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Summary Merozoite surface proteins (MSPs) are critical for parasite invasion; they represent attractive targets for antibody-based protection against clinical malaria. To identify protection-associated target MSPs, the present study analysed antibody responses to whole merozoite extract (ME) and to defined MSP recombinant antigens in hospitalized patients from a low endemic urban area as a function of disease severity (mild versus cerebral malaria). Sera from 110 patients with confirmed severe cerebral malaria (CM) and 91 patients with mild malaria (MM) were analysed (mean age = 29 years) for total and subclass immunoglobulin (Ig)G to ME and total IgG to MSP1p19, MSP2, MSP3, MSP4 and MSP5 by enzyme-linked immunosorbent assay (ELISA). Functional antibody responses were evaluated using the antibody-dependent respiratory burst (ADRB) assay in a subset of sera. There was a trend towards higher IgG1 and IgG4 levels to ME in CM compared to MM; only ME IgM responses differed significantly between fatal and surviving CM patients. Increased prevalence of IgG to individual MSPs was found in the CM compared to the MM group, including significantly higher levels of IgG to MSP4 and MSP5 in the former. Sera from fatal (24·5%) versus surviving cases showed significantly lower IgG to MSP1p19 and MSP3 (P < 0·05). ADRB assay readouts correlated with high levels of anti-MSP IgG, and trended higher in sera from patients with surviving compared to fatal CM outcome (P = 0·07). These results document strong differential antibody responses to MSP antigens as targets of protective immunity against CM and in particular MSP1p19 and MSP3 as prognostic indicators.

Background In low-malaria–transmission areas of Madagascar, annual parasite incidence (API) from routine data has been used to target indoor residual spraying at subdistrict commune level. To assess validity of this approach, we conducted school-based serological surveys and health facility (HF) data quality assessments in 7 districts to compare API to gold-standard commune-level serological measures. Methods At 2 primary schools in each of 93 communes, 60 students were randomly selected with parents and teachers. Capillary blood was drawn for rapid diagnostic tests (RDTs) and serology. Multiplex bead-based immunoassays to detect antibodies to 5 Plasmodium falciparum antigens were conducted, and finite mixture models used to characterize seronegative and seropositive populations. Reversible catalytic models generated commune-level annual seroconversion rates (SCRs). HF register data were abstracted to assess completeness and accuracy. Results RDT positivity from 12 770 samples was 0.5%. Seroprevalence to tested antigens ranged from 17.9% (MSP-1) to 59.7% (PF13). Median commune-level SCR was 0.0108 (range, 0.001–0.075). Compared to SCRs, API identified 71% (95% confidence interval, 51%–87%) of the 30% highest-transmission communes; sensitivity declined at lower levels. Routine data accuracy did not substantially affect API performance. Conclusions API performs reasonably well at identifying higher-transmission communes but sensitivity declined at lower transmission levels.

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.

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

Identification of parasite antigens targeted by immune effector mechanisms that confer protection against malaria is important for the design of a multi-component malaria vaccine. Here, the association of antibodies reacting with the Plasmodium falciparum merozoite surface protein-4 (MSP4) with protection against clinical malaria was investigated in a Senegalese community living in an area of moderate, seasonal malaria transmission. Blood samples were collected at the end of an 8-month long dry season without any recorded parasite transmission from 206 residents enrolled in a prospective follow-up study. Active daily clinical monitoring was implemented during the subsequent five months. Entomologic monitoring documented parasite transmission during the first three months of follow-up. Serum IgG levels were determined by ELISA against three MSP4 baculovirus-encoded recombinant protein constructs, namely the full-length MSP4p40, MSP4p30 devoid of a highly polymorphic sequence stretch and the conserved C-terminal EGF-containing MSP4p20, as well as against a merozoite crude extract. Community seroprevalence against all three constructs was quite high, the lowest being against MSP4p30. Seroprevalence and antibody levels against the three MSP4 constructs were age-dependent. IgG1 dominated the anti-MSP4p20 responses, while both IgG1 and IgG3 were observed against MSP4p40. Anti-MSP4 antibodies were associated with the antibody-dependent respiratory burst (ADRB) activity in a functional assay of merozoite phagocytosis by polymorphonuclear cells. Importantly, high antibody levels against each of the three MSP4 constructs at the end of the dry season were associated with reduced morbidity during the subsequent transmission season in an age-adjusted Poisson regression model (IRR = 0.65 [0.50-0.83], P < 0.001 for responses over the median values). These data are consistent with a protective role for the naturally acquired anti-MSP4 antibodies and support further development of MSP4 as a candidate component of malaria vaccine.

