Quantifying Viable M. tuberculosis Safely Obviating the Need for High Containment Facilities.

Mycobacterium tuberculosis is an infectious pathogen that requires biosafety level-3 laboratory for handling. The risk of transmission is high to laboratory staff, and to manage the organism safely, it is necessary to construct high containment laboratory facilities at great expense. This limits the application of tuberculosis diagnostics to areas where there is insufficient capital to invest in laboratory infrastructure. In this method, we describe a process of inactivating sputum samples by either heat or guanidine thiocyanate (GTC) that renders them safe without affecting the quantification of viable bacteria. This method eliminates the need for level 3 containment laboratory for the tuberculosis molecular bacterial load assay (TB-MBLA) and is applicable in low- and middle-income countries.

Spatial distribution of bioaerosols and evaluation of four ventilation method on controlling their diffusion in a typical enhanced biosafety level 2 laboratory.

Biosafety laboratories are critical in many fields. However, experimenters associated the infection risk from biological aerosols. In this study, by conducting experiments on the release and collection of bioaerosols within a typical BSL-2 + laboratory, the spatial distribution of bioaerosols was tracked. Numerical calculations were employed to obtain and visualize the airflow patterns and aerosol dispersion paths of four ventilation methods. The results indicated that equipment and tables led to uneven airflow distribution within the laboratory. The comparison results of the four evaluation indicators showed that the air age distribution of UU (Upward supply and upward return) mode and CD (Cross-supply and downward return) mode was superior, with air change efficiency values of 0.595 and 0.603, respectively. Additionally, the contaminant removal index of CD mode was 1.48, significantly higher than the other ventilation methods. The statistical results of the contaminant dispersion index also indicated that CD mode was most conducive to diluting aerosols in the spatial environment. The LD (lateral supply and downward return) mode may lead to airflow short-circuiting. The UD (upward supply and downward return) mode can provide balanced protection for laboratory. Overall, CD mode performed the best among the four ventilation methods, followed by UU mode.

Collection and transportation of SARS-CoV-2 and influenza A virus diagnostic samples: Optimizing the usage of guanidine-based chaotropic salts for enhanced biosafety and viral genome preservation.

Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.

Design and Application of Biosafe Coronavirus Engineering Systems without Virulence.

In the last twenty years, three deadly zoonotic coronaviruses (CoVs)-namely, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2-have emerged. They are considered highly pathogenic for humans, particularly SARS-CoV-2, which caused the 2019 CoV disease pandemic (COVID-19), endangering the lives and health of people globally and causing unpredictable economic losses. Experiments on wild-type viruses require biosafety level 3 or 4 laboratories (BSL-3 or BSL-4), which significantly hinders basic virological research. Therefore, the development of various biosafe CoV systems without virulence is urgently needed to meet the requirements of different research fields, such as antiviral and vaccine evaluation. This review aimed to comprehensively summarize the biosafety of CoV engineering systems. These systems combine virological foundations with synthetic genomics techniques, enabling the development of efficient tools for attenuated or non-virulent vaccines, the screening of antiviral drugs, and the investigation of the pathogenic mechanisms of novel microorganisms.

To probe outbreak, BSL-3 labs plan to infect cows with flu virus.

Novel effort comes as study finds key receptor for avian flu virus in udders, where the virus flourishes.

A novel BSL-2 Lassa virus reverse genetics system modelling the complete viral life cycle.

Lassa virus (LASV), a risk-group 4 pathogen, must be handled in biosafety level-4 (BSL-4) conditions, thereby limiting its research and antiviral development. Here, we developed a novel LASV reverse genetics system which, to our knowledge, is the first to study the complete LASV life cycle under BSL-2 conditions. Viral particles can be produced efficiently when LASV minigenomic RNA harbouring minimal viral cis-elements and reporter genes is transfected into a helper cell line stably expressing viral NP, GP, Z and L proteins. The resulting defective virions, named LASVmg, can propagate only in the helper cell line, providing a BSL-2 model to study the complete LASV life cycle. Using this model, we found that a previously reported cellular receptor α-dystroglycan is dispensable for LASVmg infection. Furthermore, we showed that ribavirin can inhibit LASVmg infection by inducing viral mutations. This new BSL-2 system should facilitate studying the LASV life cycle and screening antivirals.

