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BackgroundThe first discovery of Plasmodium vivax infections in Senegal used archived patients’ sera in place of blood pellet, the preferred specimen for the molecular diagnosis of Plasmodium species. The present study assessed the reliability of detecting P. vivax DNA in plasma in comparison to blood pellet from the same patient’s samples.MethodsA total of 616 blood samples obtained from febrile patients living in Kolda (2015 and 2020), Tambacounda (2017 and 2020), and Kedougou (2020) regions in Senegal, were first screened for Plasmodium species composition by 18S ssrRNA-based nested PCR. Paired blood pellets and plasma samples were selected from a subset of 50 P. vivax-positive patients matched by age and sex with 50 P. vivax-negative patients, and subjected to a cytochrome b-based qPCR to compare the detection and quantification of P. vivax genomic DNA between the two specimen types.Results and discussionThe study reports 1.8% and 14.77% of single and mixed P. vivax infections in the study population, and a high concordance (84%) between the qPCR detection of P. vivax genomic DNA from paired blood pellets and plasma samples. Importantly, all P. vivax negative samples from the blood pellets were also confirmed plasma-negative, and parasitaemia in blood pellets was higher compared to plasma samples. The results support investigations of P. vivax infections in archived sera or plasma collections with a high degree of confidence to generate additional data on the neglected P. vivax malaria, and ultimately guide strategies to control the disease.

Ex vivo phenotyping of P. falciparum erythrocyte invasion diversity is important in the identification and down selection of potential malaria vaccine targets. However, due to the lack of appropriate laboratory facilities in remote areas of endemic countries, direct processing of P. falciparum clinical isolates is usually not feasible. Here, we investigated the combined effect of short-term cryopreservation and thawing processes on the ex vivo invasion phenotypes of P. falciparum isolates. Ex-vivo or in vitro invasion phenotyping assays were performed with P. falciparum clinical isolates prior to or following culture adaptation, respectively. All isolates were genotyped at Day 0 for parasite clonality. Subsequently, isolates that were successfully culture-adapted were genotyped again at Days 7, 15, 21, and 28-post adaptation. Invasion phenotyping assays were performed in isogenic isolates revived at different time points (3, 6, and 12 months) post-cryopreservation and the resulting data were compared to that from ex-vivo invasion data of matched isogenic parental isolates. We also show that short-term culture adaptation selects for parasite clonality and could be a driving force for variation in invasion phenotypes as compared to ex vivo data where almost all parasite clones of a given isolate are present. Interestingly, our data show little variation in the parasites’ invasion phenotype following short-term cryopreservation. Altogether, our data suggest that short-term cryopreservation of uncultured P. falciparum clinical isolates is a reliable mechanism for storing parasites for future use.

International audience

Plasmodium falciparum erythrocyte invasion phenotyping assays are a very useful tool for assessing parasite diversity and virulence, and for characterizing the formation of ligand–receptor interactions. However, such assays need to be highly sensitive and reproducible, and the selection of labeling dyes for differentiating donor and acceptor erythrocytes is a critical factor. We investigated the suitability of cell trace far-red (CTFR) as a dye for P. falciparum invasion phenotyping assays. Using the dyes carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and dichloro dimethyl acridin one succinimidyl ester (DDAO-SE) as comparators, we used a dye-dilution approach to assess the limitations and specific staining procedures for the applicability of CTFR in P. falciparum invasion phenotyping assays. Our data show that CTFR effectively labels acceptor erythrocytes and provides a stable fluorescent intensity at relatively low concentrations. CTFR also yielded a higher fluorescence intensity relative to DDAO-SE and with a more stable fluorescence intensity over time. Furthermore, CTFR did not affect merozoites invasion of erythrocytes and was not toxic to the parasite’s intraerythrocytic development. Additionally, CTFR offers flexibility in the choice of combinations with several other DNA dyes, which broaden its usage for P. falciparum erythrocyte invasion assays, considering a wider range of flow cytometers with various laser settings. Impact statement In recent years, flow cytometry has become a cornerstone in investigating P. falciparum phenotypic diversity using multiple dyes to discriminate between donor and acceptor erythrocytes. To broaden the applicability of such assays, we optimized the staining conditions of a newly developed cytoplasmic dye, cell trace far-red (CTFR), and assessed its suitability for use in P. falciparum invasion phenotyping assays. We showed that CTFR has a very narrow emission peak excited by red lasers. Furthermore, CTFR labeling of target erythrocytes, achieved even at low concentrations, is stable over time and did not impair parasite development. P. falciparum erythrocyte invasion phenotyping assays revealed that CTFR is suitable for use in combination with several DNA dyes in multiplex assays. This will allow for high throughput phenotyping of parasites as well as facilitate the evaluation of preference of erythrocytes by merozoites. Altogether, these make screening for potential invasion-blocking interventions possible.