Malaria remains a major health problem in sub- Saharan African countries despite substantial decreases in morbidity and mortality due to sustained control programs. Vaccines candidates were mainly tested in rural endemic setting; however increasing proportion of the population is living in urban area. Evaluation of the qualitative or quantitative immune responses to key targets of anti-Plasmodium immunity requires further investigation in urban area. In a cohort of 144 patients with mild malaria living in Dakar, we analyzed IgG responses against target antigens of P. falciparum: CSP, LSA-3NR2 and GLURP by ELISA. A mean age of 15 yrs (4-65 yrs) was found and patients were separated in 59 adults (

Background. Management of clinical malaria requires the development of reliable diagnostic methods and efficient biomarkers for follow-up of patients. Protection is partly based on IgG responses to parasite antigens exposed at the surface of infected erythrocytes (iRBCs). These IgG responses appeared low during clinical infection, particularly in severe disease.Methods. We analyzed the IgG binding capacity to the surface of live erythrocytes infected by knob positive FCR3 strain. Sera from 69 cerebral malaria (CM) and 72 mild malaria (MM) cases were analyzed by ELISA for IgG responses to five antigens from iRBC and by flow cytometry for IgG binding as expressed in labeling index ratio (LIR). The relationship between IgG levels, LIR, parasitemia, age, and the clinical outcomes was evaluated.Results. We found a significant decrease of LIR in adult CM fatal cases compared to surviving patients (p=0.019). In MM, LIRs were correlated to IgG anti-iRBC and anti-PfEMP3/5 levels. In CM, no correlation was found between LIR, IgG levels, and parasitemia.Conclusion. The IgG binding assay was able to discriminate outcome of cerebral malaria cases and it deserves further development as a potential functional-associated assay for symptomatic malaria analysis.

International audience; Background: Serological markers are potentially useful tools for monitoring the progress of malaria control programs , but a better understanding of antibody response dynamics is necessary. The use of a magnetic bead-based immunoassay (MBA) is advantageous compared to ELISA, due to its multiplexing capacity, but limited information is available on the standardization and validation of this assay. Methods: Several parameters for multiplex testing of antibodies to Plasmodium antigens were analysed using a set of 4 antigens and 98 sera from Senegalese rural asymptomatic and urban symptomatic individuals. The 4 antigens included Plasmodium falciparum CSP and PfAMA1 peptides, recombinant P. falciparum MSP4p20 and a Plasmodium malariae CSP (PmCSP) peptide. Comparisons with ELISA were done using MSP4p20 and whole schizont extract (SE) antigens.Results: The use of fewer beads (1000 beads per well instead of 2000) and 5 µg of antigen per 10 6 bead were validated as lower amounts. The use of a carrier protein (BSA) was shown to be critical when using peptides and the effect of a 24 h delayed measures was evaluated (5-25% signal decrease). Analysis of Ab responses showed almost equally high levels and prevalence in all transmission settings. Clear distinctions between rural and urban malaria were noted using PmCSP and SE antigens.Conclusions: This study underlines the importance of further optimization of the MBA technique and highlights the interest of using multistage/multispecies antigens for surveillance of malaria in endemic settings.

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.

