Summary of Changes to the Biosafety in Microbiological and Biomedical Laboratories 6th Edition (BMBL-6)

This Summary of Changes was prepared by the ABSA International Technical and Regulatory Review Committee (TRR). May 2021

This Summary of Changes is not to be used as a substitute for the CDC/NIH BMBL-6 edition.

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Table of Contents

I: Introduction

II: Biological Risk Assessment

III: Principles of Biosafety

IV: Laboratory Biosafety Criteria
   Biosafety Level 1 (BSL-1)
   Biosafety Level 2 (BSL-2)
   Biosafety Level 3 (BSL-3)
   Biosafety Level 4 (BSL-4)

V: Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities
   Animal Biosafety Level 1
   Animal Biosafety Level 2
   Animal Biosafety Level 3
   Animal Biosafety Level 4

VI: Principles of Laboratory Biosecurity

VII: Occupational Health Support for Biomedical Research

VIII-A: Bacterial Agents

VIII-B: Fungal Agents

VIII-C: Parasitic Agents

VIII-D: Rickettsial Agents

VIII-E: Viral Agents

VIII-F: Viral Agents Arboviruses and Related Zoonotic Viruses

VIII-G: Toxin Agents

VIII-H: Prion Agents

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

Appendix B: Decontamination and Disinfection of Laboratory Surfaces and Items

Appendix C: Transportation of Infectious Substances

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

Appendix E: Arthropod Containment Guidelines

Appendix F: Select Agents and Toxins

Appendix G: Integrated Pest Management

Appendix H: Working with Human, NHP, and Other Mammalian Cells and Tissues

Appendix I: Guidelines for Work with Toxins of Biological Origin

Appendix J: NIH Oversight of Research Involving Recombinant Biosafety Issues

Appendix K: Inactivation and Verification

Appendix L: Sustainability

Appendix M: Large-Scale Biosafety

Appendix N: Clinical laboratories

Section VIII-B: Fungal Agents (pages 212-219)

Section VII-B comprises summaries for fungal agents that include Blastomyces dermatitidis and Blastomyces gilchristii, Coccidioides immitis and Coccidioides posadasii, Histoplasma capsulatum and Sporothrix schenckii species complex. Each agent summary details occupational infections, natural modes of infection, laboratory safety and containment recommendations and any special issues. These agents require an import permit and a domestic transfer permit.

Blastomyces dermatitidis is a dimorphic fungus that exists in nature and laboratory cultures at room temperature as a filamentous mold with spores that are infectious particles. New to the BMBL-6 is the addition of Blastomyces gilchristii which has recently been recognized as a novel species. Spores, or conidia, convert to large budding yeasts under appropriate culture conditions in the laboratory and in the parasitic phase in warm-blooded animals. Infections occur when spores are inhaled, or the yeast form is injected. LAIs have occurred through parenteral inoculation with tissues or cultures containing yeast forms, as well as through inhalation of spores. At least 11 reported LAIs with two fatalities have occurred. Natural modes of infection require common exposure from a point source from the environment and human-to-human transmission occurs rarely via perinatal or sexual transmission. BSL-3 practices are recommended for samples that contain spores. BSL-2 and ABSL-2 practices are recommended for diagnostic and yeast-form cultures.

Coccidioides species are dimorphic fungal pathogens that exist in nature and lab cultures at room temperature as filamentous molds, with asexual spores that are the infectious particles. Occupational exposure has been associated with high dust exposure in endemic regions. LAIs tend to result in clinical disease whereas infections in nature tend to be asymptomatic. Single spores can produce infections by the respiratory route, though most infections from the environment are subclinical. BSL-3 practices are recommended for propagating and manipulating spores, processing soil known or suspected to contain spores or animal studies where challenge is via inhalation or the pulmonary route. BSL-2 practices are recommended for clinical specimens and ABSL-2 practices are recommended for animal studies involving the parenteral challenge.

Histoplasma capsulatum is a dimorphic fungal pathogen that exists in the lab culture setting as filamentous mold with asexual spores. Specific hazards include increased risk to immunocompromised individuals. LAIs tend to be common among diagnostic work, from handling mold for cultures and spray to the eyes. Spores are resistant to drying and may remain viable for long periods of time. Natural modes of infection require a point source and infections are not transmissible from person-to-person. BSL-3 practices are recommended for propagating sporulating H.capsulatum in the mold form or environmental samples suspected to contain spores. BSL-2 and ABSL-2 practices are recommended for clinical specimens and a biosafety cabinet is used for any samples where dimorphic fungi are identified.

Sporothrix schenckii species are dimorphic fungal pathogens that exist in nature and in lab cultures as filamentous mold with asexual spores. Large outbreaks of sporotrichosis have been documented in environments where exposed to soil or plant material containing the fungus. Occupational exposure cases have been associated with local skin or eye infections in lab personnel. BSL-2 and ABSL-2 practices are recommended for handling of clinical specimens and a BSC is used for any culture identified as dimorphic fungi.

The BMBL-6 removed Cryptococcus neoformans and Dermatophytes agent summaries. Instead a new table, Table 1 Miscellaneous Yeast and Mold, has been added at the end of the section describing occupational and natural mode of infection and the biosafety level for Candida species, Cryptococcus neoformans and C. gattii, Dermatophyte molds, Hyaline molds, Talaromyces marneffei, Dematiaceous molds and Mucormycete molds.