Background A detailed understanding of the contribution of the asymptomatic Plasmodium reservoir to the occurrence of clinical malaria at individual and community levels is needed to guide effective elimination interventions. This study investigated the relationship between asymptomatic Plasmodium falciparum carriage and subsequent clinical malaria episodes in the Dielmo and Ndiop villages in Senegal. Methods The study used a total of 2792 venous and capillary blood samples obtained from asymptomatic individuals and clinical malaria datasets collected from 2013 to 2016. Mapping, spatial clustering of infections, and risk analysis were performed using georeferenced households. Results High incidences of clinical malaria episodes were observed to occur predominantly in households of asymptomatic P falciparum carriers. A statistically significant association was found between asymptomatic carriage in a household and subsequent episode of clinical malaria occurring in that household for each individual year (P values were 0.0017, 6 × 10–5, 0.005, and 0.008 for the years 2013, 2014, 2015, and 2016 respectively) and the combined years (P = 8.5 × 10–8), which was not found at the individual level. In both villages, no significant patterns of spatial clustering of P falciparum clinical cases were found, but there was a higher risk of clinical episodes Conclusion The findings provide strong epidemiological evidence linking the asymptomatic P falciparum reservoir to clinical malaria episodes at household scale in Dielmo and Ndiop villagers. This argues for a likely success of a mass testing and treatment intervention to move towards the elimination of malaria in the villages of Dielmo and Ndiop.

Background: Serology is a great tool to assess the level of immunity against SARS-CoV-2 in settings with limited access to molecular diagnostics. However, African populations displays a particular immunological profile with massive circulation of infectious agents from different aetiologies that can affect assays performance. Methods: We evaluated the OMEGA Diagnostics COVID-19 ELISA-IgG and the ID Screen® SARS-CoV-2-N IgG Indirect in Senegal using a panel of 636 blood samples covering several African-endemic diseases and healthy donors to determine test sensitivity and specificity. The sensitivity panel of sera includes 461 serum samples collected from 91 patients hospitalized for COVID-19 disease. COVID-19 cases were confirmed by qRT-PCR and samples were collected on an interval of three days until viral clearance. In addition, 272 sera obtained from COVID-19 negative individuals were selected from a well-documented biobank of sera collected before the COVID-19 outbreak. Finding: High-cross reactivity have been found in individuals with a history of exposure to Chikungunya, HIV, malaria (Plasmodium falciparum), rheumatoid factor as well as healthy donors with respective specificities of 55%, 41.8%, 70%, 70% and 75%. ELISA experiments with commercial assays targeting either SARS-CoV-2 Nucleocapsid protein and Spike 2 protein or nucleocapsid protein only suggest that cross-reactivity might be directed against Spike 2 protein and not Nucleocapsid protein. Further samples characterisation reveals that anti-malaria IgG is the leading cause of such poor specificities, but exposure to other diseases contributed as well. Interpretation: We anticipate that COVID-19 seroprevalence can be biased if assays are not contextualized. Since malaria is endemic in African settings, we propose that a particular attention must be given in serological surveillance of COVID-19 or anti-SARS-CoV-2 antibodies quantification as vaccines are being rolled out.FundingUK Foreign, Commonwealth and Development Office/Wellcome Trust Joint Initiative for Research in Epidemic Preparedness and Response (JIREP grant number 220764/Z/20/Z). Funding Information: UK Foreign, Commonwealth and Development Office/Wellcome Trust Joint Initiative for Research in Epidemic Preparedness and Response (JIREP grant number 220764/Z/20/Z). Declaration of Interests: JRAF was an employee of Mologic Ltd, which was the development partner of one of the ELISAs adopted in this study. The remaining authors declare no competing interest. Ethics Approval Statement: Pre-COVID-19 samples for malaria (PCR), dengue, yellow fever, Zika, Chikungunya, Influenza A/B, HIV, rheumatoid arthritis and samples tested negative for the same diseases and also Crimean Congo haemorrhagic fever, West Nile fever encephalitis and Rift Valley fever were part of national public health surveillance program of the Senegalese Ministry of Social Action and Health performed in collaboration with Institut Pasteur de Dakar. Therefore, consultation with ethics committee was not required. Pre-COVID-19 samples for malaria endemic areas were from a longitudinal cohort survey performed in Dielmo village and approved by the Senegalese National Ethics Committee for Research in Health (reference number 00000007/MSAS/CNERS/Sec 26 January 2021). Samples from COVID-19 RT-PCR positive patients were obtained from a multicentre cohort survey approved by the Senegalese National Ethics Committee for Research in Health (reference number 00000068/MSAS/CNERS/Sec, 10 April 2020).

