Microbiological Safety Cabinets Research Articles

Heat inactivation of clinical COVID-19 samples on an industrial scale for low risk and efficient high-throughput qRT-PCR diagnostic testing

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as ‘direct to PCR’ assays that do not involve RNA extraction or chemical neutralisation methods.

Dry-fog decontamination of microbiological safety cabinets after activities with SARS-CoV-2: cycle development and process validation for dry fogging with peroxyacetic acid.

Background: Technical protection measures for laboratory activities involving biological agents include biological safety cabinets (BSC) that may be contaminated. In the case of diagnostic activities with SARS-CoV-2, this may also affect BSC that are operated at protection level 2; therefore, decontamination of all contaminated surfaces of the BSC may be required. In addition to fumigation with hydrogen peroxide (H2O2), dry fogging of H2O2-stabilized peroxyacetic acid (PAA) represents another alternative to fumigation with formalin. However, to prove their efficacy, these alternatives need to be validated for each model of BSC.Methods: The validation study was performed on 4 different BSCs of Class II A2 using the “Mini Dry Fog” system.Results: An aerosol concentration of 0.03% PAA and 0.15% H2O2 during a 30 min exposure was sufficient to inactivate SARS-CoV-2. Effective concentrations of 1.0% PAA and 5% H2O2 were required to decontaminate the custom-prepared biological indicators loaded with spores of G. stearothermophilus and deployed at 9 different positions in the BSC. Commercial spore carriers were easier to inactivate by a factor of 4, which corresponded to a reduction of 106 in all localizations.Conclusions: Dry fogging with PAA is an inexpensive, robust, and highly effective decontamination method for BSCs for enveloped viruses such as SARS-CoV-2. The good material compatibility, lack of a requirement for neutralization, low pH – which increases the range of efficacy compared to H2O2 fumigation – the significantly shorter processing time, and the lower costs argue in favor of this method.

The Impact of Air Inflow and Interfering Factors on the Performance of Microbiological Safety Cabinets.

Introduction:The operator protection factor (OPF) of four biological safety cabinets (BSCs) has been measured under standard and suboptimal conditions.Methods:The OPF for one BSC1, two BSC2, and an acid-fast bacilli staining station (AFBSS) was measured using the potassium iodide method for in situ testing of BSCs (CEN12469) over a range of inflow velocities under standard conditions and with common interfering factors (fans, opening doors, and walk pasts).Results:The BSC1 and the AFBSS gave a high level of protection under standard test conditions at all airflows (down to 0.3 and 0.38 m/s, respectively). During interfering processes, the BSC1 and AFBSS gave a high level of protection (OPF >105) at the specified inward airflow. At lower airflows, there was a predictable deterioration in performance. There was a significant difference in performance between the two BSC2s tested, with one model passing all tests under all interfering conditions at all airflows. The second BSC2 failed the standard test at the lowest airflow and provided poor levels of protection (OPF <105) in all tests carried out with interfering processes.Conclusion:Although BSC2s are capable of giving a high level of performance, this is design dependent and the BSC1 and AFBSS give a more predictable level of performance due to their simpler design. In environments where BSC certification is not possible, they may provide more robust and sustainable primary containment.

Comparing the Efficacy of Formaldehyde with Hydrogen Peroxide Fumigation on Infectious Bronchitis Virus.

