In vitro comparison of viral replication and cytopathology induced by SARS-CoV-2 variants.

A myriad of coronaviruses cause diseases from a common cold to severe lung infections and pneumonia. SARS-CoV-2 was discovered to be the etiologic agent of the Coronavirus pandemic and many laboratory techniques were examined for virus culture and basic and applied research. Understanding the replication kinetics and characterizing the effect the virus has on different cell lines is crucial for developing in vitro studies. With the emergence of multiple variants of SARS-CoV-2, a comparison between their infectivity and replication in common cell lines will help give us a clear understanding of their characteristic differences in pathogenicity. In this study we compared the cytopathic effect and replication of Wild-Type (USA/WA1), Omicron (B.1.1.529), and Delta (B.1.617.2) variants on five different cell lines; VeroE6, VeroE6 cells expressing high endogenous ACE2, VeroE6 cells expressing human ACE2 and TMPRSS2, Calu3 cells highly expressing human ACE2 and A549 cells. This data will aid researchers with experimental planning and viral pathogenicity analysis and provide a baseline for testing any future variants.

Validated Methods for Effective Decontamination and Inactivation of Rift Valley Fever Virus.

The Rift Valley fever virus (RVFV) is an arthropod-borne, zoonotic, hemorrhagic fever virus that can cause severe diseases both in livestock and humans. The spread of RVFV in areas previously considered as non-endemic together with the absence of licensed vaccines for use in humans and animals poses a major health and economic threat worldwide. It is therefore crucial to make major progresses in our understanding and management of this virus and its zoonosis. RVFV is considered a bioterrorism pathogen, and, thus, only a few institutes, facilities, and personnel are legally authorized to detain it and handle it. Moreover, this virus must be manipulated in a biosafety level 3 (BSL3) laboratory following strict biosafety protocols to ensure that biosecurity's highest standards are met. Only certain attenuated strains such as the MP12 strain can be handled in BSL2 laboratories, depending on the country considered. To assist researchers in working with RVFV in the safest possible conditions, this chapter presents validated methods for effective RVFV decontamination and inactivation.

A high-throughput response to the SARS-CoV-2 pandemic.

Four years after the beginning of the COVID-19 pandemic, it is important to reflect on the events that have occurred during that time and the knowledge that has been gained. The response to the pandemic was rapid and highly resourced; it was also built upon a foundation of decades of federally funded basic and applied research. Laboratories in government, pharmaceutical, academic, and non-profit institutions all played roles in advancing pre-2020 discoveries to produce clinical treatments. This perspective provides a summary of how the development of high-throughput screening methods in a biosafety level 3 (BSL-3) environment at Southern Research Institute (SR) contributed to pandemic response efforts. The challenges encountered are described, including those of a technical nature as well as those of working under the pressures of an unpredictable virus and pandemic.

Validation of a severe acute respiratory syndrome coronavirus 2 microneutralization assay for evaluation of vaccine immunogenicity.

As variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge, assessment of vaccine immunogenicity remains a critical factor to support continued vaccination. To this end, an in vitro microneutralization (MN50) assay was validated to quantitate SARS-CoV-2 neutralizing antibodies against prototype and variant strains (Beta, Delta, Omicron BA.1, Omicron BA.5, and XBB.1.5) in human serum. For the prototype strain, the MN50 assay met acceptance criteria for inter-/intra-assay precision, specificity, linearity, and selectivity. The assay was robust against changes to virus/serum incubation time, cell seeding density, virus content per well, cell passage number, and serum interference. Analyte in serum samples was stable up to five freeze/thaw cycles and for up to 12 months of storage at -80 ± 10 °C. Similar results were observed for the variant-adapted MN50 assays. The conversion factor to convert assay result units to WHO international standard units (IU/mL) was determined to be 0.62 for the prototype strain. This MN50 assay will be useful for vaccine immunogenicity analyses in clinical trial samples, enabling assessment of vaccine immunogenicity for ancestral and variant strains as variant-adapted vaccines are developed.

