Intensive Care Unit-Like Care of Nonhuman Primates with Ebola Virus Disease.

BACKGROUND: Ebola virus disease (EVD) supportive care strategies are largely guided by retrospective observational research. This study investigated the effect of EVD supportive care algorithms on duration of survival in a controlled nonhuman primate (NHP) model. METHODS: Fourteen rhesus macaques were challenged intramuscularly with a target dose of Ebola virus (1000 plaque-forming units; Kikwit). NHPs were allocated to intensive care unit (ICU)-like algorithms (n = 7), intravenous fluids plus levofloxacin (n = 2), or a control group (n = 5). The primary outcome measure was duration of survival, and secondary outcomes included changes in clinical laboratory values. RESULTS: Duration of survival was not significantly different between the pooled ICU-like algorithm and control groups (8.2 vs 6.9 days of survival; hazard ratio; 0.50; P = .25). Norepinephrine was effective in transiently maintaining baseline blood pressure. NHPs treated with ICU-like algorithms had delayed onset of liver and kidney injury. CONCLUSIONS: While an obvious survival difference was not observed with ICU-like care, clinical observations from this model may aid in EVD supportive care NHP model refinement.

Will There Be a Cure for Ebola?

Despite the unprecedented Ebola virus outbreak response in West Africa, no Ebola medical countermeasures have been approved by the US Food and Drug Administration. However, multiple valuable lessons have been learned about the conduct of clinical research in a resource-poor, high risk-pathogen setting. Numerous therapeutics were explored or developed during the outbreak, including repurposed drugs, nucleoside and nucleotide analogues (BCX4430, brincidofovir, favipiravir, and GS-5734), nucleic acid-based drugs (TKM-Ebola and AVI-7537), and immunotherapeutics (convalescent plasma and ZMapp). We review Ebola therapeutics progress in the aftermath of the West Africa Ebola virus outbreak and attempt to offer a glimpse of a path forward.

Clinical Laboratory Values as Early Indicators of Ebola Virus Infection in Nonhuman Primates.

The Ebola virus (EBOV) outbreak in West Africa during 2013-2016 demonstrated the need to improve Ebola virus disease (EVD) diagnostics and standards of care. This retrospective study compared laboratory values and clinical features of 3 nonhuman primate models of lethal EVD to assess associations with improved survival time. In addition, the study identified laboratory values useful as predictors of survival, surrogates for EBOV viral loads, and triggers for initiation of therapeutic interventions in these nonhuman primate models. Furthermore, the data support that, in nonhuman primates, the Makona strain of EBOV may be less virulent than the Kikwit strain of EBOV. The applicability of these findings as potential diagnostic and management tools for EVD in humans warrants further investigation.

Activity of Norspermidine on Bacterial Biofilms of Multidrug-Resistant Clinical Isolates Associated with Persistent Extremity Wound Infections.

Biofilm formation is a major virulence factor for numerous pathogenic bacteria and is cited as a central event in the pathogenesis of chronic human infections, which is in large part due to excessive extracellular matrix secretion and metabolic changes that occur within the biofilm rendering them highly tolerant to antimicrobial treatments. Polyamines, including norspermidine, play central roles in bacterial biofilm development, but have also recently been shown to inhibit biofilm formation in select strains of various pathogenic bacteria. The aim of this study was to evaluate in vitro the biofilm dispersive and inhibitory activities of norspermidine against multidrug-resistant clinical isolates of Acinetobacter baumannii(n = 4), Klebsiella pneumoniae (n = 3), Pseudomonas aeruginosa (n = 5) and Staphylococcus aureus (n = 4) associated with chronic extremity wound infections using the semi-quantitative 96-well plate method and confocal laser microscopy. In addition to the antibiofilm activity, biocompatibility of norspermidine was also evaluated by measuring toxicity in vitro to human cell lines and whole porcine tissue explants using MTT viability assay and histological analysis. Norspermidine (5-20 mM) had variable dispersive and inhibitory activity on biofilms which was dependent on both the strain and species. Of the clinical bacterial species evaluated herein, A. baumannii isolates were the most sensitive to the effect of norspermidine, which was in part due to the inhibitory effects of norspermidine on bacterial motility and expression of genes involved in the production of homoserine lactones and quorum sensing molecules both essential for biofilm formation. Importantly, exposure of cell lines and whole tissues to norspermidine for prolonged periods of time (≥24 h) was observed to reduce viability and alter tissue histology in a time and concentration dependent manner, with 20 mM exposure having the greatest negative effects on both tissues and individual cell lines. Collectively our findings demonstrate that, similar to other polyamines, norspermidine displays both inhibitory and dispersive activities on biofilms of clinical multidrug-resistant bacterial isolates, in particular for strains of A. baumannii. Additionally our findings suggest that direct application may be considered on tissues, albeit for limited exposure times.

