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.

Orbivirus RNA in a Banana Serotine (Afronycteris nanus) Bat in the Republic of the Congo.

Orbiviruses are arthropod borne viruses of vertebrates, with some of them being important pathogens of veterinary, conservation and economic importance, while others are occasionally associated with human disease. Some apparently bat specific orbiviruses have been detected, but little is known about their distribution and diversity. We thus sampled and screened 52 bats living in the Congo Basin, and detected RNA indicative of a novel orbivirus in a single banana serotine (Afronycteris nanus) by PCR. The detected RNA clusters with epizootic haemorrhagic disease virus, bluetongue virus, and others. The findings highlight the need for more studies into arbovirus presence and diversity in bat species.

Laboratory capacity assessments in 25 African countries at high risk of yellow fever, August-December 2018.

INTRODUCTION: accurate and timely laboratory diagnosis of yellow fever (YF) is critical to the Eliminate Yellow Fever Epidemics (EYE) strategy. Gavi, the Vaccine Alliance recognized the need to support and build capacity in the national and regional laboratories in the Global YF Laboratory Network (GYFLN) as part of this strategy. METHODS: to better understand current capacity, gaps and needs of the GYFLN laboratories in Africa, assessments were carried out in national and regional reference laboratories in the 25 African countries at high risk for YF outbreaks that were eligible for new financial support from Gavi. RESULTS: the assessments found that the GYFLN in Africa has high capacity but 21% of specimens were not tested due to lack of testing kits or reagents and approximately 50% of presumptive YF cases were not confirmed at the regional reference laboratory due to problems with shipping. CONCLUSION: the laboratory assessments helped to document the baseline capacities of these laboratories prior to Gavi funding to support strengthening YF laboratories.

Coronavirus surveillance in wildlife from two Congo basin countries detects RNA of multiple species circulating in bats and rodents.

Coronaviruses play an important role as pathogens of humans and animals, and the emergence of epidemics like SARS, MERS and COVID-19 is closely linked to zoonotic transmission events primarily from wild animals. Bats have been found to be an important source of coronaviruses with some of them having the potential to infect humans, with other animals serving as intermediate or alternate hosts or reservoirs. Host diversity may be an important contributor to viral diversity and thus the potential for zoonotic events. To date, limited research has been done in Africa on this topic, in particular in the Congo Basin despite frequent contact between humans and wildlife in this region. We sampled and, using consensus coronavirus PCR-primers, tested 3,561 wild animals for coronavirus RNA. The focus was on bats (38%), rodents (38%), and primates (23%) that posed an elevated risk for contact with people, and we found coronavirus RNA in 121 animals, of which all but two were bats. Depending on the taxonomic family, bats were significantly more likely to be coronavirus RNA-positive when sampled either in the wet (Pteropodidae and Rhinolophidae) or dry season (Hipposideridae, Miniopteridae, Molossidae, and Vespertilionidae). The detected RNA sequences correspond to 15 alpha- and 6 betacoronaviruses, with some of them being very similar (>95% nucleotide identities) to known coronaviruses and others being more unique and potentially representing novel viruses. In seven of the bats, we detected RNA most closely related to sequences of the human common cold coronaviruses 229E or NL63 (>80% nucleotide identities). The findings highlight the potential for coronavirus spillover, especially in regions with a high diversity of bats and close human contact, and reinforces the need for ongoing surveillance.

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.

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'.

Epidemiologic and Ecologic Investigations of Monkeypox, Likouala Department, Republic of the Congo, 2017.

Monkeypox, caused by a zoonotic orthopoxvirus, is endemic in Central and West Africa. Monkeypox has been sporadically reported in the Republic of the Congo. During March 22-April 5, 2017, we investigated 43 suspected human monkeypox cases. We interviewed suspected case-patients and collected dried blood strips and vesicular and crust specimens (active lesions), which we tested for orthopoxvirus antibodies by ELISA and monkeypox virus and varicella zoster virus DNA by PCR. An ecologic investigation was conducted around Manfouété, and specimens from 105 small mammals were tested for anti-orthopoxvirus antibodies or DNA. Among the suspected human cases, 22 met the confirmed, probable, and possible case definitions. Only 18 patients had available dried blood strips; 100% were IgG positive, and 88.9% (16/18) were IgM positive. Among animals, only specimens from Cricetomys giant pouched rats showed presence of orthopoxvirus antibodies, adding evidence to this species' involvement in the transmission and maintenance of monkeypox virus in nature.

