Roots, Not Parachutes: Research Collaborations Combat Outbreaks.

Recent infectious disease epidemics illustrate how health systems failures anywhere can create disease vulnerabilities everywhere. We must therefore prioritize investments in health care infrastructure in outbreak-prone regions of the world. We describe how "rooted" research collaborations can establish capacity for pathogen surveillance and facilitate rapid outbreak responses.

Avatar Mice Underscore the Role of the T Cell-Dendritic Cell Crosstalk in Ebola Virus Disease and Reveal Mechanisms of Protection in Survivors.

Ebola virus disease (EVD) is a complex infectious disease characterized by high inflammation, multiorgan failure, the dysregulation of innate and adaptive immune responses, and coagulation abnormalities. Evidence accumulated over the last 2 decades indicates that, during fatal EVD, the infection of antigen-presenting cells (APC) and the dysregulation of T cell immunity preclude a successful transition between innate and adaptive immunity, which constitutes a key disease checkpoint. In order to better understand the contribution of the APC-T cell crosstalk to EVD pathophysiology, we have developed avatar mice transplanted with human, donor-specific APCs and T cells. Here, we show that the transplantation of T cells and APCs from Ebola virus (EBOV)-naive individuals into avatar mice results in severe disease and death and that this phenotype is dependent on T cell receptor (TCR)-major histocompatibility complex (MCH) recognition. Conversely, avatar mice were rescued from death induced by EBOV infection after the transplantation of both T cells and plasma from EVD survivors. These results strongly suggest that protection from EBOV reinfection requires both cellular and humoral immune memory responses. IMPORTANCE The crosstalk between dendritic cells and T cells marks the transition between innate and adaptive immune responses, and it constitutes an important checkpoint in EVD. In this study, we present a mouse avatar model in which T cell and dendritic cell interactions from a specific donor can be studied during EVD. Our findings indicate that T cell receptor-major histocompatibility complex-mediated T cell-dendritic cell interactions are associated with disease severity, which mimics the main features of severe EVD in these mice. Resistance to an EBOV challenge in the model was achieved via the transplantation of both survivor T cells and plasma.

Dose-Dependent Response to Infection with Ebola Virus in the Ferret Model and Evidence of Viral Evolution in the Eye.

Filoviruses cause high-consequence infections with limited approved medical countermeasures (MCMs). MCM development is dependent upon well-characterized animal models for the assessment of antiviral agents and vaccines. Following large-scale Ebola virus (EBOV) disease outbreaks in Africa, some survivors are left with long-term sequelae and persistent virus in immune-privileged sites for many years. We report the characterization of the ferret as a model for Ebola virus infection, reproducing disease and lethality observed in humans. The onset of clinical signs is rapid, and EBOV is detected in the blood, oral, and rectal swabs and all tissues studied. We identify viral RNA in the eye (a site of immune privilege) and report on specific genomic changes in EBOV present in this structure. Thus, the ferret model has utility in testing MCMs that prevent or treat long-term EBOV persistence in immune-privileged sites. IMPORTANCE Recent reemergence of Ebola in Guinea that caused over 28,000 cases between 2013 and 2016 has been linked to the original virus from that region. It appears the virus has remained in the region for at least 5 years and is likely to have been maintained in humans. Persistence of Ebola in areas of the body for extended periods of time has been observed, such as in the eye and semen. Despite the importance of reintroduction of Ebola from this route, such events are rare in the population, which makes studying medical interventions to clear persistent virus difficult. We studied various doses of Ebola in ferrets and detected virus in the eyes of most ferrets. We believe this model will enable the study of medical interventions that promote clearance of Ebola virus from sites that promote persistence.

Unique human immune signature of Ebola virus disease in Guinea.

Despite the magnitude of the Ebola virus disease (EVD) outbreak in West Africa, there is still a fundamental lack of knowledge about the pathophysiology of EVD. In particular, very little is known about human immune responses to Ebola virus. Here we evaluate the physiology of the human T cell immune response in EVD patients at the time of admission to the Ebola Treatment Center in Guinea, and longitudinally until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we identify an immune signature that is unique in EVD fatalities. Fatal EVD was characterized by a high percentage of CD4(+) and CD8(+) T cells expressing the inhibitory molecules CTLA-4 and PD-1, which correlated with elevated inflammatory markers and high virus load. Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation, despite comparable overall T cell activation. Concomitant with virus clearance, survivors mounted a robust Ebola-virus-specific T cell response. Our findings suggest that dysregulation of the T cell response is a key component of EVD pathophysiology.