International audience; Recent control scale-up has reduced malaria in many areas but new tools are needed to monitor further progress, including indicators of decreasing exposure to parasite infection. Although serology is considered a promising approach in this regard, the serological impact of control interventions has been so far studied using indirect quantification of exposure. Cohort surveys concomitantly recording entomological and malariometric indices have been conducted in two Senegalese settings where supervised control intensification implemented in 2006 shifted malaria from historically holoendemic in Dielmo and mesoendemic in Ndiop to hypoendemic in both settings by 2013. We analyse here serological signatures of declining transmission using archived blood samples. Responses against ten pre-erythrocytic and erythrocytic antigens from Plasmodium falciparum and P. malariae alongside an Anopheles gambiae salivary gland antigen were analysed. Cross-sectional surveys conducted before (2002) and after (2013) control intensification showed a major impact of control intensification in both settings. The age-associated prevalence, magnitude and breadth of the IgG responses to all antigens were village-specific in 2002. In 2013, remarkably similar patterns were observed in both villages, with marginal responses against all parasite antigens in the 0-5y children and reduced responses in all previously seropositive age groups. Waning of humoral responses of individuals who were immune at the time of control intensification was studied from 2006 to 2013 using yearly samplings. Longitudinal data were analysed using the Cochran-Armittage trend test and an age-related reversible catalytic conversion model. This showed that the antigen-specific antibody declines were more rapid in older children than adults. There was a strong association of antibody decline with the declining entomological inoculation rate. We thus identified serological markers of declining exposure to malaria parasites that should help future monitoring of progress towards malaria elimination.

International audience; Background: Advances in malaria control have reduced the burden of disease resulting from exposure to parasite infections. The consequences on naturally acquired immunity are unclear. A magnetic bead‑based immunoassay (MBA) to assess antibody levels in populations living in endemic areas was previously evaluated. In this study, the effect of clinical attacks on immunity was analysed in three sentinel sites of Ivory Coast. Methods: Recombinant proteins or peptides derived from liver or blood stage antigens of Plasmodium falciparum (CSP, LSA1 41 , LSA3, SALSA, PF13‑DBL1α 1 , GLURP, AMA1, MSP1p19, MSP4p20), the CSP of Plasmodium malariae and the salivary glands antigen of Anopheles gambiae (gSG6) were covalently linked to a colour‑coded microsphere (Luminex ™ beads) for the multiplex assay. ELISA was used for whole parasite extract antigen. Blood samples (n = 94) of patients consulting for symptomatic malaria attacks and living in three different malaria endemic settings (rural and periurban) were analysed. Results: Highly variable seroprevalence of antibody responses against parasite antigens was found ranging from 3 (gSG6) to 97 % (MSP4p20). A marked prevalence and significantly higher level of antibodies was found in patients from the rural site (Korhogo), those harbouring the lowest level of parasitaemia. The use of whole schizont extract could not discriminate immunity level, contrary to parasite‑derived recombinant proteins or peptides. Prevalence of responders to LSA1 41 and levels of antibodies to PF13 were significantly different between the three settings. Moreover, the post‑treatment clearance of parasites was clearly associated with a significantly higher level of antibody response for almost 50 % of the parasite antigens tested.Conclusion: The multiplex MBA‑Magpix technology assay provides an accurate high throughput monitoring of parasite‑specific antibodies during symptomatic malaria. The levels of antibody responses may provide a risk criterion with respect to the degree of parasitic infection. Additionally, they can be used as an indicator in the implementation of malaria prevention and local control strategies.

Background Identification of plasmodial antigens targeted by protective immune mechanisms is important for malaria vaccine development. Among functional assays, the neutrophil antibody-dependent respiratory burst (ADRB) induced by opsonized Plasmodium falciparum merozoites has been correlated with acquired immunity to clinical malaria in endemic areas, but the target merozoite antigens are unknown. Here, the contribution of antibodies to the conserved C-terminal domain of the P. falciparum merozoite surface protein-1 (PfMSP1p19) in mediating ADRB was investigated in sera from individuals living in two Senegalese villages with differing malaria endemicity. Methods Anti-PfMSP1p19 antibody levels in sera from 233 villagers were investigated and the involvement of anti-PfMSP1p19 antibodies in ADRB was explored in a subset of samples using (1) isogenic P. falciparum parasite clones expressing P. falciparum or Plasmodium chabaudi MSP1p19; (2) PfMSP1p19-coated plaque ADRB; and, (3) ADRB triggering using sera depleted from PfMSP1p19 antibodies by absorption onto the baculovirus recombinant antigen. Results ADRB activity correlated with anti-PfMSP1p19 IgG levels (P

