Seroprevalence of anti-SARS-CoV-2 antibodies in a population living in Bomassa village, Republic of Congo.

Objectives: With limited data available from Central Africa, the aim of our study was to evaluate the anti-SARS-CoV-2 Ab prevalence in indigenous residents of Bomassa, a village located in the Sangha region in the Republic of Congo. Methods: Plasma and oropharyngeal swab samples were collected from 304 healthy adult individuals, randomly recruited in May 2021 before vaccine introduction in the area. In addition, 82 plasma samples from the same area in 2019 were included as controls for the investigation of cross-reactivity against other coronaviruses. The SARS-CoV-2 virus was detected by qRT-PCR and sequenced using next-generation sequencing. ELISA was used for detecting IgG, IgM, and neutralizing Ab against SARS-CoV-2 antigens. Results: Around 4.9% (15/304) of the participants were SARS-CoV-2 positive, with B.1.631 being the only variant identified. Of 109 individuals harboring anti-SARS-CoV-2 IgG and/or IgM Ab, 45.9% (50/109) had anti-SARS-CoV-2 neutralizing Ab. Of the control samples collected before the pandemic, 3.7% (3/82) were positive for IgG, but negative for neutralizing Ab. Conclusions: Seroprevalence against SARS-CoV-2 occurred in 25% of the indigenous population sample, with almost 50% of these seropositive participants possessing neutralizing antibodies. These findings suggest that the spread of SARS-CoV-2 has been underestimated in the Republic of Congo.

Zaire ebolavirus surveillance near the Bikoro region of the Democratic Republic of the Congo during the 2018 outbreak reveals presence of seropositive bats.

On the 8th of May, 2018, an outbreak of Ebola virus disease (EVD) was declared, originating in the Bikoro region of the Democratic Republic of the Congo (DRC) near the border with neighboring Republic of the Congo (ROC). Frequent trade and migration occur between DRC and ROC-based communities residing along the Congo River. In June 2018, a field team was deployed to determine whether Zaire ebolavirus (Ebola virus (EBOV)) was contemporaneously circulating in local bats at the human-animal interface in ROC near the Bikoro EVD outbreak. Samples were collected from bats in the Cuvette and Likouala departments, ROC, bordering the Équateur Province in DRC where the Bikoro EVD outbreak was first detected. EBOV genomic material was not detected in bat-derived samples by targeted quantitative reverse transcription-polymerase chain reaction or by family-level consensus polymerase chain reaction; however, serological data suggests recent exposure to EBOV in bats in the region. We collected serum from 144 bats in the Cuvette department with 6.9% seropositivity against the EBOV glycoprotein and 14.3% seropositivity for serum collected from 27 fruit bats and one Molossinae in the Likouala department. We conclude that proactive investment in longitudinal sampling for filoviruses at the human-animal interface, coupled with ecological investigations are needed to identify EBOV wildlife reservoirs.

Development and validation of portable, field-deployable Ebola virus point-of-encounter diagnostic assay for wildlife surveillance.

Early detection of Ebola virus spillover into wildlife is crucial for rapid response. We developed and validated a portable, cold-chain independent Ebola virus RT-qPCR assay. METHODS: The field syringe-based RNA extraction method was compared with a conventional laboratory-based spin-column RNA extraction method. Next, the qPCR efficiency and limit of detection of the assay was compared to standard laboratory-based reagents and equipment. The specificity of the assay was confirmed by testing against multiple Zaire Ebolavirus (EBOV) variants and other ebolavirus species. Lastly, swabs from an EBOV-infected non-human primate carcass, stored at environmental conditions mimicking central and west Africa, were analyzed to mimic in field conditions. RESULTS: The syringe-based RNA extraction method performed comparably to a standard laboratory spin-column-based method. The developed assay was comparable in sensitivity and specificity to standard laboratory-based diagnostic assays. The assay specifically detected EBOV and not any of the other tested ebolavirus species, including Reston ebolavirus, Sudan ebolavirus, Bundibugyo ebolavirus, and Tai Forrest ebolavirus. Notably, the assays limit of detection for EBOV isolates were all below 4 genome copies/µL. The assay was able to detect EBOV in oral, nasal, thoracic cavity, and conjunctiva swabs obtained from an infected non-human primate. CONCLUSION: We developed a field-based Ebolavirus assay which is comparable in sensitivity and specificity to laboratory-based assays. Currently, the assay is being incorporated into wildlife carcass surveillance in the Republic of the Congo and is being adapted for other infectious disease agents.