Monitoring Exposure to Ebola and Health of U.S. Military Personnel Deployed in Support of Ebola Control Efforts - Liberia, October 25, 2014-February 27, 2015.

In response to the unprecedented Ebola virus disease (Ebola) outbreak in West Africa, the U.S. government deployed approximately 2,500 military personnel to support the government of Liberia. Their primary missions were to construct Ebola treatment units (ETUs), train health care workers to staff ETUs, and provide laboratory testing capacity for Ebola. Service members were explicitly prohibited from engaging in activities that could result in close contact with an Ebola-infected patient or coming in contact with the remains of persons who had died from unknown causes. Military units performed twice-daily monitoring of temperature and review of exposures and symptoms ("unit monitoring") on all persons throughout deployment, exit screening at the time of departure from Liberia, and post-deployment monitoring for 21 days at segregated, controlled monitoring areas on U.S. military installations. A total of 32 persons developed a fever during deployment from October 25, 2014, through February 27, 2015; none had a known Ebola exposure or developed Ebola infection. Monitoring of all deployed service members revealed no Ebola exposures or infections. Given their activity restrictions and comprehensive monitoring while deployed to Liberia, U.S. military personnel constitute a unique population with a lower risk for Ebola exposure compared with those working in the country without such measures.

Biofilm formation by clinical isolates and its relevance to clinical infections.

Reports of biofilms have increased exponentially in the scientific literature over the past two decades, yet the vast majority of these are basic science investigations with limited clinical relevance. Biofilm studies involving clinical isolates are most often surveys of isolate collections, but suffer from lack of standardization in methodologies for producing and assessing biofilms. In contrast, more informative clinical studies correlating biofilm formation to patient data have infrequently been reported. In this chapter, biofilm surveys of clinical isolates of aerobic and anaerobic bacteria, mycobacteria, and Candida are reviewed, as well as those pertaining to the unique situation of cystic fibrosis. In addition, the influence of host components on in vitro biofilm formation, as well as published studies documenting the clinical impact of biofilms in human infections, are presented.

Dakin solution alters macrophage viability and function.

BACKGROUND: Macrophages are important in wound defense and healing. Dakin's solution (DS), buffered sodium hypochlorite, has been used since World War I as a topical antimicrobial for wound care. DS has been shown to be toxic to host cells, but effects on immune cells are not well documented. MATERIALS AND METHODS: DS at 0.5%, 0.125%, and ten-fold serial dilutions from 0.25%-0.00025% were evaluated for cellular toxicity on murine macrophages (J774A.1). The effect of DS on macrophage adhesion, phagocytosis, and generation of reactive oxygen species was examined. Macrophage polarization following DS exposure was determined by gene expression using quantitative real-time polymerase chain reaction. RESULTS: Concentrations of DS >0.0025% reduced macrophage viability to <5% in exposure times as short as 30 s. Similarly, phagocytosis of Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus flavus were significantly reduced at all tested concentrations by macrophages pretreated with DS. H2O2 production was reduced by 8%-38% following treatment with 0.00025%-0.125% DS. Macrophage adherence was significantly increased with >0.0025% DS after 15 min of exposure compared with controls. Quantitative real-time polymerase chain reaction demonstrated that DS exposure resulted in classical macrophage activation, with increased expression of inducible nitric oxide synthase 2, interferon-γ, and interleukin (IL)-1ß. CONCLUSIONS: DS at clinically used concentrations (0.025%-0.25%) was detrimental to macrophage survival and function. For optimal clinical use, understanding the impact of DS on macrophages is important as depletion may result in impaired pathogen clearance and delayed healing. These findings indicate that 0.00025% DS is a safe starting dose; however, optimal use of DS requires further validation with in vivo models.