Diverse RNA viruses of arthropod origin in the blood of fruit bats suggest a link between bat and arthropod viromes.

Bats host diverse viruses due to their unique ecology, behavior, and immunology. However, the role of other organisms with which bats interact in nature is understudied as a contributor to bat viral diversity. We discovered five viruses in the blood of fruit bats (Hypsignathus monstrosus) from the Republic of Congo. Of these five viruses, four have phylogenetic and genomic features suggesting an arthropod origin (a dicistrovirus, a nodavirus, and two tombus-like viruses), while the fifth (a hepadnavirus) is clearly of mammalian origin. We also report the parallel discovery of related tombus-like viruses in fig wasps and primitive crane flies from bat habitats, as well as high infection rates of bats with haemosporidian parasites (Hepatocystis sp.). These findings suggest transmission between arthropods and bats, perhaps through ingestion or hyperparasitism (viral infection of bat parasites). Some "bat-associated" viruses may be epidemiologically linked to bats through their ecological associations with invertebrates.

Highly diverse and antimicrobial susceptible Escherichia coli display a naïve bacterial population in fruit bats from the Republic of Congo.

Bats are suspected to be a reservoir of several bacterial and viral pathogens relevant to animal and human health, but studies on Escherichia coli in these animals are sparse. We investigated the presence of E. coli in tissue samples (liver, lung and intestines) collected from 50 fruit bats of five different species (Eidolon helvum, Epomops franqueti, Hypsignathus monstrosus, Myonycteris torquata, Rousettus aegyptiacus) of two different areas in the Republic of Congo between 2009 and 2010. To assess E. coli pathotypes and phylogenetic relationships, we determined the presence of 59 virulence associated genes and multilocus sequence types (STs). Isolates were further tested for their susceptibility to several antimicrobial substances by agar disk diffusion test and for the presence of an Extended-Spectrum Beta-Lactamase phenotype. E. coli was detected in 60% of the bats analysed. The diversity of E. coli strains was very high, with 37 different STs within 40 isolates. Occasionally, we detected sequence types (e.g. ST69, ST127, and ST131) and pathotypes (e.g. ExPEC, EPEC and atypical EPEC), which are known pathogens in human and/or animal infections. Although the majority of strains were assigned to phylogenetic group B2 (46.2%), which is linked with the ExPEC pathovar, occurrence of virulence-associated genes in these strains were unexpectedly low. Due to this, and as only few of the E. coli isolates showed intermediate resistance to certain antimicrobial substances, we assume a rather naïve E. coli population, lacking contact to humans or domestic animals. Future studies featuring in depth comparative whole genome sequence analyses will provide insights into the microevolution of this interesting strain collection.

Diagnostic preparedness for infectious disease outbreaks.

Diagnostics are crucial in mitigating the effect of disease outbreaks. Because diagnostic development and validation are time consuming, they should be carried out in anticipation of epidemics rather than in response to them. The diagnostic response to the 2014-15 Ebola epidemic, although ultimately effective, was slow and expensive. If a focused mechanism had existed with the technical and financial resources to drive its development ahead of the outbreak, point-of-care Ebola tests supporting a less costly and more mobile response could have been available early on in the diagnosis process. A new partnering model could drive rapid development of tests and surveillance strategies for novel pathogens that emerge in future outbreaks. We look at lessons learned from the Ebola outbreak and propose specific solutions to improve the speed of new assay development and ensure their effective deployment.

Sickle-Cell Disease Healthcare Cost in Africa: Experience of the Congo.