Lek-associated movement of a putative Ebolavirus reservoir, the hammer-headed fruit bat (Hypsignathus monstrosus), in northern Republic of Congo.

The biology and ecology of Africa's largest fruit bat remains largely understudied and enigmatic despite at least two highly unusual attributes. The acoustic lek mating behavior of the hammer-headed bat (Hypsignathus monstrosus) in the Congo basin was first described in the 1970s. More recently molecular testing implicated this species and other African bats as potential reservoir hosts for Ebola virus and it was one of only two fruit bat species epidemiologically linked to the 2008 Luebo, Democratic Republic of Congo, Ebola outbreak. Here we share findings from the first pilot study of hammer-headed bat movement using GPS tracking and accelerometry units and a small preceding radio-tracking trial at an apparent lekking site. The radio-tracking revealed adult males had high rates of nightly visitation to the site compared to females (only one visit) and that two of six females day-roosted ~100 m west of Libonga, the nearest village that is ~1.6 km southwest. Four months later, in mid-April 2018, five individual bats, comprised of four males and one female, were tracked from two to 306 days, collecting from 67 to 1022 GPS locations. As measured by mean distance to the site and proportion of nightly GPS locations within 1 km of the site (percent visitation), the males were much more closely associated with the site (mean distance 1.4 km; 51% visitation), than the female (mean 5.5 km; 2.2% visitation). Despite the small sample size, our tracking evidence supports our original characterization of the site as a lek, and the lek itself is much more central to male than female movement. Moreover, our pilot demonstrates the technical feasibility of executing future studies on hammer-headed bats that will help fill problematic knowledge gaps about zoonotic spillover risks and the conservation needs of fruit bats across the continent.

Long-term wildlife mortality surveillance in northern Congo: a model for the detection of Ebola virus disease epizootics.

Ebolavirus (EBOV) has caused disease outbreaks taking thousands of lives, costing billions of dollars in control efforts and threatening great ape populations. EBOV ecology is not fully understood but infected wildlife and consumption of animal carcasses have been linked to human outbreaks, especially in the Congo Basin. Partnering with the Congolese Ministry of Health, we conducted wildlife mortality surveillance and educational outreach in the northern Republic of Congo (RoC). Designed for EBOV detection and to alert public health authorities, we established a low-cost wildlife mortality reporting network covering 50 000 km2. Simultaneously, we delivered educational outreach promoting behavioural change to over 6600 people in rural northern RoC. We achieved specimen collection by training project staff on a safe sampling protocol and equipping geographically distributed bases with sampling kits. We established in-country diagnostics for EBOV testing, reducing diagnostic turnaround time to 3 days and demonstrated the absence of EBOV in 58 carcasses. Central Africa remains a high-risk EBOV region, but RoC, home to the largest remaining populations of great apes, has not had an epidemic since 2005. This effort continues to function as an untested early warning system in RoC, where people and great apes have died from past Ebola virus disease outbreaks. This article is part of the theme issue 'Dynamic and integrative approaches to understanding pathogen spillover'.

Laboratory Findings, Compassionate Use of Favipiravir, and Outcome in Patients With Ebola Virus Disease, Guinea, 2015-A Retrospective Observational Study.

