Performance assessment and validation of a plaque reduction neutralization test (PRNT) in support to yellow fever diagnostic and vaccine clinical trials.

Yellow fever (YF) virus is a mosquito-borne virus belonging to the Flaviviridae family that circulates in tropical and subtropical areas of Africa and South America. Despite the availability of an effective vaccine, YF remains a threat to travelers, residents of endemic areas, and unvaccinated populations. YF vaccination and natural infection both induce the production of neutralizing antibodies. Serological diagnostic methods detecting YF virus-specific antibodies demonstrate high levels of cross-reactivities with other flaviviruses. To date, the plaque reduction neutralization test (PRNT) is the most specific serological test for the differentiation of flavivirus infections and is considered the reference method for detecting YF neutralizing antibodies and assessing the protective immune response following vaccination. In this study, we developed and validated a YF PRNT. We optimized different parameters including cell concentration and virus-serum neutralization time period and then assessed the intra- and inter-assay precisions, dilutability, specificity, and lower limit of quantification (LLOQ) using international standard YF serum, sera from vaccinees and human specimens collected through YF surveillance. The YF PRNT has shown good robustness and 100% of intra-assay precision, 95.6% of inter-assay precision, 100% of specificity, 100% of LLOQ, and 95.3% of dilutability. The test is, therefore, suitable for use in the YF diagnostic as well as evaluation of the YF vaccine neutralizing antibody response and risk assessment studies.

Rabies surveillance in Senegal 2001 to 2015 uncovers first infection of a honey-badger.

Despite the establishment of Rabies surveillance in animals and humans since 2008, there is a lack of data on its circulation, dynamic of transmission and real burden in Senegal. To better understand the molecular epidemiology of rabies virus in Senegal, we investigated the genetic diversity of 18 new characterized Senegalese rabies virus sequences collected over 14 years, including a honey-badger-related isolate. Phylogeographic analyses demonstrated that the Senegalese isolates belong to a monophyletic cluster into the Africa-2 clade and supported two RABV introductions in Senegal from West-African neighbour countries, 36-40 years ago. Our study is noteworthy for reporting on the first characterization of an African honey-badger-related rabies virus that did not have the N-glycosylation site NKT at position 338-G of the glycoprotein. The identified amino acid polymorphisms found in the Senegalese rabies virus sequences are worthy of further investigation. Although a strong multidisciplinary stepwise cooperation is important for the successful elimination of Rabies in dog populations in Senegal by 2030, the establishment of surveillance in wildlife could be necessary to avoid future re-introductions into domestic hosts.

Detection of Rift Valley Fever Virus Lineage H From South Africa Through the Syndromic Sentinel Surveillance Network in Senegal.

BACKGROUND: Rift Valley fever virus (RVFV) is an arbovirus that causes epizootics and epidemics among livestock population and humans. Our surveillance system has revealed multiple emergences and re-emergences of RVFV in West Africa over the last decade. METHODS: The Sentinel Syndromic Surveillance Network in Senegal (4S) has been implemented since 2011. Samples from human suspected arbovirus infection in 4S sentinel sites were sent to Institut Pasteur de Dakar (IPD), where arbovirus diagnosis by enzyme-linked immunosorbent assay (ELISA), real-time reverse transcription polymerase chain reaction (RT-PCR), and virus isolation was performed. Overall, IPD has received a total of 1149 samples from arboviral suspected patients through the 4S network from January to December 2020. These samples were screened for 7 arboviruses including RVFV. Whole-genome sequencing of positive RVFV samples by RT-PCR was performed using the Illumina Miseq platform followed by genome assembly. Phylogenetic analyses were performed using MEGA X. RESULTS: Out of the 1149 arbovirus suspected cases, 4 RVFV-positive samples were detected with RT-PCR while 5 RVFV-positive samples were detected by ELISA. Complete genome sequences were obtained for 3 strains among the 4 positive samples by RT-PCR. Phylogenetic analyses indicated an emergence of a virus first described in South Africa during a major outbreak. CONCLUSIONS: This strong surveillance system allowed the detection of an RVFV outbreak in Senegal in 2020. The obtained genomes clustered with strains from South Africa belonging to lineage H. This calls for implementation of a strong surveillance system for wild animals, humans, and livestock simultaneously in all African countries.

Detection of the Northeastern African Rift Valley Fever Virus Lineage During the 2015 Outbreak in Mauritania.

