disinfectants Appendix D—Biosafety and Biocontainment for Pathogens Affecting Agricultural Animals and Animals that are Loose-Housed or in Open Penning

Table 1. Bacteria Table 2. Fungi and Molds Table 3. Nematodes, Trematodes, Cestodes, Protozoa, and Ectoparasites Table 4. Viruses Table 5. Toxins Table 6. Prions xxvContents

Table Key 1. Natural Host Range Table Key 2. Natural Routes of Transmission Table Key 3. Environmental Stability

Appendix I—Guidelines for Work with Toxins of Biological Origin Table 1. Physical Inactivation of Toxins Table 2. Chemical Inactivation of Toxins

Appendix K—Inactivation and Verification Table 1. Advantages of Physical Inactivation Table 2. Disadvantages of physical inactivation Table 3. Advantages of chemical inactivation Table 4. Disadvantages of chemical inactivation Table 5. Advantages of chemical activated by physical treatment Table 6. Disadvantages of chemical activated by physical treatment Table 7. Advantages of natural and emerging antimicrobial strategies Table 8. Disadvantages of natural and emerging antimicrobial strategies Table 9. Advantages of combination methods Table 10. Disadvantages of combination methods Table 11. Advantages of novel methods to attenuate pathogens Table 12. Disadvantages of novel methods to attenuate pathogens xxvi Biosafety in Microbiological and Biomedical Laboratories

Figures Appendix A—Primary Containment for Biohazards: Selection, Installation, and Use of Biological Safety Cabinets

Figure 1. HEPA Filters Figure 2. The Class I BSC Figure 3. The Class II, Type A BSC Figure 4. Canopy (thimble) unit for ducting a Class II, Type A BSC Figure 5a. The Class II, Type B1 BSC (classic design) Figure 5b. The Class II, Type B1 BSC (benchtop design) Figure 6. The Class II, Type B2 BSC Figure 7a. The Class II, Type C1 BSC (not connected to building exhaust system) Figure 7b. The Class II, Type C1 BSC (connected to building exhaust system) Figure 8. The Class III BSC Figure 9a. The Horizontal Laminar flow Clean Bench Figure 9b. The Vertical Laminar Flow Clean Bench Figure 10. Clean to Dirty Figure 11. Protection of a house vacuum Figure 12. Bag-in/bag-out filter enclosure

Appendix B—Decontamination and Disinfection of Laboratory Surfaces and Items

Figure 1. Descending Order of Relative Resistance to Disinfectant Chemicals

Appendix C—Transportation of Infectious Substances Figure 1. A Category A UN Standard Triple Packaging Figure 2. A Category B Non-specification Triple Packaging

1Section 1—Introduction

Section I—Introduction Biosafety in Microbiological and Biomedical Laboratories (BMBL) has become the overarching guidance document for the practice of biosafety in the U.S.— the mechanism for addressing the safe handling and containment of infectious microorganisms and hazardous biological materials. The principles of biosafety introduced in 1984 in the first edition of BMBL1 and carried through this edition remain steadfast. These principles are containment and risk assessment.

The fundamentals of containment include the microbiological practices, safety equipment, and facility safeguards that protect laboratory workers, the environment, and the public from exposure to infectious microorganisms that are handled and stored in the laboratory. Risk assessment is the process that enables the appropriate selection of microbiological practices, safety equipment, and facility safeguards that can help prevent Laboratory-associated infections (LAI). The purpose of periodic updates of BMBL is to refine guidance based on new knowledge and experiences and to address contemporary issues that present new risks that confront laboratory workers and the public health. In this way, the guidance provided within the BMBL will continue to serve the microbio-logical and biomedical community as a relevant and valuable reference.

The uncertainty and change regarding the identification of emerging agents and the requirements for containment and safe storage of pathogens continues to accelerate since the last edition of the BMBL was published. New infectious agents and diseases have emerged. Work with infectious agents in public and private research, public health, clinical and diagnostic laboratories, and in animal care facilities has expanded. World events have demonstrated new threats of bioterrorism. For these reasons, organizations and laboratory directors are compelled to evaluate and ensure the effectiveness of their biosafety programs, the proficiency of their workers, as well as the capability of equipment, facilities, and management practices to provide containment and security of microbiological agents. Similarly, individual workers who handle pathogenic microorganisms must understand the containment conditions under which infectious agents can be safely manipulated and secured. Application of this knowledge and the use of appropriate techniques and equipment will enable the microbiological and biomedical community to help prevent personal, laboratory, and environmental exposure to potentially infectious agents or biohazards.

