Influence of Landscape Patterns on Exposure to Lassa Fever Virus, Guinea.

Lassa fever virus (LASV) is the causative agent of Lassa fever, a disease endemic in West Africa. Exploring the relationships between environmental factors and LASV transmission across ecologically diverse regions can provide crucial information for the design of appropriate interventions and disease monitoring. We investigated LASV exposure in 2 ecologically diverse regions of Guinea. Our results showed that exposure to LASV was heterogenous between and within sites. LASV IgG seropositivity was 11.9% (95% CI 9.7%-14.5%) in a coastal study site in Basse-Guinée, but it was 59.6% (95% CI 55.5%-63.5%) in a forested study site located in Guinée Forestière. Seropositivity increased with age in the coastal site. We also found significant associations between exposure risk for LASV and landscape fragmentation in coastal and forested regions. Our study highlights the potential link between environmental change and LASV emergence and the urgent need for research on land management practices that reduce disease risks.

Public Health Program for Decreasing Risk for Ebola Virus Disease Resurgence from Survivors of the 2013-2016 Outbreak, Guinea.

At the end of the 2013-2016 Ebola virus disease outbreak in Guinea, we implemented an alert system for early detection of Ebola resurgence among survivors. Survivors were asked to report health alerts in their household and provide body fluid specimens for laboratory testing. During April-September 2016, a total of 1,075 (88%) of 1,215 survivors participated in the system; follow up occurred at a median of 16 months after discharge (interquartile range 14-18 months). Of these, 784 acted as focal points and reported 1,136 alerts (including 4 deaths among survivors). A total of 372 (91%) of 408 eligible survivors had >1 semen specimen tested; of 817 semen specimens, 5 samples from 4 survivors were positive up to 512 days after discharge. No lochia (0/7) or breast milk (0/69) specimens tested positive. Our findings underscore the importance of long-term monitoring of survivors' semen samples in an Ebola-affected country.

Comparative performance study of three Ebola rapid diagnostic tests in Guinea.

BACKGROUND: The 2014/15 Ebola outbreak in West Africa resulted in 11,000 deaths and massive strain on local health systems, and the ongoing outbreak in Democratic Republic of Congo has afflicted more than 3000 people. Accurate, rapid Ebola diagnostics suitable for field deployment would enable prompt identification and effective response to future outbreaks, yet remain largely unavailable. The purpose of this study was to assess the accuracy of three novel rapid diagnostic tests (RDTs): an Ebola, an Ebola-Malaria, and a Fever Panel test that includes Ebola, all from a single manufacturer. METHODS: We evaluated the three RDTs in 109 Ebola-positive and 96 Ebola-negative stored serum samples collected during the outbreak in Guinea in 2014/15, and tested by real-time polymerase chain reaction (RT-PCR). Sensitivity, specificity, and overall percent agreement were calculated for each RDT using RT-PCR as a reference standard, stratified by Ct value ranges. RESULTS: All tests performed with high accuracy on samples with low Ct value (high viral load). The Fever Panel test performed with the highest accuracy, with a sensitivity of 89.9% and specificity of 90.6%. The Ebola and Ebola-Malaria tests performed comparably to each other: sensitivity was 77.1 and 78% respectively, and specificity was 91.7% for the Ebola test and 95.8% for the Ebola-Malaria test. CONCLUSIONS: This study evaluated the accuracy of three novel rapid diagnostic tests for Ebola. The tests may have significant public health relevance, particularly the Fever Panel test, which detects seven pathogens including Ebola. Given limitations to the study resulting from uncertain sample quality, further evaluation is warranted. All tests performed with highest accuracy on samples with low Ct value (high viral load), and the data presented here suggests that these RDTs may be useful for point-of-care diagnosis of cases in the context of an outbreak. Restrictions to their use in non-severe Ebola cases or for longitudinal monitoring, when viral loads are lower, may be appropriate. Highlighting the challenge in developing and evaluating Ebola RDTs, there were concerns regarding sample integrity and reference testing, and there is a need for additional research to validate these assays.

Lassa Virus in Pygmy Mice, Benin, 2016-2017.

Lassa virus has been identified in 3 pygmy mice, Mus baoulei, in central Benin. The glycoprotein and nucleoprotein sequences cluster with the Togo strain. These mice may be a new reservoir for Lassa virus in Ghana, Togo, and Benin.

Evaluation of Convalescent Plasma for Ebola Virus Disease in Guinea.