Cytometry in High-Containment Laboratories.

The emergence of new pathogens continues to fuel the need for advanced high-containment laboratories across the globe. Here we explore challenges and opportunities for integration of cytometry, a central technology for cell analysis, within high-containment laboratories. We review current applications in infectious disease, vaccine research, and biosafety. Considerations specific to cytometry within high-containment laboratories, such as biosafety requirements, and sample containment strategies are also addressed. We further tour the landscape of emerging technologies, including combination of cytometry with other omics, the application of automation, and artificial intelligence. Finally, we propose a framework to fast track the immersion of advanced technologies into the high-containment research setting to improve global preparedness for new emerging diseases.

Ceramic biomaterials in dentistry: chemical structure and biosafety - a review and a bibliometric visual mapping on Scopus database.

OBJECTIVE: A biomaterial is any non-pharmaceutical substance or a mixture of synthetic or natural substances used independently or as part of a system for any amount of time with the aim of mending, supporting the growth, or replacing tissues, organs, or functions of the body. It is a non-renewable material that interacts with biological systems. The purpose of this study was to assess the advances in ceramic biomaterials and perform a bibliometric mapping of the literature on the subject. MATERIALS AND METHODS: The Scopus database was used for manuscript screening (Elsevier, Amsterdam, The Netherlands). The effect of the scientific production has been assessed using scientometric citational metrics. RESULTS: A total of 2,554 pieces of literature, including 2,234 papers, 170 conference proceedings, 109 reviews, 35 book chapters, 3 editorial letters, and 3 short surveys, were retrieved. Based on the research conducted, it is noted that ceramic materials are high-performing by being porous or glassy and can, therefore, serve as fillers, covering materials, and scaffolds in medicine and biotechnology. They are frequently employed not only in orthopedic and maxillofacial surgery but also in dentistry for dental prostheses. CONCLUSIONS: Materials monitoring methods enable us to track the three-dimensional evolution of ceramics' volume, as well as flaws or micro-cracks.

Biosafety and chemical solubility studies of multiscale crystal-reinforced lithium disilicate glass-ceramics.

Lithium disilicate (Li2 Si2 O5 ) glass-ceramics are currently a more widely used all-ceramic restorative material due to their good mechanical properties and excellent aesthetic properties. However, they have a series of problems such as high brittleness and low fracture toughness, which has become the main bottleneck restricting its development. Therefore, in order to compensate for these shortcomings, we propose to prepare a reinforced glass-ceramics with better mechanical properties and to test the biosafety and chemical solubility of the material. Li2 Si2 O5 whiskers were synthesized by a one-step hydrothermal method, and multi-scale crystal-enhanced Li2 Si2 O5 glass-ceramics were prepared by reaction sintering. The biosafety of multi-scale crystal-reinforced Li2 Si2 O5 glass-ceramics was investigated by in vitro cytotoxicity test, rabbit pyrogen test, mice bone marrow micronucleus test, skin sensitization test, sub-chronic systemic toxicity test, and chronic systemic toxicity test. Additionally, the chemical solubility of multi-scale crystal-reinforced Li2 Si2 O5 glass-ceramics was investigated. The test results showed that the material was non-cytotoxic, non-thermogenic, non-mutagenic, non-sensitizing, and non-systemic. The chemical solubility, determined to be 377 ± 245 µg/cm2 , complied with the ISO 6872 standard for the maximum solubility of ceramic materials. Multi-scale crystal-reinforced Li2 Si2 O5 glass-ceramics' biosafety and chemical solubility met current normative criteria, and they can move on to mechanical property measurements (such as flexural strength test, fatigue life test, friction and wear property study, etc.) and bonding property optimization, which shows promise for future clinical applications.

Biosafety and regulatory issues of RNA therapeutics.

RNA therapy has recently emerged as a therapy targeting specific genes or proteins. With its outstanding advantages, this therapy has opened promising doors for treating and preventing diseases. The great application potential has driven the need for a comprehensive understanding of these therapies, particularly on biosafety and regulatory issues. This chapter began by discussing the risks to RNA therapy, such as off-target effects, immunogenicity and immune responses, and long-term effects. Since then, this therapy's intricate landscape of biosafety issues has been elucidated. Common biosecurity measures applied around the world have also been reviewed. In addition, this chapter emphasized the importance of regulations and laws in applying RNA therapy to prevent and treat human and animal diseases. At the same time, the current legal regulations in the world for RNA therapies have also been thoroughly discussed. To sum up, this chapter has provided a comprehensive perspective on biosafety and regulatory issues for developing RNA therapies. Understanding the biosafety and regulatory issues in RNA therapy can help researchers use this promising new technology safely and effectively in the future.