The Occurrence of Laboratory-associated infections Published reports of LAIs first appeared around the start of the 20th century. By 1978, four studies by Pike and Sulkin collectively identified 4,079 LAIs resulting in 168 deaths occurring between 1930 and 1978.2–5 These studies found that the ten most common causative agents of overt infections among workers were Brucella spp., Coxiella burnetii, hepatitis B virus (HBV), Salmonella enterica serotype Typhi, Francisella tularensis, Mycobacterium tuberculosis, Blastomyces

2 Biosafety in Microbiological and Biomedical Laboratories dermatitidis, Venezuelan equine encephalitis virus, Chlamydia psittaci, and Coccidioides immitis. The authors acknowledged that the 4,079 cases did not represent all LAIs that occurred during this period, since many laboratories chose not to report overt cases or conduct surveillance programs to identify subclinical or asymptomatic infections.

In addition, historical reports of LAIs seldom provided data sufficient to determine incidence rates, complicating quantitative assessments of risk. Similarly, there were no distinguishable accidents or exposure events identified in more than 80% of the LAIs reported before 1978. Studies did show that, in many cases, the infected person worked with a microbiological agent or was in the vicinity of another person handling an agent.2–6

During the 20 years following the Pike and Sulkin publications, a worldwide literature search by Harding and Byers revealed 1,267 overt infections with 22 deaths.7 Five deaths were of fetuses aborted as the consequence of a maternal LAI. Mycobacterium tuberculosis, Coxiella burnetii, hantavirus, arboviruses, HBV, Brucella spp., Salmonella spp., Shigella spp., hepatitis C virus, and Crypto-sporidium spp. accounted for 1,074 of the 1,267 infections. The authors also identified an additional 663 cases that presented as subclinical infections. Like Pike and Sulkin, Harding and Byers reported that only a small number of the LAI involved a documented specific incident. The non-specific associations reported most often by these authors were working with a microbiological agent, being in or around the laboratory, or being around infected animals.

The findings of Harding and Byers indicated that clinical (diagnostic) and research laboratories accounted for 45% and 51%, respectively, of the total LAIs reported.

This is a marked difference from the LAIs reported by Pike and Sulkin prior to 1979, which indicated that clinical and research laboratories accounted for 17% and 59%, respectively. The relative increase of LAIs in clinical laboratories may be due in part to improved employee health surveillance programs that are able to detect subclinical infections, or to the use of inadequate containment proce-dures during the early stages of culture identification.

Comparison of the more recent LAIs reported by Harding and Byers with those reported by Pike and Sulkin suggests that the number is decreasing. Harding and Byers note that improvements in containment equipment, engineering controls, and greater emphasis on safety training may be contributing factors to the apparent reduction in LAIs over two decades. However, due to the lack of infor-mation on the actual numbers of infections and the population at risk, it is difficult to determine the true incidence of LAIs.

Publication of the occurrence of LAIs provides an invaluable resource for the microbiological and biomedical community. For example, one report of occupa-tional exposures associated with Brucella melitensis, an organism capable of

3Section 1—Introduction transmission by the aerosol route, described how a staff member in a clinical microbiology laboratory accidentally sub-cultured B. melitensis on the open bench.8 This error and a breach in containment practices resulted in eight LAIs with B. melitensis among 26 laboratory members—an attack rate of 31%.

Reports of LAIs can serve as lessons in the importance of maintaining safe conditions in biomedical and clinical laboratories.

Evolution of National Biosafety Guidelines National biosafety guidelines evolved from the efforts of the microbiological and biomedical community to promote the use of safe microbiological practices, safety equipment, and facility safeguards that reduce LAIs and protect public health and the environment. The historical accounts of LAIs raised awareness about the hazards of infectious microorganisms and the health risks to laboratory workers who handle them. Many published accounts suggested practices and methods that might prevent LAIs.9 Arnold G. Wedum was the Director of Industrial Health and Safety at the United States Army Biological Research Laboratories, Fort Detrick, from 1944 to 1969. His pioneering work in biosafety provided the foundation for evaluating the risks of handling infectious microorganisms and for recognizing biological hazards and developing practices, equipment, and facility safeguards for their control. Fort Detrick also advanced the field by aiding the development of biosafety programs at the United States Department of Agriculture (USDA), National Animal Research Center (NARC) and the United States Department of Health and Human Services (DHHS), Centers for Disease Control and Prevention (CDC), and National Institutes of Health (NIH). These governmental organizations subsequently developed several national biosafety guidelines that preceded the first edition of BMBL.