Coronavirus Disease 2019 Causing Infection of Transplanted Lung Allograft: A Pitfall of Prolonged Shedding of Severe Acute Respiratory Syndrome Coronavirus-2 Pretransplant.

Coronavirus disease 2019 (COVID-19) pandemic has led to considerable morbidity and mortality across the world. Lung transplant is a viable option for a few with COVID-19-related lung disease. Whom and when to transplant has been the major question impacting the transplant community given the novelty of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We describe a pitfall of presumed prolonged shedding of SARS-CoV-2 in a patient with COVID-19 associated acute respiratory distress syndrome leading to COVID-19 pneumonia after lung transplant. This raises concerns that replication-competent SARS-CoV-2 virus can persist for months post-infection and can lead to re-infection of grafts in the future.

A DNA-based non-infectious replicon system to study SARS-CoV-2 RNA synthesis.

The coronavirus disease-2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seriously affected public health around the world. In-depth studies on the pathogenic mechanisms of SARS-CoV-2 is urgently necessary for pandemic prevention. However, most laboratory studies on SARS-CoV-2 have to be carried out in bio-safety level 3 (BSL-3) laboratories, greatly restricting the progress of relevant experiments. In this study, we used a bacterial artificial chromosome (BAC) method to assemble a SARS-CoV-2 replication and transcription system in Vero E6 cells without virion envelope formation, thus avoiding the risk of coronavirus exposure. Furthermore, an improved real-time quantitative reverse transcription PCR (RT-qPCR) approach was used to distinguish the replication of full-length replicon RNAs and transcription of subgenomic RNAs (sgRNAs). Using the SARS-CoV-2 replicon, we demonstrated that the nucleocapsid (N) protein of SARS-CoV-2 facilitates the transcription of sgRNAs in the discontinuous synthesis process. Moreover, two high-frequency mutants of N protein, R203K and S194L, can obviously enhance the transcription level of the replicon, hinting that these mutations likely allow SARS-CoV-2 to spread and reproduce more quickly. In addition, remdesivir and chloroquine, two well-known drugs demonstrated to be effective against coronavirus in previous studies, also inhibited the transcription of our replicon, indicating the potential applications of this system in antiviral drug discovery. Overall, we developed a bio-safe and valuable replicon system of SARS-CoV-2 that is useful to study the mechanisms of viral RNA synthesis and has potential in novel antiviral drug screening.

Hydrogen Peroxide Vapor Decontamination of Hazard Group 3 Bacteria and Viruses in a Biosafety Level 3 Laboratory.

Aim: This study aimed to validate the efficacy of hydrogen peroxide vapor (HPV) decontamination technology set up in a biosafety level 3 (BSL-3) laboratory on surrogates and hazard group 3 (HG3) agents. Methods and Results: The HPV decontamination system (Bioquell) was assessed with both qualitative and quantitative methods on (1) spore surrogates (Geobacillus stearothermophilus, Bacillus atrophaeus, and Bacillus thuringiensis) in the BSL-3 laboratory and in the material airlock and on (2) HG3 agents (Bacillus anthracis; SARS-CoV-2, Venezuelan equine encephalitis virus [VEE], and Vaccinia virus) in the BSL-3 laboratory. Other HG3 bacteria likely to be handled in the BSL-3 laboratory (Yersinia pestis, Burkholderia mallei, Brucella melitensis, and Francisella tularensis) were excluded from the HPV decontamination assays as preliminary viability tests demonstrated the total inactivation of these agents after 48 h drying on different materials. Conclusions: The efficacy of HPV decontamination was validated with a reduction in viability of 5-7 log10 for the spores (surrogates and B. anthracis), and for the enveloped RNA viruses. Vaccinia showed a higher resistance to the decontamination process, being dependent on the biological indicator location in the BSL-3 laboratory.

