Ebola Virus-Specific Neutralizing Antibody Persists at High Levels in Survivors 2 Years After Resolution of Disease in a Sierra Leonean Cohort.

Ebola virus (EBOV) infection results in Ebola virus disease (EVD), an often severe disease with a nonspecific presentation. Since its recognition, periodic outbreaks of EVD continue to occur in sub-Saharan Africa. The 2013-2016 West African EVD outbreak was the largest recorded, resulting in a substantial cohort of EVD survivors with persistent health complaints and variable immune responses. In this study, we characterize humoral immune responses in EVD survivors and their contacts in Eastern Sierra Leone. We found high levels of EBOV IgG in EVD survivors and lower yet substantial antibody levels in household contacts, suggesting subclinical transmission. Neutralizing antibody function was prevalent but variable in EVD survivors, raising questions about the durability of immune responses from natural infection with EBOV. Additionally, we found that certain discrete symptoms-ophthalmologic and auditory-are associated with EBOV IgG seropositivity, while an array of symptoms are associated with the presence of neutralizing antibody.

Novel Tools for Lassa Virus Surveillance in Peri-domestic Rodents.

Background: Lassa fever (LF) is a rodent-borne disease endemic to West Africa. In the absence of licensed therapeutics or vaccines, rodent exclusion from living spaces remains the primary method of preventing LF. Zoonotic surveillance of Lassa virus (LASV), the etiologic agent of LF, can assess the burden of LASV in a region and guide public health measures against LF. Methods: In this study, we adapted commercially available LASV human diagnostics to assess the prevalence of LASV in peri-domestic rodents in Eastern Sierra Leone. Small mammal trapping was conducted in Kenema district, Sierra Leone between November 2018-July 2019. LASV antigen was detected using a commercially available LASV NP antigen rapid diagnostic test. LASV IgG antibodies against LASV nucleoprotein (NP) and glycoprotein (GP) were tested by adapting a commercially available semi-quantitative enzyme linked immunosorbent assay (ELISA) for detection of mouse-related and rat-related species IgG. Findings: Of the 373 tested specimens, 74 (20%) tested positive for LASV antigen. 40 (11%) specimens tested positive for LASV NP IgG, while an additional 12 (3%) specimens only tested positive for LASV GP IgG. Simultaneous antigen presence and IgG antibody presence was linked in Mastomys sp. specimens (p < 0.01), but not Rattus sp. specimens (p = 1). Despite the link between antigen presence and IgG antibody presence in Mastomys sp., the strength of antigen response did not correlate with the strength of IgG response to either GP IgG or NP IgG. Interpretation: The tools developed in this study can aid in the generation of valuable public health data for rapid field assessment of LASV burden during outbreak investigations and general LASV surveillance. Funding: Funding for this work was supported by the National Institute of Allergy and Infectious Diseases National Institute of Health, Department of Health and Human Services under the following grants: International Collaboration in Infectious Disease Research on Lassa fever and Ebola - ICIDR - U19 AI115589, Consortium for Viral Systems Biology - CViSB - 5U19AI135995, West African Emerging Infectious Disease Research Center - WARN-ID - U01AI151812, West African Center for Emerging Infectious Diseases: U01AI151801.

Seroprevalence of anti-Lassa Virus IgG antibodies in three districts of Sierra Leone: A cross-sectional, population-based study.