Background: Past and recent outbreaks have highlighted the vulnerability of humans to infectious diseases, which represent serious economic and health security threats. A paradigm shift in the management of sanitary crises is urgently needed. Based on lessons from the 2014 Ebola outbreak, the Praesens Foundation has developed an all-terrain mobile biosafety laboratory (MBS-Lab) for effective field diagnostics capabilities. Objective: The aim of the study was to train African teams and run a field evaluation of the MBS-Lab, including robustness, technical and operational sustainability, biosafety, connectivity, turn-around times for testing and result delivery. Methods: The MBS-Lab was deployed in Senegal in October 2017 for a six-month field assessment under various ecological conditions and was mobilised during the dengue outbreaks in 2017 and 2018. Results: The MBS-Lab can be considered an off-grid solution that addresses field challenges with regard to working conditions, mobility, deployment, environment and personnel safety. Blood (n = 398) and nasal swab (n = 113) samples were collected from 460 study participants for molecular screening for acute febrile illnesses and respiratory infections. The results showed that malaria (particularly in Kédougou) and upper respiratory tract infections remain problematic. Suspected dengue samples were tested on board during the dengue outbreaks in 2017 (882 tests; 128 confirmed cases) and 2018 (1736 tests; 202 confirmed cases). Conclusion: The MBS-Lab is an innovative solution for outbreak response, even in remote areas. The study demonstrated successful local ownership and community engagement. The MBS-Lab can also be considered an open mobile healthcare platform that offers various opportunities for field-deployable, point-of-care technologies for surveillance programmes.

Background Combined malaria control interventions have reduced mortality rate, number of clinical cases and parasite prevalence across Africa between 2000 and 2015. As a consequence, Plasmodium infections have become mostly asymptomatic and often sub-microscopic in many endemic areas. The present study aims to evaluate the contribution of asymptomatic sub-microscopic P. falciparum carriage on antibody responses against malaria antigens. Methods A total of 353 samples from a cross-sectional sampling conducted in Ndiop (Senegal) in 2016 were tested by qPCR and microscopy to determine parasite prevalence. Plasmodium falciparum positive samples were genotyped using msp-2 marker. The IgG seroprevalence against crude schizont antigen of a local P. falciparum strain, tested by ELISA, was compared to parasite prevalence. An age-matched, under 10 years, 10-15 and over 15 years cohort of 110 positive and negative qPCR samples were used to determine the impact of sub-microscopic carriage on IgG and IgM antibody responses against schizont extract and MSP3 recombinant antigen. Results The microscopic diagnosis was negative for all thick smears defining a study cohort of submicroscopic asymptomatic individuals with an overall parasite prevalence of 22.37% (79/353) by qPCR. Submicroscopic infections were associated with significant lower IgG responses in qPCR positive samples for individuals over 15 years of age, for both crude schizont (p=0.021) and MSP3 recombinant antigen (p=0.035). IgM responses were significantly higher (p=0.034) in children under 10 years, showing their susceptibility to primary infection. Above 15 years of age, a significant difference was found between parasite prevalence of 9.3% (33/353) and IgG seroprevalence of 23.8% (84/353) (p=0.01). The overall genetic diversity detected is characterized by a complexity of infection (COI) and a number of genotypes of 2.4 and 17, respectively.Conclusion Asymptomatic submicroscopic P. falciparum carriage is prevalent in the study area and is associated with immune tolerance to parasites in adults. The difference between parasite prevalence and IgG seroprevalence results from long-lived IgG in adults point out attention for elders in endemic areas as a stable parasite reservoir. In this context, mass molecular detection followed by treatment could be a valuable method for achieving elimination.