Daptomycin-induced acute eosinophilic pneumonia: analysis of the current data and illustrative case reports.

Acute eosinophilic pneumonia (AEP) is a rare but important complication of daptomycin therapy. We describe 2 cases of daptomycin-associated AEP, compile available data from another 22 published cases, and propose a revised set of diagnostic criteria.

Gastric volvulus, Borchardt's Triad, and Endoscopy: A Rare Twist.

The authors describe a case of gastric volvulus, which is a rare cause of gastric outlet obstruction. An 85-year-old man presented with nausea, vomiting, and epigastric pain. Admission abdominal radiograph demonstrated a grossly distended stomach with air-fluid levels. Multiple attempts at nasogastric tube placement failed. Endoscopy revealed a fluid-filled, tortuous stomach with a paraesophageal hernia, and the operator was unable to locate or pass the scope through the pylorus. Traditionally Borchardt's triad is believed to be diagnostic for acute gastric volvulus and consists of unproductive retching, epigastric pain and distention, and the inability to pass a nasogastric tube. The authors propose that the following features on endoscopy are highly suggestive of the most common type of volvulus (organoaxial): tortuous stomach, paraesophageal hernia, and inability to locate or pass the scope through the pylorus.

Celiac disease presenting as severe osteopenia.

The authors describe a unique presentation of celiac disease as multiple non-traumatic fractures in a young male without gastrointestinal complaints. A 29-year-old man presented with back pain and was found to have a non-traumatic compression fracture of the lumbar and thoracic spine on plain X-ray. Dual-energy x-ray absorptiometry (DXA) confirmed osteoporosis at the L3/L4 vertebral bodies. Parathyroid hormone (PTH), calcium, and vitamin D levels were normal. He had no gastrointestinal complaints, but serologic studies were positive to include an elevated gliadin IgA Ab, gliadin IgG Ab, and an elevated tissue transglutaminase IgA Ab. He was treated with a gluten-free diet, calcium, and vitamin D supplementation as well as teriparatide. Follow up bone density showed improvement and has no further fractures to date. Primary care physicians, gastroenterologists, and endocrinologists must have a high index of clinical suspicion for celiac disease in any patient who presents with low bone density regardless of the serum 25-OH vitamin D levels or presence of gastrointestinal complaints.

Antibiotic desensitization therapy in secondary syphilis and Listeria infection: case reports and review of desensitization therapy.

Two adult cases, one of secondary syphilis and one of Listeria monocytogenes bacteremia, in which antibiotic desensitization therapy was utilized to assist treatment of active infection in the face of severe penicillin allergy. Clinical considerations are discussed that led to the decision to employ a formal desensitization procedure. Antibiotic desensitization protocols can facilitate optimal and safe antibiotic therapy in the appropriate clinical setting.

Natural History of Aerosol-Induced Ebola Virus Disease in Rhesus Macaques.