BACKGROUND: Lassa virus (LASV), the cause of the acute viral hemorrhagic illness Lassa fever (LF), is endemic in West Africa. Infections in humans occur mainly after exposure to infected excrement or urine of the rodent-host, Mastomys natalensis. The prevalence of exposure to LASV in Sierra Leone is crudely estimated and largely unknown. This cross-sectional study aimed to establish a baseline point seroprevalence of IgG antibodies to LASV in three administrative districts of Sierra Leone and identify potential risk factors for seropositivity and LASV exposure. METHODOLOGY AND PRINCIPAL FINDINGS: Between 2015 and 2018, over 10,642 participants from Kenema, Tonkolili, and Port Loko Districts were enrolled in this cross-sectional study. Previous LASV and LF epidemiological studies support classification of these districts as "endemic," "emerging," and "non-endemic", respectively. Dried blood spot samples were tested for LASV antibodies by ELISA to determine the seropositivity of participants, indicating previous exposure to LASV. Surveys were administered to each participant to assess demographic and environmental factors associated with a higher risk of exposure to LASV. Overall seroprevalence for antibodies to LASV was 16.0%. In Kenema, Port Loko, and Tonkolili Districts, seroprevalences were 20.1%, 14.1%, and 10.6%, respectively. In a multivariate analysis, individuals were more likely to be LASV seropositive if they were living in Kenema District, regardless of sex, age, or occupation. Environmental factors contributed to an increased risk of LASV exposure, including poor housing construction and proximity to bushland, forested areas, and refuse. CONCLUSIONS AND SIGNIFICANCE: In this study we determine a baseline LASV seroprevalence in three districts which will inform future epidemiological, ecological, and clinical studies on LF and the LASV in Sierra Leone. The heterogeneity of the distribution of LASV and LF over both space, and time, can make the design of efficacy trials and intervention programs difficult. Having more studies on the prevalence of LASV and identifying potential hyper-endemic areas will greatly increase the awareness of LF and improve targeted control programs related to LASV.

The prevalence of Post-Ebola Syndrome hearing loss, Sierra Leone.

BACKGROUND: Globally, hearing loss is the second leading cause of disability, affecting approximately 18.7% of the world's population. However, the burden of hearing loss is unequally distributed, with the majority of affected individuals located in Asia or Sub-Saharan Africa. Following the 2014 West African Ebola Outbreak, disease survivors began to describe hearing loss as part of the constellation of symptoms known as Post-Ebola Syndrome. The goal of this study was to more fully characterize hearing loss among Ebola Virus Disease (EVD) survivors. METHODOLOGY AND PRINCIPAL FINDINGS: EVD survivors and their household contacts were recruited (n = 1,12) from Eastern Sierra Leone. Each individual completed a symptom questionnaire, physical exam, and a two-step audiometry process measuring both air and bone conduction thresholds. In comparison to contacts, EVD survivors were more likely to have complaints or abnormal findings affecting every organ system. A significantly greater percentage of EVD survivors were found to have hearing loss in comparison to contacts (23% vs. 9%, p < 0.001). Additionally, survivors were more likely to have bilateral hearing loss of a mixed etiology. Logistic regression revealed that the presence of any symptoms of middle or inner ear (p < 0.001), eye (p = 0.005), psychiatric (p = 0.019), and nervous system (p = 0.037) increased the odds of developing hearing loss. CONCLUSIONS AND SIGNIFICANCE: This study is the first to use an objective and standardized measurement to report hearing loss among EVD survivors in a clinically meaningful manner. In this study it was found that greater than 1/5th of EVD survivors develop hearing loss. The association between hearing impairment and symptoms affecting the eye and nervous system may indicate a similar mechanism of pathogenesis, which should be investigated further. Due to the quality of life and socioeconomic detriments associated with untreated hearing loss, a greater emphasis must be placed on understanding and mitigating hearing loss following survival to aid in economic recovery following infectious disease epidemics.

Immunomodulatory potential of in vivo natural killer T (NKT) activation by NKTT320 in Mauritian-origin cynomolgus macaques.

Invariant natural killer T-lymphocytes (iNKT) are unique immunomodulatory innate T cells with an invariant TCRα recognizing glycolipids presented on MHC class-I-like CD1d molecules. Activated iNKT rapidly secrete pro-and anti-inflammatory cytokines, potentiate immunity, and modulate inflammation. Here, we report the effects of in vivo iNKT activation in Mauritian-origin cynomolgus macaques by a humanized monoclonal antibody, NKTT320, that binds to the invariant region of the iNKT TCR. NKTT320 led to rapid iNKT activation, increased polyfunctionality, and elevation of multiple plasma analytes within 24 hours. Flow cytometry and RNA-Seq confirmed downstream activation of multiple immune subsets, enrichment of JAK/STAT and PI3K/AKT pathway genes, and upregulation of inflammation-modulating genes. NKTT320 also increased iNKT frequency in adipose tissue and did not cause iNKT anergy. Our data indicate that NKTT320 has a sustained effect on in vivo iNKT activation, potentiation of innate and adaptive immunity, and resolution of inflammation, which supports its future use as an immunotherapeutic.