Background:The recent reclassification of formaldehyde as a presumed carcinogen prompted the investigation into the comparative efficacy of hydrogen peroxide as a fumigant in microbiological safety cabinets.Introduction:The aim of the study was to quantify the biocidal efficacy of formaldehyde fumigation, including variables such as exposure time and concentration, and then to compare this to the biocidal efficacy achieved from a hydrogen peroxide vapor fumigation system. The study also investigated the ability of both fumigants to permeate the microbiological safety cabinet (MBSC), including the workspace, under the work tray, and after the HEPA filters. Furthermore, the effect of organic soiling on efficacy was also assessed. Infectious bronchitis virus (IBV) was used as the biological target to develop this study model.Methods:A model using IBV was developed to determine the efficacy of formaldehyde and hydrogen peroxide as fumigants. Virus was dried on stainless steel discs, and variables including concentration, time, protein soiling, and location within an MBSC were assessed.Results:It was demonstrated that formaldehyde fumigation could achieve a 6-log reduction in the titer of the virus throughout the cabinet, and high protein soiling in the presentation did not affect efficacy. Appropriate cycle parameters for the hydrogen peroxide system were developed, and when challenged with IBV, it was shown that vaporized hydrogen peroxide could achieve an equal 6-log titer reduction as formaldehyde within the cabinet workspace and overcome the presence of soiling.Conclusion:Hydrogen peroxide was demonstrated to be a viable alternative to formaldehyde under most situations tested. However, the hydrogen peroxide system did not achieve an equal titer reduction above the cabinet’s first HEPA filter using the cabinet workspace cycle, and further optimization of the hydrogen peroxide cycle parameters, including pulsing of the cabinet fans, may be required to achieve this.

Peculiarities of Disinfection of Microbiological Safety Cabinets and Premises of Virological Laboratories when Carrying out Works with the Smallpox Virus

Objective. Applying microbiological method to determine the effectiveness of disinfection treatments with formaldehyde fumigation of the hidden cavities and the reverse side of the microbiological safety cabinet filters (MSC) located in the “infectious” areas of an isolated to maximum extent laboratory. Evaluation of the effectiveness of experimental unit for automation of the formaldehyde treatment of laboratory premises. Materials and methods. Assessment of effectiveness of disinfection treatments of MSCs and the premises of virological laboratory was carried out using cambric test objects contaminated with the spore-forming test culture of B. thuringiensis. Results and discussion. The disinfection time sufficient for the complete inactivation of particularly dangerous pathogenic microorganisms potentially located in hidden cavities and on the back side of microbiological safety cabinet filters, placed in the “infectious” zone of the most isolated laboratory has been determined experimentally. As a result of the studies, it was possible to achieve complete inactivation of the spore-forming test microorganism located on test objects situated in hidden cavities and hardly accessible places of MSC at concentration of 1·106 cells/cm3 only with disinfection time equal to 24 hours. Effective disinfection of test objects contaminated with B. thuringiensis spore-forming test culture, placed in hard-toreach places and on the back of the equipment in a boxed virological room, using an experimental plant producing gaseous formaldehyde, was possible only when formalin was used in a concentration of 40 ml/m3 of the room. It was established that the amount of formalin required to achieve 100% efficiency of the disinfection treatment of the virological boxed room using an experimental unit producing gaseous formaldehyde depends on the operating mode of the general supply and exhaust ventilation, the configuration of the laboratory room, and the furnishing with laboratory and engineering equipment.

Regulatory Framework for Biological Safety Provision When Working with Microorganisms of the III–IV Pathogenicity Groups

Objective of the study was to review the current normative-methodological documentation on the matters of safe handling of pathogenic biological agents (PBA) of the III–IV pathogenicity groups. Materials and methods. We used analytical method for the study. We carried out comparative analysis of the provisions contained in the current regulations approved at different times for different agencies on the matters of air filtering system equipping, usage of microbiological safety cabinets, ensuring hand washing station provision for laboratories, inspection of disinfectants entering a lab, and waste decontamination procedures. Results and discussion. We have considered the provisions of the current documentation that require further refinement and clarification for the future editions of sanitary regulations. Two aspects draw increasing attention: the problem of regulating the inlet and exhaust ventilation system setup in the laboratories of microbiological specialization for work with PBA and the need to devise a consistent coordinated regulatory framework. Examples provided in the paper demonstrate the necessity of adhering to the integrated unified approach to the requirements for biosafety provision in the documents of various agencies; clear wording; harmonized system of normative and technical specifications for laboratory equipment, stating the basic characteristics of microbiological safety cabinets and ventilation systems. The documents should be in compliance with the requirements of various agencies. The maintenance of technical-engineering systems should be performed by the specialists with an appropriate qualification on a regular basis.