In 1974, the CDC published Classification of Etiologic Agents on the Basis of Hazard.10 This report introduced the concept for establishing ascending levels of containment that correspond to risks associated with handling infectious microor-ganisms that present similar hazardous characteristics. Human pathogens were grouped into four classes according to mode of transmission and the severity of disease they caused. A fifth class included non-indigenous animal pathogens whose entry into the United States was restricted by USDA policy.

The NIH published National Cancer Institute Safety Standards for Research Involving Oncogenic Viruses in 1974.11 These guidelines established three levels of containment based on an assessment of the hypothetical risk of cancer in humans from exposure to animal oncogenic viruses or a suspected human oncogenic virus isolate.12,13 In 1976, NIH first published the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines).14 The current NIH Guidelines described in detail the microbiological practices, equipment, and facility safeguards that correspond to four ascending levels of physical

4 Biosafety in Microbiological and Biomedical Laboratories containment and established criteria for assigning experiments to a containment level based on an assessment of potential hazards of this continually evolving technology.15 The evolution of these guidelines set the foundation for developing a code of practice for biosafety in microbiological and biomedical laboratories.

Led by the CDC and NIH, a broad collaborative initiative involving scientists, laboratory directors, occupational physicians, epidemiologists, public health officials, and health and safety professionals developed the first edition of BMBL in 1984.16 The BMBL provided the technical content not previously available in biosafety guidelines by adding summary statements conveying guidance pertinent to infectious microorganisms that had caused LAIs. The sixth edition of BMBL is also the product of a broad collaborative initiative committed to perpetuate the value of this national biosafety code of practice.

Risk Criteria for Establishing Ascending Levels of Containment The primary risk criteria used to define the four ascending levels of containment, referred to as Biosafety Levels 1 through 4, are infectivity, severity of disease, transmissibility, and the nature of the work being conducted. Another important risk factor for agents that cause moderate to severe disease is the origin of the agent, whether indigenous or exotic. Each level of containment describes the microbiological practices, safety equipment, and facility safeguards for the corre-sponding level of risk associated with handling an agent. The facility safeguards associated with Biosafety Levels 1 through 4 help protect non-laboratory occupants of the facility, the public health, and the environment.

Biosafety Level 1 (BSL-1) is the basic level of protection and is appropriate for defined and characterized strains of viable biological agents that are not known to cause disease in immunocompetent adult humans. Biosafety Level 2 (BSL-2) is appropriate for handling moderate-risk agents that cause human disease of varying severity by ingestion or through percutaneous or mucous membrane exposure. Biosafety Level 3 (BSL-3) is appropriate for agents with a known potential for aerosol transmission, for agents that may cause serious and poten-tially lethal infections, and that are indigenous or exotic in origin. Exotic agents that pose a high individual risk of life-threatening disease by infectious aerosols and for which no treatment is available are restricted to high containment labora-tories that meet Biosafety Level 4 (BSL-4) guidelines.

It is important to emphasize that the causative incident for most LAIs is unknown.7,8 Less obvious exposures such as the inhalation of infectious aerosols or direct contact of broken skin or mucous membranes with droplets containing an infectious microorganism or surfaces contaminated by droplets may possibly explain the incident responsible for a number of LAIs. Manipulations of liquid suspensions of microorganisms may produce aerosols and droplets.

Small-particle aerosols have respirable size particles that may contain one or several microorganisms. These small particles stay airborne and easily disperse

5Section 1—Introduction throughout the laboratory. When inhaled, the human lung will retain these particles. Larger particle droplets rapidly fall out of the air, contaminating gloves, the immediate work area, and the mucous membranes of unprotected workers.

A procedure’s potential to release microorganisms into the air as aerosols and droplets is the most important operational risk factor that supports the need for containment equipment and facility safeguards.

Agent Summary Statements The sixth edition, as in all previous editions, includes agent summary statements that describe the hazards, recommended precautions, and levels of containment appropriate for handling specific human and zoonotic pathogens in the laboratory and in facilities that house laboratory vertebrate animals. Agent summary statements are included for agents that meet one or more of the following three criteria:

1. The agent is a proven hazard to laboratory personnel working with infectious materials;

2. The agent is suspected to have a high potential for causing LAIs even though no documented cases exist; and

3. The agent causes grave disease or presents a significant public health hazard.

Scientists, clinicians, and biosafety professionals prepared the statements by assessing the risks of handling the agents using standard protocols followed in many laboratories. No one should conclude that the absence of an agent summary statement for a human pathogen means that the agent is safe to handle at BSL-1 or without a risk assessment to determine the appropriate level of containment. Laboratory directors should also conduct independent risk assessments before beginning work with an agent or procedure new to the laboratory, even though an agent summary statement is available. There may be situations where a laboratory director should consider modifying the precau-tionary measures or recommended practices, equipment, and facility safeguards described in an agent summary statement. In addition, laboratory directors should seek guidance when conducting risk assessments. Knowledgeable colleagues, institutional safety committees, institutional biosafety committees, biosafety officers, and public health, biosafety, and scientific associations are excellent resources.

