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Lassa fever (LF) causes multisystem disease and has a fatality rate <70%. Severe cases exhibit abnormal coagulation, endothelial barrier disruption, and dysfunctional platelet aggregation but the underlying mechanisms remain poorly understood. In Sierra Leone during 2015-2018, we assessed LF patients' day-of-admission plasma samples for levels of proteins necessary for coagulation, fibrinolysis, and platelet function. P-selectin, soluble endothelial protein C receptor, soluble thrombomodulin, plasminogen activator inhibitor 1, ADAMTS-13, von Willebrand factor, tissue factor, soluble intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 were more elevated in LF patients than in controls. Endothelial protein C receptor, thrombomodulin, intercellular adhesion molecule 1, plasminogen activator inhibitor 1, D-dimer, and hepatocyte growth factor were higher in fatal than nonfatal LF cases. Platelet disaggregation occurred only in samples from fatal LF cases. The impaired homeostasis and platelet dysfunction implicate alterations in the protein C pathway, which might contribute to the loss of endothelial barrier function in fatal infections.

Situated in southeastern Sierra Leone, Kenema Government Hospital (KGH) maintains one of the world's only Lassa fever isolation wards and was a strategic Ebola virus disease (EVD) treatment facility during the 2014 EVD outbreak. Since 2006, the Viral Hemorrhagic Fever Consortium (VHFC) has carried out research activities at KGH, capturing clinical and laboratory data for suspected cases of Lassa fever. Here we describe the approach, progress, and challenges in designing and maintaining a data capture and management system (DCMS) at KGH to assist infectious disease researchers in building and sustaining DCMS in low-resource environments. Results on screening patterns and case-fatality rates are provided to illustrate the context and scope of the DCMS covered in this study. A medical records system and DCMS was designed and implemented between 2010 and 2016 linking historical and prospective Lassa fever data sources across KGH Lassa fever units and its peripheral health units. Data were captured using a case report form (CRF) system, enzyme-linked immunosorbent assay (ELISA) plate readers, polymerase chain reaction (PCR) machines, blood chemistry analyzers, and data auditing procedures. Between 2008 and 2016, blood samples for 4,229 suspected Lassa fever cases were screened at KGH, ranging from 219 samples in 2008 to a peak of 760 samples in 2011. Lassa fever case-fatality rates before and following the Ebola outbreak were 65.5% (148/226) and 89.5% (17/19), respectively, suggesting that fewer, but more seriously ill subjects with Lassa fever presented to KGH following the 2014 EVD outbreak (p = .040). DCMS challenges included weak specificity of the Lassa fever suspected case definition, limited capture of patient survival outcome data, internet costs, lapses in internet connectivity, low bandwidth, equipment and software maintenance, lack of computer teaching laboratories, and workload fluctuations due to variable screening activity. DCMS are the backbone of international research efforts and additional literature is needed on the topic for establishing benchmarks and driving goal-based approaches for its advancement in developing countries.

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modify putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 and 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.

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.

Monoclonal antibodies can mediate protection against Ebola virus (EBOV) infection through direct neutralization as well as through the recruitment of innate immune effector functions. However, the antibody functional response following survival of acute EBOV disease has not been well characterized. In this study, serum antibodies from Ebola virus disease (EVD) survivors from Sierra Leone were profiled to capture variation in overall subclass/isotype abundance, neutralizing activity, and innate immune effector functions. Antibodies from EVD survivors exhibited robust innate immune effector functions, mediated primarily by IgG1 and IgA1. In conclusion, development of functional antibodies follows survival of acute EVD.

The recent Ebola epidemic exemplified the importance of understanding and controlling emerging infections. Despite the importance of T cells in clearing virus during acute infection, little is known about Ebola-specific CD8⁺ T cell responses. We investigated immune responses of individuals infected with Ebola virus (EBOV) during the 2013–2016 West Africa epidemic in Sierra Leone, where the majority of the >28,000 EBOV disease (EVD) cases occurred. We examined T cell memory responses to seven of the eight Ebola proteins (GP, sGP, NP, VP24, VP30, VP35, and VP40) and associated HLA expression in survivors. Of the 30 subjects included in our analysis, CD8⁺ T cells from 26 survivors responded to at least one EBOV antigen. A minority, 10 of 26 responders (38%), made CD8⁺ T cell responses to the viral GP or sGP. In contrast, 25 of the 26 responders (96%) made response to viral NP, 77% to VP24 (20 of 26), 69% to VP40 (18 of 26), 42% (11 of 26) to VP35, with no response to VP30. Individuals making CD8⁺ T cells to EBOV VP24, VP35, and VP40 also made CD8⁺ T cells to NP, but rarely to GP. We identified 34 CD8⁺ T cell epitopes for Ebola. Our data indicate the immunodominance of the EBOV NP-specific T cell response and suggest that its inclusion in a vaccine along with the EBOV GP would best mimic survivor responses and help boost cell-mediated immunity during vaccination.

