ABSTRACT
Purpose Global efforts to combat the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have placed renewed focus on the use of transmission electron microscopy (EM) for infectious disease diagnosis and detection. Recently, attempts to identify SARS-CoV-2 directly in human autopsy and animal necropsy tissues have inaccurately identified normal subcellular structures, including coated vesicles, multivesicular bodies, and rough endoplasmic reticulum, as coronavirus particles. Working with SARS-CoV-2 positive autopsy and necropsy tissues, the Infectious Diseases Pathology Branch at CDC sought to use EM to accurately detect coronavirus particles. Methods & Materials Two sample types were used, formalin-fixed wet tissue and formalin-fixed paraffin embedded (FFPE) tissue blocks. Wet tissue samples provide the best preservation of ultrastructure but require a time-intensive search for viral particles. FFPE tissues enable a targeted approach to finding viral particles but with deteriorated ultrastructure. Areas of formalin-fixed wet tissue showing evident disease pathology were selected for EM, while areas of interest from FFPE blocks were selected based on results from SARS-CoV-2 immunohistochemistry and in situ hybridization results. All samples were post-fixed with 1% osmium tetroxide, en-bloc stained with uranyl acetate, dehydrated, and embedded in Epon-Araldite resin. Results A multifaceted approach for SARS-CoV-2 detection in autopsy and necropsy tissues allowed for swift and accurate determination of the localization of coronavirus and correlation of histopathological and ultrastructural features of SARS-CoV-2 infection. Coronavirus particles were found associated with degenerating cells in the alveolar space, in pneumocytes, and near collagen of the heart in fetal tissue as well as in the syncytiotrophoblast of the placenta. In animal tissues, virus was found in the bronchiolar epithelium and type 1 pneumocytes. Conclusion Comprehensive studies of SARS-CoV-2 infection, and all emerging pathogens, are crucial to improving the understanding of pathogenesis and for the formulation of clinical treatments and transmission prevention measures. An important part of this process is providing robust EM evidence of SARS-CoV-2 localization within tissues to ensure that misinterpretations of subcellular structures as virus are reduced, enabling more accurate conclusions concerning COVID-19 pathology and disease.
ABSTRACT
Background: It is standard practice in our centre for patients to be given permanent skin marks during breastradiotherapy planning, for use as reliable landmarks in daily reproduction of their positioning for treatment. However,these permanent marks (tattoos) may have a significant psychological impact on patients (1). In recent years, therehave been technological advances in surface-guided radiotherapy techniques (SGRT) which may provide improvedset-up accuracy compared to permanent markers. Aims: 1. To evaluate if surface-guided set-up is as good, if notbetter, than set-up with permanent markers alone.2. To safely implement a permanent marker (PM) free, surface-guided set-up technique. Methods: A pilot study was conducted with tangents-only breast patients treated in freebreathing (FB). All treatments were delivered on Varian TrueBeam linear accelerators, with patients immobilised ona couch indexed breast board. The study group (n=20) were set up using PMs with adjustments guided by theAlignRT SGRT system to optimise patient positioning. Imaging (MV tangent images) was performed as perstandard protocol on fractions 1-3, 8 and 12. Additional imaging was performed if indicated. The translational androtational displacements calculated by the TrueBeam verification system for this group were compared to averagedisplacements calculated for patients set up using PMs alone (the control group, n=20). Encouraged by the resultsof the pilot study, the centre moved to safely roll-out the PM-free technique to include additional applications. Thestep-wise approach taken will be described. Results: The mean displacements calculated from the verificationimaging are shown in Table 1 below: SGRT set-up demonstrated statistically-significant improvements compared to PM set-up alone with respect tolateral, vertical and total translational displacements. Longitudinal displacements favoured PM set-up alone but thisdid not reach statistical significance. Rotational displacements favoured SGRT set-up and the results reachedstatistical significance.Given the improved set-up accuracy with SGRT, and the wish to reduce the psychologicalmorbidity of radiotherapy for our breast cancer patients, the centre moved to safely implement a PM free SGRTtechnique along agreed timelines as shown in Table 2 : Conclusions: Our pilot study demonstrated that surface-guided radiotherapy (SGRT) set-up is as good, if not better,than set-up with permanent markers (PM) alone in tangent-only free-breathing patients. Subsequent further analysisshowed improved consistency of set-up guided by PM plus SGRT for all breast set-ups (to include nodal regionsand DIBH). We have described our step-wise approach to setting up PM free radiotherapy delivery for breast cancerpatients, which has additional advantages in maintaining social distancing in the COVID19 era.