Resilience and Protection of Health Care and Research Laboratory Workers During the SARS-CoV-2 Pandemic: Analysis and Case Study From an Austrian High Security Laboratory.

The SARS-CoV-2 pandemic has highlighted the interdependency of healthcare systems and research organizations on manufacturers and suppliers of personnel protective equipment (PPE) and the need for well-trained personnel who can react quickly to changing working conditions. Reports on challenges faced by research laboratory workers (RLWs) are rare in contrast to the lived experience of hospital health care workers. We report on experiences gained by RLWs (e.g., molecular scientists, pathologists, autopsy assistants) who significantly contributed to combating the pandemic under particularly challenging conditions due to increased workload, sickness and interrupted PPE supply chains. RLWs perform a broad spectrum of work with SARS-CoV-2 such as autopsies, establishment of virus cultures and infection models, development and verification of diagnostics, performance of virus inactivation assays to investigate various antiviral agents including vaccines and evaluation of decontamination technologies in high containment biological laboratories (HCBL). Performance of autopsies and laboratory work increased substantially during the pandemic and thus led to highly demanding working conditions with working shifts of more than eight hours working in PPE that stressed individual limits and also the ergonomic and safety limits of PPE. We provide detailed insights into the challenges of the stressful daily laboratory routine since the pandemic began, lessons learned, and suggest solutions for better safety based on a case study of a newly established HCBL (i.e., BSL-3 laboratory) designed for autopsies and research laboratory work. Reduced personal risk, increased resilience, and stress resistance can be achieved by improved PPE components, better training, redundant safety measures, inculcating a culture of safety, and excellent teamwork.

Zika, Nipah and Kala-azar: Emerging lethal infectious diseases amid COVID-19 as an escalating public health threat in South India.

As of 6 June 2022, a sum 25,782 of active cases and 524,701 deaths due to Coronavirus disease-19 (COVID-19) have been recorded in India. Stewing in the flares of the pandemic, Kerala is entwined in the wrath of multiple emerging infectious diseases. India, a home to 1.3 billion people, recently faced a devastating second wave of COVID-19 during May of 2021, with a ruckus of chronic shortage of medicine, oxygen supplies, ventilators, besides, being challenged by secondary infections and chronic health ailments. The state of Kerala, alone contributes to 50% COVID-19 caseload, besides, recent simultaneous outbreaks of Zika Virus Disease (ZVD), Nipah Virus Disease (NiVD) and Kala-azar (black fever) on July 8, September 5 and 8, 2021 respectively. Syndemicity and a high case fatality rates of these highly contagious diseases coupled with post infection sequelae, overwhelm the already fragile healthcare system. Thus, these lethal infectious diseases along with an anticipated third wave of COVID-19 pose a serious public health threat in and around South India. With this narrative review, we aim to discuss the challenges that the emergence of intersecting outbreaks of Zika, Nipah, Kala-azar presents with, in the nation, amidst the global pandemic of COVID-19 and provide recommendations so as to help alleviate the situation. The syndemicity of COVID-19 with other infectious diseases, calls for adequate surveillance and monitoring of diseases' outbreaks. To avoid the worst situations like pandemic, the health ministry, public and private health stakeholders in India should strengthen the public healthcare delivery system and providence of quick medical facilities to control the rate of mortality and morbidity during outbreaks.

Bactericidal efficacy of a low concentration of vaporized hydrogen peroxide with validation in a BSL-3 laboratory.