Ebola virus disease (EVD) is a serious global health concern because case fatality rates are approximately 50% due to recent widespread outbreaks in Africa. Well-defined nonhuman primate (NHP) models for different routes of Ebola virus exposure are needed to test the efficacy of candidate countermeasures. In this natural history study, four rhesus macaques were challenged via aerosol with a target titer of 1000 plaque-forming units per milliliter of Ebola virus. The course of disease was split into the following stages for descriptive purposes: subclinical, clinical, and decompensated. During the subclinical stage, high levels of venous partial pressure of carbon dioxide led to respiratory acidemia in three of four of the NHPs, and all developed lymphopenia. During the clinical stage, all animals had fever, viremia, and respiratory alkalosis. The decompensatory stage involved coagulopathy, cytokine storm, and liver and renal injury. These events were followed by hypotension, elevated lactate, metabolic acidemia, shock and mortality similar to historic intramuscular challenge studies. Viral loads in the lungs of aerosol-exposed animals were not distinctly different compared to previous intramuscularly challenged studies. Differences in the aerosol model, compared to intramuscular model, include an extended subclinical stage, shortened clinical stage, and general decompensated stage. Therefore, the shortened timeframe for clinical detection of the aerosol-induced disease can impair timely therapeutic administration. In summary, this nonhuman primate model of aerosol-induced EVD characterizes early disease markers and additional details to enable countermeasure development.

The utilization of advance telemetry to investigate critical physiological parameters including electroencephalography in cynomolgus macaques following aerosol challenge with eastern equine encephalitis virus.

Most alphaviruses are mosquito-borne and can cause severe disease in humans and domesticated animals. In North America, eastern equine encephalitis virus (EEEV) is an important human pathogen with case fatality rates of 30-90%. Currently, there are no therapeutics or vaccines to treat and/or prevent human infection. One critical impediment in countermeasure development is the lack of insight into clinically relevant parameters in a susceptible animal model. This study examined the disease course of EEEV in a cynomolgus macaque model utilizing advanced telemetry technology to continuously and simultaneously measure temperature, respiration, activity, heart rate, blood pressure, electrocardiogram (ECG), and electroencephalography (EEG) following an aerosol challenge at 7.0 log10 PFU. Following challenge, all parameters were rapidly and substantially altered with peak alterations from baseline ranged as follows: temperature (+3.0-4.2°C), respiration rate (+56-128%), activity (-15-76% daytime and +5-22% nighttime), heart rate (+67-190%), systolic (+44-67%) and diastolic blood pressure (+45-80%). Cardiac abnormalities comprised of alterations in QRS and PR duration, QTc Bazett, T wave morphology, amplitude of the QRS complex, and sinoatrial arrest. An unexpected finding of the study was the first documented evidence of a critical cardiac event as an immediate cause of euthanasia in one NHP. All brain waves were rapidly (~12-24 hpi) and profoundly altered with increases of up to 6,800% and severe diffuse slowing of all waves with decreases of ~99%. Lastly, all NHPs exhibited disruption of the circadian rhythm, sleep, and food/fluid intake. Accordingly, all NHPs met the euthanasia criteria by ~106-140 hpi. This is the first of its kind study utilizing state of the art telemetry to investigate multiple clinical parameters relevant to human EEEV infection in a susceptible cynomolgus macaque model. The study provides critical insights into EEEV pathogenesis and the parameters identified will improve animal model development to facilitate rapid evaluation of vaccines and therapeutics.

Safety and immunogenicity of an inactivated eastern equine encephalitis virus vaccine.

