Detection of SARS-CoV-2 within the healthcare environment: a multi-centre study conducted during the first wave of the COVID-19 outbreak in England.

BACKGROUND: Understanding how severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is spread within the hospital setting is essential in order to protect staff, implement effective infection control measures, and prevent nosocomial transmission. METHODS: The presence of SARS-CoV-2 in the air and on environmental surfaces around hospitalized patients, with and without respiratory symptoms, was investigated. Environmental sampling was undertaken within eight hospitals in England during the first wave of the coronavirus disease 2019 outbreak. Samples were analysed using reverse transcription polymerase chain reaction (PCR) and virus isolation assays. FINDINGS: SARS-CoV-2 RNA was detected on 30 (8.9%) of 336 environmental surfaces. Cycle threshold values ranged from 28.8 to 39.1, equating to 2.2 x 105 to 59 genomic copies/swab. Concomitant bacterial counts were low, suggesting that the cleaning performed by nursing and domestic staff across all eight hospitals was effective. SARS-CoV-2 RNA was detected in four of 55 air samples taken <1 m from four different patients. In all cases, the concentration of viral RNA was low and ranged from <10 to 460 genomic copies/m3 air. Infectious virus was not recovered from any of the PCR-positive samples analysed. CONCLUSIONS: Effective cleaning can reduce the risk of fomite (contact) transmission, but some surface types may facilitate the survival, persistence and/or dispersal of SARS-CoV-2. The presence of low or undetectable concentrations of viral RNA in the air supports current guidance on the use of specific personal protective equipment for aerosol-generating and non-aerosol-generating procedures.

The effect of a non-denaturing detergent and a guanidinium-based inactivation agent on the viability of Ebola virus in mock clinical serum samples.

The 2014 Ebola outbreak in West Africa required the rapid testing of clinical material for the presence of potentially high titre Ebola virus (EBOV). Safe, fast and effective methods for the inactivation of such clinical samples are required so that rapid diagnostic tests including downstream analysis by RT-qPCR or nucleotide sequencing can be carried out. One of the most commonly used guanidinium - based denaturing agents, AVL (Qiagen) has been shown to fully inactivate EBOV once ethanol is added, however this is not compatible with the use of automated nucleic acid extraction systems. Additional inactivation agents need to be identified that can be used in automated systems. A candidate inactivation agent is Triton X-100, a non-denaturing detergent that is frequently used in clinical nucleic acid extraction procedures and has previously been used for inactivation of EBOV. In this study the effect of 0.1% and 1.0% Triton X-100 (final concentration 0.08% and 0.8% respectively) alone and in combination with AVL on the viability of EBOV (106 TCID50/ml) spiked into commercially available pooled negative human serum was tested. The presence of viable EBOV in the treated samples was assessed by carrying out three serial passages of the samples in Vero E6 cells (37°C, 5% CO2, 1 week for each passage). At the end of each passage the cells were observed for evidence of cytopathic effect and samples were taken for rRT-PCR analysis for the presence of EBOV RNA. Before cell culture cytotoxic components of AVL and Triton X-100 were removed from the samples using size exclusion spin column technology or a hydrophobic adsorbent resin. The results of this study showed that EBOV spiked into human serum was not fully inactivated when treated with either 0.1% (v/v) Triton X-100 for 10 mins or 1.0% (v/v) Triton X-100 for 20 mins (final concentrations 0.08% and 0.8% Triton X-100 respectively). AVL alone also did not consistently provide complete inactivation. Samples treated with both AVL and 0.1% Triton X-100 for 10 or 20 mins were shown to be completely inactivated. This treatment is compatible with downstream analysis by RT-qPCR and next generation sequencing.

Effective binding of a phosphatidylserine-targeting antibody to Ebola virus infected cells and purified virions.

Ebola virus is responsible for causing severe hemorrhagic fevers, with case fatality rates of up to 90%. Currently, no antiviral or vaccine is licensed against Ebola virus. A phosphatidylserine-targeting antibody (PGN401, bavituximab) has previously been shown to have broad-spectrum antiviral activity. Here, we demonstrate that PGN401 specifically binds to Ebola virus and recognizes infected cells. Our study provides the first evidence of phosphatidylserine-targeting antibody reactivity against Ebola virus.

Predicting coronary heart disease in remote settings: a prospective, cross-sectional observational study.