Pro‐inflammatory cytokines induced by glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum contribute to malaria pathogenesis and hence, the naturally acquired anti‐GPI antibody thought to provide protection against severe malaria (SM) by neutralizing the stimulatory activity of GPIs. In previous studies, the anti‐GPI antibody levels increased with age in parallel with the development of acquired immunity, and high levels of anti‐GPI antibodies were associated with mild malaria (MM) cases. In the present study, the relationship between the levels of pro‐inflammatory cytokines and anti‐GPI IgG antibody responses, parasitemia, and the clinical outcomes were evaluated in SM and mild malaria (MM) patients. Sera from a total of 110 SM and 72 MM cases after excluding of ineligible patients were analyzed for the levels of anti‐GPI antibodies, IgG subclasses, and cytokine responses by ELISA. While the total anti‐GPI antibody levels were similar in overall SM and MM groups, they were significantly higher in surviving SM patients than in fatal SM cases. In the case of cytokines, the TNF‐α and IL‐6 levels were significantly higher in SM compared to MM, whereas the IL‐10 levels were similar in both groups. The data presented here demonstrate that high levels of the circulatory pro‐inflammatory, TNF‐α, and IL‐6, are indicators of malaria severity, whereas anti‐inflammatory cytokine IL‐10 level does not differentiate SM and MM cases. Further, among SM patients, relatively low levels of anti‐GPI antibodies are indicators of fatal outcomes compared to survivors, suggesting that anti‐GPI antibodies provide some level of protection against SM fatality.

International audience; Background: Multiplex assays are currently used to facilitate the evaluation of antibody (Ab) responses to multiple Plasmodium falciparum antigens from large field-based epidemiological studies. The present study aimed at (i) optimizing parameters of a novel cost-effective, compact and reliable magnetic bead-based multiplex immunoassay (MBA) carried out with the MAGPIX ®-Luminex system and (ii) comparing the results with those obtained using standard ELISA technology. Methods: Several MBA parameters including antigen amount for coupling, plasma dilution, type of plates, buffers, washing procedure to minimize bead loss, and bead quantity for testing were optimized. Antibody responses to two recombinant and two peptidic P. falciparum antigens, one Anopheles gambiae salivary gland peptide gSG6 and Bovine Serum Albumin as negative control were tested by MBA and ELISA using sera from 14 villagers from an hyper-endemic Senegalese village. Results: The MBA procedures were developed to reflect responses observed in the standard ELISA protocols used in previous studies. Using the finalized MBA protocol, a strong significant positive correlation (P

Summary: Drug resistance to Plasmodium falciparum contributes to major health problems in central Africa and, as a consequence, poverty. We have analyzed the efficacy of three currently available antimalarial drugs to treat symptomatic, uncomplicated P. falciparum malaria in semiimmune adults living in Bangui, Central Republic of Africa. 210 consecutive individuals were enrolled in the survey, of which 45 were excluded. Those having received dihydroartemisin proved significantly less parasitemic than those having received quinine per os or sulfadoxin-pyrimethamin (χ2 = 16.93 ; p < 0.05), and 75 % recovered in two days compared to 57 and 44 %, respectively. The 25 % who did not recover benefited from a second cure with dihydroartemisin, which proved 100 % efficient. The most accurate protocol remains to be established by analyzing clinical and parasitological data and taking into account the economics of the country.

International audience; Antibodies to Plasmodium falciparum C-terminal merozoite surface protein 1 (PfMSP-1p19) have been correlated with protection against malaria, but this association may apply to many merozoite antigens. To address this question, we conducted a prospective serological study of 205 individuals in an active 5-month clinical survey in a Senegalese village where malaria is mesoendemic. Before the 2000 rainy season, antibody responses specific for recombinant baculovirus PfMSP-1p19 or merozoite extracts were compared with 2 in vitro functional antibody activities (inhibition of parasite grown and erythrocyte invasion) and with the number of clinical episodes during 5 months of follow-up. Antibody levels to PfMSP-1p19 and merozoite extract correlated, respectively, with erythrocyte invasion and parasite growth inhibition. Although antibody levels to both antigen preparations were associated with age, functional parameters were not. High levels of anti-PfMSP-1p19 immunoglobulin G were associated with reduced malaria in an age-adjusted multivariate analysis. These results support baculovirus PfMSP-1p19-based vaccine development.