BACKGROUND: Eastern equine encephalitis virus (EEEV) is a mosquito borne alphavirus spread primarily in Atlantic and Gulf Coast regions of the United States. EEEV is the causative agent of a devastating meningoencephalitis syndrome, with approximately 30% mortality and significant morbidity. There is no licensed human vaccine against EEEV. An inactivated EEEV vaccine has been offered under investigational new drug (IND) protocols at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) since 1976. METHODS: Healthy at-risk laboratory personnel received inactivated PE-6 strain EEEV (TSI-GSD 104) vaccine under two separate IND protocols. Protocol FY 99-11 (2002-2008) had a primary series consisting of doses on day 0, 7, and 28. Protocol FY 06-31 (2008-2016) utilized a primary series with doses on day 0 and 28, and month 6. Participants with an inadequate immune response, plaque reduction neutralization test with 80% cut-off (PRNT80) titer < 40, received booster vaccination. Volunteers with prior EEEV vaccination were eligible to enroll for booster doses based on annual titer evaluation. RESULTS: The FY06-31 dosing schema resulted in significantly greater post-primary series immune response (PRNT80 ≥ 40) rates (84% vs 54%) and geometric mean titers (184.1 vs 39.4). The FY 06-31 dosing schema also resulted in significantly greater cumulative annual immune response rates from 1 to up to 7 years post vaccination (75% vs 59%) and geometric mean of titers (60.1 vs 43.0). The majority of probably or definitely related adverse events were mild and local; there were no probably or definitely related serious adverse events. CONCLUSIONS: Inactivated PE-6 EEEV vaccine is safe and immunogenic in at-risk laboratory personnel. A prolonged primary series, with month 6 dose, significantly improved vaccine immunogenicity both post-primary series and longitudinally on annual titers. Despite decades of safe use under IND, full licensure is not planned due to manufacturing constraints, and ongoing development of alternatives.

The Western Equine Encephalitis Lyophilized, Inactivated Vaccine: An Update on Safety and Immunogenicity.

Background: Western Equine Encephalitis (WEE) is a naturally acquired infection and potentially devastating bioweapon, with no specific human countermeasures. An experimental inactivated Western Equine Encephalitis Vaccine (WEEV; WEE TSI-GSD 210) has been used under an IND (investigational New Drug) protocol at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) since 1976. Methods: Over 24 years from 1987 to 2011, 876 subjects received 3 primary vaccine doses under 3 studies with 1,537 booster doses administered (FY87-8, phase 2, laboratory workers, vaccine lots 1-81-1, 1-81-2, and 2-1-91; FY99-12, phase 2 laboratory workers, lot 2-1-91; and FY09-02, phase 1 healthy volunteer, lot 3-1-92). Post-vaccination safety and immunogenicity [plaque reduction neutralization test 80% (PRNT80) > 1:40] were analyzed. Results: Overall PRNT80 response to the primary series in FY87-8 was 42% (326/770) but dropped to 16% (14/87) in FY99-12, prompting study FY09-02, which achieved 89% (17/19). The first booster response rate was 68% (814/1194) in FY87-8, 53% (171/324) in FY99-12, and 100% (10/10) in FY09-02. The majority of definitely related adverse reactions (AEs) were mild and local with no definitely related serious AEs. No laboratory acquired WEE infection was documented during this period despite 4 reported exposures in vaccinated subjects. Conclusion: The TSI-GSD 210 WEE vaccine was immunogenic, safe and well tolerated. Use of this vaccine could be considered in an emergency setting. Despite decades of safe and effective use under IND, full licensure is not planned due to manufacturing constraints, and a strategic decision to develop alternatives. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT01159561.

Pre-positioned Outbreak Research: The Joint Medical Emerging Diseases Intervention Clinical Capability Experience in Uganda.

The West Africa Ebola virus disease outbreak of 2014-2016 demonstrated that responses to viral hemorrhagic fever epidemics must go beyond emergency stopgap measures and should incorporate high-quality medical care and clinical research. Optimal patient management is essential to improving outcomes, and it must be implemented regardless of geographical location or patient socioeconomic status. Coupling clinical research with improved care has a significant added benefit: Improved data quality and management can guide the development of more effective supportive care algorithms and can support regulatory approvals of investigational medical countermeasures (MCMs), which can alter the cycle of emergency response to reemerging pathogens. However, executing clinical research during outbreaks of high-consequence pathogens is complicated and comes with ethical and research regulatory challenges. Aggressive care and excellent quality control must be balanced by the requirements of an appropriate infection prevention and control posture for healthcare workers and by overcoming the resource limitations inherent in many outbreak settings. The Joint Mobile Emerging Disease Intervention Clinical Capability was established in 2015 to develop a high-quality clinical trial capability in Uganda to support rigorous evaluation of MCMs targeting high-consequence pathogens like Ebola virus. This capability assembles clinicians, laboratorians, clinical researchers, logisticians, and regulatory professionals trained in infection prevention and control and in good clinical and good clinical laboratory practices. The resulting team is prepared to provide high-quality medical care and clinical research during high-consequence outbreaks.