BACKGROUND: Highly infective pathogens are cultured and studied in biosafety laboratories. It is critical to disinfect these laboratories thoroughly in order to prevent laboratory infection. A whole-room, non-contact, reduced corrosion disinfection strategy using hydrogen peroxide was proposed and evaluated. AIM: To evaluate the bactericidal efficacy of 8% and 10% vaporized hydrogen peroxide (VHP) in a laboratory setting, with spores and bacteria used as bioindicators. METHODS: Spores of Bacillus atrophaeus and Bacillus stearothermophilus, along with Escherichia coli, Staphylococcus aureus and Staphylococcus albus bacteria were placed in pre-selected locations in a sealed laboratory, and an OXY-PHARM NOCOSPRAY2 VHP generator was applied. Spore killing efficacy was evaluated qualitatively, bactericidal efficacy was analysed quantitatively, and the mean log10 reduction was determined. Finally, the optimized disinfection strategy was verified in a biosafety level 3 (BSL-3) laboratory. FINDINGS: Significant reductions in microbial load were obtained for each of the selected spores and bacteria when exposed to VHP 8% and 10% for 2-3 h. S. aureus was found to be more resistant than E. coli and S. albus. Tests with VHP 8% and exposure for >3 h showed a 100% kill rate for B. atrophaeus on surfaces and equipment in the BSL-3 laboratory. CONCLUSION: The VHP generator has good diffusivity and low corrosiveness, and is a time-saving method for removal of disinfectant residue. This study provides reference for the precise disinfection of air and the surfaces of objects in biosafety laboratories under various conditions.

Biorisk Management for SARS-CoV-2 Research in a Biosafety Level-3 Core Facility.

The emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents hazards to researchers and other laboratory personnel in research settings where the live virus is stored and handled. The Biosafety Level-3 (BSL-3) Core Facility (CF) at Yong Loo Lin School of Medicine in National University of Singapore (NUS Medicine) has implemented a biorisk management (BRM) system to ensure that biorisk to employees, the public, or the environment are consistently minimized to an acceptable level while working with SARS-CoV-2. This chapter summarizes how a BRM system can be implemented in academic institutions based on international standards in the context of existing local legislations/regulations and institutional policies/guidelines to minimize the risk of laboratory-acquired infections and deliberate misuse of the newly emerged virus, SARS-CoV-2 in BSL-3 laboratories. The BRM programs prioritize performing risk assessments prior to implementation of work processes and reassessing the risk portfolio of the facilities from time to time, determining root causes and prevention of recurrences. Focusing on awareness-raising and educating the laboratory users in biosafety and biosecurity, and identifying opportunities for improvement are the other key factors for a sustainable and successful BRM system in the NUS Medicine BSL-3 CF.

Preclinical coronavirus studies and pathology: Challenges of the high-containment laboratory.

The COVID-19 pandemic has highlighted the critical role that animal models play in elucidating the pathogenesis of emerging diseases and rapidly analyzing potential medical countermeasures. Relevant pathologic outcomes are paramount in evaluating preclinical models and therapeutic outcomes and require careful advance planning. While there are numerous guidelines for attaining high-quality pathology specimens in routine animal studies, preclinical studies using coronaviruses are often conducted under biosafety level-3 (BSL3) conditions, which pose unique challenges and technical limitations. In such settings, rather than foregoing pathologic outcomes because of the inherent constraints of high-containment laboratory protocols, modifications can be made to conventional best practices of specimen collection. Particularly for those unfamiliar with working in a high-containment laboratory, the authors describe the logistics of conducting such work, focusing on animal experiments in BSL3 conditions. To promote scientific rigor and reproducibility and maximize the value of animal use, the authors provide specific points to be considered before, during, and following a high-containment animal study. The authors provide procedural modifications for attaining good quality pathologic assessment of the mouse lung, central nervous system, and blood specimens under high-containment conditions while being conscientious to maximize animal use for other concurrent assays.

Impact of a PMMA tube on performances of a Vereos PET/CT system adapted for BSL-3 environment according to the NEMA NU2-2012 standard.