Biological Validation and Observations of Formaldehyde Fumigation in Operational and Representative Scenarios in High-Containment Laboratories.

The effectiveness of formaldehyde as a fumigant in laboratories, for equipment, and for containment barrier decontamination applications was assessed, in particular the ability to reproduce biological inactivation (6-log reduction) of commercially available rapid biological indicators in representative operational scenarios and their relative sensitivity to other biological and chemical indicators. The primary objective of this study was to describe observations and results of formaldehyde fumigation efficacy in high-containment laboratories. Biological indicators placed throughout laboratory spaces, including ventilation ductwork at distances up to 15 meters, inside pieces of equipment and in lengths of pipe, were mostly negative, demonstrating the ability of formaldehyde to reach the interior and external surfaces tested. Dwell times as short as 10 minutes were shown to be sufficient in barrier decontamination equipment for the fumigation of personal computers. Furthermore, a pipework bore:length ratio of 1:1500 was proven too great a challenge. Indicators placed after extracting HEPA filters in microbiological safety cabinets (MBSCs) were also successfully fumigated (at room temperature) relying solely on diffusion and in the cabinet workspace at 10°C to 12°C. In addition, pressures of up to 900 Pa were experienced in low-leakage laboratory spaces during fumigation. Formaldehyde fumigation was shown to be effective in a variety of scenarios representing operational activities thereby giving process assurance.

A CD34+ Cell Enrichment Protocol of Hematopoietic Stem Cells in a Well-Established Quality Management System

Allogeneic stem cell transplantation applications have improved tremendously over the past quarter of a century. The use of new immunosuppressive protocols and elimination of T cells by CD34+ cell enrichment or T cell depletion on apheresis products increases the chance of using partially matched or haploidentical grafts. This is without increasing the risk of graft-versus-host disease, which is observed as a major complication of hematopoietic stem cell transplantation. The aim of this protocol is to evaluate the results obtained from 6 different process cycles performed on 6 different days. We used the CliniMACS Plus system located in our Cell and Tissue Manufacturing Center Quality Control Unit which is already calibrated as a class D room and includes a class A microbiological safety cabinet inside. The average purity of the end products was 95.66%, excluding only one end product which was 70%; this was higher than the values in current studies in the field. Superior to the reported studies, the CD3 quantity in each end product was below the dedicated thresholds. Bactec^TM FX40 blood culture system test results were detected as negative for each end product. Endotoxin testing suggested the absence of endotoxin within the products. The consistent outcomes obtained from these 6 different process cycles confirmed that the CliniMACS® Plus process cycles performed in accordance with our well-defined quality management system procedure is sufficient for the routine application of high-quality and safe CD34+ enrichment processes within our clean room area.

Use and reliability of multiplex bead-based assays (Luminex) at Containment Level 4.

In the UK, research on hazard group 4 (HG4) pathogens requires specialised Containment Level 4 (CL4) facilities. These differ from Biosafety Level 4 (BSL4) conditions in that work is conducted in class III microbiological safety cabinets for primary containment instead of using positive pressure suits. This presents unique challenges associated with the physical restrictions of working in a limited space, and prohibits the use of many techniques and specialist equipment. In consequence, detailed studies on the biology of HG4 pathogens and in particular their immunological relationships with the host are understudied in the UK; for example, the majority of immunological assays with which the immune system is interrogated require specialist equipment that is unsuitable for CL4. Multiplexing to simultaneously measure multiple analytes is increasingly being used in immunological studies. This assay is attractive for CL4 work because it reduces the time spent in the laboratory whilst maximising the use of valuable sample volume. The Luminex microsphere approach allows for the determination of many cytokines and chemokines, however, the detection system uses fixed aligned lasers and integrated computer systems which are unsuitable for use at CL4. Therefore, we have developed an approach in which the Luminex assay is conducted within the CL4 laboratory and a formalin-fixation stage is introduced to allow for analysis to be undertaken outside of containment. Quality control preparations allow the assay characteristics to be monitored and analysis of assay performance to be evaluated. Our data demonstrate that Luminex is an applicable tool for use at CL4 and that assays can be run reliably to generate reproducible standardised data across different plates and individual experiments.

