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The emergence of SARS-CoV-2 and the subsequent pandemic has highlighted the need for animal models that faithfully replicate the salient features of COVID-19 disease in humans. These models are necessary for the rapid selection, testing, and evaluation of potential medical countermeasures. Here, we performed a direct comparison of two distinct routes of SARS-CoV-2 exposure-combined intratracheal/intranasal and small particle aerosol-in two nonhuman primate species, rhesus and cynomolgus macaques. While all four experimental groups displayed very few outward clinical signs, evidence of mild to moderate respiratory disease was present on radiographs and at necropsy. Cynomolgus macaques exposed via the aerosol route also developed the most consistent fever responses and had the most severe respiratory disease and pathology. This study demonstrates that while all four models produced suitable representations of mild COVID-like illness, aerosol exposure of cynomolgus macaques to SARS-CoV-2 produced the most severe disease, which may provide additional clinical endpoints for evaluating therapeutics and vaccines.

Objectives: To examine population changes in 5-year survival for people in South Australia diagnosed with acute leukaemia during 1980-2016, by socio-demographic characteristics., Design, Setting: Retrospective analysis of South Australian Cancer Registry data for the period 1980-2016., Participants: All South Australian residents diagnosed with primary acute lymphoblastic leukaemia (ALL) or acute myeloid leukaemia (AML) during 1980-2016., Main Outcome Measures: 5-year disease-specific survival and disease-specific mortality., Results: Crude 5-year disease-specific survival was 58% (95% CI, 54-61%) for the 1035 people diagnosed with ALL during 1980-2016, and 18% (95% CI, 17-20%) for the 2814 people diagnosed with AML. Survival improved steadily across the study period: from 44% (95% CI, 35-52%) for people with ALL diagnosed during 1980-1984 to 69% (95% CI, 63-75%) for those diagnosed during 2010-2016; and from 9% (95% CI, 5-15%) to 23% (95% CI, 20-26%) for people diagnosed with AML. Disease-specific mortality increased with age, but was not influenced by socio-economic status or remoteness of residence. After adjusting for other factors, rates of change in risk of leukaemia-related death were greater for younger than older patients with ALL (for interaction: P = 0.004) or AML (P = 0.005), but were not significantly influenced by socio-economic status or remoteness., Conclusion: Five-year survival for people with acute leukaemia in South Australia continuously improved during 1980-2016, and socio-economic status and remoteness did not influence survival. It improved markedly for younger patients (under 50 years of age). However, survival is still relatively poor, especially for people over 50 years with AML., (© 2022 AMPCo Pty Ltd.)

A new coronavirus (SARS-CoV-2) emerged in the winter of 2019 in Wuhan, China, and rapidly spread around the world. The extent and efficiency of SARS-CoV-2 pandemic is far greater than previous coronaviruses that emerged in the 21st Century. Here, we modeled stability of SARS-CoV-2 on skin, paper currency, and clothing to determine if these surfaces may factor in the fomite transmission dynamics of SARS-CoV-2. Skin, currency, and clothing samples were exposed to SARS-CoV-2 under laboratory conditions and incubated at three different temperatures (4°C± 2°C, 22°C± 2°C, and 37°C ± 2°C). We evaluated stability at 0 hours (h), 4 h, 8 h, 24 h, 72 h, 96 h, 7 days, and 14 days post-exposure. SARS-CoV-2 was stable on skin through the duration of the experiment at 4°C (14 days). Virus remained stable on skin for at least 96 h at 22°C and for at least 8h at 37°C. There were minimal differences between the tested currency samples. The virus remained stable on the $1 U.S.A. Bank Note for at least 96 h at 4°C while we did not detect viable virus on the $20 U.S.A. Bank Note samples beyond 72 h. The virus remained stable on both Bank Notes for at least 8 h at 22°C and 4 h at 37°C. Clothing samples were similar in stability to the currency. Viable virus remained for at least 96 h at 4°C and at least 4 h at 22°C. We did not detect viable virus on clothing samples at 37°C after initial exposure. This study confirms the inverse relationship between virus stability and temperature. Furthermore, virus stability on skin demonstrates the need for continued hand hygiene practices to minimize fomite transmission both in the general population as well as in workplaces where close contact is common., Competing Interests: The authors have declared that no competing interests exist.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China, in December 2019. The virus rapidly spread globally, resulting in a public health crisis including almost 5 million cases and 323,256 deaths as of May 21, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected FFPE cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross-detection of the respective SARS-CoV-2 proteins by IHC and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex FISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. It is hoped that these reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus within the family Bunyaviridae. Infection can result in general myalgia, fever, and headache with some patients developing hemorrhagic fever with mortality rates ranging from 5% to 30%. CCHFV has a wide geographic range that includes Africa, Asia, the Middle East, and Europe with nucleotide sequence variation approaching 20% across the three negative-sense RNA genome segments. While phylogenetic clustering generally aligns with geographic origin of individual strains, distribution can be wide due to tick/CCHFV dispersion via migrating birds. This sequence diversity negatively impacts existing molecular diagnostic assays, leading to false negative diagnostic results. Here, we updated a previously developed CCHFV real-time reverse transcription polymerase chain reaction (RT-PCR) assay to include strains not detected using that original assay. Deep sequencing of eight different CCHFV strains, including three that were not detectable using the original assay, identified sequence variants within this assay target region. New primers and probe based on the sequencing results and newly deposited sequences in GenBank greatly improved assay sensitivity and inclusivity with the exception of the genetically diverse strain AP92. For example, we observed a four log improvement in IbAr10200 detection with a new limit of detection of 256 PFU/mL. Subsequent comparison of this assay to another commonly used CCHFV real-time RT-PCR assay targeting a different region of the viral genome showed improved detection, and both assays could be used to mitigate CCHFV diversity for diagnostics. Overall, this work demonstrated the importance of continued viral sequencing efforts for robust diagnostic assay development.

