Procedures for Flow Cytometry-Based Sorting of Unfixed Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infected Cells and Other Infectious Agents.

In response to the recent COVID-19 pandemic, many laboratories are involved in research supporting SARS-CoV-2 vaccine development and clinical trials. Flow cytometry laboratories will be responsible for a large part of this effort by sorting unfixed antigen-specific lymphocytes. Therefore, it is critical and timely that we have an understanding of risk assessment and established procedures of infectious cell sorting. Here we present procedures covering the biosafety aspects of sorting unfixed SARS-CoV-2-infected cells and other infectious agents of similar risk level. These procedures follow the ISAC Biosafety Committee guidelines and were recently approved by the National Institutes of Health Institutional Biosafety Committee for sorting SARS-CoV-2-infected cells. © 2020 International Society for Advancement of Cytometry.

International Society for the Advancement of Cytometry cell sorter biosafety standards.

Flow cytometric cell sorting of biological specimens has become prevalent in basic and clinical research laboratories. These specimens may contain known or unknown infectious agents, necessitating precautions to protect instrument operators and the environment from biohazards arising from the use of sorters. To this end the International Society of Analytical Cytology (ISAC) was proactive in establishing biosafety guidelines in 1997 (Schmid et al., Cytometry 1997;28:99-117) and subsequently published revised biosafety standards for cell sorting of unfixed samples in 2007 (Schmid et al., Cytometry Part A J Int Soc Anal Cytol 2007;71A:414-437). Since their publication, these documents have become recognized worldwide as the standard of practice and safety precautions for laboratories performing cell sorting experiments. However, the field of cytometry has progressed since 2007, and the document requires an update. The new Standards provides guidance: (1) for laboratory design for cell sorter laboratories; (2) for the creation of laboratory or instrument specific Standard Operating Procedures (SOP); and (3) on procedures for the safe operation of cell sorters, including personal protective equipment (PPE) and validation of aerosol containment.

Implementing an Organizational Culture of Biosafety and Biosecurity in the SAP Institute.

An organizational culture of biosafety and biosecurity is critical for effective management of transboundary animal diseases. One essential aspect of this work is keeping important pathogens studied in veterinary laboratories under control. Türkiye is among the countries that are both endemic and disease-free for foot-and-mouth disease (FMD) virus, and it has a unique institute dedicated to FMD diagnosis, control, and vaccine production. To build an organizational safety culture within this institute and strengthen awareness of the importance of safe and secure handling of FMD, 4 staff members previously trained in biorisk management developed and provided trainings to all institute staff. The institute's 173 personnel were divided into 3 groups by job description based on direct or indirect work with FMD virus. All 3 groups received training that addressed biosecurity, biosafety, biorisk awareness, and insider threat; the trainings varied in length by group. Three-quarters (n=130, 75%) of all institute staff completed their training and were asked to complete knowledge surveys using a Likert scale survey before and after their training. A majority (n=104, 80%) of those participants completed both the pretraining and posttraining surveys. All 3 training groups' posttraining surveys showed improved awareness above baseline scores, and all 3 groups scores reached the targeted threshold goal. Group 2 demonstrated a realization that some of the knowledge and habits they had acquired through experience were incorrect. Scores for several individual questions decreased at posttraining, and these results will need further evaluation. The overall training results prompted the institute to provide periodic updates to employees to sustain the organizational safety culture. With this study, the institute now has a dedicated group of biorisk management representatives. This work serves as a wake-up call for established institutions that rely on staff experience to foster an organizational culture of biosafety and biosecurity.

Biorisk Management Features of a Temporary COVID-19 Hospital.

Introduction: Yale University designed and constructed a temporary field hospital for 100 COVID-19 symptomatic patients. Conservative biocontainment decisions were made in design and operational practices. Objectives of the field hospital included the safe flow of patients, staff, equipment and supplies, and obtaining approval by the Connecticut Department of Public Health (CT DPH) for opening as a field hospital. Methods: The CT DPH regulations for mobile hospitals were used as primary guidance for the design, equipment, and protocols. References for BSL-3 and ABSL-3 design from the National Institutes of Health (NIH) and Tuberculosis isolation rooms from the United States Centers for Disease Control and Prevention (CDC) were also utilized. The final design involved an array of experts throughout the university. Results and Conclusion: Vendors tested and certified all High Efficiency Particulate Air (HEPA) filters and balanced the airflows inside the field hospital. Yale Facilities designed and constructed positive pressure access and exit tents within the field hospital, established appropriate pressure relationships between zones, and added Minimum Efficiency Reporting Value 16 exhaust filters. The BioQuell ProteQ Hydrogen Peroxide decontamination unit was validated with biological spores in the rear sealed section of the biowaste tent. A ClorDiSys Flashbox UV-C Disinfection Chamber was also validated. Visual indicators were placed the doors of the pressurized tents and spaced throughout the facility to verify airflows. The plans created to design, construct and operate the field hospital provide a blueprint for recreating and reopening a field hospital in the future if ever needed at Yale University.

Short-Term Use Biocontainment Bubbles: Innovative Source Containment of Potentially Infectious SARS-CoV-2 Aerosols.