BACKGROUND: In the wake of the recent outbreak of Ebola virus disease (EVD) in several African countries, the World Health Organization prioritized the evaluation of treatment with convalescent plasma derived from patients who have recovered from the disease. We evaluated the safety and efficacy of convalescent plasma for the treatment of EVD in Guinea. METHODS: In this nonrandomized, comparative study, 99 patients of various ages (including pregnant women) with confirmed EVD received two consecutive transfusions of 200 to 250 ml of ABO-compatible convalescent plasma, with each unit of plasma obtained from a separate convalescent donor. The transfusions were initiated on the day of diagnosis or up to 2 days later. The level of neutralizing antibodies against Ebola virus in the plasma was unknown at the time of administration. The control group was 418 patients who had been treated at the same center during the previous 5 months. The primary outcome was the risk of death during the period from 3 to 16 days after diagnosis with adjustments for age and the baseline cycle-threshold value on polymerase-chain-reaction assay; patients who had died before day 3 were excluded. The clinically important difference was defined as an absolute reduction in mortality of 20 percentage points in the convalescent-plasma group as compared with the control group. RESULTS: A total of 84 patients who were treated with plasma were included in the primary analysis. At baseline, the convalescent-plasma group had slightly higher cycle-threshold values and a shorter duration of symptoms than did the control group, along with a higher frequency of eye redness and difficulty in swallowing. From day 3 to day 16 after diagnosis, the risk of death was 31% in the convalescent-plasma group and 38% in the control group (risk difference, -7 percentage points; 95% confidence interval [CI], -18 to 4). The difference was reduced after adjustment for age and cycle-threshold value (adjusted risk difference, -3 percentage points; 95% CI, -13 to 8). No serious adverse reactions associated with the use of convalescent plasma were observed. CONCLUSIONS: The transfusion of up to 500 ml of convalescent plasma with unknown levels of neutralizing antibodies in 84 patients with confirmed EVD was not associated with a significant improvement in survival. (Funded by the European Union's Horizon 2020 Research and Innovation Program and others; ClinicalTrials.gov number, NCT02342171.).

Ebola Virus Persistence in Breast Milk After No Reported Illness: A Likely Source of Virus Transmission From Mother to Child.

A 9-month-old infant died from Ebola virus (EBOV) disease with unknown epidemiological link. While her parents did not report previous illness, laboratory investigations revealed persisting EBOV RNA in the mother's breast milk and the father's seminal fluid. Genomic analysis strongly suggests EBOV transmission to the child through breastfeeding.

Emergence of Zaire Ebola virus disease in Guinea.

In March 2014, the World Health Organization was notified of an outbreak of a communicable disease characterized by fever, severe diarrhea, vomiting, and a high fatality rate in Guinea. Virologic investigation identified Zaire ebolavirus (EBOV) as the causative agent. Full-length genome sequencing and phylogenetic analysis showed that EBOV from Guinea forms a separate clade in relationship to the known EBOV strains from the Democratic Republic of Congo and Gabon. Epidemiologic investigation linked the laboratory-confirmed cases with the presumed first fatality of the outbreak in December 2013. This study demonstrates the emergence of a new EBOV strain in Guinea.

Deployment of a Reverse Transcription Loop-Mediated Isothermal Amplification Test for Ebola Virus Surveillance in Remote Areas in Guinea.

To strengthen the laboratory diagnostic capacity for Ebola virus disease (EVD) in the remote areas of Guinea, we deployed a mobile field laboratory and implemented reverse transcription loop-mediated isothermal amplification (RT-LAMP) for postmortem testing. We tested 896 oral swab specimens and 21 serum samples, using both RT-LAMP and reverse transcription-polymerase chain reaction (RT-PCR). Neither test yielded a positive result, and the results from RT-LAMP and RT-PCR were consistent. More than 95% of the samples were tested within 2 days of sample collection. These results highlight the usefulness of the RT-LAMP assay as an EVD diagnostic testing method in the field or remote areas.

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

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

Resurgence of Ebola Virus Disease in Guinea Linked to a Survivor With Virus Persistence in Seminal Fluid for More Than 500 Days.

We report on an Ebola virus disease (EVD) survivor who showed Ebola virus in seminal fluid 531 days after onset of disease. The persisting virus was sexually transmitted in February 2016, about 470 days after onset of symptoms, and caused a new cluster of EVD in Guinea and Liberia.