Antimicrobial stewardship hindered by inadequate biosecurity and biosafety practices, and inappropriate antibiotics usage in poultry farms of Nepal-A pilot study.

Nepal's poultry industry has experienced remarkable growth in the last decade, but farm biosafety and biosecurity measures are often overlooked by farmers. As a result, farms often suffer from sporadic and regular outbreaks of many diseases, impacting production and creating public health challenges. Poor management practices, including overuse of antibiotics for prophylaxis and therapeutics, can enhance the spread of poultry diseases by propagating antimicrobial resistance (AMR) that is threatening poultry and human health. We assessed biosafety, biosecurity risks and AMR stewardship in sixteen poultry farms located in four districts: Ramechhap, Nuwakot, Sindhupalchowk, and Kavre. Risk assessment and AMR stewardship evaluation questionnaires were administered to formulate biosafety and biosecurity compliance matrix (BBCM). Risk assessment checklist assessed facility operations, personnel and standard operating procedures, water supply, cleaning and maintenance, rodent/pest control and record keeping. Oral and cloacal samples from the poultry were collected, pooled, and screened for eight poultry pathogens using Polymerase Chain Reaction (PCR) tests. Based on BBCM, we identified the highest BBCM score of 67% obtained by Sindhupalchowk farm 4 and the lowest of 12% by Kavre farm 3. Most of the farms (61.6%) followed general poultry farming practices, only half had clean and well-maintained farms. Lowest scores were obtained for personnel safety standard (42.4%) and rodent control (3.1%). At least one of the screened pathogens were detected in all farms. Mycoplasma gallisepticum was the most common pathogen detected in all but three farms, followed by Mycoplasma synoviae. More than half of the farmers considered AMR a threat, over 26% of them used antibiotics as a preventive measure and 81% did not consider withdrawal period for antibiotics prior to processing of their meat products. Additionally, antibiotics classified as "Watch" and "Restrict" by the WHO were frequently used by the farmers to treat bacterial infections in their farms.

Biosafety consideration of nanocellulose in biomedical applications: A review.

Nanocellulose-based biomaterials have gained significant attention in various fields, especially in medical and pharmaceutical areas, due to their unique properties, including non-toxicity, high specific surface area, biodegradability, biocompatibility, and abundant feasible and sophisticated strategies for functional modification. The biosafety of nanocellulose itself is a prerequisite to ensure the safe and effective application of biomaterials as they interact with living cells, tissues, and organs at the nanoscale. Potential residual endogenous impurities and exogenous contaminants could lead to the failure of the intended functionalities or even serious health complications if they are not adequately removed and assessed before use. This review summarizes the sources of impurities in nanocellulose that may pose potential hazards to their biosafety, including endogenous impurities that co-exist in the cellulosic raw materials themselves and exogenous contaminants caused by external exposure. Strategies to reduce or completely remove these impurities are outlined and classified as chemical, physical, biological, and combined methods. Additionally, key points that require careful consideration in the interpretation of the biosafety evaluation outcomes were discussed to ensure the safety and effectiveness of the nanocellulose-based biomaterials in medical applications.

Chapter 10: Planetary Protection-History, Science, and the Future.

Planetary protection is a principle in the design of interplanetary missions that aims to prevent biological cross contamination between the target body and Earth. Planetary protection policies and procedures have worked to mitigate forward contamination (from Earth) and back contamination (to Earth) since the beginning of the space age. Today, planetary protection policy is guided by international agreements, nongovernmental advisory councils, and national space agencies. The landscape of planetary protection science and policy is changing rapidly, as new technologies, crewed missions to Mars and the Moon, and even orbital settlements are being developed. Space exploration, whether specifically targeted toward questions in astrobiology or not, must consider planetary protection concerns to minimize contamination that poses a risk to both astrobiological investigations as well as Earth's biosphere. In this chapter, we provide an introduction to and overview of the history, motivations, and implementation of planetary protection in the United States.