Neurological, Cognitive, and Psychological Findings Among Survivors of Ebola Virus Disease From the 1995 Ebola Outbreak in Kikwit, Democratic Republic of Congo: A Cross-sectional Study.

BACKGROUND: Clinical sequelae of Ebola virus disease (EVD) have not been described more than 3 years postoutbreak. We examined survivors and close contacts from the 1995 Ebola outbreak in Kikwit, Democratic Republic of Congo (DRC), and determined prevalence of abnormal neurological, cognitive, and psychological findings and their association with EVD survivorship. METHODS: From August to September 2017, we conducted a cross-sectional study in Kikwit, DRC. Over 2 decades after the EVD outbreak, we recruited EVD survivors and close contacts from the outbreak to undergo physical examination and culturally adapted versions of the Folstein mini-mental status exam (MMSE) and Goldberg anxiety and depression scale (GADS). We estimated the strength of relationships between EVD survivorship and health outcomes using linear regression models by comparing survivors versus close contacts, adjusting for age, sex, educational level, marital status, and healthcare worker status. RESULTS: We enrolled 20 EVD survivors and 187 close contacts. Among the 20 EVD survivors, 4 (20%) reported at least 1 abnormal neurological symptom, and 3 (15%) had an abnormal neurological examination. Among the 187 close contacts, 14 (11%) reported at least 1 abnormal neurologic symptom, and 9 (5%) had an abnormal neurological examination. EVD survivors had lower mean MMSE and higher mean GADS scores as compared to close contacts (MMSE: adjusted coefficient: -1.85; 95% confidence interval [CI]: -3.63, -0.07; GADS: adjusted coefficient: 3.91; 95% CI: 1.76, 6.04). CONCLUSIONS: EVD survivors can have lower cognitive scores and more symptoms of depression and anxiety than close contacts more than 2 decades after Ebola virus outbreaks.

Ebola virus glycoprotein directly triggers T lymphocyte death despite of the lack of infection.

Fatal outcomes of Ebola virus (EBOV) infections are typically preceded by a 'sepsis-like' syndrome and lymphopenia despite T cells being resistant to Ebola infection. The mechanisms that lead to T lymphocytes death remain largely unknown; however, the degree of lymphopenia is highly correlative with fatalities. Here we investigated whether the addition of EBOV or its envelope glycoprotein (GP) to isolated primary human CD4+ T cells induced cell death. We observed a significant decrease in cell viability in a GP-dependent manner, which is suggestive of a direct role of GP in T cell death. Using immunoprecipitation assays and flow cytometry, we demonstrate that EBOV directly binds to CD4+ T cells through interaction of GP with TLR4. Transcriptome analysis revealed that the addition of EBOV to CD4+ T cells results in the significant upregulation of pathways associated with interferon signaling, pattern recognition receptors and intracellular activation of NFκB signaling pathway. Both transcriptome analysis and specific inhibitors allowed identification of apoptosis and necrosis as mechanisms associated with the observed T cell death following exposure to EBOV. The addition of the TLR4 inhibitor CLI-095 significantly reduced CD4+ T cell death induced by GP. EBOV stimulation of primary CD4+ T cells resulted in a significant increase in secreted TNFα; inhibition of TNFα-mediated signaling events significantly reduced T cell death while inhibitors of both necrosis and apoptosis similarly reduced EBOV-induced T cell death. Lastly, we show that stimulation with EBOV or GP augments monocyte maturation as determined by an overall increase in expression levels of markers of differentiation. Subsequently, the increased rates of cellular differentiation resulted in higher rates of infection further contributing to T cell death. These results demonstrate that GP directly subverts the host's immune response by increasing the susceptibility of monocytes to EBOV infection and triggering lymphopenia through direct and indirect mechanisms.