Background. Lack of medical coverage in Africa leads to inappropriate care that has an impact on the mortality rate. In this study, we aimed to evaluate the cost of severe acute sickle-cell related complications in Brazzaville. Methods. A retrospective study was conducted in 2014 in the Paediatric Intensive Care Unit. It concerned 94 homozygote sickle-cell children that developed severe acute sickle-cell disease related complications (average age 69 months). For each patient, we calculated the cost of care complication. Results. The household income was estimated as low (

First reported chikungunya fever outbreak in the republic of Congo, 2011.

BACKGROUND: Chikungunya is an Aedes -borne disease characterised by febrile arthralgia and responsible for massive outbreaks. We present a prospective clinical cohort study and a retrospective serological study relating to a CHIK outbreak, in the Republic of Congo in 2011. METHODOLOGY AND FINDINGS: We analysed 317 suspected cases, of which 308 (97.2%) lived in the city of Brazzaville (66.6% in the South area). Amongst them, 37 (11.7%) were CHIKV+ve patients (i.e., biologically confirmed by a real-time RT-PCR assay), of whom 36 (97.3%) had fever, 22 (66.7%) myalgia and 32 (86.5%) arthralgia. All tested negative for dengue. The distribution of incident cases within Brazzaville districts was compared with CHIKV seroprevalence before the outbreak (34.4% in 517 blood donors), providing evidence for previous circulation of CHIKV. We applied a CHIK clinical score to 126 patients recruited within the two first day of illness (including 28 CHIKV+ves (22.2%)) with sensitivity (78.6%) and specificity (72.4%) values comparing with those of the referent study in Reunion Island. The negative predictive value was high (92%), but the positive predictive value (45%) indicate poor potential contribution to medical practice to identify CHIKV+ve patients in low prevalence outbreaks. However, the score allowed a slightly more accurate follow-up of the evolution of the outbreak than the criterion "fever+arthralgia". The complete sequencing of a Congolase isolate (Brazza_MRS1) demonstrated belonging to the East/Central/South African lineage and was further used for producing a robust genome-scale CHIKV phylogenetic analysis. CONCLUSIONS/SIGNIFICANCE: We describe the first Chikungunya outbreak declared in the Republic of Congo. The seroprevalence study conducted amongst blood donors before outbreak provided evidence for previous CHIKV circulation. We suggest that a more systematic survey of the entomological situation and of arbovirus circulation is necessary in Central Africa for better understanding the environmental, microbiological and sociological determinants of emergence.

In vitro evaluation of antiplasmodial activity of extracts of Acanthospermum hispidum DC (Asteraceae) and Ficus thonningii Blume (Moraceae), two plants used in traditional medicine in the Republic of Congo.

The aim of this study was to evaluate extracts from two medicinal plants, Acanthospermum hispidum and Ficus thonningii, used in traditional medicine in Congo Brazzaville, for in vitro antiplasmodial activities against two laboratory strains of Plasmodium falciparum: the chloroquine sensitive 3D7 and the chloroquine resistant Dd2. ELISA HRP2 assay was used to evaluate the in vitro inhibitory activity of the extracts alone or in combination with chloroquine. Cytotoxicity was assessed on human HeLa cell line and reflected by the selectivity index. Methanolic extract of Acanthospermum hispidum exhibited a strong and a moderate inhibitory activity on the growth of Dd2 and 3D7 at 2.8 µg/ml and 9.2 µg/ml concentrations respectively with a selectivity index >10. The combination of the most active extract (methanolic extract of Acanthospermum hispidum) with chloroquine showed a synergistic interaction on both strains. The good selectivity index of Acanthospermum hispidum on HeLa cells reflects the safety of this plant. Extracts from Ficus thonningii did not show any promising antiplasmodial activity on both 3D7 and Dd2. Except the methanolic extract which exhibited a slight antiplasmodial activity with inhibitory concentration and selectivity index corresponding to 9.61 µg/ml and 11.16 respectively. Methanolic extract of Acanthospermum hispidum exhibited moderate to high inhibitory activity on 3D7 and Dd2 laboratory strains and a synergistic antimalarial effect when combined with chloroquine. Ficus thonningii seems to have no antimalarial activity. Phytochemical analysis, in vivo investigations using animal models and later clinical trials in collaboration with traditional practitioners are necessary to clarify the potential antimalarial activity of both plants.