Cross-Reactive Antibodies to SARS-CoV-2 and MERS-CoV in Pre-COVID-19 Blood Samples from Sierra Leoneans.

Many countries in sub-Saharan Africa have experienced lower COVID-19 caseloads and fewer deaths than countries in other regions worldwide. Under-reporting of cases and a younger population could partly account for these differences, but pre-existing immunity to coronaviruses is another potential factor. Blood samples from Sierra Leonean Lassa fever and Ebola survivors and their contacts collected before the first reported COVID-19 cases were assessed using enzyme-linked immunosorbent assays for the presence of antibodies binding to proteins of coronaviruses that infect humans. Results were compared to COVID-19 subjects and healthy blood donors from the United States. Prior to the pandemic, Sierra Leoneans had more frequent exposures than Americans to coronaviruses with epitopes that cross-react with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), SARS-CoV, and Middle Eastern respiratory syndrome coronavirus (MERS-CoV). The percentage of Sierra Leoneans with antibodies reacting to seasonal coronaviruses was also higher than for American blood donors. Serological responses to coronaviruses by Sierra Leoneans did not differ by age or sex. Approximately a quarter of Sierra Leonian pre-pandemic blood samples had neutralizing antibodies against SARS-CoV-2 pseudovirus, while about a third neutralized MERS-CoV pseudovirus. Prior exposures to coronaviruses that induce cross-protective immunity may contribute to reduced COVID-19 cases and deaths in Sierra Leone.

The Origins and Future of Sentinel: An Early-Warning System for Pandemic Preemption and Response.

While investigating a signal of adaptive evolution in humans at the gene LARGE, we encountered an intriguing finding by Dr. Stefan Kunz that the gene plays a critical role in Lassa virus binding and entry. This led us to pursue field work to test our hypothesis that natural selection acting on LARGE-detected in the Yoruba population of Nigeria-conferred resistance to Lassa Fever in some West African populations. As we delved further, we conjectured that the "emerging" nature of recently discovered diseases like Lassa fever is related to a newfound capacity for detection, rather than a novel viral presence, and that humans have in fact been exposed to the viruses that cause such diseases for much longer than previously suspected. Dr. Stefan Kunz's critical efforts not only laid the groundwork for this discovery, but also inspired and catalyzed a series of events that birthed Sentinel, an ambitious and large-scale pandemic prevention effort in West Africa. Sentinel aims to detect and characterize deadly pathogens before they spread across the globe, through implementation of its three fundamental pillars: Detect, Connect, and Empower. More specifically, Sentinel is designed to detect known and novel infections rapidly, connect and share information in real time to identify emerging threats, and empower the public health community to improve pandemic preparedness and response anywhere in the world. We are proud to dedicate this work to Stefan Kunz, and eagerly invite new collaborators, experts, and others to join us in our efforts.

Space-Time Trends in Lassa Fever in Sierra Leone by ELISA Serostatus, 2012-2019.

Lassa fever (LF) is a viral hemorrhagic disease found in Sub-Saharan Africa and is responsible for up to 300,000 cases and 5000 deaths annually. LF is highly endemic in Sierra Leone, particularly in its Eastern Province. Kenema Government Hospital (KGH) maintains one of only a few LF isolation facilities in the world with year-round diagnostic testing. Here we focus on space-time trends for LF occurring in Sierra Leone between 2012 and 2019 to provide a current account of LF in the wake of the 2014-2016 Ebola epidemic. Data were analyzed for 3277 suspected LF cases and classified as acute, recent, and non-LF or prior LF exposure using enzyme-linked immunosorbent assays (ELISAs). Presentation rates for acute, recent, and non-LF or prior LF exposure were 6.0% (195/3277), 25.6% (838/3277), and 68.4% (2244/3277), respectively. Among 2051 non-LF or prior LF exposures, 33.2% (682/2051) tested positive for convalescent LF exposure. The overall LF case-fatality rate (CFR) was 78.5% (106/135). Both clinical presentations and confirmed LF cases declined following the Ebola epidemic. These declines coincided with an increased duration between illness onset and clinical presentation, perhaps suggesting more severe disease or presentation at later stages of illness. Acute LF cases and their corresponding CFRs peaked during the dry season (November to April). Subjects with recent (but not acute) LF exposure were more likely to present during the rainy season (May to October) than the dry season (p < 0.001). The findings here suggest that LF remains endemic in Sierra Leone and that caseloads are likely to resume at levels observed prior to the Ebola epidemic. The results provide insight on the current epidemiological profile of LF in Sierra Leone to facilitate LF vaccine studies and accentuate the need for LF cohort studies and continued advancements in LF diagnostics.