Kinetics of Soluble Mediators of the Host Response in Ebola Virus Disease.

Background: The pathophysiology of Ebola virus disease (EVD) is still poorly understood. This study aimed at identifying soluble biomarkers that inform on disease mechanisms. Methods: Fifty-four soluble mediators of the immune, coagulation, and endothelial system were measured in baseline and follow-up samples from hospitalized patients with EVD, using Luminex technology. Cross-sectional expression levels and changes over time were correlated with outcome. Results: Levels of circulating proinflammatory cytokines and chemokines, as well as markers of endothelial dysfunction and coagulopathy, were elevated on admission to hospital in patients who died from EVD as compared to survivors. These markers further increased in patients who died and/or decreased over time in survivors. In contrast, markers of gut integrity and T-cell response were higher in survivors and increased until discharge. Conclusions: Inflammatory response, endothelial integrity, gastric tissue protection, and T cell immunity play a role in EVD pathophysiology.

Musculoskeletal manifestations of Ebola virus.

The 2014 West African Ebola virus disease outbreak shocked the world as it swept through the region leaving Guinea, Liberia and Sierra Leone struggling to gain control. As the largest Ebola virus disease outbreak to date, there are more survivors in its wake than ever before, with a spectrum of health problems requiring management. Here we review various musculoskeletal manifestations of the virus that can occur both during and after the infection, and consider possible pathogenesis.

Ebola Virus Disease in Humans: Pathophysiology and Immunity.

Viruses of the Ebolavirus genus cause sporadic epidemics of severe and systemic febrile disease that are fueled by human-to-human transmission. Despite the notoriety of ebolaviruses, particularly Ebola virus (EBOV), as prominent viral hemorrhagic fever agents, and the international concern regarding Ebola virus disease (EVD) outbreaks, very little is known about the pathophysiology of EVD in humans and, in particular, about the human immune correlates of survival and immune memory. This lack of basic knowledge about physiological characteristics of EVD is probably attributable to the dearth of clinical and laboratory data gathered from past outbreaks. The unprecedented magnitude of the EVD epidemic that occurred in West Africa from 2013 to 2016 has allowed, for the first time, evaluation of clinical, epidemiological, and immunological parameters in a significant number of patients using state-of-the-art laboratory equipment. This review will summarize the data from the literature regarding human pathophysiologic and immunologic responses to filoviral infection.

Characteristics and Outcomes of Pediatric Patients With Ebola Virus Disease Admitted to Treatment Units in Liberia and Sierra Leone: A Retrospective Cohort Study.

BACKGROUND: The clinical and virologic characteristics of Ebola virus disease (EVD) in children have not been thoroughly documented. METHODS: Consecutive children aged <18 years with real-time polymerase chain reaction (RT-PCR)-confirmed EVD were enrolled retrospectively in 5 Ebola treatment units in Liberia and Sierra Leone in 2014/2015. Data collection and medical management were based on standardized International Medical Corps protocols. We performed descriptive statistics, multivariate logistic regression, and Kaplan-Meier survival analyses. RESULTS: Of 122 children enrolled, the median age was 7 years and one-third were aged <5 years. The female-to-male ratio was 1.3. The most common clinical features at triage and during hospitalization were fever, weakness, anorexia, and diarrhea, although 21% of patients were initially afebrile and 6 patients remained afebrile. Bleeding was rare at presentation (5%) and manifested subsequently in fewer than 50%. The overall case fatality rate was 57%. Factors associated with death in bivariate analyses were age <5 years, bleeding at any time during hospitalization, and high viral load. After adjustment with logistic regression modeling, the odds of death were 14.8-fold higher if patients were aged <5 years, 5-fold higher if the patient had any evidence of bleeding, and 5.2-fold higher if EVD RT-PCR cycle threshold value was ≤20. Plasmodium parasitemia had no impact on EVD outcomes. CONCLUSIONS: Age <5 years, bleeding, and high viral loads were poor prognostic indicators of children with EVD. Research to understand mechanisms of these risk factors and the impact of dehydration and electrolyte imbalance will improve health outcomes.

Symptom- and Laboratory-Based Ebola Risk Scores to Differentiate Likely Ebola Infections.