BACKGROUND: Rift Valley fever (RVF) is an acute viral anthropozoonosis that causes epizootics and epidemics among livestock population and humans. Multiple emergences and reemergences of the virus have occurred in Mauritania over the last decade. This article describes the outbreak that occurred in 2015 in Mauritania and reports the results of serological and molecular investigations of blood samples collected from suspected RVF patients. METHODS: An RVF outbreak was reported from 14 September to 26 November 2015 in Mauritania. Overall, 184 suspected cases from different localities were identified by 26 health facilities. Blood samples were collected and tested by enzyme-linked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) at the Institut Pasteur de Dakar (IPD). Sequencing of partial genomes and phylogenetic analyses were performed on RT-PCR-positive samples. As part of routine surveillance at IPD, samples were also screened for dengue, yellow fever, West Nile, Crimean Congo hemorrhagic fever, Zika, and Chikungunya viruses by ELISA and RT-PCR. RESULTS: Of the 184 suspected cases, there were 57 confirmed cases and 12 deaths. Phylogenetic analysis of the sequences indicated an emergence of a virus that originated from Northeastern Africa. Our results show co-circulation of other arboviruses in Mauritania-dengue, Crimean Congo hemorrhagic fever, and West Nile viruses. CONCLUSION: The Northeastern Africa lineage of RVF was responsible for the outbreak in Mauritania in 2015. Co-circulation of multiples arboviruses was detected. This calls for systematic differential diagnosis and highlights the need to strengthen arbovirus surveillance in Africa.

Widespread Rift Valley Fever Emergence in Senegal in 2013-2014.

Rift Valley fever (RVF), which caused epizootics and epidemics among human and livestock populations, occurred in Senegal in 2013-2014. A multidisciplinary field investigation was carried out in 3 regions of Senegal. We found 11 confirmed human cases of Rift Valley fever, including severe cases with encephalitis and retinitis, 1 pool of mosquito (Aedes ochraceus), and 52 animals tested positive for the disease. Symptoms such as encephalitis and macular retinitis were the most severe cases reported so far in Senegal. The outbreak was widespread due to animals' movements, leading to the largest RVF outbreak in Senegal in terms of geographic spreading and reaching areas that never reported RVF activity previously.

Vector competence of Aedes vexans (Meigen), Culex poicilipes (Theobald) and Cx. quinquefasciatus Say from Senegal for West and East African lineages of Rift Valley fever virus.

BACKGROUND: Rift Valley fever virus (RVFV; Phlebovirus, Bunyaviridae) is a mosquito-borne, zoonotic pathogen. In Senegal, RVFV was first isolated in 1974 from Aedes dalzieli (Theobald) and thereafter from Ae. fowleri (de Charmoy), Ae. ochraceus Theobald, Ae. vexans (Meigen), Culex poicilipes (Theobald), Mansonia africana (Theobald) and Ma. uniformis (Theobald). However, the vector competence of these local species has never been demonstrated making hypothetical the transmission cycle proposed for West Africa based on serological data and mosquito isolates. METHODS: Aedes vexans and Cx. poicilipes, two common mosquito species most frequently associated with RVFV in Senegal, and Cx. quinquefasciatus, the most common domestic species, were assessed after oral feeding with three RVFV strains of the West and East/central African lineages. Fully engorged mosquitoes (420 Ae. vexans, 563 Cx. quinquefasciatus and 380 Cx. poicilipes) were maintained at 27 ± 1 °C and 70-80% relative humidity. The saliva, legs/wings and bodies were tested individually for the RVFV genome using real-time RT-PCR at 5, 10, 15 and 20 days post exposure (dpe) to estimate the infection, dissemination, and transmission rates. Genotypic characterisation of the 3 strains used were performed to identify factors underlying the different patterns of transmission. RESULTS: The infection rates varied between 30.0-85.0% for Ae. vexans, 3.3-27% for Cx. quinquefasciatus and 8.3-46.7% for Cx. poicilipes, and the dissemination rates varied between 10.5-37% for Ae. vexans, 9.5-28.6% for Cx. quinquefasciatus and 3.0-40.9% for Cx. poicilipes. However only the East African lineage was transmitted, with transmission rates varying between 13.3-33.3% in Ae. vexans, 50% in Cx. quinquefasciatus and 11.1% in Cx. poicilipes. Culex mosquitoes were less susceptible to infection than Ae. vexans. Compared to other strains, amino acid variation in the NSs M segment proteins of the East African RVFV lineage human-derived strain SH172805, might explain the differences in transmission potential. CONCLUSION: Our findings revealed that all the species tested were competent for RVFV with a significant more important role of Ae. vexans compared to Culex species and a highest potential of the East African lineage to be transmitted.

A molecular survey of acute febrile illnesses reveals Plasmodium vivax infections in Kedougou, southeastern Senegal.