Post-Ebola Syndrome Presents With Multiple Overlapping Symptom Clusters: Evidence From an Ongoing Cohort Study in Eastern Sierra Leone.

BACKGROUND: Following the 2013-2016 West African Ebola outbreak, distinct, persistent health complaints were recognized in Ebola virus disease (EVD) survivors. Here we provide an in-depth characterization of post-Ebola syndrome >2.5 years after resolution of disease. Additionally, we report subphenotypes of post-Ebola syndrome with overlapping symptom clusters in survivors from Eastern Sierra Leone. METHODS: Participants in Eastern Sierra Leone were identified by the Sierra Leone Association of Ebola survivors. Survivors and their contacts were administered a questionnaire assessing self-reported symptoms and a physical examination. Comparisons between survivors and contacts were conducted using conditional logistic regression. Symptom groupings were identified using hierarchical clustering approaches. Simplified presentation of incredibly complex evaluations (SPICE), correlation analysis, logistic regression, and principal component analysis (PCA) were performed to explore the relationships between symptom clusters. RESULTS: Three hundred seventy-five EVD survivors and 1040 contacts were enrolled into the study. At enrollment, EVD survivors reported significantly more symptoms than their contacts in all categories (P < .001). Symptom clusters representing distinct organ systems were identified. Correlation and logistic regression analysis identified relationships between symptom clusters, including stronger relationships between clusters including musculoskeletal symptoms (r = 0.63, P < .001; and P < .001 for correlation and logistic regression, respectively). SPICE and PCA further highlighted subphenotypes with or without musculoskeletal symptoms. CONCLUSIONS: This study presents an in-depth characterization of post-Ebola syndrome in Sierra Leonean survivors >2.5 years after disease. The interrelationship between symptom clusters indicates that post-Ebola syndrome is a heterogeneous disease. The distinct musculoskeletal and non-musculoskeletal phenotypes identified likely require targeted therapies to optimize long-term treatment for EVD survivors.

SARS-CoV-2 seroprevalence rates of children seeking medical care in Louisiana during the state stay at home order.

Serologic testing of residual blood samples from 812 children from a hospital in New Orleans, LA, between March and May 2020, demonstrated a SARS-CoV-2 seroprevalence of 6.8% based on S and N protein IgG; Black and Hispanic children, and children living in zip codes with lower household incomes were over-represented.

Pericytes as novel targets for HIV/SIV infection in the lung.

Antiretroviral therapy in HIV patients has lengthened lifespan but led to an increased risk for secondary comorbidities, such as pulmonary complications characterized by vascular dysfunction. In the lung, PDGFRß+ mesenchymal cells known as pericytes intimately associate with endothelial cells and are key for their survival both structurally and through the secretion of prosurvival factors. We hypothesize that in HIV infection there are functional changes in pericytes that may lead to destabilization of the microvasculature and ultimately to pulmonary abnormalities. Our objective in this study was to determine whether lung pericytes could be directly infected with HIV. We leveraged lung samples from macaque lungs with or without SIV infection and normal human lung for in vitro experiments. Pericytes were isolated based on the marker platelet-derived growth factor receptor-ß (PDGFRß). We determined that lung PDGFRß-positive (PDGFRß+) pericytes from both macaques and humans express CD4, the primary receptor for SIV/HIV, as well as the major coreceptors CXCR4 and CCR5. We found cells positive for both PDGFRß and SIV in lungs from infected macaques. Lung pericytes isolated from these animals also harbored detectable SIV. To confirm relevance to human disease, we demonstrated that human lung pericytes are capable of being productively infected by HIV in vitro, with the time course of infection suggesting development of viral latency. In summary, we show for the first time that SIV/HIV directly infects lung pericytes, implicating these cells as a novel target and potential reservoir for the virus in vivo.

SARS-CoV-2 Seroprevalence Rates of Children in Louisiana During the State Stay at Home Order.

