Two multiplex PCR screening capabilities (TaqMan Array Cards and FilmArray) were evaluated for their ability to detect Bacillus anthracis, Francisella tularensis and Yersinia pestis in blood samples obtained from respective murine infection models. Blood samples were obtained from infected mice at 24 h intervals after exposure. Multiplex PCR results were compared with standard blood culture and singleplex real-time PCR. Across all three models, 71 mice were tested in total, within which a subset of 43 samples was shown to contain an infecting agent by at least one of the detection technologies. Within this subset of positive samples, for each model studied, the detection rates of each technology were compared. The B. anthracis model blood culture (14 of 15 agent-containing samples tested) and FilmArray PCR (12 of 15) were shown to have equivalent detection rates, which were significantly higher (at the 95 % confidence level) than singleplex (five of 14) or Array Card (two of 14) PCRs. The F. tularensis model blood culture (12 of 12) was shown to have a significantly higher (at 95 % confidence level) detection rate than all PCR technologies, with FilmArray (seven of 11) and singleplex (seven of 12) PCRs shown to have significantly higher (at 95 % confidence level) detection rates than the Array Card PCR (two of 11). Within the Y. pestis model, there was no significant difference in detection rates between blood culture (10 of 16), singleplex PCR (14 of 16), Array Card PCR (10 of 16) and FilmArray PCR (10 of 13).

Spores of an acrystalliferous derivative of Bacillus thuringiensis subsp. kurstaki, termed Btcry؊, are morphologically, aerodynamically, and structurally indistinguishable from Bacillus anthracis spores. Btcry؊ spores were dispersed in a large, openended barn together with spores of Bacillus atrophaeus subsp. globigii, a historically used surrogate for Bacillus anthracis. Spore suspensions (2 ؋ 10 12 CFU each of B. atrophaeus subsp. globigii and Btcry؊) were aerosolized in each of five spray events using a backpack misting device incorporating an air blower; a wind of 4.9 to 7.6 m s ؊1 was also flowing through the barn in the same direction. Filter air samplers were situated throughout the barn to assess the aerosol density of the spores during each release. Trays filled with a surfactant in aqueous buffer were placed on the floor near the filter samplers to assess spore deposition. Spores were also recovered from arrays of solid surfaces (concrete, aluminum, and plywood) that had been laid on the floor and set up as a wall at the end of the barn. B. atrophaeus subsp. globigii spores were found to remain airborne for significantly longer periods, and to be deposited on horizontal surfaces at lower densities, than Btcry؊ spores, particularly near the spray source. There was a 6-fold-higher deposition of Btcry؊ spores than of B. atrophaeus subsp. globigii spores on vertical surfaces relative to the surrounding airborne density. This work is relevant for selecting the best B. anthracis surrogate for the prediction of human exposure, hazard assessment, and hazard management following a malicious release of B. anthracis. IMPORTANCEThere is concern that pathogenic bacteria could be maliciously disseminated in the air to cause human infection and disruption of normal life. The threat from spore-forming organisms, such as the causative agent of anthrax, is particularly serious. In order to assess the extent of this risk, it is important to have a surrogate organism that can be used to replicate the dispersal characteristics of the threat agent accurately. This work compares the aerosol dispersal and deposition behaviors of the surrogates Btcry؊ and B. atrophaeus subsp. globigii. Btcry؊ spores remained in the air for a shorter time, and were markedly more likely to adhere to vertical surfaces, than B. atrophaeus subsp. globigii spores. Bacillus anthracis is an important bacterial agent of concern (1). It is pathogenic to humans, particularly by inhalational exposure (2). The organism's endospores manifest greater environmental persistence and higher resistance to chemical decontaminants than those of other bacteria. Understanding of the threat posed by malicious use of B. anthracis and the development of effective countermeasures require a comparable, nonpathogenic surrogate. Bacillus atrophaeus subsp. globigii has, for many decades, been a B. anthracis surrogate (3). B. atrophaeus subsp. globigii and other surrogates related to Bacillus subtilis have generated useful information regarding spore dispersal (4, 5)....

Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QIAcube HT and the Thermo Fisher KingFisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type: SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer mix from the MagMAX Pathogen RNA/DNA kit (Thermo Fisher), used on the KingFisher Flex, which included guanidinium isothiocyanate (GITC), a detergent, and isopropanol, demonstrated a minimum inactivation efficacy of 1 × 105 tissue culture infectious dose (TCID)50/ml. Alternative lysis buffer mixes from the MagMAX Viral/Pathogen Nucleic Acid kit (Thermo Fisher) also used on the KingFisher Flex and from the QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QIAcube HT (both of which contained GITC and a detergent) reduced titres by 1 × 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 min, 68°C) did not completely inactivate the virus, demonstrating a reduction of 1 × 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory.

BackgroundIn the face of the COVID-19 pandemic, the Defence Science and Technology Laboratory (Dstl) and Defence Pathology combined to form the Defence Clinical Lab (DCL), an accredited (ISO/IEC 17025:2017) high-throughput SARS-CoV-2 PCR screening capability for military personnel.Laboratory structure and resourceThe DCL was modular in organisation, with laboratory modules and supporting functions combining to provide the accredited SARS-CoV-2 (envelope (E)-gene) PCR assay. The DCL was resourced by Dstl scientists and military clinicians and biomedical scientists.Laboratory resultsOver 12 months of operation, the DCL was open on 289 days and tested over 72 000 samples. Six hundred military SARS-CoV-2-positive results were reported with a median E-gene quantitation cycle (Cq) value of 30.44. The lowest Cq value for a positive result observed was 11.20. Only 64 samples (0.09%) were voided due to assay inhibition after processing started.ConclusionsThrough a sustained effort and despite various operational issues, the collaboration between Dstl scientific expertise and Defence Pathology clinical expertise provided the UK military with an accredited high-throughput SARS-CoV-2 PCR test capability at the height of the COVID-19 pandemic. The DCL helped facilitate military training and operational deployments contributing to the maintenance of UK military capability. In offering a bespoke capability, including features such as testing samples in unit batches and oversight by military consultant microbiologists, the DCL provided additional benefits to the UK Ministry of Defence that were potentially not available from other SARS-CoV-2 PCR laboratories. The links between Dstl and Defence Pathology have also been strengthened, benefitting future research activities and operational responses.

A transduced mouse model of SARS-CoV-2 infection was established using Balb/c mice. This was achieved through the adenovirus-vectored delivery of the hACE2 gene, to render the mice transiently susceptible to the virus. The model was characterised in terms of the dissemination of hACE2 receptor expression, the dissemination of three SARS-CoV-2 virus variants in vivo up to 10 days following challenge, the resulting histopathology and the clinical signs induced in the mice. In transduced mice, the infection was short-term, with a rapid loss in body weight starting at day 2 with maximum weight loss at day 4, followed by subsequent recovery until day 10. The induced expression of the hACE2 receptor was evident in the lungs, but, upon challenge, the SARS-CoV-2 virus disseminated beyond the lungs to spleen, liver and kidney, peaking at day 2 post infection. However, by day 10 post infection, the virus was undetectable. The lung histopathology was characterised by bronchial and alveolar inflammation, which was still present at day 10 post infection. Transduced mice had differential responses to viral variants ranking CVR-Glasgow 1 > Victoria-1 > England-2 isolates in terms of body weight loss. The transduced mouse model provides a consistent and manipulatable model of SARS-CoV-2 infection to screen viral variants for their relative virulence and possible interventions.