PET imaging of TSPO expression in immune cells can assess organ-level pathophysiology in high-consequence viral infections.

Ebola virus (EBOV) disease is characterized by lymphopenia, breach in vascular integrity, cytokine storm, and multiorgan failure. The pathophysiology of organ involvement, however, is incompletely understood. Using [18F]-DPA-714 positron emission tomography (PET) imaging targeting the translocator protein (TSPO), an immune cell marker, we sought to characterize the progression of EBOV-associated organ-level pathophysiology in the EBOV Rhesus macaque model. Dynamic [18F]-DPA-714 PET/computed tomography imaging was performed longitudinally at baseline and at multiple time points after EBOV inoculation, and distribution volumes (Vt) were calculated as a measure of peripheral TSPO binding. Using a mixed-effect linear regression model, spleen and lung Vt decreased, while the bone marrow Vt increased over time after infection. No clear trend was found for liver Vt. Multiple plasma cytokines correlated negatively with lung/spleen Vt and positively with bone marrow Vt. Multiplex immunofluorescence staining in spleen and lung sections confirmed organ-level lymphoid and monocytic loss/apoptosis, thus validating the imaging results. Our findings are consistent with EBOV-induced progressive monocytic and lymphocytic depletion in the spleen, rather than immune activation, as well as depletion of alveolar macrophages in the lungs, with inefficient reactive neutrophilic activation. Increased bone marrow Vt, on the other hand, suggests hematopoietic activation in response to systemic immune cell depletion and leukocytosis and could have prognostic relevance. In vivo PET imaging provided better understanding of organ-level pathophysiology during EBOV infection. A similar approach can be used to delineate the pathophysiology of other systemic infections and to evaluate the effectiveness of newly developed treatment and vaccine strategies.

Rhesus Macaque CODEX Multiplexed Immunohistochemistry Panel for Studying Immune Responses During Ebola Infection.

Non-human primate (NHP) animal models are an integral part of the drug research and development process. For some biothreat pathogens, animal model challenge studies may offer the only possibility to evaluate medical countermeasure efficacy. A thorough understanding of host immune responses in such NHP models is therefore vital. However, applying antibody-based immune characterization techniques to NHP models requires extensive reagent development for species compatibility. In the case of studies involving high consequence pathogens, further optimization for use of inactivated samples may be required. Here, we describe the first optimized CO-Detection by indEXing (CODEX) multiplexed tissue imaging antibody panel for deep profiling of spatially resolved single-cell immune responses in rhesus macaques. This 21-marker panel is composed of a set of 18 antibodies that stratify major immune cell types along with a set three Ebola virus (EBOV)-specific antibodies. We validated these two sets of markers using immunohistochemistry and CODEX in fully inactivated Formalin-Fixed Paraffin-Embedded (FFPE) tissues from mock and EBOV challenged macaques respectively and provide an efficient framework for orthogonal validation of multiple antibody clones using CODEX multiplexed tissue imaging. We also provide the antibody clones and oligonucleotide tag sequences as a valuable resource for other researchers to recreate this reagent set for future studies of tissue immune responses to EBOV infection and other diseases.

Longitudinal in vivo imaging of acute neuropathology in a monkey model of Ebola virus infection.

Ebola virus (EBOV) causes neurological symptoms yet its effects on the central nervous system (CNS) are not well-described. Here, we longitudinally assess the acute effects of EBOV on the brain, using quantitative MR-relaxometry, 18F-Fluorodeoxyglucose PET and immunohistochemistry in a monkey model. We report blood-brain barrier disruption, likely related to high cytokine levels and endothelial viral infection, with extravasation of fluid, Gadolinium-based contrast material and albumin into the extracellular space. Increased glucose metabolism is also present compared to the baseline, especially in the deep gray matter and brainstem. This regional hypermetabolism corresponds with mild neuroinflammation, sporadic neuronal infection and apoptosis, as well as increased GLUT3 expression, consistent with increased neuronal metabolic demands. Neuroimaging changes are associated with markers of disease progression including viral load and cytokine/chemokine levels. Our results provide insight into the pathophysiology of CNS involvement with EBOV and may help assess vaccine/treatment efficacy in real time.

Multimodal Imaging and Spatial Analysis of Ebola Retinal Lesions in 14 Survivors of Ebola Virus Disease.