Survey of Safety Practices among Hospital Laboratories in Oromia Regional State, Ethiopia

Unsafe working practices, working environments, disposable waste products, and chemicals in clinical laboratories contribute to infectious and non-infectious hazards. Staffs, the community, and patients are less safe. Furthermore, such practices compromise the quality of laboratory services. We conducted a study to describe safety practices in public hospital laboratories of Oromia Regional State, Ethiopia. Randomly selected ten public hospital laboratories in Oromia Regional State were studied from Oct 2011- Feb 2012. Self-administered structured questionnaire and observation checklists were used for data collection. The respondents were heads of the laboratories, senior technicians, and safety officers. The questionnaire addressed biosafety label, microbial hazards, chemical hazards, physical/mechanical hazards, personal protective equipment, first aid kits and waste disposal system. The data was analyzed using descriptive analysis with SPSS version16 statistical software. All of the respondents reported none of the hospital laboratories were labeled with the appropriate safety label and safety symbols. These respondents also reported they may contain organisms grouped under risk group IV in the absence of microbiological safety cabinets. Overall, the respondents reported that there were poor safety regulations or standards in their laboratories. There were higher risks of microbial, chemical and physical/mechanical hazards. Laboratory safety in public hospitals of Oromia Regional State is below the standard. The laboratory workers are at high risk of combined physical, chemical and microbial hazards. Prompt recognition of the problem and immediate action is mandatory to ensure safe working environment in health laboratories.

Survey of Safety Practices among Hospital Laboratories in Oromia Regional State, Ethiopia

Background: Unsafe working practices, working environments, disposable waste products, and chemicals in clinical laboratories contribute to infectious and non-infectious hazards. Staffs, the community, and patients are less safe. Furthermore, such practices compromise the quality of laboratory services. We conducted a study to describe safety practices in public hospital laboratories of Oromia Regional State, Ethiopia.Method: Randomly selected ten public hospital laboratories in Oromia Regional State were studied from Oct 2011- Feb 2012. Self-administered structured questionnaire and observation checklists were used for data collection. The respondents were heads of the laboratories, senior technicians, and safety officers. The questionnaire addressed biosafety label, microbial hazards, chemical hazards, physical/mechanical hazards, personal protective equipment, first aid kits and waste disposal system. The data was analyzed using descriptive analysis with SPSS version16 statistical software.Result: All of the respondents reported none of the hospital laboratories were labeled with the appropriate safety label and safety symbols. These respondents also reported they may contain organisms grouped under risk group IV in the absence of microbiological safety cabinets. Overall, the respondents reported that there were poor safety regulations or standards in their laboratories. There were higher risks of microbial, chemical and physical/mechanical hazards.Conclusion: Laboratory safety in public hospitals of Oromia Regional State is below the standard. The laboratory workers are at high risk of combined physical, chemical and microbial hazards. Prompt recognition of the problem and immediate action is mandatory to ensure safe working environment in health laboratories.Keywords: laboratory safety, district hospital, Oromia Region

Assessing the Containment Efficiency of a Microbiological Safety Cabinet During the Simultaneous Generation of a Nanoaerosol and a Tracer Gas

The intention of this article is to compare the containment performance of a Type II microbiological safety cabinet (MSC) confronted with the simultaneous generation of a saline nanoparticle aerosol and a tracer gas (SF(6)). The back dissemination coefficient, defined as the ratio of the pollutant concentration measured outside the enclosure to the pollutant flow rate emitted inside the enclosure, is calculated in order to quantify the level of protection of each airborne contaminant tested for three enclosure operating configurations: an initial configuration (without perturbations), a configuration exposing a dummy in front of the enclosure (simulation of an operator), and a configuration employing the movement of a plate in front of the enclosure (simulation of human movement). Based on the results of this study, we observed that nanoparticulate and gaseous behaviours are strongly correlated, thus showing the predominance of air-driven transport over particle-specific behaviour. The average level of protection afforded by the MSC was found systematically slightly higher for the nanoaerosol than for the gas in the studied configurations (emission properties of the source, operating conditions, and measurement protocols). This improved protection efficiency, however, cannot be considered as a warrant of protection for operators since operating condition and ventilation parameters are still more influential on the containment than the pollutant nature (i.e. nanoaerosol or gas).