Rapidly identifying likely Ebola patients is difficult because of a broad case definition, overlap of symptoms with common illnesses, and lack of rapid diagnostics. However, rapid identification is critical for care and containment of contagion. We analyzed retrospective data from 252 Ebola-positive and 172 Ebola-negative patients at a Sierra Leone Ebola treatment center to develop easy-to-use risk scores, based on symptoms and laboratory tests (if available), to stratify triaged patients by their likelihood of having Ebola infection. Headache, diarrhea, difficulty breathing, nausea/vomiting, loss of appetite, and conjunctivitis comprised the symptom-based score. The laboratory-based score also included creatinine, creatine kinase, alanine aminotransferase, and total bilirubin. This risk score correctly identified 92% of Ebola-positive patients as high risk for infection; both scores correctly classified >70% of Ebola-negative patients as low or medium risk. Clinicians can use these risk scores to gauge the likelihood of triaged patients having Ebola while awaiting laboratory confirmation.

Sequelae and Other Conditions in Ebola Virus Disease Survivors, Sierra Leone, 2015.

We rapidly assessed the health of Ebola virus disease (EVD) survivors in Kenema, Sierra Leone, by reviewing medical charts of all patients attending the Survivor Clinic of Kenema Government Hospital. Data were abstracted on signs and symptoms at every attendance. As of November 2015, a total of 621 attendances by 115 survivors with laboratory-confirmed EVD were made to the Survivor Clinic. Most (60.9%) survivors were women. Survivors' median age was 28 years (range 0.25-70 years). Survivors attended the clinic a median of 5 times (range 1-21 times) each, and the median time from EVD discharge to attendance was 261 days (range 4-504 days). The most commonly reported signs and symptoms among the 621 attendances were headache (63.1%), fever (61.7%), and myalgia (43.3%). Because health needs of EVD survivors are complex, rapid chart reviews at survivor clinics should be repeated regularly to assess the extent of illness and prioritize service delivery.

Characteristics of the musculoskeletal symptoms observed among survivors of Ebola virus disease in the Postebogui cohort in Guinea.

Objective: Previous studies show that arthralgia is the most common symptom experienced by Ebola virus disease (EVD) survivors. Nevertheless, specific analyses of rheumatological sequelea are still lacking. Methods: The Postebogui study is a prospective, multicentre cohort aiming to evaluate the long-term outcomes of EVD survivors infected during the 2014-15 outbreak in Guinea. Of the 216 participants enrolled in the study in October 2015, 44 patients with arthralgia/myalgia underwent a physical examination by a rheumatologist (the Eborheum cohort). Data were collected using a standardized questionnaire. Results: In the Eborheum cohort, 61% of patients were female, the median age was 31.1 years, and the median time from Ebola Treatment Centre discharge was 8.8 months. Pain manifestation started after Ebola infection in all but one patient. Patients had mainly both mechanical and inflammatory pain (45%) and low back pain (77%). All patients reported pain in at least one peripheral joint. Pain in large joints was more frequently reported than in small joints (73 vs 41%). Oligo- and polyarticular presentations were similar, with symmetrical pain distribution. Furthermore, 36 patients had at least one painful 18-tender point count, most of whom reported extensive pain (n = 19) and symmetrical distribution (91%). Diagnoses were mainly non-specific musculoskeletal disorders (59%) and mechanical back pain (52%). No polyarthritis was observed. We found a higher percentage of depressed patients compared with the remaining Postebogui group (42 vs 11%; P < 0.001). Conclusion: Results from the study come from the first complete rheumatological examination of a cohort of EVD survivors, nearly 9 months after Ebola Treatment Centre discharge. Importantly, we found that patients with arthralgia/myalgia included in the Eborheum cohort were more likely to experience depression than survivors not reporting these symptoms, highlighting the impact of pain symptoms among survivors.

Ebola Virus Disease Is Characterized by Poor Activation and Reduced Levels of Circulating CD16+ Monocytes.

