Applied Biosafety

The study of and experimentation with zoonotic pathogens such as highly pathogenic avian influenza (HPAI) requires risk mitigation strategies including laboratory engineering controls and safety equipment, personal protective equipment (PPE), and proper practices and techniques. Incidences of potential exposure should be investigated promptly and corrective actions taken.

Two incidences occurred at ABSL-3E facilities in 2014. These involved a tear in a breathing tube and separation of a breathing tube from a fitting joint on powered air purifying respirators (PAPRs). The events, as well as the occupational health response, outcomes, and investigations into the incidents, are described in this article. While no exposure-associated infections occurred, changes and improvements were implemented to prevent future incidents. The changes and improvements to PAPRs included the addition of breathing tube covers, reinforcement of breathing tube and fitting joints, and notifying the manufacturer of product deficiencies. Improvements to animal isolators were also completed to prevent potential snags or tears to breathing tubes. Updated standard procedures for PAPR assembly and use were instituted and thoroughly emphasized in the annual training of BSL-3E personnel.

These incidents demonstrate the need for continuous evaluation and updating of biosafety procedures and equipment. While thorough investigation coupled with corrective actions and improvements exemplifies proper procedures for prevention, the prompt and expedient response of personnel during these incidents is a reflection of routine and comprehensive biosafety training.

International standards as well as national regulations often require the incorporation of HEPA filters in the exhaust of BSL-3 (unless work with non-airborne organisms is performed—Swiss Containment Ordinance [Federal Council, 2012]) and certainly BSL-4 laboratories (single HEPA filter on supply and double HEPA filter on exhaust). These may need to be decontaminated from time to time, for example prior to filter integrity testing (protection of the testing equipment in case filters or seals are damaged and thus leaking) or when exchanging filters (bag-in/bag-out may offer an alternative or an additional safety measure, respectively). However, HEPA filter housings are not generally equipped with sample ports that allow for placement of indicators to confirm a successful fumigation process. Additionally, a site-specific risk assessment concluded that since the filter boxes are situated in an area deemed to remain non-contaminated at all times (HEPA filter floor above laboratory suites), indicators may not be placed inside the filter housings as opening them prior to fumigation would open containment and thus pose a threat to the people working on the filter boxes as well as to the environment.

In this article the authors describe the use of the IndicatorSafe, a simple and cheap solution to the above-mentioned problem. Placing the IndicatorSafe in the return line following the filter housing was shown to be an efficient alternative when fumigating filter housings with vaporized hydrogen peroxide (VHP), as shown during the validation of filter housing fumigation cycles, and may likely be adapted to other fumigation methods on the market.

In high-containment laboratories and animal facilities common practice is to decontaminate the facilities prior to maintenance or in an emergency situation. Many laboratories use commercially available biological indicators (BIs) to validate the decontamination procedure. In this study the focus was to evaluate the reliability of four different commercial BIs in comparison to control microorganisms that are commonly used in laboratories. Two different fumigation decontamination procedures were chosen: formaldehyde (FA) and vaporized hydrogen peroxide (VHP). The control microorganisms were Salmonella typhimurium, Brucella melitensis, Bacillus thuringiensis spores, porcine parvovirus, equine rhinitis A virus, bovine viral diarrhea virus, and low pathogenic avian influenza virus.

Exposure to formaldehyde caused a sufficient reduction of all the control microorganisms, including B. thuringiensis spores, whereas only one of the four commercial BIs was completely negative for growth. The VHP decontamination procedure was not able to reduce any of the control microorganisms more than 4 log₁₀, except for the enveloped viruses, whereas the BIs with the lowest concentration (10⁵ microorganisms per spore strip) indicated a satisfactory decontamination procedure.

These results indicate that commercial BIs could be unreliable as general indicators of decontamination effectiveness. To ensure a reliable decontamination process, BIs have to be evaluated for each protocol in parallel with the microorganisms used in the laboratory.

The Chordopoxvirinae subfamily (Family Poxviridae) includes human and animal pathogens with zoonotic potential. Among them are viruses that are commonly used as viral and vaccination vectors, some of them bearing oncolytic or oncotropic potential for anti-tumor therapy. The authors have developed a versatile triplex real-time PCR assay that facilitates differentiation between the genera (i) Orthopoxvirus, (ii) Avipoxvirus, and (iii) Parapoxvirus and Molluscipoxvirus. Hydrolysis probes labelled with Yakima Yellow, Cy5, and FAM specifically bind to Ortho-, Avi-, Para- or Molluscipoxviruses, respectively. The authors detected the expected four poxvirus genera in purified DNA from 47 poxviral samples, whereas four samples belonging to the genera Yatapoxvirus and Capripoxvirus were not detected. Taken together, a set of 24 negative controls comprising other viruses including non-target poxvirus-genera as well as bacteria, yeast, mammal, and avian cells were tested to proof the specificity of the assay. Probit analysis revealed a detection limit of three copies per reaction (95% confidence level) for the Ortho-, Avi-, and Para-/Molluscipoxvirus-PCR using two independent Cycler-Master mix combinations.

Over the past 30 years, several microbiology laboratories in Singapore General Hospital developed extensive collections of Risk Group 3 (RG) agents. With the opening of a new, central diagnostic laboratory, the pathogenic specimens had to be relocated to the state-of-the-art Biosafety Level 3 (BSL-3) facility without disruption of laboratory diagnostic work. A risk assessment of all aspects of the task was conducted and communicated, and the planning, preparation, and dry run are described.