Importance: Differentiation between Ebola retinal lesions and other retinal pathologies in West Africa is important, and the pathogenesis of Ebola retinal disease remains poorly understood. Objective: To describe the appearance of Ebola virus disease (EVD) retinal lesions using multimodal imaging to enable inferences on potential pathogenesis. Design, Setting, and Participants: This prospective case series study was carried out at 34 Military Hospital in Freetown, Sierra Leone. Ophthalmological images were analyzed from 14 consecutively identified survivors of EVD of Sierra Leonean origin who had identified Ebola retinal lesions. Main Outcomes and Measures: Multimodal imaging findings including ultra-widefield scanning laser ophthalmoscopy, fundus autofluorescence, swept-source optical coherence tomography (OCT), Humphrey visual field analysis, and spatial analysis. Results: The 14 study participants had a mean (SD) age of 37.1 (8.8) years; 6 (43%) were women. A total of 141 Ebola retinal lesions were observed in 22 of 27 eyes (81%) of these 14 survivors on ultra-widefield imaging. Of these, 41 lesions (29.1%) were accessible to OCT imaging. Retinal lesions were predominantly nonpigmented with a pale-gray appearance. Peripapillary lesions exhibited variable curvatures in keeping with the retinal nerve fiber layer projections. All lesions respected the horizontal raphe and spared the fovea. The OCT imaging demonstrated a V-shaped hyperreflectivity of the outer nuclear layer overlying discontinuities of the ellipsoid zone and interdigitation zone in the smaller lesions. Larger lesions caused a collapse of the retinal layers and loss of retinal thickness. Lesion shapes were variable, but sharp angulations were characteristic. Perilesional areas of dark without pressure (thinned ellipsoid zone hyporeflectivity) accompanied 125 of the 141 lesions (88.7%) to varying extents. Conclusions and Relevance: We demonstrate OCT evidence of localized pathological changes at the level of the photoreceptors in small lesions among survivors of EVD with retinal lesions. The relevance of associated areas of dark without pressure remains undetermined.

Development and Deployment of the OpenMRS-Ebola Electronic Health Record System for an Ebola Treatment Center in Sierra Leone.

BACKGROUND: Stringent infection control requirements at Ebola treatment centers (ETCs), which are specialized facilities for isolating and treating Ebola patients, create substantial challenges for recording and reviewing patient information. During the 2014-2016 West African Ebola epidemic, paper-based data collection systems at ETCs compromised the quality, quantity, and confidentiality of patient data. Electronic health record (EHR) systems have the potential to address such problems, with benefits for patient care, surveillance, and research. However, no suitable software was available for deployment when large-scale ETCs opened as the epidemic escalated in 2014. OBJECTIVE: We present our work on rapidly developing and deploying OpenMRS-Ebola, an EHR system for the Kerry Town ETC in Sierra Leone. We describe our experience, lessons learned, and recommendations for future health emergencies. METHODS: We used the OpenMRS platform and Agile software development approaches to build OpenMRS-Ebola. Key features of our work included daily communications between the development team and ground-based operations team, iterative processes, and phased development and implementation. We made design decisions based on the restrictions of the ETC environment and regular user feedback. To evaluate the system, we conducted predeployment user questionnaires and compared the EHR records with duplicate paper records. RESULTS: We successfully built OpenMRS-Ebola, a modular stand-alone EHR system with a tablet-based application for infectious patient wards and a desktop-based application for noninfectious areas. OpenMRS-Ebola supports patient tracking (registration, bed allocation, and discharge); recording of vital signs and symptoms; medication and intravenous fluid ordering and monitoring; laboratory results; clinician notes; and data export. It displays relevant patient information to clinicians in infectious and noninfectious zones. We implemented phase 1 (patient tracking; drug ordering and monitoring) after 2.5 months of full-time development. OpenMRS-Ebola was used for 112 patient registrations, 569 prescription orders, and 971 medication administration recordings. We were unable to fully implement phases 2 and 3 as the ETC closed because of a decrease in new Ebola cases. The phase 1 evaluation suggested that OpenMRS-Ebola worked well in the context of the rollout, and the user feedback was positive. CONCLUSIONS: To our knowledge, OpenMRS-Ebola is the most comprehensive adaptable clinical EHR built for a low-resource setting health emergency. It is designed to address the main challenges of data collection in highly infectious environments that require robust infection prevention and control measures and it is interoperable with other electronic health systems. Although we built and deployed OpenMRS-Ebola more rapidly than typical software, our work highlights the challenges of having to develop an appropriate system during an emergency rather than being able to rapidly adapt an existing one. Lessons learned from this and previous emergencies should be used to ensure that a set of well-designed, easy-to-use, pretested health software is ready for quick deployment in future.