BACKGROUNDChildren ([≤]18 years) account for [~]20% of the US population but currently represent <2% of coronavirus disease 2019 (COVID-19) cases. Because infected children often have few or no symptoms and may not be tested, the extent of infection in children is poorly understood. METHODSDuring the March 18th-May 15th 2020 Louisiana Stay At Home Order, 1690 blood samples from 812 individuals from a Childrens Hospital were tested for antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Demographics, COVID-19 testing, and clinical presentation abstracted from medical records were compared with local COVID-19 cases. RESULTSIn total, 62 subjects (7.6%) were found to be seropositive. The median age was 11 years with 50.4% female. The presenting complaint of seropositive patients was chronic illness (43.5%). Only 18.2% had a previous positive COVID-19 PCR or antibody test. Seropositivity was significantly associated with parish (counties), race, and residence in a low-income area. Importantly, seropositivity was linearly correlated with cumulative COVID-19 case number for all ages by parish. CONCLUSIONIn a large retrospective study, the seropositivity prevalence for SARS-CoV-2 in children in Louisiana during the mandated Stay At Home Order was 7.6%. Residence location, race, and lower socioeconomic factors were linked to more frequent seropositivity in children and correlated to regional COVID-19 case rates. Thus, a significant number of children in Louisiana had SARS-CoV-2 infections that went undetected and unreported and may have contributed to virus transmission.

Partnering to harmonize IRBs for community-engaged research to reduce health disparities.

Emerging advances in health disparities research include controlled trials and comparative effectiveness studies that are frequently conducted at multiple community and academic sites. Review by different institutional review boards (IRBs) presents a major impediment to the timely and effective conduct of such research. When research involves minority and underserved communities as well as multiple geographic regions, institutional requirements and interpretation of ethical standards may vary substantially. Such variations can complicate the informed consent process and research protocol, and may undermine participant respect and trial quality. In addition, multiple IRB review can lead to unnecessary delays, jeopardizing funding and capacity to perform collaborative projects. In response to these issues, the Research Centers in Minority Institutions (RCMI) Translational Research Network (RTRN) is developing a community-partnered approach to streamlining IRB review across its consortium of 18 RCMI grantee institutions that will ensure compliance while enhancing the quality of health disparities research.

Oral terbinafine (Lamisil) in the short-term treatment of fungal infections of the skin: results of a post-marketing surveillance study.

Oral terbinafine (Lamisil, Novartis Pharma AG, Basel, Switzerland) is an effective therapy for fungal infections of the skin and nails. A post-marketing surveillance study was undertaken to evaluate the clinical efficacy and safety of oral terbinafine. A total of 454 patients with clinically and mycologically confirmed superficial fungal infections of the skin were enrolled from 79 dermatology clinics. Patients received oral terbinafine (250 mg day-1) for 2 weeks. Specific signs and symptoms were assessed by standard questionnaire before, immediately after, and 4 weeks after treatment. Observed improvements in patients after 2 weeks treatment were: erythema 81%, blistering 33%, exudation 50%, scaling 89%, pruritus 83%. After 4 weeks treatment, erythema was absent in 85% of patients, blistering and exudation in 99.7%, scaling in 82%, and pruritus in 94%. Overall clinical efficacy was assessed as good to excellent in 97% of patients. Adverse effects--mainly gastrointestinal and minor skin rashes--were reported in 5.3% of patients. The results of this study confirm that oral terbinafine is safe and highly effective for the short-term treatment of fungal skin infections.

Benazepril, an angiotensin converting enzyme inhibitor: drug interaction with salbutamol and bronchial response to histamine in normal subjects.

AIMS: To investigate the effect of the angiotensin converting enzyme inhibitor, benazepril, on pulmonary function. METHODS: We investigated the influence of benazepril, on lung function and the interaction with inhaled salbutamol (0.1 to 6.6 mg) and histamine (0.03 to 30.69 g l-1) in normal subjects. Benazepril 20 mg, salbutamol 8 mg, propranolol 160 mg, and placebo were given orally once daily over 10 days. RESULTS: On day 8, there was no difference in the area under the salbutamol dose-response curves between benazepril, placebo and oral salbutamol (P > 0.05), propranolol shifted the curves to the right (P < 0.05). On day 10, histamine challenge resulted in following PD35sGaw values (geometric mean and 95% CI): with placebo 1.02 (0.95-1.09) g l-1, benazepril 1.04 (0.99-1.08), salbutamol 1.19 (1.13-1.25), propranolol 0.57 (0.50-0.65). CONCLUSIONS: Benazepril had no influence on baseline lung function, caused no interaction with inhaled salbutamol and the bronchial response to histamine was similar to placebo. However, our findings in normal subjects cannot be extrapolated automatically to asthmatics.