BACKGROUND: In 2015, the laboratory at the Ebola treatment center in Coyah, Guinea, confirmed Ebola virus disease (EVD) in 286 patients. The cycle threshold (Ct) of an Ebola virus-specific reverse transcription-polymerase chain reaction assay and 13 blood chemistry parameters were measured on admission and during hospitalization. Favipiravir treatment was offered to patients with EVD on a compassionate-use basis. METHODS: To reduce biases in the raw field data, we carefully selected 163 of 286 patients with EVD for a retrospective study to assess associations between potential risk factors, alterations in blood chemistry findings, favipiravir treatment, and outcome. RESULTS: The case-fatality rate in favipiravir-treated patients was lower than in untreated patients (42.5% [31 of 73] vs 57.8% [52 of 90]; P = .053 by univariate analysis). In multivariate regression analysis, a higher Ct and a younger age were associated with survival (P < .001), while favipiravir treatment showed no statistically significant effect (P = .11). However, Kaplan-Meier analysis indicated a longer survival time in the favipiravir-treated group (P = .015). The study also showed characteristic changes in blood chemistry findings in patients who died, compared with survivors. CONCLUSIONS: Consistent with the JIKI trial, this retrospective study revealed a trend toward improved survival in favipiravir- treated patients; however, the effect of treatment was not statistically significant, except for its influence on survival time.

Persistence and clearance of Ebola virus RNA from seminal fluid of Ebola virus disease survivors: a longitudinal analysis and modelling study.

BACKGROUND: By January, 2016, all known transmission chains of the Ebola virus disease (EVD) outbreak in west Africa had been stopped. However, there is concern about persistence of Ebola virus in the reproductive tract of men who have survived EVD. We aimed to use biostatistical modelling to describe the dynamics of Ebola virus RNA load in seminal fluid, including clearance parameters. METHODS: In this longitudinal study, we recruited men who had been discharged from three Ebola treatment units in Guinea between January and July, 2015. Participants provided samples of seminal fluid at follow-up every 3-6 weeks, which we tested for Ebola virus RNA using quantitative real-time RT-PCR. Representative specimens from eight participants were then inoculated into immunodeficient mice to test for infectivity. We used a linear mixed-effect model to analyse the dynamics of virus persistence in seminal fluid over time. FINDINGS: We enrolled 26 participants and tested 130 seminal fluid specimens; median follow up was 197 days (IQR 187-209 days) after enrolment, which corresponded to 255 days (228-287) after disease onset. Ebola virus RNA was detected in 86 semen specimens from 19 (73%) participants. Median duration of Ebola virus RNA detection was 158 days after onset (73-181; maximum 407 days at end of follow-up). Mathematical modelling of the quantitative time-series data showed a mean clearance rate of Ebola virus RNA from seminal fluid of -0·58 log units per month, although the clearance kinetic varied greatly between participants. Using our biostatistical model, we predict that 50% and 90% of male survivors clear Ebola virus RNA from seminal fluid at 115 days (90% prediction interval 72-160) and 294 days (212-399) after disease onset, respectively. We also predicted that the number of men positive for Ebola virus RNA in affected countries would decrease from about 50 in January 2016, to fewer than 1 person by July, 2016. Infectious virus was detected in 15 of 26 (58%) specimens tested in mice. INTERPRETATION: Time to clearance of Ebola virus RNA from seminal fluid varies greatly between individuals and could be more than 13 months. Our predictions will assist in decision-making about surveillance and preventive measures in EVD outbreaks. FUNDING: This study was funded by European Union's Horizon 2020 research and innovation programme, Directorate-General for International Cooperation and Development of the European Commission, Institut national de la santé et de la recherche médicale (INSERM), German Research Foundation (DFG), and Innovative Medicines Initiative 2 Joint Undertaking.

Analysis of Diagnostic Findings From the European Mobile Laboratory in Guéckédou, Guinea, March 2014 Through March 2015.