Evaluation of the Operator Protection Factors Offered by Positive Pressure Air Suits against Airborne Microbiological Challenge

Laboratories throughout the world that perform work with Risk Group 4 Pathogens generally adopt one of two approaches within BSL-4 environments: either the use of positive pressure air-fed suits or using Class III microbiological safety cabinets and isolators for animal work. Within the UK at present, all laboratories working with Risk Group 4 agents adopt the use of Class III microbiological safety cabinet lines and isolators. Operator protection factors for the use of microbiological safety cabinets and isolators are available however; there is limited published data on the operator protection factors afforded by the use of positive pressure suits. This study evaluated the operator protection factors provided by positive pressure air suits against a realistic airborne microbiological challenge. The suits were tested, both intact and with their integrity compromised, on an animated mannequin within a stainless steel exposure chamber. The suits gave operator protection in all tests with an intact suit and with a cut in the leg. When compromised by a cut in the glove, a very small ingress of the challenge was seen as far as the wrist. This is likely to be due to the low airflow in the gloves of the suit. In all cases no microbiological penetration of the respiratory tract was observed. These data provide evidence on which to base safety protocols for use of positive pressure suits within high containment laboratories.

Testing the performance of microbiological safety cabinets used in microbiology laboratories in South Korea

To test a performance of the microbiological safety cabinets (MSCs) according to the type of MSCs in microbial laboratories. Tests were carried out to assess the performance of 31 MSCs in 14 different facilities, including six different biological test laboratories in six hospitals and eight different laboratories in three universities. The following tests were performed on the MSCs: the downflow test, intake velocity test, high-efficiency particulate air filter leak test and the airflow smoke pattern test. These performance tests were carried out in accordance with the standard procedures. Only 23% of Class II A1 (8), A2 (19) and unknown MSCs (4) passed these performance tests. The main reasons for the failure of MSCs were inappropriate intake velocity (65%), leakage in the HEPA filter sealing (50%), unbalanced airflow smoke pattern in the cabinets (39%) and inappropriate downflow (27%). This study showed that routine checks of MSCs are important to detect and strengthen the weak spots that frequently develop, as observed during the evaluation of the MSCs of various institutions. Routine evaluation and maintenance of MSCs are critical for optimizing performance.

Non-Attenuation Of Highly Pathogenic Avian Influenza H5N1 By Laboratory Exposure To Ultraviolet Rays

Avian influenza H5N1 represents one of the most researched viruses in laboratories world-wide in recent times with regards to its epidemiology, ecology, biology and geography. The virus has caused 409 human cases and 256 human fatalities to date. Some laboratory activities and other lab related works predispose certain workers to exposure to this virus. In this work, we assessed the effect of exposure of HPAI infective allantoic fluid to ultraviolet rays for between 15 and 180 minutes. No significant difference was found between the unexposed and exposed viruses. The ability of the virus to haemagglutinate chicken red blood cells, the haemagglutination titre and its pathogenicity in embryonating eggs did not change despite this prolong exposure to UV-light. We call for caution in the handling of HPAI viruses in laboratory inside the microbiological safety cabinet despite sterilization using UV-light.