Disposition of [14C]pinacidil in humans.

Pinacidil [(+/-)-2-cyano-1-(4-pyridyl)-3-(1,2,2-trimethylpropyl)guanidine monohydrate] is a novel, direct-acting vasodilator antihypertensive agent. The cyano 14C-labeled drug is rapidly and completely absorbed after an oral 12.5-mg dose in solution. The blood:plasma concentration ratios (0.8-0.9) indicate transient penetration of radioactivity into blood cells. Blood and plasma tmax (0.5 h) and t 1/2 (4 h) of [14C]pinacidil equivalents are similar. Pinacidil (51%), pinacidil N-oxide (28%), and unidentified polar metabolites (21%) comprise the plasma radioactivity. The plasma t 1/2 of pinacidil is 2-3 h, and that of pinacidil N-oxide is 4-5 h. Renal excretion of radioactivity is the major route (80-90% dose) of drug elimination; fecal elimination accounted for 4% of the dose. Renal clearance of the N-oxide is 10 times the renal clearance of the parent drug and exceeds the creatinine clearance. Biotransformation products in 0-24-h urine samples include pinacidil (10%), pinacidil N-oxide (60%), and free and conjugated analogues of pinacidil and metabolites (30%). Stereoselective metabolism is not a major biotransformation pathway of pinacidil or the N-oxide metabolite.

Adaptation of electrolyte transport in rat large intestine after proximal resection. II. Colon after 50% jejunoilectomy combined with cecectomy.

Electrolyte transport was studied in rat colon adapting to 50% small intestinal resection with cecectomy. Four weeks after surgery, colonic gross surface area, dry and wet weight were increased compared to sham-operated controls. Net absorption in vivo of sodium, chloride and volume was stimulated per organ and per unit tissue mass, and net potassium secretion was diminished. The electrical potential difference, mucosal Na-K-ATPase specific activity and cAMP concentration remained unaffected. In vitro, measurements of unidirectional fluxes and electrical parameters across isolated mucosa indicated enhanced electrically neutral sodium chloride absorption in the proximal and possibly diminished bicarbonate secretion in the distal colon. Thus, removal of the cecum in addition to partial jejunoilectomy induces profound adaptive changes in the colon, involving not only mucosal growth but also a functional reaction of the individual epithelial cell.

Mechanism of action of diphenolic laxatives: the role of adenylate cyclase and mucosal permeability.

Net fluid movement and mucosal 14C-erythritol clearance were measured in ligated colonic loops of the rat in vivo. The diphenolic laxatives, oxyphenisatin and bisacodyl, dose-dependently inhibited net fluid absorption or caused secretion, both increased the 14C-erythritol clearance. Pretreatment with the adenylate cyclase inhibitor, RMI 12 330 A, did not change these results. It is concluded that diphenolic laxatives mainly influence intestinal fluid transport not by stimulation of mucosal adenylate cyclase but rather by augmenting epithelial permeability.

Differentiation of secretagogue drugs by chlorpromazine in rat intestine in vivo.

The effect of chlorpromazine (CPZ) on passive epithelial permeability and net fluid movement induced by secretagogues was tested in the rat intestine in vivo. CPZ, in a dose of 20 mg/kg intramuscularly, did not alter colonic permeability either in control conditions or during increased permeability caused by deoxycholic acid (DOC) or bisacodyl. Fluid secretion induced by cholera toxin and theophylline was strongly reduced by CPZ. The effects of oxyphenisatin and bisacodyl were only slightly but significantly inhibited by CPZ, whereas the action of DOC was unaffected. It is concluded, that the increase of the epithelial permeability is the main reason for the augmented fluid secretion caused by DOC. Bisacodyl and oxyphenisatin seem to act partly via an increase in permeability and to some degree via an induction of an active secretory process.