Background Ongoing efforts to fight Plasmodium falciparum malaria has reduced malaria in many areas, but new tools are needed to monitor further progress, including indicators of decreasing exposure to parasite infection. Sero-surveillance is considered promising to monitor exposure, transmission and immunity. Methods IgG responses to three antigen biomarkers were evaluated in a retrospective study involving: (i) surveys of 798 asymptomatic villagers from 2 Senegalese endemic settings conducted before 2002 and after the 2013 intensification of control measures, and (ii) in 105 symptomatic individuals from different settings in Côte d’Ivoire. Response to up to eight P. falciparum antigens, including recombinant MSP1p9 antigen and LSA141 peptide, were analysed using multiplex technology and responses to whole P. falciparum schizont extract (SE, local strain adapted to culture) were measured by ELISA. Results MSP1p9 and LSA141 IgG responses were shown to be relevant indicators monitoring immune status in the different study sites both from Côte d’Ivoire and Senegal. Between 2002 and 2013, individuals participating in both studies showed higher decline of sero-positivity in young ( 15 years: no decline to 15%) individuals from Dielmo and Ndiop. A mathematical sero-catalytic model from the complete Dielmo/Ndiop survey was used to reconstruct declining levels of sero-positivity in more detail, demonstrating that anti-SE seroprevalence levels most accurately reflected malaria exposure in the two villages. Conclusion For standard screening of population immune status at sites envisaging elimination, the use of ELISA-based assays targeting selected antigens can contribute to provide important epidemiologic surveillance data to aid malaria control programmes.

Additional file 1: Table S1. Estimates from sero-catalytic models. Statistical inference was implemented in a Bayesian framework with uniform priors. Parameters are presented as median and 95% credible intervals of the estimated posterior distributions.

Background Malaria in Senegal is due essentially to infections by Plasmodium falciparum and, to a lesser extent to Plasmodium malariae and Plasmodium ovale. By the use of molecular methods, detection of Plasmodium vivax has been recently reported in the region of Kedougou, raising the question of appraisal of its potential prevalence in this setting. Methods A retrospective serological study was carried out using 188 samples taken from 2010 to 2011 in a longitudinal school survey during which 48 asymptomatic children (9–11 years) were recruited. Four collections of samples collected during two successive dry and rainy seasons were analysed for antibody responses to P. vivax and P. falciparum. Recombinant P. falciparum and P. vivax MSP1 antigens and total P. falciparum schizont lysate from African 07/03 strain (adapted to culture) were used for ELISA. Nested PCR amplification was used for molecular detection of P. vivax. Results A surprising high prevalence of IgG responses against P. vivax MSP1 was evidenced with 53% of positive samples and 58% of the individuals that were found positive to this antigen. There was 77% of responders to P. falciparum outlined by 63% of positive samples. Prevalence of responders did not differ as function of seasons. Levels of antibodies to P. falciparum fluctuated with significant increasing between dry and rainy season (P

Additional file 4: Fig. S2. Box plot of parasitaemia in samples from asymptomatic children infected with Plasmodium falciparum. The eight outliers removed prior to comparison of monoclonal and polyclonal infections are shown.

IntroductionSubmicroscopic Plasmodium infections are common in malaria endemic countries, but very little studies have been done in Senegal. This study investigates the genetic diversity and complexity of submicroscopic P. falciparum infections among febrile patients in low transmission areas in Senegal.Materials and methodsHundred and fifty blood samples were collected from febrile individuals living in Dielmo and Ndiop (Senegal) between August 2014 and January 2015, tested for microscopic and sub-microscopic P. falciparum infections and characterized for their genetic diversity and complexity of infections using msp-1 and msp-2 genotyping.ResultsSubmicroscopic P. falciparum infections were 19.6% and 25% in Dielmo and Ndiop, respectively. K1 and 3D7 were the predominant msp-1 and msp-2 allelic types with respective frequencies of 67.36% and 67.10% in microscopic isolates and 58.24% and 78% in submicroscopic ones. Frequencies of msp-1 allelic types were statistically comparable between the studied groups (p>0.05), and were respectively 93.54% vs 87.5% for K1, 60% vs 54.83% for MAD20 and 41.93% vs 22.5% for RO33 while frequencies of msp-2 allelic types were significantly highest in the microscopy group for FC27 (41.93% vs 10%, Fisher's Exact Test, p = 0.001) and 3D7 (61.29% vs 32.5%, Fisher's Exact Test, p = 0.02). Multiplicities of infection were lowest in submicroscopic P. falciparum isolates.ConclusionsThe study revealed a high submicroscopic P. falciparum carriage among patients in the study areas, and that submicroscopic P. falciparum isolates had a lower genetic diversity and complexity of malaria infections.