A method for accurate quantitation of virus particles has long been sought, but a perfect method still eludes the scientific community. Electron Microscopy (EM) quantitation is a valuable technique because it provides direct morphology information and counts of all viral particles, whether or not they are infectious. In the past, EM negative stain quantitation methods have been cited as inaccurate, non-reproducible, and with detection limits that were too high to be useful. To improve accuracy and reproducibility, we have developed a method termed Scanning Transmission Electron Microscopy - Virus Quantitation (STEM-VQ), which simplifies sample preparation and uses a high throughput STEM detector in a Scanning Electron Microscope (SEM) coupled with commercially available software. In this paper, we demonstrate STEM-VQ with an alphavirus stock preparation to present the method's accuracy and reproducibility, including a comparison of STEM-VQ to viral plaque assay and the ViroCyt Virus Counter., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Crimean-Congo hemorrhagic fever virus (CCHFV) is a geographically widespread RNA virus with a high degree of genomic diversity that complicates sequence-based diagnostics. Here, we sequenced eight CCHFV strains for improved assay design and deposition into FDA-ARGOS, the FDA's pathogen database for development and verification of next generation sequencing assays., (Copyright © 2017 Koehler et al.)

Viral preparations are essential components in diagnostic research and development. The production of large quantities of virus traditionally is done by infecting numerous tissue culture flasks or roller bottles, which require large incubators and/or roller bottle racks. The Corning HYPERFlask ® is a multilayer flask that uses a gas permeable film to provide gas exchange between the cells and culture medium and the atmospheric environment. This study evaluated the suitability of the HYPERFlask for production of Lassa, Ebola, Bundibugyo, Reston, and Marburg viruses and compared it to more traditional methods using tissue culture flasks and roller bottles. The HYPERFlask produced cultures were equivalent in virus titer and indistinguishable in immunodiagnostic assays. The use of the Corning HYPERFlask for viral production is a viable alternative to traditional tissue culture flasks and roller bottles. HYPERFlasks allow for large volumes of virus to be produced in a small space without specialized equipment., (Copyright © 2016. Published by Elsevier B.V.)

Development and evaluation of medical countermeasures for diagnostics, vaccines, and therapeutics requires production of standardized, reproducible, and well characterized virus preparations. For filoviruses this includes plaque assay for quantitation of infectious virus, transmission electron microscopy (TEM) for morphology and quantitation of virus particles, and real-time reverse transcription PCR for quantitation of viral RNA (qRT-PCR). The ViroCyt® Virus Counter (VC) 2100 (ViroCyt, Boulder, CO, USA) is a flow-based instrument capable of quantifying virus particles in solution. Using a proprietary combination of fluorescent dyes that stain both nucleic acid and protein in a single 30 min step, rapid, reproducible, and cost-effective quantification of filovirus particles was demonstrated. Using a seed stock of Ebola virus variant Kikwit, the linear range of the instrument was determined to be 2.8E+06 to 1.0E+09 virus particles per mL with coefficient of variation ranging from 9.4% to 31.5% for samples tested in triplicate. VC particle counts for various filovirus stocks were within one log of TEM particle counts. A linear relationship was established between the plaque assay, qRT-PCR, and the VC. VC results significantly correlated with both plaque assay and qRT-PCR. These results demonstrated that the VC is an easy, fast, and consistent method to quantify filoviruses in stock preparations.