BACKGROUND: Control efforts towards malaria due to Plasmodium falciparum significantly decreased the incidence of the disease in many endemic countries including Senegal. Surprisingly, in Kedougou (southeastern Senegal) P. falciparum malaria remains highly prevalent and the relative contribution of other Plasmodium species to the global malaria burden is very poorly documented, partly due to the low sensitivity of routine diagnostic tools. Molecular methods offer better estimate of circulating Plasmodium species in a given area. A molecular survey was carried out to document circulating malaria parasites in Kedougou region. METHODS: A total of 263 long-term stored sera obtained from patients presenting with acute febrile illness in Kedougou between July 2009 and July 2013 were used for malaria parasite determination. Sera were withdrawn from a collection established as part of a surveillance programme of arboviruses infections in the region. Plasmodium species were characterized by a nested PCR-based approach targeting the 18S small sub-unit ribosomal RNA genes of Plasmodium spp. RESULTS: Of the 263 sera screened in this study, Plasmodium genomic DNA was amplifiable by nested PCR from 62.35% (164/263) of samples. P. falciparum accounted for the majority of infections either as single in 85.97% (141/164) of Plasmodium-positive samples or mixed with Plasmodium ovale (11.58%, 19/164) or Plasmodium vivax (1.21%, 2/164). All 19 (11.58%) P. ovale-infected patients were mixed with P. falciparum, while no Plasmodium malariae was detected in this survey. Four patients (2.43%) were found to be infected by P. vivax, two of whom were mixed with P. falciparum. P. vivax infections originated from Bandafassi and Ninefesha villages and concerned patients aged 4, 9, 10, and 15 years old, respectively. DNA sequences alignment and phylogenetic analysis demonstrated that sequences from Kedougou corresponded to P. vivax, therefore confirming the presence of P. vivax infections in Senegal. CONCLUSION: The results confirm the high prevalence of P. falciparum in Kedougou and provide the first molecular evidence of P. vivax infections in Senegal. These findings pave the ways for further investigations of P. vivax infections in Senegal and its contribution to the global burden of malaria disease before targeted strategies can be deployed.

STEVOR is a Plasmodium falciparum erythrocyte binding protein that mediates merozoite invasion and rosetting.

Variant surface antigens play an important role in Plasmodium falciparum malaria pathogenesis and in immune evasion by the parasite. Although most work to date has focused on P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1), two other multigene families encoding STEVOR and RIFIN are expressed in invasive merozoites and on the infected erythrocyte surface. However, their role during parasite infection remains to be clarified. Here we report that STEVOR functions as an erythrocyte-binding protein that recognizes Glycophorin C (GPC) on the red blood cell (RBC) surface and that its binding correlates with the level of GPC on the RBC surface. STEVOR expression on the RBC leads to PfEMP1-independent binding of infected RBCs to uninfected RBCs (rosette formation), while antibodies targeting STEVOR in the merozoite can effectively inhibit invasion. Our results suggest a PfEMP1-independent role for STEVOR in enabling infected erythrocytes at the schizont stage to form rosettes and in promoting merozoite invasion.

Identification of a new export signal in Plasmodium yoelii: identification of a new exportome.

Development of the erythrocytic malaria parasite requires targeting of parasite proteins into multiple compartments located within and beyond the parasite confine. Beyond the PEXEL/VTS pathway and its characterized players, increasing amount of evidence has highlighted the existence of proteins exported using alternative export-signal(s)/pathway(s); hence, the exportomes currently predicted are incomplete. The nature of these exported proteins which could have a prominent role in most of the Plasmodium species remains elusive. Using P. yoelii variant proteins, we identified a signal associated to lipophilic region that mediates export of P. yoelii proteins. This non-PEXEL signal termed PLASMED is defined by semi-conserved residues and possibly a secondary structure. In vivo characterization of exported-proteins indicated that PLASMED is a bona fide export-signal that allowed us to identify an unseen P. yoelii exportome. The repertoire of the newly predicted exported proteins opens up perspectives for unravelling the remodelling of the host-cell by the parasite, against which new therapies could be elaborated.

Parasite polymorphism and severe malaria in Dakar (Senegal): a West African urban area.

BACKGROUND: Transmission of malaria in West African urban areas is low and healthcare facilities are well organized. However, malaria mortality remains high. We conducted a survey in Dakar with the general objective to establish who died from severe malaria (SM) in urban areas (particularly looking at the age-groups) and to compare parasite isolates associated with mild or severe malaria. METHODOLOGY/PRINCIPAL FINDINGS: The current study included mild- (MM) and severe malaria (SM) cases, treated in dispensaries (n = 2977) and hospitals (n = 104), We analysed Pfdhfr/Pfcrt-exon2 and nine microsatellite loci in 102 matched cases of SM and MM. Half of the malaria cases recorded at the dispensaries and 87% of SM cases referred to hospitals, occurred in adults, although adults only accounted for 26% of all dispensary consultations. This suggests that, in urban settings, whatever the reason for this adult over-representation, health-workers are forced to take care of increasing numbers of malaria cases among adults. Inappropriate self treatment and mutations in genes associated with drug resistance were found associated with SM in adults. SM was also associated with a specific pool of isolates highly polymorphic and different from those associated with MM. CONCLUSION: In this urban setting, adults currently represent one of the major groups of patients attending dispensaries for malaria treatment. For these patients, despite the low level of transmission, SM was associated with a specific and highly polymorphic pool of parasites which may have been selected by inappropriate treatment.