WeChat mini program in laboratory biosafety education among medical students at Guangzhou Medical University: a mixed method study of feasibility and usability.

BACKGROUND: Laboratory biosafety should be a priority in all healthcare institutions. In traditional laboratory safety teaching students typically receive knowledge passively from their teachers without active involvement. The combination of experiential learning and mobile learning may provide students with greater engagement, retention, and application of knowledge. To address this issue, we developed and conducted a convergent mixed methods study to assess the feasibility and usability of a WeChat mini program (WMP) named WeMed for laboratory biosafety education for medical laboratory students at Guangzhou Medical University (GMU). METHODS: The study was conducted between November 2022 and October 2023 among second-year undergraduate students at GMU. It involved the concurrent collection, analysis, and interpretation of both qualitative and quantitative data to assess feasibility and usability. In the quantitative strand, two evaluations were conducted via online surveys from students (n = 67) after a four-week study period. The System Usability Scale (SUS) was used to evaluate usability, while self-developed questions were used to assess feasibility. Additionally, a knowledge test was administered 6 months after the program completion. In the qualitative strand, fourteen semi-structured interviews were conducted, whereby a reflexive thematic analysis was utilized to analyze the interview data. RESULTS: The overall SUS score is adequate (M = 68.17, SD = 14.39). The acceptability of the WeMed program is in the marginal high range. Most students agreed that WeMed was useful for learning biosafety knowledge and skills (13/14, 93%), while 79% (11/14) agreed it was easy to use and they intended to continue using it. After 6 months, a significant difference in the knowledge test scores was observed between the WeMed group (n = 67; 2nd year students) and the traditional training group (n = 90; 3rd year students). However, the results should be interpreted cautiously due to the absence of a pretest. CONCLUSION: The combination of experiential learning and mobile learning with WMP is a feasible tool for providing laboratory biosafety knowledge and skills. Ongoing improvements should be made in order to increase long-term acceptance.

Planetary Protection Knowledge Gap Closure Enabling Crewed Missions to Mars.

As focus for exploration of Mars transitions from current robotic explorers to development of crewed missions, it remains important to protect the integrity of scientific investigations at Mars, as well as protect the Earth's biosphere from any potential harmful effects from returned martian material. This is the discipline of planetary protection, and the Committee on Space Research (COSPAR) maintains the consensus international policy and guidelines on how this is implemented. Based on National Aeronautics and Space Administration (NASA) and European Space Agency (ESA) studies that began in 2001, COSPAR adopted principles and guidelines for human missions to Mars in 2008. At that point, it was clear that to move from those qualitative provisions, a great deal of work and interaction with spacecraft designers would be necessary to generate meaningful quantitative recommendations that could embody the intent of the Outer Space Treaty (Article IX) in the design of such missions. Beginning in 2016, COSPAR then sponsored a multiyear interdisciplinary meeting series to address planetary protection "knowledge gaps" (KGs) with the intent of adapting and extending the current robotic mission-focused Planetary Protection Policy to support the design and implementation of crewed and hybrid exploration missions. This article describes the outcome of the interdisciplinary COSPAR meeting series, to describe and address these KGs, as well as identify potential paths to gap closure. It includes the background scientific basis for each topic area and knowledge updates since the meeting series ended. In particular, credible solutions for KG closure are described for the three topic areas of (1) microbial monitoring of spacecraft and crew health; (2) natural transport (and survival) of terrestrial microbial contamination at Mars, and (3) the technology and operation of spacecraft systems for contamination control. The article includes a KG data table on these topic areas, which is intended to be a point of departure for making future progress in developing an end-to-end planetary protection requirements implementation solution for a crewed mission to Mars. Overall, the workshop series has provided evidence of the feasibility of planetary protection implementation for a crewed Mars mission, given (1) the establishment of needed zoning, emission, transport, and survival parameters for terrestrial biological contamination and (2) the creation of an accepted risk-based compliance approach for adoption by spacefaring actors including national space agencies and commercial/nongovernment organizations.

Cas9-assisted biological containment of a genetically engineered human commensal bacterium and genetic elements.