Introduction: This article will review the processes utilized to develop simple effective containment engineering controls. Short-Term Use Biocontainment Bubbles-Yale (STUBB-Ys), as Yale refers to them, were designed, built, tested, and implemented to protect members of the Yale University community from exposure to SARS-CoV-2 aerosols. STUBB-Ys were designed and created in conjunction with end users, constructed by Environmental Health and Safety (EHS) or partner groups, and tested onsite after installation to verify effective operation and containment. Methods: A wide variety of devices in different settings were developed and installed. STUBB-Ys were used at COVID-19 indoor test centers, laboratories, and clinics. The devices were pursued to create infection prevention measures where existing processes could not be utilized or were inadequate. Each STUBB-Y was tested with a C-Breeze Condensed Moisture Airflow Visualizer to generate smoke and a Fluke 985 Particle Counter, which gives the particle counts from 0.3 to 10 µm to measure particle escape visually and quantitatively. Airflow rates were also tested where applicable with a TSI VelociCalc 9525 Air Velocity Meter. Results: Students and faculty were able to safely continue vital research or clinical study in the targeted areas with the addition of these simple containment devices to confine aerosols. Conclusion: From a biorisk management point of view, EHS was able to confine aerosols at their potential source using simple designs and equipment and adhering to the hierarchy of controls. This article demonstrates how a straightforward design process can be used to enhance worker protection during a pandemic.

Neuroinvasion of SARS-CoV-2 in human and mouse brain.

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus on the consequences of CNS infections. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in infected and neighboring neurons. However, no evidence for type I interferon responses was detected. We demonstrate that neuronal infection can be prevented by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate SARS-CoV-2 neuroinvasion in vivo. Finally, in autopsies from patients who died of COVID-19, we detect SARS-CoV-2 in cortical neurons and note pathological features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV-2 and an unexpected consequence of direct infection of neurons by SARS-CoV-2.

Neuroinvasion of SARS-CoV-2 in human and mouse brain.

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus whether the virus can infect the brain, or what the consequences of CNS infection are. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in the infected and neighboring neurons. However, no evidence for the type I interferon responses was detected. We demonstrate that neuronal infection can be prevented either by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate in vivo that SARS-CoV-2 neuroinvasion, but not respiratory infection, is associated with mortality. Finally, in brain autopsy from patients who died of COVID-19, we detect SARS-CoV-2 in the cortical neurons, and note pathologic features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV2, and an unexpected consequence of direct infection of neurons by SARS-CoV-2.

Update of Arthropod Containment Guidelines.

The American Society of Tropical Medicine and Hygiene's American Committee of Medical Entomology has released their updated edition (Version 3.2) of the Arthropod Containment Guidelines. The Guidelines were written to provide pertinent risk assessment and risk management information for the safe handling and rearing of arthropods used in research. The format of the Guidelines is like the outline used in the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories. Four Arthropod Containment Levels (ACLs) are described, with increasing requirements for safety and security from ACL-1 to ACL-4. Each containment level provides information on standard practices, including standard and special practices, storage, labeling, monitoring and trapping escaped arthropods, training, medical surveillance, safety equipment, and facility design.

So You Want to Start an Institutional Biosafety Committee.

Introduction: The number of institutions engaging in research with potentially biohazardous materials has increased, indicating a need for newly formed Institutional Biosafety Committees (IBCs) in the United States and for similar biorisk management committees located outside the United States. Our institution identified the need for an IBC due to the growth of pertinent activities on campus. Objectives: This article shares our experiences creating a new IBC at our institution from September 2017 to April 2019. Our lessons learned and approaches to the challenges faced may be helpful to others finding themselves with similar needs. Methods: In this case study, we outline IBC membership, documents, relationships with federal agencies and within the institution, creation of registration forms, and the review process. Along with our account, we have included links to helpful resources from federal agencies. Results: At the time of the submission of this article, we have established our IBC and reviewed two registrations. Conclusion: This case report demonstrates the successful creation of an IBC that works for our current institutional needs.

Containment capabilities of animal transfer stations.

Animal-transfer and cage-changing stations are portable downdraft-filtered clean benches that have been specifically modified for small-rodent handling and cage changing from two or more sides, and that are advertised by their manufacturers as providing improved laboratory animal allergen control. The authors evaluated the dust containment capability of three such devices under exaggerated challenge conditions, compared design features, and conclude that animal-transfer stations can be a useful addition to an institutional laboratory animal allergy control program.

Institutional responsibilities in contamination control in research animals and occupational health and safety for animal handlers.

The mechanisms for controlling microbial contamination in research animals are similar to those for preventing exposure among animal handlers to naturally occurring pathogens, research-related biohazards, or animal allergens. Research and resource preservation are the primary goals of each approach, and an appropriate assessment of risk is their foundation. The identification of potential risks enables the implementation of relevant risk management or control measures. This article summarizes the components of an occupational health and safety program for animal handlers, including screening, training, work practices, effective use of engineering controls, selection and use of personal protective equipment, and emergency response protocols. The features of a risk assessment and risk management program and the level of interaction and training required to implement and sustain the program correlate well with programs designed to control microbial contamination in laboratory animals. This article includes an explanation of the five Ps of risk assessment and risk management. Pathogens and the proposed experimental procedures account for the first two Ps; the other three focus on training and awareness of the personnel involved in the experiment, protective equipment and work practices, and factors associated with the place (or facility) where the research will be conducted. Animal handlers comprehension and knowledge determine the success of any containment program, and so this review also includes a discussion of critical teaching points for animal handlers and of the importance of evaluating personnel to verify their proficiency and competence in required protocols.

General guidelines for experimenting with HIV.

Performing experiments using human immunodeficiency virus (HIV)-infected materials represents a potential biological hazard for the investigator. An intensive training program for laboratory personnel must always precede the actual execution of research involving manipulation of HIV. In addition, appropriate sterile tissue culture techniques are absolute prerequisites for producing meaningful experiments and for achieving safe working conditions. In most circumstances more than one investigator uses the same HIV research facility; therefore, careful training in biosafety is mandatory not only for self-protection, but for the safety of other investigators as well. The first protocol in this unit establishes a general framework for working safely with HIV and the second describes the proper storage of HIV stocks and test supernatants.