Sequestration of the mature Plasmodium falciparum forms complicates detection, quantification and molecular analysis of human infections. Whether the circulating parasites represent all or only a subset of co-infecting genotypes is unclear. We have investigated this issue and compared placenta and peripheral blood msp1 and msp2 genotypes in 58 women delivering with an ICT-positive placenta in Guediawaye, Senegal. Most placenta (91%) and blood samples (98%) were multiply infected. Multiplicity of infection was positively correlated in both tissues. However, the placental and circulating genotype profiles differed markedly. Only 10% of matched peripheral blood/placenta samples had identical genotypes, whereas 74% had only partially concordant genotypes, with some alleles detected in both tissues, together with additional allele(s) detected in one tissue only. Eight women (14%) had totally discordant placental and peripheral blood genotypes. Thus, in the vast majority of cases, some sequestered genotypes remain hidden, undetected in the peripheral circulation, indicating that analysis of peripheral parasites generates a partial picture of a P. falciparum infection.

Summary Background A better understanding of the effect of malaria control interventions on vector and parasite populations, acquired immunity, and burden of the disease is needed to guide strategies to eliminate malaria from highly endemic areas. We monitored and analysed the changes in malaria epidemiology in a village community in Senegal, west Africa, over 22 years. Methods Between 1990 and 2012, we did a prospective longitudinal study of the inhabitants of Dielmo, Senegal, to identify all episodes of fever and investigate the relation between malaria host, vector, and parasite. Our study included daily medical surveillance with systematic parasite detection in individuals with fever. We measured parasite prevalence four times a year with cross-sectional surveys. We monitored malaria transmission monthly with night collection of mosquitoes. Malaria treatment changed over the years, from quinine (1990–94), to chloroquine (1995–2003), amodiaquine plus sulfadoxine-pyrimethamine (2003–06), and finally artesunate plus amodiaquine (2006–12). Insecticide-treated nets (ITNs) were introduced in 2008. Findings We monitored 776 villagers aged 0–101 years for 2 378 150 person-days of follow-up. Entomological inoculation rate ranged from 142·5 infected bites per person per year in 1990 to 482·6 in 2000, and 7·6 in 2012. Parasite prevalence in children declined from 87% in 1990 to 0·3 % in 2012. In adults, it declined from 58% to 0·3%. We recorded 23 546 fever episodes during the study, including 8243 clinical attacks caused by Plasmodium falciparum , 290 by Plasmodium malariae , and 219 by Plasmodium ovale . Three deaths were directly attributable to malaria, and two to severe adverse events of antimalarial drugs. The incidence of malaria attacks ranged from 1·50 attacks per person-year in 1990 to 2·63 in 2000, and to only 0·046 in 2012. The greatest changes were associated with the replacement of chloroquine and the introduction of ITNs. Interpretation Malaria control policies combining prompt treatment of clinical attacks and deployment of ITNs can nearly eliminate parasite carriage and greatly reduce the burden of malaria in populations exposed to intense perennial malaria transmission. The choice of drugs seems crucial. Rapid decline of clinical immunity allows rapid detection and treatment of novel infections and thus has a key role in sustaining effectiveness of combining artemisinin-based combination therapy and ITNs despite increasing pyrethroid resistance. Funding Pasteur Institutes of Dakar and Paris, Institut de Recherche pour le Developpement, and French Ministry of Cooperation.

International audience; BACKGROUND: Effective vaccines to combat malaria are urgently needed, but have proved elusive in the absence of validated correlates of natural immunity. Repeated blood stage infections induce antibodies considered to be the main arbiters of protection from pathology, but their essential functions have remained speculative. METHODOLOGY/PRINCIPAL FINDINGS: This study evaluated antibody dependent respiratory burst (ADRB) activity in polymorphonuclear neutrophils (PMN) induced by Plasmodium falciparum merozoites and antibodies in the sera of two different African endemic populations, and investigated its association with naturally acquired clinical protection. Respiratory bursts by freshly isolated PMN were quantified by chemiluminescence readout in the presence of isoluminol, which preferentially detects extra-cellular reactive oxygen species (ROS). Using a standardized, high throughput protocol, 230 sera were analyzed from individuals of all age groups living in meso- (Ndiop) or holo-endemic (Dielmo) Senegalese villages, and enrolled in a cross-sectional prospective study with intensive follow-up. Statistical significance was determined using non-parametric tests and Poisson regression models. The most important finding was that PMN ADRB activity was correlated with acquired clinical protection from malaria in both high and low transmission areas (P = 0.006 and 0.036 respectively). Strikingly, individuals in Dielmo with dichotomized high ADRB indexes were seventeen fold less susceptible to malaria attacks (P = 0.006). Complementary results showed that ADRB activity was (i) dependent on intact merozoites and IgG opsonins, but not parasitized erythrocytes, or complement, (ii) correlated with merozoite specific cytophilic IgG1 and IgG3 antibody titers (P