INTRODUCTION: A Vereos PET/CT device was adapted to be compatible with the experimentation in large animals within BSL-3 environment. The aim of this study was to investigate the impact of this modification on the performance according to NEMA NU2-2012 standard. METHODS: Spatial resolution, sensitivity, count rate performance, accuracies of corrections and image quality were assessed using the NEMA NU2-2012 standards before and after installation of a transparent poly-methyl methacrylate tube of 8 mm thickness, 680 mm diameter and 2800 mm long inside the tunnel of the system. In addition, CT performance tests were performed according to manufacturer standard procedure. RESULTS: Although the presence of the tube led to a slight decrease in sensitivity, performance measurements were in accordance with manufacturer preconisation ranges and comparable to previous performance published data. CONCLUSION: Modifications of Vereos PET/CT system allowing its use in BSL-3 conditions did not affect significantly its performance according to NEMA NU2-2012 standard. KEY POINTS: Question. Does a BSL-3 compatible modification alter Philips Vereos PET/CT performances according to NEMA NU2-2012 standards? Pertinent findings. Our Vereos PET/CT system was modified by a wall separating BSL-1 and BSL-3 sides and an 8 mm thickness PMMA tube inserted into the bore of the camera in order to extend the BSL-3 containment along the bed movement. The performances of our modified system according to NEMA NU2-2012 standards were not significantly impacted by the modifications and were in accordance with the values prescribed by the manufacturer. Implications for patients care. Our clinical PET/CT device was modified for human infectious diseases studies in Non-Human Primates. This unusual set up may then provide truly transposable data from preclinical studies into clinical application in infected patients.

Scaling Biosafety Up During and Down After the COVID-19 Pandemic.

Purpose: The aim of this work was to review and analyze changes to the practice of biosafety imposed by pandemics. Methods: A narrative review of the COVID-19 pandemic that began in 2020 and prior pandemics from the perspective of a working virologist. Results: By definition, pandemics, outbreaks, and other emergencies are transient phenomena. They manifest as waves of events that induce unforeseen needs and present unknown challenges. After a pandemic, the return to normality is as crucial as the scale-up during the exponential growth phase. The COVID-19 pandemic presents an example to study operational biosafety and biocontainment issues during community transmission of infectious agents with established pandemic potential, the propensity to induce severe disease, and the ability to disrupt aspects of human society. Conclusions: Scaling down heightened biocontainment measures after a pandemic is as important as scaling up during a pandemic. The availability of preventive vaccines, and therapeutic drug regimens, should be considered in risk assessments for laboratory studies. There exists the need to preserve situational memory at the personal and institutional levels that can be served by professional societies.

The Evaluation of the Containment Efficacy of Semi-Rigid Isolators for Housing Cages of Laboratory Animals Infected With BSL-3 Agents.

Research animals models infected with Biosafety Level-3 (BSL-3) agents need to be housed in specialized biocontainment caging. Most of these specialized cages have input and exhaust that is high efficiency particulate air filtered and sealed to prevent escape of the BSL-3 agent. An alternative to the use of the above BSL-3 biocontainment caging is the use of a flexible film or modified semi-rigid plastic film isolator that has its own high efficiency particulate air-filtered input and exhaust and is sealed with respect to the animal room environment, thus preventing BSL-3 agent escape. Standard caging can be housed within such an isolator. Computational fluid dynamics was used to evaluate the integrity of modified semi-rigid isolators for containment of aerosolized BSL-3 agents. Three isolators were located inside an animal BSL-3 room to provide an extra tier of protection and to permit different infectious studies within the same room while reducing or eliminating the risk of cross-contamination. The isolators were sized to house caging for rabbits and smaller non-human primates such as marmosets, African greens, and macaques. Multiple case studies of failure scenarios were investigated, including isolator breaches through the plastic membrane seam separation and rips, and exhaust fan failure. Breaching the level of containment provided by the isolators required the improbable simultaneous event of a plastic membrane rip in addition to the rare malfunction of the back-up exhaust fans. Each isolator was equipped with 2 blower motors connected in parallel to a common exhaust plenum and a battery backup. Even with this rare double (simultaneous) event, the animal BSL-3 room air exhaust system was able to contain the few droplets released in the simulated computational fluid dynamics breach. The modified semi-rigid isolators with negative airflow proved safe and effective for aerosol studies using BSL-3 agents, even in the unlikely event of a breach in containment.