Sophisticated gene circuits built by synthetic biology can enable bacteria to sense their environment and respond predictably. Engineered biosensing bacteria outfitted with such circuits can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. To provide robust biocontainment for engineered bacteria, we devised a Cas9-assisted auxotrophic biocontainment system combining thymidine auxotrophy, an Engineered Riboregulator (ER) for controlled gene expression, and a CRISPR Device (CD). The CD prevents the engineered bacteria from acquiring thyA via horizontal gene transfer, which would disrupt the biocontainment system, and inhibits the spread of genetic elements by killing bacteria harboring the gene cassette. This system tunably controlled gene expression in the human gut commensal bacterium Bacteroides thetaiotaomicron, prevented escape from thymidine auxotrophy, and blocked transgene dissemination. These capabilities were validated in vitro and in vivo. This biocontainment system exemplifies a powerful strategy for bringing genetically engineered microorganisms safely into biomedicine.

Acetic Acid Enables Molecular Enumeration of Mycobacterium tuberculosis from Sputum and Eliminates the Need for a Biosafety Level 3 Laboratory.

BACKGROUND: Improved monitoring of Mycobacterium tuberculosis response to treatment is urgently required. We previously developed the molecular bacterial load assay (MBLA), but it is challenging to integrate into the clinical diagnostic laboratory due to a labor-intensive protocol required at biosafety level 3 (BSL-3). A modified assay was needed. METHODS: The rapid enumeration and diagnostic for tuberculosis (READ-TB) assay was developed. Acetic acid was tested and compared to 4 M guanidine thiocyanate to be simultaneously bactericidal and preserve mycobacterial RNA. The extraction was based on silica column technology and incorporated low-cost reagents: 3 M sodium acetate and ethanol for the RNA extraction to replace phenol-chloroform. READ-TB was fully validated and compared directly to the MBLA using sputa collected from individuals with tuberculosis. RESULTS: Acetic acid was bactericidal to M. tuberculosis with no significant loss in 16S rRNA or an unprotected mRNA fragment when sputum was stored in acetic acid at 25°C for 2 weeks or -20°C for 1 year. This novel use of acetic acid allows processing of sputum for READ-TB at biosafety level 2 (BSL-2) on sample receipt. READ-TB is semiautomated and rapid. READ-TB correlated with the MBLA when 85 human sputum samples were directly compared (R2 = 0.74). CONCLUSIONS: READ-TB is an improved version of the MBLA and is available to be adopted by clinical microbiology laboratories as a tool for tuberculosis treatment monitoring. READ-TB will have a particular impact in low- and middle-income countries (LMICs) for laboratories with no BSL-3 laboratory and for clinical trials testing new combinations of anti-tuberculosis drugs.

Gaps and inconsistencies in the current knowledge and implementation of biosafety and biosecurity practices for rickettsial pathogens.

INTRODUCTION: Rickettsia spp. and Orientia spp. are the causes of neglected infections that can lead to severe febrile and systemic illnesses in humans. Implementing proper biosafety practices when handling these pathogens is crucial to ensure a safe and sustainable work environment. It is essential to assess the current knowledge and identify any potential gaps to develop effective measures that minimise the risk of exposure to these pathogens. By doing so, we can establish a comprehensive framework that promotes safety, mitigates hazards, and safeguards the well-being of personnel and the surrounding community. METHODS AND RESULTS: This review aimed to synthesise and determine the evidence base for biosafety precautions for Rickettsia spp. and Orientia spp. pathogens. Enhancing our understanding of the relative infectious risk associated with different strains of Rickettsia and Orientia spp. requires identifying the infectious dose of these pathogens that can cause human disease. The application of risk groups for Rickettsia and Orientia spp. is inconsistent across jurisdictions. There is also incomplete evidence regarding decontamination methods for these pathogens. With regards to Orientia spp. most of the available information is derived from experiments conducted with Rickettsia spp. CONCLUSIONS: Rickettsia and Orientia spp. are neglected diseases, as demonstrated by the lack of evidence-based and specific biosafety information about these pathogens. In the case of Orientia spp., most of the available information is derived from Rickettsia spp., which may not be appropriate and overstate the risks of working with this pathogen. The advent of effective antibiotic therapy and a better understanding of the true hazards and risks associated with pathogen manipulation should inform decisions, allowing a sustainable and safe work environment.