BACKGROUND: Coronary heart disease (CHD) places a major burden on the Australian health care system. Determining the likelihood of CHD in a patient presenting with chest pain can be particularly difficult in a remote setting where access to transportation and specialised investigations including myocardial stress studies and coronary angiography can be difficult and delayed. The objective is to develop a predictive model for determining the risk of CHD, including the value of high sensitivity C-reactive protein (hsCRP), in patients presenting with chest pain with a particular emphasis on resources and information likely to be available in a remote primary health care setting. METHODS: A prospective, cross-sectional observational study of patients with no prior diagnosis of CHD presenting to a specialist chest pain assessment clinic at Cairns Hospital from November 2012 to May 2013. RESULTS: Out of the 163 participants included in the study analyses, a total of 38 were classified as CHD likely (23.3% (95% CI 17.1-30.6)). Logistic regression modelling identified two factors that were independently associated with likely CHD, namely the presence of typical chest pain (OR 83.7 (95% CI 21.7-322.1)) and an abnormal baseline ECG (OR 12.8 (95% CI 1.9-86.0)). CONCLUSION: In this study, it was demonstrated that the presence of typical chest pain and an abnormal resting ECG, remain the cornerstone of predicting a subsequent diagnosis of CHD. This information is easily accessible in remote primary health care and should be utilised to expedite assessment in patients presenting with symptoms suggestive of CHD.

Human immunodeficiency virus infection of xenografted SCID-beige mice.

The application of C.B-17 SCID-Beige mice as an experimental animal system for the acceptance of human leukocyte xenografts, the establishment of functional human immune responses and infection with HIV has been assessed. Reconstitution efficiencies approaching 100% could be obtained by using 2 x 10(7) human peripheral blood lymphocytes (PBLs). Typical levels of human immunoglobulin in mouse blood reached 120 micrograms/ml within 2 weeks of reconstitution rising to a maximum in excess of 3 mg/ml by 5 weeks. Immunohistological examination of lung, spleen, lymph node and thymus tissue, derived from reconstituted mice, with human leukocyte specific monoclonal antibodies revealed the presence of human macrophages (CD68+), T cells (CD43+) and B cells (CD20+). The establishment of a functional immune system was demonstrated by the ability of reconstituted mice to respond to immunisation with KLH. Finally, reconstituted Hu-PBL-SCID-Beige mice were susceptible to infection with HIV-1 by intraperitoneal injection. These results indicate that SCID-Beige mice are a valuable tool for the generation of human/mouse chimeras and for the establishment of an in vivo HIV infection model. The results are compared with other similar model systems and are discussed in the context of animal models of HIV vaccine studies.

Rapid vaccination protocols for commercial vaccines against tick-borne encephalitis.

Although inactivated viral vaccines have been dramatically successful in controlling many of the world's most devastating diseases, they frequently need several injections to ensure high levels of protection, and thus their efficacy is reduced in many situations. We have developed several rapid vaccination protocols for two commercial vaccine preparations against tick-borne encephalitis virus and studied their efficacy in an experimental murine model. Vaccination protocols as brief as two doses given over two days elicit efficient protection against challenge with potentially fatal doses of virus and this protection is afforded as soon as 5 or as long as 100 days after the first vaccination. The very rapid induction of protection and the poor antibody responses observed would suggest that cell-mediated immune responses are the most important mechanisms for the protection elicited by conventional inactivated vaccines against tick-borne encephalitis.

The NS1 protein of tick-borne encephalitis virus forms multimeric species upon secretion from the host cell.

Flaviviruses elicit a humoral immune response to two virus-encoded, membrane-associated glycoproteins. One is the major virion surface envelope protein (E), which is recognized by antibody, whereas the other is a secreted, heavily glycosylated non-structural protein (NS1). Inoculation with either protein can give rise to a protective immune response, as can the passive transfer of E and NS1 monospecific monoclonal antibodies. Experiments reported here demonstrate that the secreted form of NS1, whether from cells infected with tick-borne encephalitis virus (TBEV) or from cells infected with a defective recombinant adenovirus containing the NS1 gene, occurs chiefly as a pentamer or hexamer and occasionally as a decamer or dodecamer. Intracellular forms of this protein however occur only as dimers. The higher M(r) forms secreted from the cell are exquisitely sensitive to detergent, suggesting they are held together by hydrophobic bonds. Both intracellular and extracellular forms of the dimer can be dissociated by heat, but at different temperatures. Unlike similar proteins from mosquito-borne viruses. NS1 from TBEV-infected cells cannot be dissociated at ambient temperatures by extremes of pH. Studies on the antigenic structure of this protein show it to have several highly conserved epitopes, confirming similar earlier conclusions from amino acid sequence analyses.