International audience; Background: In the southeastern Senegal, the report of Plasmodium vivax infections among febrile patients in Kedougou constitutes a new emerging health problem.Methods: Samples from 48 asymptomatic schoolchildren sampled twice a year over 2 years were used to explore the reservoir of P. vivax parasite infections in this region. Both Duffy genotyping and Plasmodium species diagnostic assays were performed.Results: PCR assays detected Plasmodium genomic DNA in 38.5% (74/192) of samples. Pure P. falciparum and P. vivax infections were identified in 79.7% (59/74) and 20.3% (15/74) of samples, respectively. All schoolchildren were classified as Duffy-negative by genotyping. P. vivax infections were detected in five children: in two children during both years, in one child in 2010 and on May 2011, and only in 2010 for the remaining two children.Conclusions: This unexpectedly high proportion of P. vivax infections in asymptomatic Duffy-negative children highlights to consider vivax malaria as an emerging problem in Senegal.

International audience; Dramatic changes in transmission intensity can impact Plasmodium population diversity. Using samples from 2 distant time-points in the Dielmo/Ndiop longitudinal cohorts from Senegal, we applied a molecular barcode tool to detect changes in parasite genotypes and complexity of infection that corresponded to changes in transmission intensity. We observed a striking statistically significant difference in genetic diversity between the 2 parasite populations. Furthermore, we identified a genotype in Dielmo and Ndiop previously observed in Thies, potentially implicating imported malaria. This genetic surveillance study validates the molecular barcode as a tool to assess parasite population diversity changes and track parasite genotypes.

Background Evaluation of local Plasmodium falciparum malaria transmission has been investigated previously using the reversible catalytic model based on prevalence of antibody responses to single antigen to estimate seroconversion rates. High correlations were observed between seroconversion rates and entomological inoculation rates (EIR). However, in this model, the effects of malaria control interventions and clinical episodes on serological measurements were not assessed. This study monitors the use of antibody responses to P. falciparum crude extracts for assessing malaria transmission, compares seroconversion rates estimated from longitudinal data to those derived from cross-sectional surveys and investigates the effects of malaria control interventions on these measures in an area of declining malaria transmission. In addition, the validity of this model was evaluated by comparison with the alternative model. Methods Five cross-sectional surveys were carried out at the end of the wet season in Dielmo, a malaria-endemic Senegalese rural area in 2000, 2002, 2008, 2010 and 2012. Antibodies against schizonts crude extract of a local P. falciparum strain adapted to culture (Pf 07/03) were measured by ELISA. Age-specific seroprevalence model was used both for cross-sectional surveys and longitudinal data (combined data of all surveys). Results A total of 1504 plasma samples obtained through several years follow-up of 350 subjects was used in this study. Seroconversion rates based on P. falciparum schizonts crude extract were estimated for each cross-sectional survey and were found strongly correlated with EIR. High variability between SCRs from cross-sectional and longitudinal surveys was observed. In longitudinal studies, the alternative catalytic reversible model adjusted better with serological data than the catalytic model. Clinical malaria attacks and malaria control interventions were found to have significant effect on seroconversion. Discussion The results of the study suggested that crude extract was a good serological tool that could be used to assess the level of malaria exposure in areas where malaria transmission is declining. However, additional parameters such as clinical malaria and malaria control interventions must be taken into account for determining serological measurements for more accuracy in transmission assessment. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-2052-0) contains supplementary material, which is available to authorized users.

Additional file 1. This supporting information contains statistical details. It includes information about models used in the analysis and the estimates methodologies.