Lassa virus infects hundreds of thousands of people each year across rural West Africa, resulting in a high number of cases of Lassa fever (LF), a febrile disease associated with high morbidity and significant mortality. The lack of approved treatments or interventions underscores the need for an effective vaccine. At least four viral lineages circulate in defined regions throughout West Africa with substantial interlineage nucleotide and amino acid diversity. An effective vaccine should be designed to elicit Lassa virus specific humoral and cell mediated immunity across all lineages. Most current vaccine candidates use only lineage IV antigens encoded by Lassa viruses circulating around Sierra Leone, Liberia, and Guinea but not Nigeria where lineages I-III are found. As previous infection is known to protect against disease from subsequent exposure, we sought to determine whether LF survivors from Nigeria and Sierra Leone harbor memory T cells that respond to lineage IV antigens. Our results indicate a high degree of cross-reactivity of CD8+ T cells from Nigerian LF survivors to lineage IV antigens. In addition, we identified regions within the Lassa virus glycoprotein complex and nucleoprotein that contributed to these responses while T cell epitopes were not widely conserved across our study group. These data are important for current efforts to design effective and efficient vaccine candidates that can elicit protective immunity across all Lassa virus lineages.

Lassa fever, a hemorrhagic fever caused by Lassa virus (LASV), is endemic in West Africa. It is difficult to distinguish febrile illnesses that are common in West Africa from Lassa fever based solely on a patient’s clinical presentation. The field performance of recombinant antigen-based Lassa fever immunoassays was compared to that of quantitative polymerase chain assays (qPCRs) using samples from subjects meeting the case definition of Lassa fever presenting to Kenema Government Hospital in Sierra Leone. The recombinant Lassa virus (ReLASV) enzyme-linked immunosorbant assay (ELISA) for detection of viral antigen in blood performed with 95% sensitivity and 97% specificity using a diagnostic standard that combined results of the immunoassays and qPCR. The ReLASV rapid diagnostic test (RDT), a lateral flow immunoassay based on paired monoclonal antibodies to the Josiah strain of LASV (lineage IV), performed with 90% sensitivity and 100% specificity. ReLASV immunoassays performed better than the most robust qPCR currently available, which had 82% sensitivity and 95% specificity. The performance characteristics of recombinant antigen-based Lassa virus immunoassays indicate that they can aid in the diagnosis of LASV Infection and inform the clinical management of Lassa fever patients.

Background. The 2013-2016 West African Ebola virus disease (EVD) epidemic is the largest recorded. Triage on the basis of clinical signs had limited success, and the time to diagnosis by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) could exceed 5 days. Here we describe the development and field validation of the ReEBOV Antigen Rapid Test (ReEBOV RDT) to aid triage of individuals with suspected EVD. Methods. Samples from patients with suspected EVD were submitted to Kenema Government Hospital, Sierra Leone, for Lassa fever and EVD screening throughout 2014. Banked residual clinical samples were tested in November 2014 and January 2015 in a blinded field trial to estimate the clinical effectiveness of the ReEBOV RDT, compared with EBOV-specific qRT-PCR. Results. Preliminary ReEBOV RDT performance demonstrated a positive percentage agreement (PPA) of 91.1% (195 of 214 results; 95% confidence interval [CI], 86.5%-94.6%) and a negative percentage agreement (NPA) of 90.2% (175 of 194; 95% CI, 85.1%-94.0%). The final estimates used by the Food and Drug Administration to determine whether to grant emergency use authorization for the test, which excluded a qRT-PCR reference method threshold cutoff, were a PPA of 62.1% (72 of 116 results; 95% CI, 52.6%-70.9%) and a NPA of 96.7% (58 of 60; 95% CI, 88.5%-99.6%), with a diagnostic likelihood of 18.6. A subsequent, independent evaluation by the World Health Organization generated results consistent with the preliminary performance estimates. Conclusions. The ReEBOV RDT demonstrated the potential to provide clinically effective rapid and accurate point-of-care test results and, thus, to be a powerful tool for increasing triage efficiency.

Background. Ebola virus disease (EVD) is a severe viral illness caused by Ebola virus (EBOV). The 2013-2016 EVD outbreak in West Africa is the largest recorded, with > 11 000 deaths. Development of the ReEBOV Antigen Rapid Test (ReEBOV RDT) was expedited to provide a point-of-care test for suspected EVD cases. Methods. Recombinant EBOV viral protein 40 antigen was used to derive polyclonal antibodies for RDT and enzyme-linked immunosorbent assay development. ReEBOV RDT limits of detection (LOD), specificity, and interference were analytically validated on the basis of Food and Drug Administration (FDA) guidance. Results. The ReEBOV RDT specificity estimate was 95% for donor serum panels and 97% for donor whole-blood specimens. The RDT demonstrated sensitivity to 3 species of Ebolavirus (Zaire ebolavirus, Sudan ebolavirus, and Bundibugyo ebolavirus) associated with human disease, with no cross-reactivity by pathogens associated with non-EBOV febrile illness, including malaria parasites. Interference testing exhibited no reactivity by medications in common use. The LOD for antigen was 4.7 ng/test in serum and 9.4 ng/test in whole blood. Quantitative reverse transcription-polymerase chain reaction testing of nonhuman primate samples determined the range to be equivalent to 3.0 × 10⁵ -9.0 × 10⁸ genomes/mL. Conclusions. The analytical validation presented here contributed to the ReEBOV RDT being the first antigen-based assay to receive FDA and World Health Organization emergency use authorization for this EVD outbreak, in February 2015.