A number of previous studies have identified antigen-presenting cells (APCs) as key targets of Ebola virus (EBOV), but the role of APCs in human Ebola virus disease (EVD) is not known. We have evaluated the phenotype and kinetics of monocytes, neutrophils, and dendritic cells (DCs) in peripheral blood of patients for whom EVD was diagnosed by the European Mobile Laboratory in Guinea. Acute EVD was characterized by reduced levels of circulating nonclassical CD16+ monocytes with a poor activation profile. In survivors, CD16+ monocytes were activated during recovery, coincident with viral clearance, suggesting an important role of this cell subset in EVD pathophysiology.

Necrotizing Scleritis, Conjunctivitis, and Other Pathologic Findings in the Left Eye and Brain of an Ebola Virus-Infected Rhesus Macaque (Macaca mulatta) With Apparent Recovery and a Delayed Time of Death.

A 3.5-year-old adult female rhesus macaque (Macaca mulatta) manifested swelling of the left upper eyelid and conjunctiva and a decline in clinical condition 18 days following intramuscular challenge with Ebola virus (EBOV; Kikwit-1995), after apparent clinical recovery. Histologic lesions with strong EBOV antigen staining were noted in the left eye (scleritis, conjunctivitis, and peri-optic neuritis), brain (choriomeningoencephalitis), stomach, proximal duodenum, and pancreas. Spleen, liver, and adrenal glands, common targets for acute infection, appeared histologically normal with no evidence of EBOV immunoreactivity. These findings may provide important insight for understanding sequelae seen in West African survivors of Ebola virus disease.

Kinetic Analysis of Biomarkers in a Cohort of US Patients With Ebola Virus Disease.

BACKGROUND: Ebola virus (EBOV) infection causes a severe and often fatal disease. Despite the fact that more than 30 000 individuals have acquired Ebola virus disease (EVD), the medical and scientific community still does not have a clear understanding of the mechanisms by which EBOV causes such severe disease. METHODS: In this study, 54 biomarkers in plasma samples serially collected from 7 patients with EVD were analyzed in an attempt to define the kinetics of inflammatory modulators. Two clinical disease groups were defined (moderate and severe) based on the need for clinical support. Biomarkers were evaluated for correlation with viremia and clinical disease in an effort to identify pathways that could be useful targets of therapeutic intervention. RESULTS: Patients with severe disease had higher viremia than those with moderate disease. Several biomarkers of immune activation and control were significantly elevated in patients with moderate disease. A series of pro-inflammatory cytokines and chemokines were significantly elevated in patients with severe disease. CONCLUSIONS: Biomarkers that were associated with severe EVD were proinflammatory and indicative of endothelial or coagulation cascade dysfunction, as has been seen historically in patients with fatal outcomes. In contrast, biomarkers that were associated with moderate EVD were suggestive of a strong interferon response and control of both innate and adaptive responses. Therefore, clinical interventions that modulate the phenotype and magnitude of immune activation may be beneficial in treating EVD.

Ebola Virus Disease in Children, Sierra Leone, 2014-2015.

Little is known about potentially modifiable factors in Ebola virus disease in children. We undertook a retrospective cohort study of children <13 years old admitted to 11 Ebola holding units in the Western Area, Sierra Leone, during 2014-2015 to identify factors affecting outcome. Primary outcome was death or discharge after transfer to Ebola treatment centers. All 309 Ebola virus-positive children 2 days-12 years old were included; outcomes were available for 282 (91%). Case-fatality was 57%, and 55% of deaths occurred in Ebola holding units. Blood test results showed hypoglycemia and hepatic/renal dysfunction. Death occurred swiftly (median 3 days after admission) and was associated with younger age and diarrhea. Despite triangulation of information from multiple sources, data availability was limited, and we identified no modifiable factors substantially affecting death. In future Ebola virus disease epidemics, robust, rapid data collection is vital to determine effectiveness of interventions for children.

Severe Ebola virus disease with vascular leakage and multiorgan failure: treatment of a patient in intensive care.