The one health landscape in Sub-Saharan African countries.

OBJECTIVES: One Health is transiting from multidisciplinary to transdisciplinary concepts and its viewpoints should move from 'proxy for zoonoses', to include other topics (climate change, nutrition and food safety, policy and planning, welfare and well-being, antimicrobial resistance (AMR), vector-borne diseases, toxicosis and pesticides issues) and thematic fields (social sciences, geography and economics). This work was conducted to map the One Health landscape in Africa. METHODS: An assessment of existing One Health initiatives in Sub-Saharan African (SSA) countries was conducted among selected stakeholders using a multi-method approach. Strengths, weaknesses, opportunities and threats to One Health initiatives were identified, and their influence, interest and impacts were semi-quantitatively evaluated using literature reviews, questionnaire survey and statistical analysis. RESULTS: One Health Networks and identified initiatives were spatiotemporally spread across SSA and identified stakeholders were classified into four quadrants. It was observed that imbalance in stakeholders' representations led to hesitation in buying-in into One Health approach by stakeholders who are outside the main networks like stakeholders from the policy, budgeting, geography and sometimes, the environment sectors. CONCLUSION: Inclusion of theory of change, monitoring and evaluation frameworks, and tools for standardized evaluation of One Health policies are needed for a sustained future of One Health and future engagements should be outputs- and outcomes-driven and not activity-driven. National roadmaps for One Health implementation and institutionalization are necessary, and proofs of concepts in One Health should be validated and scaled-up. Dependence on external funding is unsustainable and must be addressed in the medium to long-term. Necessary policy and legal instruments to support One Health nationally and sub-nationally should be implemented taking cognizance of contemporary issues like urbanization, endemic poverty and other emerging issues. The utilization of current technologies and One Health approach in addressing the ongoing pandemic of COVID-19 and other emerging diseases are desirable. Finally, One Health implementation should be anticipatory and preemptive, and not reactive in containing disease outbreaks, especially those from the animal sources or the environment before the risk of spillover to human.

Significance of High-Containment Biological Laboratories Performing Work During the COVID-19 Pandemic: Biosafety Level-3 and -4 Labs.

High containment biological laboratories (HCBL) are required for work on Risk Group 3 and 4 agents across the spectrum of basic, applied, and translational research. These laboratories include biosafety level (BSL)-3, BSL-4, animal BSL (ABSL)-3, BSL-3-Ag (agriculture livestock), and ABSL-4 laboratories. While SARS-CoV-2 is classified as a Risk Group 3 biological agent, routine diagnostic can be handled at BSL-2. Scenarios involving virus culture, potential exposure to aerosols, divergent high transmissible variants, and zoonosis from laboratory animals require higher BSL-3 measures. Establishing HCBLs especially those at BSL-4 is costly and needs continual investments of resources and funding to sustain labor, equipment, infrastructure, certifications, and operational needs. There are now over 50 BSL-4 laboratories and numerous BSL-3 laboratories worldwide. Besides technical and funding challenges, there are biosecurity and dual-use risks, and local community issues to contend with in order to sustain operations. Here, we describe case histories for distinct HCBLs: representative national centers for diagnostic and reference, nonprofit organizations. Case histories describe capabilities and assess activities during COVID-19 and include capacities, gaps, successes, and summary of lessons learned for future practice.

Evaluation of Hydrogen Peroxide Fumigation and Heat Treatment for Standard Emergency Arthropod Inactivation in BSL-3 Insectaries.