Background In the progress towards malaria elimination, the accurate diagnosis of low-density asymptomatic infections is critical. Low-density asymptomatic submicroscopic malaria infections may act as silent reservoirs that maintain low-level residual malaria transmission in the community. Light microscopy, the gold standard in malaria diagnosis lacks the sensitivity to detect low-level parasitaemia. In this study, the presence and prevalence of submicroscopic Plasmodium carriage were investigated to estimate the parasites reservoir among asymptomatic individuals living in low transmission areas in Dielmo and Ndiop, Senegal during the dry season. Methods A total of 2,037 blood samples were collected during cross-sectional surveys prior the malaria transmission season in July 2013 (N = 612), June 2014 (N = 723) and June 2015 (N = 702) from asymptomatic individuals living in Dielmo and Ndiop, Senegal. Samples were used to determine the prevalence of submicroscopic Plasmodium carriage by real time PCR (qPCR) in comparison to microscopy considered as gold standard. Results The prevalence of submicroscopic Plasmodium carriage was 3.75% (23/612), 12.44% (90/723) and 6.41% (45/702) in 2013, 2014 and 2015, respectively. No Plasmodium carriage was detected by microscopy in 2013 while microscopy-based prevalence of Plasmodium carriage accounted for only 0.27% (2/723) and 0.14% (1/702) in 2014 and 2015, respectively. Plasmodium falciparum accounted for the majority of submicroscopic infections and represented 86.95% (20/23), 81.11% (73/90) and 95.55 (43/45) of infections in 2013, 2014 and 2015 respectively. Conclusion Low-density submicroscopic asymptomatic Plasmodium carriage is common in the study areas during the dry season indicating that traditional measures are insufficient to assess the scale of parasite reservoir when transmission reaches very low level. Control and elimination strategies may wish to consider using molecular methods to identify parasites carriers to guide Mass screening and Treatment strategies.

BackgroundThe global decline of malaria incidence over the past decade has led to the thought that elimination could be achieved. This has resulted in an increased interest to design strategies to target the hidden reservoir of asymptomatic infections among populations and interrupt on-going residual local malaria transmission. This study explored the reservoir of asymptomatic Plasmodium infections and its relationship with subsequent clinical malaria infections in low-transmission areas in Senegal.MethodsCross-sectional surveys were carried out in 2013, 2014, 2015, and 2016 and combined with longitudinal follow-up to determine and geolocalise both asymptomatic and clinical malaria episodes in Dielmo and Ndiop, Senegal. The prevalence of asymptomatic Plasmodium carriage in the community was investigated by real-time PCR while clinical malaria attacks were identified at health facilities during the transmission season. All households were georeferenced to spatially map asymptomatic and clinical infections.ResultsThe study revealed substantial asymptomatic infections with average parasite carriage of 8.11% and 7% in Dielmo and Ndiop, respectively. P. falciparum accounted for most asymptomatic infections (more than 90%). In Dielmo, 95% of asymptomatic infections clustered within the same geographical areas while infections were disparate in Ndiop. Preliminary fine-scale mapping of asymptomatic and clinical malaria infections identified clusters of higher malaria incidence interpreted as foci of transmission across the four-year study period with 95%–98% of clinical infections occurring in households where an asymptomatic malaria infection existed.ConclusionThis study revealed substantial asymptomatic Plasmodium infections in both settings throughout the four-year study period and spatial clusters of malaria infections at the microepidemiological level. Together, these findings could offer a feasible approach for a rational targeting of malaria control interventions to achieve elimination.

The intracellular malaria parasites extensively modify host erythrocytes to allow nutrient uptake, ensure homeostasis, and evade the host's immune response. To achieve this, the parasite exports several proteins to the erythrocyte surface. In Plasmodium falciparum, the parasite responsible for the most severe form of human malaria, three major variant surface antigen families - PfEMP1, STEVOR, and RIFIN - have been implicated in contributing to immune evasion, parasite sequestration, and parasite-mediated rosetting of uninfected erythrocytes. Sequestration and rosetting have been linked to parasite-mediated pathology, making the variant surface antigens of P. falciparum major virulence factors. Here we review our current understanding of rosetting mechanism, recent findings of STEVOR, RIFIN-mediated rosetting, and their implication on the severity and pathology of the disease.