BACKGROUND: A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. METHODS: The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcription-polymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. RESULTS: The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged <15 years had the highest proportion of Ebola virus-malaria parasite coinfections. The case-fatality ratio was high in patients aged <5 years (80%) and those aged >74 years (90%) and low in patients aged 10-19 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of <5 and ≥45 years, and, among children aged 5-14 years, malaria parasite coinfection were independent determinants of a poor EVD outcome. CONCLUSIONS: Virus load, age, and malaria parasite coinfection play a role in the outcome of EVD.

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.

Real-time, portable genome sequencing for Ebola surveillance.

The Ebola virus disease epidemic in West Africa is the largest on record, responsible for over 28,599 cases and more than 11,299 deaths. Genome sequencing in viral outbreaks is desirable to characterize the infectious agent and determine its evolutionary rate. Genome sequencing also allows the identification of signatures of host adaptation, identification and monitoring of diagnostic targets, and characterization of responses to vaccines and treatments. The Ebola virus (EBOV) genome substitution rate in the Makona strain has been estimated at between 0.87 × 10(-3) and 1.42 × 10(-3) mutations per site per year. This is equivalent to 16-27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions. Genomic surveillance during the epidemic has been sporadic owing to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities. To address this problem, here we devise a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. We present sequence data and analysis of 142 EBOV samples collected during the period March to October 2015. We were able to generate results less than 24 h after receiving an Ebola-positive sample, with the sequencing process taking as little as 15-60 min. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.

Dilemmas in Managing Pregnant Women With Ebola: 2 Case Reports.

We report 2 cases of Ebola viral disease (EVD) in pregnant women who survived, initially with intact pregnancies. Respectively 31-32 days after negativation of the maternal blood EVD-polymerase chain reaction (PCR) both patients delivered a stillborn fetus with persistent EVD-PCR amniotic fluid positivity.

Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trial.

BACKGROUND: A recombinant, replication-competent vesicular stomatitis virus-based vaccine expressing a surface glycoprotein of Zaire Ebolavirus (rVSV-ZEBOV) is a promising Ebola vaccine candidate. We report the results of an interim analysis of a trial of rVSV-ZEBOV in Guinea, west Africa. METHODS: For this open-label, cluster-randomised ring vaccination trial, suspected cases of Ebola virus disease in Basse-Guinée (Guinea, west Africa) were independently ascertained by Ebola response teams as part of a national surveillance system. After laboratory confirmation of a new case, clusters of all contacts and contacts of contacts were defined and randomly allocated 1:1 to immediate vaccination or delayed (21 days later) vaccination with rVSV-ZEBOV (one dose of 2 × 10(7) plaque-forming units, administered intramuscularly in the deltoid muscle). Adults (age ≥18 years) who were not pregnant or breastfeeding were eligible for vaccination. Block randomisation was used, with randomly varying blocks, stratified by location (urban vs rural) and size of rings (≤20 vs >20 individuals). The study is open label and masking of participants and field teams to the time of vaccination is not possible, but Ebola response teams and laboratory workers were unaware of allocation to immediate or delayed vaccination. Taking into account the incubation period of the virus of about 10 days, the prespecified primary outcome was laboratory-confirmed Ebola virus disease with onset of symptoms at least 10 days after randomisation. The primary analysis was per protocol and compared the incidence of Ebola virus disease in eligible and vaccinated individuals in immediate vaccination clusters with the incidence in eligible individuals in delayed vaccination clusters. This trial is registered with the Pan African Clinical Trials Registry, number PACTR201503001057193. FINDINGS: Between April 1, 2015, and July 20, 2015, 90 clusters, with a total population of 7651 people were included in the planned interim analysis. 48 of these clusters (4123 people) were randomly assigned to immediate vaccination with rVSV-ZEBOV, and 42 clusters (3528 people) were randomly assigned to delayed vaccination with rVSV-ZEBOV. In the immediate vaccination group, there were no cases of Ebola virus disease with symptom onset at least 10 days after randomisation, whereas in the delayed vaccination group there were 16 cases of Ebola virus disease from seven clusters, showing a vaccine efficacy of 100% (95% CI 74·7-100·0; p=0·0036). No new cases of Ebola virus disease were diagnosed in vaccinees from the immediate or delayed groups from 6 days post-vaccination. At the cluster level, with the inclusion of all eligible adults, vaccine effectiveness was 75·1% (95% CI -7·1 to 94·2; p=0·1791), and 76·3% (95% CI -15·5 to 95·1; p=0·3351) with the inclusion of everyone (eligible or not eligible for vaccination). 43 serious adverse events were reported; one serious adverse event was judged to be causally related to vaccination (a febrile episode in a vaccinated participant, which resolved without sequelae). Assessment of serious adverse events is ongoing. INTERPRETATION: The results of this interim analysis indicate that rVSV-ZEBOV might be highly efficacious and safe in preventing Ebola virus disease, and is most likely effective at the population level when delivered during an Ebola virus disease outbreak via a ring vaccination strategy. FUNDING: WHO, with support from the Wellcome Trust (UK); Médecins Sans Frontières; the Norwegian Ministry of Foreign Affairs through the Research Council of Norway; and the Canadian Government through the Public Health Agency of Canada, Canadian Institutes of Health Research, International Development Research Centre, and Department of Foreign Affairs, Trade and Development.