Section VIII-C: Parasitic Agents (pages 223-237)

The BMBL-6 includes the same five subsections of parasites included in BMBL-5. The first two subsections include taxonomically diverse protozoal groups categorized according to lifestyle/ mode of infection. These two subsections include Microsporidia, which are noted actually to be fungi but are grouped with protozoal parasites for historical reasons. The last three categories represent distinct taxonomic groups from the phyla Platyhelminthes (trematodes and cestodes) and Nematoda respectively. A history of LAIs is noted for most subgroups. Known and possible routes of LAIs (e.g. ingestion, percutaneous exposure, arthropod vector bite, etc) are noted for each category. For all parasite groups except nematodes, BSL-2 and ABSL-2 are specifically recommended when handling infective stages; a specific biocontainment level is not recommended for nematode parasites (but BSL-2 / ABSL-2 may be inferred). The need for arthropod containment per Appendix E when infected vector arthropods are present is noted. Specific inactivation procedures using bleach, peroxide, etc are recommended for certain organisms. Special CDC, USDA and/ or DoC permits may be required for importation and shipping for most parasites.

The BMBL-6 includes additional species of Free-Living Amoeba (FLA) and describes the medical conditions associated with FLA, and potential sources of infection in the workplace.

Section VIII-D: Rickettsial Agents (pages 239-244)

This section is in the same order as in the BMBL-5, immediately following Parasitic Agents. All headings found within are also in the same order as the previous edition.

The Rickettsial Agents section begins with Coxiella burnetti, an intracellular pathogen and Select Agent. There is a brief description of C. burnetti followed by the same headers used throughout Section VIII. There are two noticeable modifications to the BMBL-6 from the BMBL-5. First, the header Laboratory Safety and Containment Recommendations now includes a paragraph regarding cell-free media that has been recently developed and more widely disseminated. This header also contains a brief statement regarding a plaque purified clonal strain that is Select Agent exempt and can be used safely following BSL-2 conditions. The second modification to the Coxiella burnetti section is that the mention of a Q-fever vaccine in the IND phase has been removed.

Following C. burnetti, the authors coalesce all other Rickettsia spp. into a single remaining section. There were some minor modifications to this section to reflect newly identified Rickettsial species and safety practices. Specifically, R. philipii and R. parkeri have been added. In terms of the Safety heading, a large paragraph describing BSL-2 with enhanced practices when performing laboratory-based research using Rickettsial species has been added.

Section VIII-E: Viral Agents (pages 247-280)

The Laboratory and Safety Recommendations include the use of these viruses in rodent species inducing chronic infections should be conducted at ABSL-4. Similarly, cell-culture propagation should be conducted at BSL-3 following BSL-3 practices, and handling of serum or tissue samples should be handled at BSL-2 following BSL-3 practices, containment equipment, and procedures. Lastly, potentially infected samples should be handled at BSL-2 following BSL-3 procedures and practices. The special Issues subsection added the potential permit required by the Department of Commerce for exporting this virus.

The BMBL-6 contains updated information about transmission of this virus and the new henipa virus, Cedar virus. Human-to-human transmission is included as a natural route of transmission. Laboratory and Safety Recommendations are provided for Cedar virus. Special Issues include mention of the Hendra vaccine available for horses in Australia

This section includes updates for Hepatitis E virus (HEV) as its genus Orthohepevirus has been assigned to the Hepeviridae family and recognizes four genotypes infecting humans. Occupational infections now have expanded HEV genotype 1 as a risk for pregnant women. The update also acknowledges the potential risk of exposure to HEV-infected pigs, rabbits, or macaques. The Natural Modes of Infection was updated to include the modes of transmission for each HEV genotype. The Vaccine section was updated to include a HEV vaccine currently available in China.

The BMBL-6 removed the alternate name “non A non B” virus for Hepatitis C (HCV). The special Issues subsection added the potential permit requirement by the Department of Commerce for exporting this virus to another country.

The name of the Herpes B virus was updated in BMBL-6 to reflect its current scientific name. Herpes B virus was removed from the Select Agent list, thus the notation of it being a Select Agent was removed in the BMBL-6, however, the permit requirement by the Department of Commerce was added.

This section has been updated in the BMBL-6 to reflect current knowledge related to clinical presentation, modes of natural transmission and treatment. Importantly, the following sentence was added for handling Epstein Barr Virus: “Autologous transformation of B cells using EBV should not be performed”.

The Influenza summary has been significantly modified to reflect newly identified serotypes and current research. Laboratory Safety and Containment recommendations are provided for Seasonal Human Influenza Viruses and Zoonotic and Animal Influenza A Viruses in addition to the existing Non-Contemporary Human Influenza Viruses, 1918 Influenza A (Pandemic Virus), Highly Pathogenic Avian Influenza A Viruses (HPAI) and other Influenza Recombinant or Reassortant Viruses. The need for risk assessment is emphasized when handling wild-type human influenza A (H1N1) and A (H3N2) as they may not circulate all the time, a case in point is discussed with the pre-2009A(H1N1) and the A(H1N1)pdm09, as pre-pandemic and pandemic viruses, respectively.

Added to this section in the BMBL-6 is the inclusion that women in childbearing age should be made aware of risks posed by LCMV or rodents potentially infected by LCMV.

The Poliovirus section now includes WHO GAPIII requirements for Type 2 and WPV3 and Type 1 and OPV3.

BMBL-5 stated that the NIH Guidelines assessed the risk for handling certain attenuated strains at BSL-1, if no other human orthopoxviruses were being used. The BMBL-6 indicates that lower containment for attenuated poxviruses and vectors may be considered. It also added that for research subject to the NIH Guidelines, approval to lower containment from BSL-2 must be requested from the NIH Office of Science Policy.

A new table has been included in this section including Lyssavirus species and the recommended biosafety level (page 272).

The BMBL-6 contains updated pathogen information for HIV, SIV, Simian foamy viruses (SFV), Simian Retrovirus (SRV), and the SIV/HIV genetic recombinant virus (SHIV) (page 276).

MERS has been included in this section. The pathogen information and occupational infections for SARS has been updated.