BackgroundThe time necessary for malaria parasite to re-appear in the blood following treatment (re-infection time) is an indirect method for evaluating the immune defences operating against pre-erythrocytic and early erythrocytic malaria stages. Few longitudinal data are available in populations in whom malaria transmission level had also been measured.MethodsOne hundred and ten individuals from the village of Ndiop (Senegal), aged between one and 72 years, were cured of malaria by quinine (25 mg/day oral Quinimax™ in three equal daily doses, for seven days). Thereafter, thick blood films were examined to detect the reappearance ofPlasmodium falciparumevery week, for 11 weeks after treatment. Malaria transmission was simultaneously measured weekly by night collection of biting mosquitoes.ResultsMalaria transmission was on average 15.3 infective bites per person during the 77 days follow up. The median reappearance time for the whole study population was 46.8 days, whereas individuals would have received an average one infective bite every 5 days. At the end of the follow-up, after 77 days, 103 of the 110 individuals (93.6%; CI 95% [89.0–98.2]) had been re-infected withP. falciparum. The median reappearance time ('re-positivation') was longer in subjects with patent parasitaemia at enrolment than in parasitologically-negative individuals (58 days vs. 45.9; p = 0.03) and in adults > 30 years than in younger subjects (58.6 days vs. 42.7; p = 0.0002). In a multivariate Cox PH model controlling for the sickle cell trait, G6PD deficiency and the type of habitat, the presence of parasitaemia at enrolment and age ≥ 30 years were independently predictive of a reduced risk of re-infection (PH = 0.5 [95% CI: 0.3–0.9] and 0.4; [95% CI: 0.2–0.6] respectively).ConclusionResults indicate the existence of a substantial resistance to sporozoites inoculations, but which was ultimately overcome in almost every individual after 2 1/2 months of natural challenges. Such a study design and the results obtained suggest that, despite a small sample size, this approach can contribute to assess the impact of intervention methods, such as the efficacy vector-control measures or of malaria pre-erythrocytic stages vaccines.

International audience; Glycosylphosphatidylinositol (GPI) membrane anchors of Plasmodium falciparum surface proteins are thought to be important factors contributing to malaria pathogenesis, and anti-GPI antibodies have been suggested to provide protection by neutralizing the toxic activity of GPIs. In this study, IgG responses against P. falciparum GPIs and a baculovirus recombinant MSP1p19 antigen were evaluated in two distinct groups of 70 patients each, who were hospitalized with malaria. Anti-GPI IgGs were significantly lower in patients hospitalized with confirmed cerebral malaria compared to those with mild malaria (P < 0.01) but did not discriminate for fatal outcome. In contrast, a specific marker of the anti-parasite immunity, as monitored by the anti-MSP1p19 IgG response, was similar in both cerebral and mild malaria individuals, although it was significantly lower in a subgroup with fatal outcomes. These results are consistent with a potential anti-toxin role for anti-GPI antibodies associated with protection against cerebral malaria.

an example of the power of re-infection studies for preclinical analysys of vaccine candidates against P falciparum; International audience; Plasmodium falciparum expresses many antigens, which elicit various immune responses in exposed individuals, but no simple surrogate marker for protection has yet been developed. In this prospective survey, we looked for immune responses predictive of protection at various stages of progression from parasite inoculation to onset of disease. We studied 110 Senegalese volunteers from an area in which malaria is mesoendemic after they had received eradication therapy. We evaluated 4 protection-related outcomes (reappearance of parasitemia, duration of asymptomatic carriage, time to first clinical episode, and incidence of clinical episodes) in terms of levels of immunoglobulin G (IgG) against 3 crude parasite extracts and 5 conserved antigens during a 5-month period. Kaplan-Meier estimates and age-adjusted regression models showed these 4 outcomes to be associated with different patterns of IgG response to PfEMP3-cl5 (derived from P. falciparum erythrocyte membrane protein 3), PfEB200, MSP-1(19) (derived from merozoite surface protein-1), [NANP]10, infected red blood cell membrane, and merozoite and schizont extracts. It should, therefore, be possible to develop surrogate markers for each end point on the basis of IgG response to a limited number of conserved antigens.