Electron Microscopy of Ebola Virus-Infected Cells.

Ebola virus (EBOV) replicates in host cells, where both viral and cellular components show morphological changes during the process of viral replication from entry to budding. These steps in the replication cycle can be studied using electron microscopy (EM), including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), which is one of the most useful methods for visualizing EBOV particles and EBOV-infected cells at the ultrastructural level. This chapter describes conventional methods for EM sample preparation of cultured cells infected with EBOV.

Visualizing Ebolavirus Particles Using Single-Particle Interferometric Reflectance Imaging Sensor (SP-IRIS).

This chapter describes an approach for the label-free imaging and quantification of intact Ebola virus (EBOV) and EBOV viruslike particles (VLPs) using a light microscopy technique. In this technique, individual virus particles are captured onto a silicon chip that has been printed with spots of virus-specific capture antibodies. These captured virions are then detected using an optical approach called interference reflectance imaging. This approach allows for the detection of each virus particle that is captured on an antibody spot and can resolve the filamentous structure of EBOV VLPs without the need for electron microscopy. Capture of VLPs and virions can be done from a variety of sample types ranging from tissue culture medium to blood. The technique also allows automated quantitative analysis of the number of virions captured. This can be used to identify the virus concentration in an unknown sample. In addition, this technique offers the opportunity to easily image virions captured from native solutions without the need for additional labeling approaches while offering a means of assessing the range of particle sizes and morphologies in a quantitative manner.

Maritime Radiology on Operation GRITROCK.

RFA ARGUS deployed on Operation (Op) GRITROCK between Oct 2014 and Apr 2015 to provide support to the United Kingdom (UK) response to the Ebola crisis. This article describes the radiology capability on board ARGUS within the Primary Casualty Receiving Facility (PCRF).

The Frankfurt Ebola patient.

UNLABELLED: Since the Ebola virus was discovered in 1976, the largest outbreak to date is the ongoing epidemic in West Africa based on the number of cases. The number of infected people is high among aid workers, some of whom have been treated at intensive care units in specialized centers in Europe and the USA. A 38-year-old patient who got infected with the Ebola virus was treated in a special isolation ward at the Frankfurt University Hospital from 10/3/14 to 11/19/14. During intensive care of the patient, X-rays were essential for control of the cardiopulmonary system and for follow-up. Special guidelines had to be considered for performing X-rays due to the risk of transmitting the virus. These are presented and discussed in the following. KEY POINTS: Chest radiographs are essential in the intensive care monitoring of Ebola patients. Chest radiographs help to assess the extent of pulmonary edema and capillary leak syndrome. With careful observance of hygiene guidelines, he risk of transmission can be virtually eliminated.

Performance of bedside diagnostic ultrasound in an Ebola isolation unit: the Emory University Hospital experience.

OBJECTIVE: Individuals with Ebola virus disease, a contagious and potentially lethal infection, are now being treated in specialized units in the United States. We describe Emory University's initial experience, current operating procedures, and ongoing planning with diagnostic ultrasound in the isolation unit. CONCLUSION: Ultrasound use has been limited to date. Future planning considerations include deciding what types of ultrasound studies will be performed, which personnel will acquire the images, and which ultrasound machine will be used.

Radiographic imaging for patients with contagious infectious diseases: how to acquire chest radiographs of patients infected with the Ebola virus.

OBJECTIVE: Contagious infectious diseases add a new dimension to radiology and pose many unanswered questions. In particular, what is the safest way to image patients with contagious and potentially lethal infectious diseases? Here, we describe protocols used by Emory University to successfully acquire chest radiographs of patients with Ebola virus disease. CONCLUSION: Radiology departments need to develop new protocols for various modalities used in imaging patients with contagious and potentially lethal infectious diseases.

Ebola virus disease: radiology preparedness.

At present, there is a major emphasis on Ebola virus disease (EVD) preparedness training at medical facilities throughout the United States. Failure to have proper EVD procedures in place was cited as a major reason for infection of medical personnel in the United States. Medical imaging does not provide diagnosis of EVD, but patient assessment in the emergency department and treatment isolation care unit is likely to require imaging services. The purpose of this article is to present an overview of relevant aspects of EVD disease and preparedness relevant to the radiologic community.