Climate change and global movements of people and goods have accelerated the spread of invasive species, including insects that vector infectious diseases, which threaten the health of more than half of the world's population. Increasing research efforts to control these diseases include the study of vector - pathogen interactions, involving the handling of pathogen-infected vector insects under biosafety level (BSL) 2 and 3 conditions. Like microbiology BSL-3 laboratories, BSL-3 insectaries are usually subjected to fixed-term or emergency room decontamination using recognized methods such as hydrogen peroxide (H2O2) or formaldehyde fumigation. While these procedures have been standardized and approved for the inactivation of diverse pathogens on surfaces, to date, there are no current standards for effective room-wide inactivation of insects in BSL-3 facilities in case of an emergency such as the accidental release of a large number of infected vectors. As H2O2 is often used for standard room decontamination in BSL-3 facilities, we evaluated H2O2 fumigation as a potential standard method for the safe, room-wide deactivation of insects in BSL-3 insectaries in comparison to heat treatment. To account for physiological diversity in vector insect species, six species from three different orders were tested. For the H2O2 fumigation we observed a strong but also varying resilience across all species. Lethal exposure time for the tested dipterans ranged from nine to more than 24 h. Furthermore, the coleopteran, Tribolium castaneum, did not respond to continuous H2O2 exposure for 48 h under standard room decontamination conditions. In contrast, temperatures of 50°C effectively killed all the tested species within 2 to 10 min. The response to lower temperatures (40-48°C) again showed a strong variation between species. In summary, results suggest that H2O2 fumigation, especially in cases where a gas generator is part of the laboratory equipment, may be used for the inactivation of selected species but is not suitable as a general emergency insect inactivation method under normal room decontamination conditions. In contrast, heat treatment at 48 to 50°C has the potential to be developed as an approved standard procedure for the effective inactivation of insects in BSL-3 facilities.

Pressure Optimized PowEred Respirator (PROPER): A miniaturized wearable cleanroom and biosafety system for aerially transmitted viral infections such as COVID-19.

The supply of Personal Protective Equipment (PPE) in hospitals to keep the Health Care Professionals (HCP) safe taking care of patients may be limited, especially during the outbreak of a new disease. In particular, the face and body protective equipment is critical to prevent the wearer from exposure to pathogenic biological airborne particulates. This situation has been now observed worldwide during the onset of the COVID-19 pandemic. As concern over shortages of PPE at hospitals grows, we share with the public and makers' community the Pressure Optimized PowEred Respirator (PROPER) equipment, made out of COTS components. It is functionally equivalent to a Powered Air Purifying Respirator (PAPR). PROPER, a hood-based system which uses open source and easily accessible components is low-cost, relatively passive in terms of energy consumption and mechanisms, and easy and fast to 3D print, build and assemble. We have adapted our experience on building clean room environments and qualifying the bioburden of space instruments to this solution, which is in essence a miniaturized, personal, wearable cleanroom. PROPER would be able to offer better protection than an N95 respirator mask, mainly because it is insensitive to seal fit and it shields the eyes as well. The PROPER SMS fabric is designed for single-use and not intended for reuse, as they may start to tear and fail but the rest of the parts can be disinfected and reused. We provide a set of guidelines to build a low-cost 3D printed solution for an effective PAPR system and describe the procedures to validate it to comply with the biosafety level 3 requirements. We have validated the prototype of PROPER unit for air flow, ISO class cleanliness level, oxygen and carbon-dioxide gas concentrations during exhalation, and present here these results for illustration. We demonstrate that the area inside the hood is more than 200 times cleaner than the external ambient without the operator and more than 175 times with the operator and in an aerosol exposed environment. We also include the procedure to clean and disinfect the equipment for reuse. PROPER may be a useful addition to provide protection to HCPs against the SARS-CoV-2 virus or other potential future viral diseases that are transmitted aerially.

Validated Methods for Removing Select Agent Samples from Biosafety Level 3 Laboratories.

The Federal Select Agent Program dictates that all research entities in the United States must rigorously assess laboratory protocols to sterilize samples being removed from containment areas. We validated procedures using sterile filtration and methanol to remove the following select agents: Francisella tularensis, Burkholderia pseudomallei, B. mallei, Yersinia pestis, and Bacillus anthracis. We validated methanol treatment for B. pseudomallei. These validations reaffirm safety protocols that enable researchers to keep samples sufficiently intact when samples are transferred between laboratories.