Seroprevalence of Antibody-Mediated, Complement-Dependent Opsonophagocytic Activity against Neisseria meningitidis Serogroup B in England.

The correlate of protection for the licensure of meningococcal vaccines is serum bactericidal activity. However, evidence indicates that a complex situation and other mechanisms, such as antibody-mediated, complement-dependent opsonophagocytosis (OP), may play a role in protection and should be investigated in order to understand immunity to this disease. In this study, a high-throughput flow cytometric opsonophagocytic assay (OPA) was optimized. The assay measures the presence of killed fluorescently labeled Neisseria meningitidis within human granulocytes (differentiated HL60 cells) by flow cytometry, using IgG-depleted pooled human plasma as an exogenous source of complement. This method was found to be reliable and correlated with the results of an opsonophagocytic killing assay. The OPA was used to measure OP activity in 1,878 serum samples from individuals ranging from 0 to 99 years of age against N. meningitidis strain NZ98/254 (B:4:P1.7-2,4). The levels of OP activity in individual serum samples varied greatly. OP activity showed an initial peak in the 6- to 12-month age group corresponding to a peak in disease incidence. The OP activity dropped in childhood until the late teenage years, although there was still a higher percentage of individuals with OP activity than with protective bactericidal antibody titers. OP activity reached a peak in the 30- to 39-year age group and then declined. This later peak in OP activity did not coincide with the young adults in whom peak serum bactericidal activity and disease incidence occurred. The demonstration of OP activity when disease incidence is low and when protective bactericidal antibody titers are not detected may indicate a role for OP in protection from meningococcal disease in these age groups.

Development of a large scale human complement source for use in bacterial immunoassays.

The serum bactericidal assay is the correlate of protection for meningococcal disease but the use and comparison of functional immunological assays for the assessment of meningococcal vaccines is complicated by the sourcing of human complement. This is due to high levels of immunity in the population acquired through natural meningococcal carriage and means that many individuals must be screened to find donors with suitably low bactericidal titres against the target strain. The use of different donors for each meningococcal strain means that comparisons of assay responses between strains and between laboratories is difficult. We have developed a method for IgG-depletion of 300 ml batches of pooled human lepirudin-derived plasma using Protein G sepharose affinity chromatography that retains complement activity. However, IgG-depletion also removed C1q. This was also eluted from the affinity matrix, concentrated and added to the complement source. The final complement source retained mean alternative pathway activity of 96.8% and total haemolytic activity of 84.2% in four batches. Complement components C3, C5, properdin and factor H were retained following the process and the IgG-depleted complement was shown to be suitable for use in antibody-mediated complement deposition and serum bactericidal activity assays against serogroup B meningococci. The generation of large IgG-depleted batches of pooled human plasma allows for the comparison of immunological responses to diverse meningococcal strain panels in large clinical trials.