Use of Viremia to Evaluate the Baseline Case Fatality Ratio of Ebola Virus Disease and Inform Treatment Studies: A Retrospective Cohort Study.

BACKGROUND: The case fatality ratio (CFR) of Ebola virus disease (EVD) can vary over time and space for reasons that are not fully understood. This makes it difficult to define the baseline CFRs needed to evaluate treatments in the absence of randomized controls. Here, we investigate whether viremia in EVD patients may be used to evaluate baseline EVD CFRs. METHODS AND FINDINGS: We analyzed the laboratory and epidemiological records of patients with EVD confirmed by reverse transcription PCR hospitalized in the Conakry area, Guinea, between 1 March 2014 and 28 February 2015. We used viremia and other variables to model the CFR. Data for 699 EVD patients were analyzed. In the week following symptom onset, mean viremia remained stable, and the CFR increased with viremia, V, from 21% (95% CI 16%-27%) for low viremia (V < 104.4 copies/ml) to 53% (95% CI 44%-61%) for intermediate viremia (104.4 ≤ V < 105.2 copies/ml) and 81% (95% CI 75%-87%) for high viremia (V ≥ 105.2 copies/ml). Compared to adults (15-44 y old [y.o.]), the CFR was larger in young children (0-4 y.o.) (odds ratio [OR]: 2.44; 95% CI 1.02-5.86) and older adults (≥ 45 y.o.) (OR: 2.84; 95% CI 1.81-4.46) but lower in children (5-14 y.o.) (OR: 0.46; 95% CI 0.24-0.86). An order of magnitude increase in mean viremia in cases after July 2014 compared to those before coincided with a 14% increase in the CFR. Our findings come from a large hospital-based study in Conakry and may not be generalizable to settings with different case profiles, such as with individuals who never sought care. CONCLUSIONS: Viremia in EVD patients was a strong predictor of death that partly explained variations in CFR in the study population. This study provides baseline CFRs by viremia group, which allow appropriate adjustment when estimating efficacy in treatment studies. In randomized controlled trials, stratifying analysis on viremia groups could reduce sample size requirements by 25%. We hypothesize that monitoring the viremia of hospitalized patients may inform the ability of surveillance systems to detect EVD patients from the different severity strata.

Development and deployment of a rapid recombinase polymerase amplification Ebola virus detection assay in Guinea in 2015.

In the absence of a vaccine or specific treatments for Ebola virus disease (EVD), early identification of cases is crucial for the control of EVD epidemics. We evaluated a new extraction kit (SpeedXtract (SE), Qiagen) on sera and swabs in combination with an improved diagnostic reverse transcription recombinase polymerase amplification assay for the detection of Ebola virus (EBOV-RT-RPA). The performance of combined extraction and detection was best for swabs. Sensitivity and specificity of the combined SE and EBOV-RT-RPA were tested in a mobile laboratory consisting of a mobile glovebox and a Diagnostics-in-a-Suitcase powered by a battery and solar panel, deployed to Matoto Conakry, Guinea as part of the reinforced surveillance strategy in April 2015 to reach the goal of zero cases. The EBOV-RT-RPA was evaluated in comparison to two real-time PCR assays. Of 928 post-mortem swabs, 120 tested positive, and the combined SE and EBOV-RT-RPA yielded a sensitivity and specificity of 100% in reference to one real-time RT-PCR assay. Another widely used real-time RT-PCR was much less sensitive than expected. Results were provided very fast within 30 to 60 min, and the field deployment of the mobile laboratory helped improve burial management and community engagement.

Rodent control strategies and Lassa virus: some unexpected effects in Guinea, West Africa.

The Natal multimammate mouse (Mastomys natalensis) is the host of Lassa mammarenavirus, causing Lassa haemorrhagic fever in West Africa. As there is currently no operational vaccine and therapeutic drugs are limited, we explored rodent control as an alternative to prevent Lassa virus spillover in Upper Guinea, where the disease is highly endemic in rural areas. In a seven-year experiment, we distributed rodenticides for 10-30 days once a year and, in the last year, added intensive snap trapping for three months in all the houses of one village. We also captured rodents both before and after the intervention period to assess their effectiveness by examining alterations in trapping success and infection rates (Lassa virus RNA and IgG antibodies). We found that both interventions reduced the rodent population by 74-92% but swiftly rebounded to pre-treatment levels, even already six months after the last snap-trapping control. Furthermore, while we observed that chemical control modestly decreased Lassa virus infection rates annually (a reduction of 5% in seroprevalence per year), the intensive trapping unexpectedly led to a significantly higher infection rate (from a seroprevalence of 28% before to 67% after snap trapping control). After seven years, we conclude that annual chemical control, alone or with intensive trapping, is ineffective and sometimes counterproductive in preventing Lassa virus spillover in rural villages. These unexpected findings may result from density-dependent breeding compensation following culling and the survival of a small percentage of chronically infected rodents that may spread the virus to a new susceptible generation of mice.