Objective: To determine survival rates of patients with lymphoma in South Australia., Design and Setting: De-identified data from the SA Cancer Registry on all patients with lymphoma were analysed, as well as the subgroup treated at the Royal Adelaide Hospital (RAH). For non-Hodgkin lymphoma (NHL), we used the International Working Formulation (IWF) grading. SA and RAH data on survival rates were compared with those for the whole of Australia and the United States., Patients: All patients diagnosed with lymphoma and treated in SA in 1977-2007., Outcome Measures: 5-year survival rates for patients with lymphoma, by type of lymphoma and age., Results: Of the total of 8651 patients with lymphoma, 939 were classified as having Hodgkin lymphoma (HL) and 7712 as having NHL. Of those with NHL, 1805 had low-grade, 3576 intermediate-grade, and 510 high-grade NHL. In another 1821 patients, the data were insufficient to make an IWF grading. There was a substantial increase in 5-year survival rates for patients with lymphoma between 1977 and 2007 in SA. While the increase in 5-year survival rates for HL was 7.6 percentage points, survival rates peaked at 88%. For NHL, there was an 18.7 percentage points increase in 5-year survival rates. The first significant increase of 7 percentage points was associated with the introduction of bone marrow transplantation; this was maintained with the increase in 5-year survival rates reaching 14 percentage points by 1995-1999. Since 1999, there has been a further increase of 5 percentage points in 5-year survival rates with the introduction of rituximab., Conclusion: Outcomes in patients with NHL have improved significantly, most likely because of the use of bone marrow transplantation and rituximab. Hospital- and state-based cancer registry data reflect the reality of population outcomes and the impact of new technologies.

A novel recombinant single-chain fragment variable (scFv) antibody against western equine encephalitis (WEE) virus has been previously constructed and partially characterized. The RS10B5huFc antibody was made by fusing an anti-WEE scFv to a human heavy-chain IgG1 constant region. The RS10B5huFc antibody was functional in binding to WEE virus in enzyme-linked immunosorbent assays (ELISAs), and the Fc domain of the antibody was capable of effector functions, such as binding to protein G and human complement. In this study, the RS10B5huFc antibody was further characterized by BIAcore analyses and was found to possess a binding affinity to a WEE virus epitope (K[D] = 9.14 x 10(-6) M), 4.5-fold lower than its parental mouse monoclonal antibody (MAb) 10B5 E7E2 (K[D] = 2 x 10(-6) M). No cross-reactivity was found between the RS10B5huFc antibody and three other alphaviruses (Sindbis virus [SIN], Venezuelan equine encephalitis [VEE] virus, and eastern equine encephalitis [EEE] virus). Pharmacokinetics studies showed that the RS10B5huFc antibody (free and encapsulated) was found to be retained in the lungs of mice for greater than 48 h when administered intranasally. In contrast, when administered intramuscularly to mice, the RS10B5huFc antibody was not detected in the lungs and only found in the liver and kidneys.

Objective: To evaluate the effectiveness of a commercially available ELISA kit for detecting antibodies against Borrelia burgdorferi in dogs., Sample Population: Banked sera from 440 military working dogs were used for serologic analyses., Procedure: Serum samples were analyzed for antibodies against B burgdorferi by use of a commercially available ELISA and subsequently by western blot analysis as a confirmatory test., Results: Results from the ELISA indicated that 89 (20%) samples were positive for exposure to B burgdorferi or canine Lyme disease vaccine, and 351 (80%) were negative. Follow-up testing by western blot analysis indicated that results for 109 (25%) samples were positive and 331 (75%) were negative for exposure. All samples that had positive results on the ELISA also had positive results on western blot analysis (true positives). Of the 351 samples that had negative results on the ELISA, only 331 had negative results on western blot analysis (true negatives). The remaining 20 samples had positive results on western blot analysis. By use of a standard 2 x 2 table, it was determined that the ELISA had a sensitivity of 82%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 94%., Conclusions and Clinical Relevance: The commercial ELISA kit evaluated in this study appeared to lack adequate sensitivity for detecting all potential cases of borreliosis in dogs. The ELISA was also unable to discriminate natural exposure from exposure attributable to vaccination, which could complicate interpretation of positive results and treatment of dogs with clinical signs.

In spite of rigorous government programs for control of the pricing and dissemination of pharmaceutical products in Australia, the list of new drugs continues to grow and prices to increase. To regain control over drug usage at Royal Adelaide Hospital, the Hospital Drug Committee developed a rating method that judged drugs on the basis of their cost-benefit to patients. The ratio of a total quality score to a total cost score becomes the determinant of additions to the hospital formulary. The background for the Australian approach to pharmaceuticals and the new evaluation technique at the teaching hospital are described in this report.