International audience; Immunity against the blood stage of Plasmodium falciparum malaria is associated with protective-type antibodies of certain classes and subclasses. Field studies have demonstrated the differential regulation of various IgG subclasses depending on the dynamics of parasite transmission and on the immune status of the individuals tested. The intrinsic properties of each IgG subclass has a crucial role in protection, both because immunoglobulin levels are dependent on their production and clearance from blood and because antibodies are actively used for parasite clearance. In vitro models using B cells obtained from P. falciparum-immune adults have enabled study of the production of various antibody subclasses depending on the individual and on the antigens used. Ex vivo and in vitro observations from immune donors have helped to extend our understanding of the development and regulation of the antibody response and to design more effective vaccine strategies.

This study examined the evolution of immunoglobulin (Ig) G1 and IgG3 antibodies against the asexual stage Plasmodium falciparum protein, MSP1(19), before and after a heavy malaria transmission period in clinically immune Senegalese subjects living under different epidemiological conditions. Plasma was tested for antibodies to a yeast-produced, recombinant PfMSP1(19) antigen (the Q-KNG allelic variant) that has previously been demonstrated to react with IgG1, IgG3, or both in the majority of these people. Anti-P. falciparum antibodies of the IgG1 and IgG3 subclasses, previously reported to be associated with protection, were shown to evolve independently one from another after the transmission period in both settings. These results suggest differential regulation of MSP1(19)-specific IgG1 and IgG3. The precise role of these antibody isotypes in maintaining malaria immunity remains to be determined.

International audience; The recombinant R23, PfEB200, and GST-5 antigens derive from conserved antigens associated with the Plasmodium falciparum-infected erythrocyte membrane. They were identified as targets of protective antibodies in the Saimiri sciureus model. We have assessed here the humoral response to these antigens in humans. Cross-sectional surveys were conducted in two Senegalese villages with different levels of endemicity. The prevalence of specific IgG and IgM was similar and influenced by age in both localities. The anti-R23 antibodies decreased after the rainy season, particularly in the children less than ten years old. The anti-PfEB200 response did not show significant seasonal variation. The anti-GST-5 response increased in both the less-than 10-year-old and the greater-than 10-year-old groups after the rainy season in Dielmo, but only in the Ndiop villagers who were more than 10-years-old. Thus, antigen-specific seasonal variations of antibody levels were influenced differently by age in both villages. The isotype distribution was antigen-specific and differed for both seasons.

In malaria endemic areas, pregnancy is assumed to be associated with a specific reduction in immunity to Plasmodium falciparum malaria. To understand some of the mechanisms which underlie such a poor immunity, we have attempted to examine the frequency and distribution of IgM and IgG antibodies to a crude antigenic extract of parasitized erythrocytes and to the merozoite surface protein-1 (MSP1), in a population of mothers compared to control non-pregnant women, all living in Dakar and suburbs. Specifically, this work describes: (i) the responses of mothers and control women; (ii) the balance between IgM and IgG responses; and (iii) responses to malarial antigen and to MSP1. An unexpected balance between P. falciparum-specific IgM and IgG is shown, associated with a substantial increase in anti-MSP1 IgM, and a decrease in anti-MSP1 IgG in parturients.