Spatio-temporal spread of Lassa virus and a new rodent host in the Mano River Union area, West Africa.

The spread of Lassa virus (LASV) in Guinea, Liberia and Sierra Leone, which together are named the Mano River Union (MRU) area, was examined phylogeographically. To provide a reliable evolutionary scenario, new rodent-derived, whole LASV sequences were included. These were generated by metatranscriptomic next-generation sequencing from rodents sampled between 2003 and 2020 in 21 localities of Guinea and Sierra Leone. An analysis was performed using BEAST to perform continuous phylogeographic inference and EvoLaps v36 to visualize spatio-temporal spread. LASV was identified as expected in its primary host reservoir, the Natal multimammate mouse (Mastomys natalensis), and also in two Guinean multimammate mice (Mastomys erythroleucus) in northern Sierra Leone and two rusty-bellied brush-furred mice (Lophuromys sikapusi) in southern Sierra Leone. This finding is consistent with the latter two species being secondary host reservoirs. The strains in these three species were very closely related in LASV lineage IV. Phylogenetic analysis indicated that the most recent common ancestor of lineage IV existed 316-374 years ago and revealed distinct, well-supported clades from Sierra Leone (Bo, Kabala and Kenema), Guinea (Faranah, Kissidougou-Guekedou and Macenta) and Liberia (Phebe-Ganta). The phylogeographic scenario suggests southern Guinea as the point of origin of LASV in the MRU area, with subsequent spread to towards Mali, Liberia and Sierra Leone at a mean speed of 1.6 to 1.1 km/year.

Serological evidence of zoonotic filovirus exposure among bushmeat hunters in Guinea.

Human Ebola virus (EBOV) outbreaks caused by persistent EBOV infection raises questions on the role of zoonotic spillover in filovirus epidemiology. To characterise filovirus zoonotic exposure, we collected cross-sectional serum samples from bushmeat hunters (n = 498) in Macenta Prefecture Guinea, adjacent to the index site of the 2013 EBOV-Makona spillover event. We identified distinct immune signatures (20/498, 4.0%) to multiple EBOV antigens (GP, NP, VP40) using stepwise ELISA and Western blot analysis and, live EBOV neutralisation (5/20; 25%). Using comparative serological data from PCR-confirmed survivors of the 2013-2016 EBOV outbreak, we demonstrated that most signatures (15/20) were not plausibly explained by prior EBOV-Makona exposure. Subsequent data-driven modelling of EBOV immunological outcomes to remote-sensing environmental data also revealed consistent associations with intact closed canopy forest. Together our findings suggest exposure to other closely related filoviruses prior to the 2013-2016 West Africa epidemic and highlight future surveillance priorities.

Correction: Hunting and consumption of rodents by children in the Lassa fever endemic area of Faranah, Guinea.

[This corrects the article DOI: 10.1371/journal.pntd.0009212.].

Association of hepatitis C virus genotype 2 spread with historic slave trade and commerce routes in Western Africa.

The hepatitis C virus genotype 2 (HCV2) is endemic in Western and Central Africa. The HCV2 evolutionary origins remain uncertain due to the paucity of available genomes from African settings. In this study, we investigated the molecular epidemiology of HCV infections in rural Guinea, Western Africa, during 2004 and 2014. Broadly reactive nested reverse transcription polymerase chain reaction (RT-PCR)-based screening of sera from 1,571 asymptomatic adults resulted in the detection of 25 (1.5 per cent; 95 per cent confidence interval 0.9-2.3) positive samples, with a median viral load of 2.54E + 05 IU/ml (interquartile range 6.72E + 05). HCV-infected persons had a median age of 47 years, and 62.5 per cent were male and 37.5 per cent were female. The full polyprotein-encoding genes were retrieved by a combination of high throughput and Sanger sequencing from 17 samples showing sufficiently high viral loads. Phylogenetic analysis and sequence distances ≥13 per cent averaged over the polyprotein genes compared to other HCV2 subtypes revealed nine previously unknown HCV2 subtypes. The time to the most recent common ancestor of the Guinean HCV2 strains inferred in a Bayesian framework was 493 years (95 per cent Highest posterior density (HPD) 453-532). Most of the Guinean strains clustered poorly by location on both the level of sampling sites within Guinea and the level of countries in the phylogenetic reconstructions. Ancestral state reconstruction provided decisive support (Bayes factor > 100) for an origin of HCV2 in Western Africa. Phylogeographic reconstructions in a Bayesian framework pointed to a radial diffusion of HCV2 from Western African regions encompassing today's countries like Ghana, Guinea Bissau, or Burkina Faso, to Central and Northern African regions that took place from the 16th century onwards. The spread of HCV2 coincided in time and space with the main historic slave trade and commerce routes, supported by Bayesian tip-association significance testing (P = 0.01). Our study confirms the evolutionary origins of HCV2 in Western Africa and provides a potential link between historic human movements and HCV2 dispersion.