BACKGROUND: In the current epidemic of Ebola virus disease in western Africa, many aid workers have become infected. Some of these aid workers have been transferred to specialised hospitals in Europe and the USA for intensified treatment, providing the potential for unique insight into the clinical course of Ebola virus disease under optimised supportive measures in isolation units. METHODS: A 38-year-old male doctor who had contracted an Ebola virus infection in Sierra Leone was airlifted to University Hospital Frankfurt, Germany, on day 5 after disease onset. Within 72 h of admission to the hospital's high-level isolation unit, the patient developed signs of severe multiorgan failure, including lungs, kidneys, and gastrointestinal tract. In addition to clinical parameters, the diagnostic work-up included radiography, ultrasound, pulse contour cardiac output technology, and microbiological and clinical chemistry analyses. Respiratory failure with pulmonary oedema and biophysical evidence of vascular leak syndrome needed mechanical ventilation. The patient received a 3 day treatment course with FX06 (MChE-F4Pharma, Vienna, Austria), a fibrin-derived peptide under clinical development for vascular leak syndrome. After FX06 administration and concurrent detection of Ebola-virus-specific antibodies and a fall in viral load, vascular leak syndrome and respiratory parameters substantially improved. We gave broad-spectrum empiric antimicrobial therapy and the patient needed intermittent renal replacement therapy. The patient fully recovered. FINDINGS: This case report shows the feasibility of delivery of successful intensive care therapy to patients with Ebola virus disease under biosafety level 4 conditions. INTERPRETATION: The effective treatment of vascular leakage and multiorgan failure by combination of ventilatory support, antibiotic treatment, and renal replacement therapy can sustain a patient with severe Ebola virus disease until virological remission. FX06 could potentially be a valuable agent in contribution to supportive therapy. FUNDING: University Hospital of Frankfurt.

Lessons learned: critical care management of patients with Ebola in the United States.

OBJECTIVE: This report will describe the preparations for and the provision of care of two patients with Ebola virus disease in the biocontainment unit at the University of Nebraska Medical Center. DATA SOURCES: Patient medical records. STUDY SELECTION: Not applicable. DATA EXTRACTION: Not applicable. DATA SYNTHESIS: Not applicable. CONCLUSIONS: Safe and effective care of patients with Ebola virus disease requires significant communication and planning. Adherence to a predetermined isolation protocol is essential, including proper donning and doffing of personal protective equipment. Location of the patient care area and the logistics of laboratory testing, diagnostic imaging, and the removal of waste must be considered. Patients with Ebola virus disease are often dehydrated and need adequate vascular access for fluid resuscitation, nutrition, and phlebotomy for laboratory sampling. Advanced planning for acute life-threatening events and code status must be considered. Intensivist scheduling should account for the significant amount of time required for the care of patients with Ebola virus disease. With appropriate precautions and resources, designated hospitals in the United States can safely provide care for patients with Ebola virus disease.

Clinical characteristics of 154 patients suspected of having Ebola virus disease in the Ebola holding center of Jui Government Hospital in Sierra Leone during the 2014 Ebola outbreak.

This article sought to analyze the clinical features of 154 patients suspected of having Ebola virus disease (EVD) in an Ebola holding center in Sierra Leone from October 1 through November 9, 2014. We found that 108 of the 154 patients were confirmed with EVD. Eighty-five had known outcomes. Forty-nine of the 85 patients had been exposed to EVD. The average mortality rate was 60%. The mean interval between the onset of symptoms and hospitalization was 5.8 ± 3.3 days. The mean incubation period was 9.2 ± 6.7 days. Common symptoms of the EVD patients on admission were fatigue (85.2%), anorexia (84.3%), fever (75.9%), and headache (72.2%). Our data showed that the total symptoms of confirmed EVD patients were significantly higher than those of non-EVD patients (9 vs. 5.5; p < 0.001). The likelihood of EVD was 87.6% when a patient presented more than 6 out of 21 symptoms on admission. The survivors were significantly younger than non-survivors (24.0 ± 10.0 years vs. 31.3 ± 15.3 years; p = 0.016). The real-time polymerase chain reaction (PCR) analysis showed that, in the survivors, the virus load was significantly lower (Ct value: 25.2 ± 4.1 vs. 28.7 ± 5.7; p = 0.002). Multivariate analysis showed that age, fever, and viral load were independent predictors of mortality. Taken together, our data suggested that a cutoff of six symptoms could be used to predict patients with high or low risk of EVD. It seemed that age, fever, and viral load were the main risk factors associated with EVD mortality.