P. falciparum malaria continues as the serial killer of over a million lives yearly, mainly for children in sub-Saharan Africa. For severe malaria, we are still on the quest for a prognostic marker of fatal outcome. We analysed the association between serum levels of Procalcitonin (PCT), a marker of septic inflammation, and clinical outcome in Senegalese patients admitted with confirmed cerebral malaria in the intensive care facility of Hopital Principal. A total of 98 patients living in the hypoendemic urban area of Dakar, Senegal, were enrolled during transmission seasons. Levels of PCT were compared between surviving vs the 26.5 % fatal cases in blood samples of the 3 days following hospitalisation. Mean PCT levels were elevated in patients with active infection, with a large range of values (0.1 to 280 nanog per mL), significantly higher on day 0 in fatal cases than in surviving (53.6 vs 27.3; P=0.01). No exact individual threshold level could indicate occurrence of fatality, however mortality could be most accurately predicted by PCT level above 69 nanog per ML and there was a very clear different profile of evolution of PCT levels on the 3 days of observation decreasing early from day 1 in surviving patients (P

We report here on functional aspects of squirrel monkey T cells isolated either from peripheral blood mononuclear cells or in the presence of accessory cells or accessory-cell-secreted cytokines. The T-cell response triggered by polyclonal activators was measured through proliferation, T-cell growth factor production and transient expression of receptors to growth factors. We also sought to delineate mechanisms by which antimitogenic molecules might prevent lymphocytes from being able to express "activation antigens" and/or from proliferating. Under these conditions, we mainly explored the inducible IL2/IL2R pathway and phenomena occurring in lymphocyte "non-activation" in terms of early and late cellular events; indeed, defects in the IL2/IL2R pathway represent one of the dysregulation events presumed to occur during the acute phase of human malaria, disease for which. Saimiri monkeys have been selected as experimental hosts.

The squirrel monkey Saimiri sciureus is one of the World Health Organization recommended experimental models of Plasmodium falciparum blood stage infection. Anti-malaria antibodies developed by this host after a drug-controlled infection play an important part in the acquired protection against the P. falciparum blood stages. Furthermore, the use of two anti-Saimiri immunoglobulin (Ig) monoclonal antibodies (mAb) has permitted the differentiation between protective (mAb 3A2/G6) and non-protective (mAb 3E4/H8) antibodies, as shown by transfer experiments to recipient monkeys infected with blood stage parasites. In the present study we have established that protection conferred by the 3A2/G6+ protective Ig preparation is strictly associated with an in vitro opsonic activity. Such an opsonic activity is not detectable in the 3E4/H8+ non-protective Ig population. In addition, results indicate that the 3E4/H8+ non-protective Ig population competes with protective opsonic 3A2/G6+ Ig antibodies when co-incubated with parasitized red blood cells. Thus, it follows that protection can be directly correlated to the quantitative and qualitative fluctuation of the two Ig populations. When challenged with 1 x 10(8) P. falciparum-infected Saimiri red blood cells parasitemia occurred in 5 out of 12 Saimiri who were lacking detectable 3A2/G6+ opsonic antibodies in their sera. By employing antibodies against the human Fc receptor for IgG (Fc gamma R) in an in vitro phagocytic assay, we have been able to show that the principal receptor is Fc gamma RIII. Finally, we also show that in contrast to the situation in man, this receptor is present on circulating monocytes. These findings could lead to a different strategy in designing malaria vaccine candidates and also allow the possibility of predicting the outcome of immunization trials in Saimiri monkeys.

Characterization and functional aspects of squirrel monkey peripheral blood mononuclear cells (PBMC), and mainly T cells, are described in the present paper; this should enable the study of cellular immune responses in an experimental model for malaria. PBMC were obtained from Ficoll-Hypaque gradient separation and fractionated into T cells and non-T cells by means of E-rosetting techniques and adherence to plastic dishes. PBMC subset phenotypes were characterized by means of monoclonal antibodies (mAb) directed against human leukocyte differentiation antigens (Ag), fluoresceinated lectins, anti-surface Ig (squirrel-monkey-specific) antibodies (Ab) and latex bead ingestion assays. PBMC functions were assayed through lymphoblastic transformation tests (LTT) in the presence of either numerous mitogenic, comitogenic and anti-mitogenic lectins or anti-human leukocyte differentiation Ag mAb. We sought to standardize reference values for lymphocyte phenotypes and functions in normal squirrel monkeys (prior to experimental infection). We also present evidence that splenectomy (generally rendered necessary for experimental human malaria infection) performed six months prior to the present investigation did not modify PBMC numbers and functions in the tested animals.

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.