The characterization of multiple novel paramyxoviruses highlights the diverse nature of the subfamily Orthoparamyxovirinae.

The subfamily Orthoparamyxovirinae is a group of single-stranded, negative-sense RNA viruses that contains many human, animal, and zoonotic pathogens. While there are currently only forty-two recognized species in this subfamily, recent research has revealed that much of its diversity remains to be characterized. Using a newly developed nested PCR-based screening assay, we report here the discovery of fifteen orthoparamyxoviruses in rodents and shrews from Belgium and Guinea, thirteen of which are believed to represent new species. Using a combination of nanopore and sanger sequencing, complete genomes could be determined for almost all these viruses, enabling a detailed evaluation of their genome characteristics. While most viruses are thought to belong to the rapidly expanding genus Jeilongvirus, we also identify novel members of the genera Narmovirus, Henipavirus, and Morbillivirus. Together with other recently discovered orthoparamyxoviruses, both henipaviruses and the morbillivirus discovered here appear to form distinct rodent-/shrew-borne clades within their respective genera, clustering separately from all currently classified viruses. In the case of the henipaviruses, a comparison of the different members of this clade revealed the presence of a secondary conserved open reading frame, encoding for a transmembrane protein, within the F gene, the biological relevance of which remains to be established. While the characteristics of the viruses described here shed further light on the complex evolutionary origin of paramyxoviruses, they also illustrate that the diversity of this group of viruses in terms of genome organization appears to be much larger than previously assumed.

Complement-Mediated Neutralisation Identified in Ebola Virus Disease Survivor Plasma: Implications for Protection and Pathogenesis.

The 2013-2016 Ebola virus (EBOV) epidemic in West Africa was unprecedented in case numbers and fatalities, and sporadic outbreaks continue to arise. Antibodies to the EBOV glycoprotein (GP) are strongly associated with survival and their use in immunotherapy is often initially based on their performance in neutralisation assays. Other immune effector functions also contribute to EBOV protection but are more complex to measure. Their interactions with the complement system in particular are comparatively under-researched and commonly excluded from cellular immunoassays. Using EBOV convalescent plasma samples from the 2013-2016 epidemic, we investigated antibody and complement-mediated neutralisation and how these interactions can influence immunity in response to EBOV-GP and its secreted form (EBOV-sGP). We defined two cohorts: one with low-neutralising titres in relation to EBOV-GP IgG titres (LN cohort) and the other with a direct linear relationship between neutralisation and EBOV-GP IgG titres (N cohort). Using flow cytometry antibody-dependent complement deposition (ADCD) assays, we found that the LN cohort was equally efficient at mediating ADCD in response to the EBOV-GP but was significantly lower in response to the EBOV-sGP, compared to the N cohort. Using wild-type EBOV neutralisation assays with a cohort of the LN plasma, we observed a significant increase in neutralisation associated with the addition of pooled human plasma as a source of complement. Flow cytometry ADCD was also applied using the GP of the highly virulent Sudan virus (SUDV) of the Sudan ebolavirus species. There are no licensed vaccines or therapeutics against SUDV and it overlaps in endemicity with EBOV. We found that the LN plasma was significantly less efficient at cross-reacting and mediating ADCD. Overall, we found a differential response in ADCD between LN and N plasma in response to various Ebolavirus glycoproteins, and that these interactions could significantly improve EBOV neutralisation for selected LN plasma samples. Preservation of the complement system in immunoassays could augment our understanding of neutralisation and thus protection against infection.