Multiplex cytokine profiling with highly pathogenic material: Use of formalin solution in luminex analysis

Work with highly pathogenic material mandates the use of biological containment facilities, involving microbiological safety cabinets and specialist laboratory engineering structures typified by containment level 3 (CL3) and CL4 laboratories. Consequences of working in high containment are the practical difficulties associated with containing specialist assays and equipment often essential for experimental analyses. In an era of increased interest in biodefence pathogens and emerging diseases, immunological analysis has developed rapidly alongside traditional techniques in virology and molecular biology. For example, in order to maximise the use of small sample volumes, multiplexing has become a more popular and widespread approach to quantify multiple analytes simultaneously, such as cytokines and chemokines. The luminex microsphere system allows for the detection of many cytokines and chemokines in a single sample, but the detection method of using aligned lasers and fluidics means that samples often have to be analysed in low containment facilities. In order to perform cytokine analysis in materials from high containment (CL3 and CL4 laboratories), we have developed an appropriate inactivation methodology after staining steps, which although results in a reduction of median fluorescent intensity, produces statistically comparable outcomes when judged against non-inactivated samples. This methodology thus extends the use of luminex technology for material that contains highly pathogenic biological agents.

Performance of open-fronted microbiological safety cabinets: the value of operator protection tests during routine servicing.

The performance of class I and II microbiological safety cabinets over 7 years, employed in a force-ventilated containment level 3 (CL-3) laboratory, is described. Operator Protection (OP) provided by the cabinets, assessed by still and latterly limited 'in-use' KI-Discus tests, showed no overall deterioration during the review period. Comparisons show that a selected class II unit, but not a second, and a new class II MSC in a recently commissioned, similar CL-3 facility, provide the same order of OP as a class I cabinet. From the experiences described, it is strongly recommended that OP tests (OPTs) should be part of the routine servicing regime to ensure that cabinets meet required performance levels, and additionally to allow detection and rectification of poor containment, particularly where induced by environmental factors. The value of OPTs is discussed with reference to certain national standards.

29.P.08 A new method for determining the effectiveness of personal protection when using type II microbiological safety cabinets (M.S.C.)

29.P.08 A new method for determining the effectiveness of personal protection when using type II microbiological safety cabinets (M.S.C.)

‘Containment’—recent developments and BS 5726-1992 (microbiological safety cabinets)

A recent resolution of the Parliamentary Assembly of Europe (No. 986–1992) emphasizes that technical innovation is an important and continuing feature of modern society and that it will act as the driving force in commercial and industrial competition for a long while to come. The public draws substantial benefits from this technological progress but has also developed a keen awareness of the supposed effects of certain technologies on the ethical values on which society is based, on health and on the environment. In this context, the issue of risks (particularly those present in certain new technologies) becomes more complex. Despite a general improvement in safety levels and a substantial reduction in traditional risks, new types of risks, far more difficult to calculate and predict, are emerging. This is especially true in the chemical, pharmaceutical and biotechnology industries where these difficulties have been recognized and where safe systems of work and equipment are therefore being developed that can effectively contain potentially hazardous material. Of particular significance over the last 10 years in this area has been the marked improvement in the design and performance of safety cabinets and related containment systems for microbiological use. In the UK this has been due to a number of factors including the implementation of the requirements of BS 5726 1979 (Microbiological safety cabinets) (Anon. 1979) which have been complimentary to the COSHH (Control of Substances Hazardous to Health) Regulations (Anon. 1988) which themselves reinforced the Health and Safety at Work Act (Anon. 1974). Taken together, this framework has been responsible for significant improvements to the manufacturing technologies for safety systems, the management of containment systems within laboratories and the awareness by users of the functional requirements that all containment systems must now have.

Continued successful operation of open-fronted microbiological safety cabinets in a force-ventilated laboratory

The considerable refinements necessary to enable Class I and II microbiological safety cabinets to operate in a force-ventilated laboratory and to meet appropriate safety criteria have been reported previously. The continued successful operation of such cabinets without a deterioration of operator protection is described. The performance of two Class II units, one meeting and one failing the current British Standard applied to four head KI-discus testing, is compared and discussed. In addition, some further potential difficulties within the environment, which could compromise cabinet containment, are highlighted.