Assessing and improving the validity of COVID-19 autopsy studies - a multi center approach to establish essential standards for immunohistochemical and ultrastructural analyses

BackgroundAutopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, e.g. by an overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing. MethodsWe assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 spike protein and nucleocapsid, dsRNA, and non-structural protein Nsp3 in cultured cell lines and COVID-19 autopsy tissues. In a multicenter study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 nucleocapsid staining. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2. FindingsPublications show high variability in the detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. In our study, we show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (r=-0.83, p-value <0.0001). For EM, we refined criteria for virus identification and also provide recommendations for optimized sampling and analysis. 116 of 122 publications misinterpret cellular structures as virus using EM or show only insufficient data. We provide publicly accessible digitized EM and IHC sections as a reference and for training purposes. InterpretationSince detection of SARS-CoV-2 in human autopsy tissues by IHC and EM is difficult and frequently incorrect, we propose criteria for a re-evaluation of available data and guidance for further investigations of direct organ effects by SARS-CoV-2. Key messagesO_LIDetection of SARS-CoV-2 proteins by IHC in autopsy tissues is less sensitive in comparison to SARS-CoV-2 RNA detection by RT-qPCR. C_LIO_LIFor determination of SARS-CoV-2 protein positive cells by IHC in autopsy tissues, detection of spike protein is less sensitive than nucleocapsid protein. C_LIO_LICorrect identification of SARS-CoV-2 particles in human samples by EM is limited to the respiratory system. C_LIO_LIInterpretation of IHC and EM should follow substantiated consensus criteria to enhance accuracy. C_LIO_LIExisting datasets describing SARS-CoV-2 presence in human autopsy tissues need to be critically re-evaluated. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/22269205v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@eafd97org.highwire.dtl.DTLVardef@1aed770org.highwire.dtl.DTLVardef@1c21ab9org.highwire.dtl.DTLVardef@68a101_HPS_FORMAT_FIGEXP M_FIG C_FIG

The phosphorus mass balance: identifying 'hotspots' in the food system as a roadmap to phosphorus security.

Phosphorus is a critical element on which all life depends. Global crop production depends on fertilisers derived from phosphate rock to maintain high crop yields. Population increase, changing dietary preferences towards more meat and dairy products, and the continuing intensification of global agriculture supporting this expansion will place increasing pressure on an uncertain, but finite supply of high-quality phosphate rock. Growing concern about phosphorus scarcity and security, coupled with the environmental impact of phosphorus pollution, has encouraged an increase in research exploring how phosphorus is used and lost in the food system-from mine to field to fork. An assessment of recent phosphorus flows analyses at different geographical scales identifies the key phosphorus 'hotspots', for example within the mining, agriculture or food processing sectors, where efficiency and reuse can be substantially improved through biotechnological approaches coupled with policy changes.

Understanding consumption-related sucralose emissions - A conceptual approach combining substance-flow analysis with sampling analysis.

This paper explores the potential of combining substance-flow modelling with water and wastewater sampling to trace consumption-related substances emitted through the urban wastewater. The method is exemplified on sucralose. Sucralose is a chemical sweetener that is 600 times sweeter than sucrose and has been on the European market since 2004. As a food additive, sucralose has recently increased in usage in a number of foods, such as soft drinks, dairy products, candy and several dietary products. In a field campaign, sucralose concentrations were measured in the inflow and outflow of the local wastewater treatment plant in Linköping, Sweden, as well as upstream and downstream of the receiving stream and in Lake Roxen. This allows the loads emitted from the city to be estimated. A method consisting of solid-phase extraction followed by liquid chromatography and high resolution mass spectrometry was used to quantify the sucralose in the collected surface and wastewater samples. To identify and quantify the sucralose sources, a consumption analysis of households including small business enterprises was conducted as well as an estimation of the emissions from the local food industry. The application of a simple model including uncertainty and sensitivity analysis indicates that at present not one large source but rather several small sources contribute to the load coming from households, small business enterprises and industry. This is in contrast to the consumption pattern seen two years earlier, which was dominated by one product. The inflow to the wastewater treatment plant decreased significantly from other measurements made two years earlier. The study shows that the combination of substance-flow modelling with the analysis of the loads to the receiving waters helps us to understand consumption-related emissions.