Background Concurrent malaria and arbovirus infections are common and represent an important public health concern in regions where both diseases are endemic. The present study investigates the genetic diversity and complexity of Plasmodium falciparum infection in concurrent malaria-arbovirus infections in Kedougou region, southeastern Senegal. Methods Parasite DNA was extracted from 60 to 27 sera samples collected from P. falciparum isolates of malaria and concurrent malaria-arbovirus infected patients, respectively, and followed by PCR-genotyping targeting the msp-1 (block2) and msp-2 (block3) allelic families. Results The mean number of genotype per allelic family was comparable between the two groups. K1 was the predominant msp-1 allelic type both in malaria (94.91 %) and arbovirus-malaria (92.59 %) groups, whereas IC/3D7 was the most prevalent msp-2 allelic type in malaria (94.91 %) and arbovirus-malaria (96.29 %) groups. Frequencies of msp-1 and msp-2 allelic types were statistically comparable between the two groups (Fisher exact test, P > 0.05) and were not associated with age. FC27 was strikingly the least prevalent in both groups and was absent in children under 5 years of age. The proportions of P. falciparum isolates from malaria-infected patients carrying the three msp-1 allelic types (67.44 %) or the two msp-2 allelic types (76.47 %) were significantly higher than those from arbovirus-malaria co-infected patients (Exact binomial test, P

Objectives: Chemoprophylaxis is recommended during pregnancy to reduce the risk of placental infection. However, in areas with increasing drug resistance, it can trigger selection of resistant parasites in the placenta and increase the frequency of placental malaria. The objective of this study was to analyse the selection of drug-resistant parasites in the placenta in an area where chloroquine was still recommended as prophylaxis. Patients and methods: We analysed the polymorphism of parasites from matched placental and venous blood samples at the time of delivery from women in Dakar. Polymorphism of the isolates was studied using nested PCR typing of MSA1 and MSA2 genes, and full sequence of PfCRT exon 2. Results: Of 692 women recruited at delivery, 72 had placental malaria. Two Pfcrt exon 2 genotypes were found, and 86% of the placentas had monoallelelic CVIET infection compared with 39% that had peripheral blood infection. Mixed parasite populations of CVIET/CVMNK occurred in 53% of the peripheral blood samples but only in 7% of the infected placentas. This selection of CVIET in placenta was not related to a decreased polymorphism of the parasites, as a large diversity of MSA1 and MSA2 was found in both placenta and venous blood. This diversity confirms that a multiplicity of circulation isolates can occur at low parasite transmission. msp1 and msp2 genotyping revealed mostly distinct populations of parasites in venous and placental blood. Conclusions: These data suggest that, even in low transmission areas, diverse parasite populations can accumulate in the placenta during pregnancy despite strong selection at the PfCRT locus due to chemoprophylaxis with chloroquine.

Attachment of erythrocytes infected by Plasmodium falciparum to receptors of the microvasculature is a major contributor to the pathology and morbidity associated with malaria. Adhesion is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP-1), which is expressed at the surface of infected erythrocytes and is linked to both antigenic variation and cytoadherence. PfEMP-1 contains multiple adhesive modules, including the Duffy binding-like domain and the cysteine-rich interdomain region (CIDR). The interaction between CIDRα and CD36 promotes stable adherence of parasitized erythrocytes to endothelial cells. Here we show that a segment within the C-terminal region of CIDRα determines CD36 binding specificity. Antibodies raised against this segment can specifically block the adhesion to CD36 of erythrocytes infected with various parasite strains. Thus, small regions of PfEMP-1 that determine binding specificity could form suitable components of an antisequestration malaria vaccine effective against different parasite strains.

FKBP35, FK506 binding protein family member, in Plasmodium species displays a canonical peptidyl-prolyl isomerase (PPIase) activity and is intricately involved in the protein folding process. Inhibition of PfFKBP35 by FK506 or its analogues were shown to interfere with the in vitro growth of Plasmodium falciparum. In this study, we have synthesized adamantyl derivatives, Supradamal (SRA/4a) and its analogues SRA1/4b and SRA2/4c, which demonstrate submicromolar inhibition of Plasmodium falciparum FK506 binding domain 35 (FKBD35) PPIase activity. SRA and its analogues not only inhibit the in vitro growth of Plasmodium falciparum 3D7 strain but also show stage specific activity by inhibiting the trophozoite stage of the parasite. SRA/4a also inhibits the Plasmodium vivax FKBD35 PPIase activity and our crystal structure of PvFKBD35 in complex with the SRA provides structural insights in achieving selective inhibition against Plasmodium FKBPs.