The agent summary statements in the fifth edition of BMBL were reviewed in the course of preparing the sixth edition. There are new and updated agent summary statements including those for agents classified as Select Agents. For example, there is an updated section on arboviruses and related zoonotic viruses including new agent summary statements as well as statements for recently emerged agents such as Middle East Respiratory Syndrome coronavirus (MERS-CoV).

6 Biosafety in Microbiological and Biomedical Laboratories

The sixth edition includes a substantially revised section on risk assessment that emphasizes the critical importance of this process in selecting the appropriate practices and level of containment. That section intentionally follows this intro-duction because risk assessment is the core principle that supports a code of practice for safe handling of infectious agents in microbiological and biomedical laboratories.

Laboratory Biosecurity The nation also continues to face a challenge in safeguarding the public health from potential domestic or international bioterrorism. Existing standards and practices may require adaptation to ensure protection from such hostile actions.

Federal regulations mandate increased security within the microbiological and biomedical community in order to protect high consequence biological pathogens and toxins from theft, loss, or misuse. The sixth edition of BMBL includes an update on laboratory biosecurity—the discipline addressing the security of microbiological agents and toxins and the threats posed to human and animal health, the environment, and the economy by deliberate misuse or release. A careful review of the laboratory biosecurity concepts and guidelines in Section VI is essential for all laboratory workers.

Using Biosafety in Microbiological and Biomedical Laboratories

BMBL is a code of practice and an authoritative reference. Knowledge sufficient to work safely with hazardous microorganisms requires a careful review of multiple sections of the BMBL. This will offer the reader an understanding of the biosafety principles that serve as the basis for the concepts and recommendations included in this reference. Reading only selected sections will not adequately prepare even an experienced laboratory worker to handle potentially infectious agents safely.

The recommended practices, safety equipment, and facility safeguards described in the BMBL are advisory. The intent was and is to establish a voluntary code of practice, one that all members of a laboratory community will together embrace to safeguard themselves and their colleagues, and to protect the public health and environment.

Additional appendices have been added to the sixth edition of the BMBL, including: Appendix K—Inactivation and Verification; Appendix L—Sustainability;

Appendix M—Large Scale Biosafety; and Appendix N—Clinical Laboratories. In Appendix K, content has been added on inactivation verification, as recent events have demonstrated that it may be insufficient to follow a published inactivation procedure and assume that it is capable of providing complete inactivation of all pathogenic organisms present in a sample. In Appendix L, content has been added to assist laboratories with finding methods to reduce the significant operating costs associated with laboratories. In Appendix M, biosafety consider-ations for large-scale production of agents has been added, in recognition of the

7Section 1—Introduction interest in the use of biological agents in the generation of biopharmaceuticals.

Finally, in Appendix N, content on the safe handling of biological materials in clinical laboratories has been added, as the risk assessment of handling specimens with unconfirmed but suspected high-risk agents can be significantly different from the assessment traditionally generated in microbiology laboratories.

The BMBL should not be used as a single source of biosafety information; it provides the basis for a rational risk assessment to be developed and reviewed by the competent stakeholders at an institution. Inclusion of all relevant stake-holders, including the biosafety office or officer, animal care staff, facilities staff, management, and the Institutional Biosafety Committee, or equivalent resource, is needed to ensure all relevant parties provide input and reach consensus on the risk assessment.

Looking Ahead Although Laboratory-associated infections are infrequent, it is critical that the microbiological and biomedical communities continue their resolve to remain vigilant and avoid complacency. The widely reported incidents of accidental shipments of or potential exposures to high-consequence pathogens over the last several years demonstrate that accidents and unrecognized exposures continue to occur. The absence of clear evidence of the means of transmission in most documented LAIs should motivate persons at risk to be alert to all potential routes of exposure. The accidental release of microbial aerosols is a probable cause of many LAIs,17 which demonstrates the importance of worker training and the ability to recognize potential hazards and correct unsafe habits.