Natural History of Aerosol Induced Lassa Fever in Non­Human Primates.

Lassa virus (LASV), an arenavirus causing Lassa fever, is endemic to West Africa with up to 300,000 cases and between 5000 and 10,000 deaths per year. Rarely seen in the United States, Lassa virus is a CDC category A biological agent inasmuch deliberate aerosol exposure can have high mortality rates compared to naturally acquired infection. With the need for an animal model, specific countermeasures remain elusive as there is no FDA-approved vaccine. This natural history of aerosolized Lassa virus exposure in Macaca fascicularis was studied under continuous telemetric surveillance. The macaque response to challenge was largely analogous to severe human disease with fever, tachycardia, hypotension, and tachypnea. During initial observations, an increase trend of activated monocytes positive for viral glycoprotein was accompanied by lymphocytopenia. Disease uniformly progressed to high viremia followed by low anion gap, alkalosis, anemia, and thrombocytopenia. Hypoproteinemia occurred late in infection followed by increased levels of white blood cells, cytokines, chemokines, and biochemical markers of liver injury. Viral nucleic acids were detected in tissues of three non­survivors at endpoint, but not in the lone survivor. This study provides useful details to benchmark a pivotal model of Lassa fever in support of medical countermeasure development for both endemic disease and traditional biodefense purposes.

An Emerging Biothreat: Crimean-Congo Hemorrhagic Fever Virus in Southern and Western Asia.

Tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV) is endemic in numerous countries, but the epidemiology and epizoology of Crimean-Congo hemorrhagic fever (CCHF) remain to be defined for most regions of the world. Using a broad database search approach, we reviewed the literature on CCHF and CCHFV in Southern and Western Asia to better define the disease burden in these areas. We used a One Health approach, moving beyond a focus solely on human disease burden to more comprehensively define this burden by reviewing CCHF case reports, human and animal CCHFV seroprevalence studies, and human and animal CCHFV isolations. In addition, we used published literature to estimate the distribution of Hyalomma ticks and infection of these ticks by CCHFV. Using these data, we propose a new classification scheme for organizing the evaluated countries into five categories by level of evidence for CCHF endemicity. Twelve countries have reported CCHF cases, five from Southern Asia and seven from Western Asia. These were assigned to level 1 or 2. Eleven countries that have evidence of vector circulation but did not report confirmed CCHF cases were assigned to level 3 or 4. This classification scheme was developed to inform policy toward strengthening CCHF disease surveillance in the Southern and Western Asia regions. In particular, the goal of this review was to inform international organizations, local governments, and health-care professionals about current shortcomings in CCHFV surveillance in these two high-prevalence regions.

The Joint Mobile Emerging Disease Clinical Capability (JMEDICC) laboratory approach: Capabilities for high-consequence pathogen clinical research.

Following the 2013-2016 Ebola virus outbreak in West Africa, numerous groups advocated for the importance of executing clinical trials in outbreak settings. The difficulties associated with obtaining reliable data to support regulatory approval of investigational vaccines and therapeutics during that outbreak were a disappointment on a research and product development level, as well as on a humanitarian level. In response to lessons learned from the outbreak, the United States Department of Defense established a multi-institute project called the Joint Mobile Emerging Disease Intervention Clinical Capability (JMEDICC). JMEDICC's primary objective is to establish the technical capability in western Uganda to execute clinical trials during outbreaks of high-consequence pathogens such as the Ebola virus. A critical component of clinical trial execution is the establishment of laboratory operations. Technical, logistical, and political challenges complicate laboratory operations, and these challenges have been mitigated by JMEDICC to enable readiness for laboratory outbreak response operations.