Malaria parasite strains have emerged to tolerate the therapeutic effects of the prophylactics and drugs presently available. This resistance now poses a serious challenge to researchers in the bid to overcome malaria parasitic infection. Recent studies have shown that FK520 and its analogs inhibit malaria parasites growth by binding to FK506 binding proteins (FKBPs) of the parasites. Structure based drug screening efforts based on three-dimensional structural information of FKBPs from Plasmodium falciparum led us to identify new chemical entities that bind to the parasite FKBP35 and inhibit its growth. Our experimental results verify that this novel compound (D44) modulate the PPIase activity of Plasmodium FKBP35 and demonstrate the stage-specific growth inhibition of Plasmodium falciparum strains. Here, we present the X-ray crystallographic structures of FK506 binding domains (FKBDs) of PfFKBP35 and PvFKBP35 in complex with the newly identified inhibitor providing molecular insights into its mode of action.

Achievement of malaria elimination requires development of novel strategies interfering with parasite transmission, including targeting the parasite sexual stages (gametocytes). The formation of Plasmodium falciparum gametocytes in the human host takes several days during which immature gametocyte-infected erythrocytes (GIEs) sequester in host tissues. Only mature stage GIEs circulate in the peripheral blood, available to uptake by the Anopheles vector. Mechanisms underlying GIE sequestration and release in circulation are virtually unknown. We show here that mature GIEs are more deformable than immature stages using ektacytometry and microsphiltration methods, and that a switch in cellular deformability in the transition from immature to mature gametocytes is accompanied by the deassociation of parasite-derived STEVOR proteins from the infected erythrocyte membrane. We hypothesize that mechanical retention contributes to sequestration of immature GIEs and that regained deformability of mature gametocytes is associated with their release in the bloodstream and ability to circulate. These processes are proposed to play a key role in P falciparum gametocyte development in the host and to represent novel and unconventional targets for interfering with parasite transmission.

Antigenic variation at the Plasmodium-infected erythrocyte surface plays a critical role in malaria disease severity and host immune evasion. Our current understanding of the role of Plasmodium variant surface antigens in antigenic variation and immune evasion is largely limited to the extensive work carried out on the Plasmodium falciparum var gene family. Although homologues of var genes are not present in other malaria species, small variant gene families comprising the rif and stevor genes in P. falciparum and the pir genes in Plasmodium vivax, Plasmodium knowlesi and the rodent malaria Plasmodium chabaudi, Plasmodium berghei and Plasmodium yoelii also show features suggesting a role in antigenic variation and immune evasion. In this article, we highlight our current understanding of these variant antigens and provide insights on the mechanisms developed by malaria parasites to effectively avoid the host immune response and establish chronic infection.

BackgroundPlacental malaria (PM) is associated with poor foetal development, but the pathophysiological processes involved are poorly understood. Cyclooxygenase (COX) and lipoxygenase (LOX) which convert fatty acids to prostaglandins and leukotrienes, play important roles in pregnancy and foetal development. COX-2, currently targeted by specific drugs, plays a dual role as it associates with both pre-eclampsia pathology and recovery during infection. The role of COX during PM was questioned by quantifying at delivery COX-1, COX-2, 15-LOX, and IL-10 expression in two groups of malaria infected and uninfected placenta.MethodsPlacental biopsies were collected at delivery for mRNA isolation and quantification, using real time PCR.ResultsCOX-2 and IL-10 mRNAs increased mainly during chronic infections (nine- and five-times, respectively), whereas COX-1 transcripts remained constant. COX-2 over-expression was associated with a higher birth weight of the baby, but with a lower rate of haemoglobin of the mother. It was associated with a macrophage infiltration of the placenta and with a low haemozoin infiltration. In the opposite way, placental infection was associated with lower expression of 15-LOX mRNA. A high degree of haemozoin deposition correlates with low birth weight and decreased expression of COX-2.ConclusionThese data provide evidence that COX-2 and IL-10 are highly induced during chronic infection of the placenta, but were not associated with preterm delivery or low birth weight. The data support the involvement of COX-2 in the recovery phase of the placental infection.